Standard methods for Apis mellifera honey research

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Journal of Apicultural Research

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Standard methods for Apis mellifera honeyresearch

Ligia Bicudo de Almeida-Muradian Ortrud Monika Barth VincentDietemann Michael Eyer Alex da Silva de Freitas Anne-Claire Martel GianLuigi Marcazzan Carla Marina Marchese Carla Mucignat-Caretta AnaPascual-Mateacute Wim Reybroeck M Teresa Sancho amp Joseacute Augusto GasparottoSattler

To cite this article Ligia Bicudo de Almeida-Muradian Ortrud Monika Barth Vincent DietemannMichael Eyer Alex da Silva de Freitas Anne-Claire Martel Gian Luigi Marcazzan Carla MarinaMarchese Carla Mucignat-Caretta Ana Pascual-Mateacute Wim Reybroeck M Teresa Sancho amp JoseacuteAugusto Gasparotto Sattler (2020) Standard methods for Apismellifera honey research Journal ofApicultural Research 593 1-62 DOI 1010800021883920201738135

To link to this article httpsdoiorg1010800021883920201738135

Published online 10 Jun 2020

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REVIEW ARTICLE

Standard methods for Apis mellifera honey research

Ligia Bicudo de Almeida-Muradiana Ortrud Monika Barthb Vincent Dietemannc Michael Eyerc Alex da Silva de Freitasd Anne-Claire Martele Gian Luigi Marcazzanf Carla Marina Marcheseg Carla Mucignat-Carettah Ana Pascual-Mateij Wim Reybroeckk M Teresa Sanchoi andJose Augusto Gasparotto Sattlera

aPharmaceutical Science School University of S~ao Paulo S~ao Paulo Brazil bInstituto Oswaldo Cruz Fiocruz Rio de Janeiro BrazilcAgroscope Swiss Bee Research Centre Bern Switzerland dInstituto de Geociencias Universidade Federal Fluminense Niteroi BrazileSophia Antipolis Laboratory Honeybee Pathology Unit ANSES Sophia France fCREA ndash Council for Agricultural Research and EconomicsResearch Centre for Agriculture and Environment Bologna Italy gRed Bee Honey American Honey Tasting Society Weston FL USAhDepartment of Molecular Medicine University of Padova Padua Italy iDepartamento de Biotecnologıa y Ciencia de los AlimentosNutricion y Bromatologıa Universidad de Burgos Burgos Spain jFacultad de Ciencias de la Salud Universidad Isabel I Spain kResearchInstitute for Agriculture Fisheries and Food Technology and Food Science Unit Melle Belgium

(Received 3 May 2018 accepted 27 February 2020)

Honey is an important food for man and has been used as a natural sweetener since ancient times It is a viscous andaromatic product made by honey bees using the nectar of flowers or honeydew Honey is composed of a complex mix-ture of carbohydrates and other substances such as organic acids amino acids proteins minerals vitamins lipids aromacompounds flavonoids pigments waxes pollen grains several enzymes and other phytochemicals This chapterpresents some properties of Apis mellifera honey as well as the main methods of honey analysis All methods are basedon specialized literature including the Codex Alimentarius AOAC and publications of the International HoneyCommission Herein we describe methods related to honey authenticity botanical origin geographical origin physico-chemical analysis radioentomology pesticide and antibiotic contamination chemotherapeutics and sensory analysis Allmethods are described in a step-by-step model in order to facilitate their use

La miel es un alimento importante para el hombre y se ha utilizado como edulcorante natural desde la antigeurouedad Esun producto viscoso y aromatico hecho por las abejas de la miel usando el nectar de las flores o el rocıo de miel Lamiel esta compuesta por una mezcla compleja de carbohidratos y otras sustancias como acidos organicos aminoacidosproteınas minerales vitaminas lıpidos compuestos aromaticos flavonoides pigmentos ceras granos de polen variasenzimas y otros fitoquımicos En este capıtulo se presentan algunas propiedades de la miel de Apis mellifera ası comolos principales metodos de analisis de la miel Todos los metodos se basan en literatura especializada incluido el CodexAlimentarius AOAC y publicaciones de la Comision Internacional de la Miel A continuacion describimos metodos rela-cionados con la autenticidad de la miel el origen botanico el origen geografico el analisis fisicoquımico la radio-entomologıa la contaminacion por pesticidas y antibioticos la quimioterapia y el analisis sensorial Todos los metodosse describen en un modelo paso a paso para facilitar su uso

蜂蜜是人类重要的食品自古以来就被用作天然甜味剂它是蜜蜂使用花蜜或蜜露酿造而成的粘性芳香产品蜂蜜由碳水化合物和其他物质（例如有机酸氨基酸蛋白质矿物质维生素脂质香气化合物类黄酮色素蜡花粉粒几种酶和其他植物化学物质）组成的复杂混合物本章介绍西方蜜蜂蜂蜜的一些特性以及主要分析方法所有方法均基于专业文献包括食品法典 AOAC和国际蜂蜜委员会的出版物在此我们描述与蜂蜜真实性蜜源植物地理来源理化分析放射昆虫学农药和抗生素污染化学治疗和感官分析有关的方法为了便于使用所有方法都采用分步描述的方式加以介绍

Keywords Apis mellifera honey physicochemical analysis honey authenticity botanical and geographical origindiagnostic radioentomology pesticides sensory antibiotics and chemotherapeutics

Corresponding author Email ligiabiuspbrPlease refer to this paper as de Almeida-Muradian L B Barth O M Dietemann V Eyer M da Silva de Freitas A MartelA-C Marcazzan G L Marchese C M Mucignat-Caretta C Pascual-Mate A Reybroeck W Sancho M Teresa amp Sattler J AG (2020) Standard methods for Apis mellifera honey research In V Dietemann P Neumann N Carreck amp J D Ellis (Eds) TheCOLOSS BEEBOOK ndash Volume III Part 2 Standard methods for Apis mellifera product research Journal of Apicultural Research 59(3) 1ndash62httpsdoiorg1010800021883920201738135

2020 International Bee Research Association

Journal of Apicultural Research 2020Vol 59 No 3 1ndash62 httpsdoiorg1010800021883920201738135

Table of contents

PageNo

1 Introduction 6

2 Honey authenticitymdashbotanicalgeographical and entomo-logical origin

6

21 Botanical origin 6

211 Pollen grains 6

212 Other structured elements 7

22 Methodology to prepare honey sedi-ment following Louveauxet al (1970a)

7

23 The acetolysis technique 7

24 Additional techniques 7

25 Honey sample evaluation 8

251 Super and subrepresentation of pol-len types

8

252 Monofloral and heterofloral honeys 9

253 Honeydew 9

26 Geographical origin 9

27 Entomological origin 9

3 Standard honey physicochemicalanalysis for nutritional compos-ition chemical composition andantioxidant activity

9

31 Water content determin-ation (moisture)

10

311 Principle 10

312 Apparatus and reagents 10

313 Procedure 10

3131 Samples and standards preparationand determination

10

32 Sugar content determination(HPLC method)

10


322 Procedure 11

3221 Sample preparation and determination 11

3222 Chromatographic conditions 11

3223 Calculation and expression of results 11

33 Reducing sugars 12


Table of contents

PageNo

332 Procedure 12

3321 Solutions preparation 12

3322 Standardization of Fehling solutions 12

3323 Preparation of the sample 12

3324 Titration 12

3325 Calculation 12

34 Apparent sucrose 13


342 Procedure 13

3421 Sodium hydroxide solution (5 molL) 13

3422 Sample preparation 13

35 Proteins (proline) 13

351 Principle 13


353 Procedure 14

3531 Preparation of the sample solution 14

3532 Determination 14


36 Vitamins (HPLC) 14


362 Procedure 15


3622 Standards preparation 15

3623 Chromatographic analysis 15

37 Minerals 15


372 Procedure 16

3721 Sample extraction 16

2 L B de Almeida-Muradian et al

Table of contents

PageNo


38 Calories 16

39 Hydroxymethylfurfural 17

391 Principle 17


393 Procedure 17

3931 Samples standards preparation anddetermination

17

3932 Calculations 17

310 Ashes 18

3101 Principle 18

3102 Apparatus 18

3103 Procedure 18


18


311 Free acidity 18

3111 Principle 18


3113 Procedure 18

31131 Samples and standards preparation 18


312 Insoluble solids 18


3122 Procedure 19

3123 Calculation 19

313 Diastase activity 19


3132 Standard preparation 19

3133 Experimental procedure 20

314 Electrical conductivity 20

Table of contents

PageNo

3141 Principle 20


3143 Procedure 20




315 Trolox equivalent antioxidant cap-acity (TEAC)

21


3152 Solutions 21

31521 Preparing the solutions 21

31522 Generation and dilution of ABTS rad-ical cation (ABTSthorn)

21




316 Color of honey by theCIELab system

22

3161 Apparatus 22

3162 Procedure 22

4 Investigating honey productionwith diagnostic radioentomology

23

41 Experimental conditions andmaterial required to conduct diag-nostic radioentomology trials

23

411 Hive to scanner size ratio 23

412 Material to be scanned 23

413 Monitoring the storage of artificial diets 24

414 Studying the storage of natural diets 24

415 Labeling of the diet to increase contrast 24

416 Calibration 24

4161 Artificial diet 25

4162 Natural diet 25

4163 Labeled diet 25

42 Computer tomography scanning 25

Standard methods for Apis mellifera honey research 3

Table of contents

PageNo

421 Handling and transporting of the combs 25

422 Scanning settings 25

423 Performing a scan 25

43 Data analysis 26

431 Analysis of density patterns in individualcell

26

432 Measuring density of cell content 26

433 Measuring cell filling status 27

434 Analyzing content of individual cellsover time

27

435 Spatial analyses at comb level 28

44 Pros and cons 28

45 Perspectives 28

5 Pesticide residues in honey 29

51 Chemical families of pesticides 29

511 Herbicides 29

512 Fungicides 29

513 Insecticides and acaricides 29

52 Analytical methods 29

521 Sampling 30

522 General requirements for pesticide resi-due analyses

30

523 Sample preparation (extraction andclean-up)

32

5231 Liquid-liquid extraction (LLE) 32

5232 Solid phase extraction (SPE) and dis-persive solid phase extraction (dSPE)

32

5233 Quick Easy Cheap Effective Ruggedand Safe (QuEChERS)

33

5234 Solid phase microextraction (SPME) 33

5235 Stir bar sorptive extraction (SBSE) 35

Table of contents

PageNo

5236 Dispersive liquid-liquid microextrac-tion (DLLME)

36

524 Chromatographic detection 36

6 Antibiotics and chemotherapeu-tics in honey

37

61 Legislation regarding residues ofveterinary drugs in honey

38

62 Determination of residues of antibi-otics and chemotherapeuticsin honey

39

621 Sampling 39

622 Sample pretreatment 39

623 General remarks for honey testing onantimicrobial residues

39

624 Microbiological screening tests 39

63 Immunological and receptor assays 41

631 Lateral flow devices 41

632 Enzyme-linked immunosorbentassays (ELISA)

43

633 Enzyme-linked aptamer assays (ELAA) 43

634 Radio-labeled receptorantibody techni-ques (Charm II tests)

43

635 Biochip-based methods 45

636 Methods using MIPs and otherimmunotechniques

45

64 Physico-chemical methods (chroma-tographic techniques)

45

7 Standard method for honey sen-sory analysis

49

71 Methods 49

72 General requirements 49

721 Test room 49

722 Selection and training of assessors andchoice of panel

50

723 Product storage 50

724 Preparation and presentation ofthe samples

50

7241 Sampling glasses 50


Table of contents

PageNo

7242 Preparation of the samples 50

73 Tasting procedures 51

731 Guidelines 51

7311 Session protocol 51

732 Evaluation of the visual characteristics 51

733 Evaluation of the olfactory characteristics 51

734 Evaluation of the olfactory-gustatory andtactile characteristics

51

Table of contents

PageNo

74 Descriptive semiquantita-tive analysis

51

741 Appearance and visual descriptors 51

742 Olfactory descriptors 51

743 Flavor descriptors 53

744 Texture and other tactile descriptors 54

Acknowledgments 54

References 54


1 Introduction

The honey chapter is divided into seven parts describingthe main properties of honey as well as their methodsof analysis Each method is described step-by-step Theauthors come from many countries such as BelgiumBrazil France Italy Spain and Switzerland with manyof the authors being members of the InternationalHoney Commission Honey is the most important prod-uct from the apiary and is consumed globally making iteconomically important (Almeida-Muradian et al 2012)As the only natural sweetener honey historically wasan important food for man Honey was mentioned inmany ancient cultures (the holy books of ancient India(the Vedas) book of songs Shi Jing (written in the sixthcentury BC in ancient China) the Holy Bible just toname a few) and depicted in many wall drawings inancient Egypt Rome (mentioned by the writers VergilVarro and Plinius) and also in medieval high cultures Inancient Greece the honey bee a sacred symbol ofArtemis was an important design on Ephesian coins foralmost six centuries (Bogdanov 2011)

Honey is a viscous and aromatic product widelyappreciated by man and prepared by honey bees(Hymenoptera Apidae Apis spp) from the nectar offlowers or honeydew the latter being producedthrough an intermediary generally an aphid(Ouchemoukh et al 2007 Almeida-Muradian et al2013 Ferreira et al 2009) This natural product is gen-erally composed of a complex mixture of carbohydratesand other less frequent substances such as organicacids amino acids proteins minerals vitamins lipidsaroma compounds flavonoids vitamins pigmentswaxes pollen grains several enzymes and other phyto-chemicals (Almeida-Muradian et al 2013 Almeida-Muradian amp Penteado 2015 Gomes et al 2010Lazarevic et al 2010)

All methods related to honey research cited hereinare based on the specialized literature and the authorsrecommend reading the primary references such asCodex Alimentarius Association of Official AgriculturalChemists (AOAC) and International Honey Commissionpublications Herein we include the main methodsrelated to research with honey from the hive to the finalproduct consumed

2 Honey authenticitymdashbotanical geographicaland entomological origin

Pollen analysis is the initial technique used to determinethe botanical origin of the honey produced by beesThis knowledge enables one to characterize the honeyproducing area by means of pollen grain identificationApis and Meliponini bees show different preferenceswhen foraging plant products

The management of honey harvesting must followquality procedures aiming not only at its efficient col-lection but mainly at maintaining its original

characteristics and the quality of the final product Thebeekeeper when handling the harvest must be wearappropriate clothing for beekeeping It is recommendednot to collect honey on rainy days or with high relativehumidity as it could lead to an increase in the moisturecontent of the product Harvesting should take placeselectively during the opening of the beehive The bee-keeper must inspect each frame giving priority toremoving only the frames that have at least 90 of theiroperculated storage cells this being indicative of thematurity of the honey in relation to the percentage ofmoisture In order to guarantee the quality of the finalproduct during the extraction of honey all the equip-ment and utensils used in the various stages of handlingmust be specific to this activity and there is no roomfor adaptation In the case of equipment and utensilsthat will have direct contact with the product all mustbe stainless steel specific for food products

Pollen analysis is the initial technique used to determinethe botanical origin of the honey produced by bees Thisknowledge enables one to characterize the honey produc-ing area by means of pollen grain identification Apis andMeliponini bees show different preferences when foragingplant products For palynological analysis honey samples ofApis have to be received in glass vials and kept at roomtemperature In case of stingless bees the honey has to bekept in a refrigerator at about 8 C to avoid fermentation

For more details in sampling please seeldquoSamplingrdquo sections

21 Botanical origin

Pollen grains and other structured elements that com-pose honey sediments are indicative of the botanicalorigin (Barth 1989 Vit et al 2018) A complete palyno-logical analysis reveals the real provenance of the rawmaterial Pollen grains are introduced into honey by dif-ferent means (Barth 1989) Besides the nectariferousplants that secrete nectar in addition to producing pol-len the polliniferous plants mainly produce pollen grainsand have low concentrations of nectar (Barth 1989)

211 Pollen grains

The first universal technique aiming at the recognitionand evaluation of the pollen in honey was elaborated bythe French Swiss and Dutch specialists on honey ana-lysis This started with Zanderrsquos work in 1935 whichwas published by Louveaux et al (1970a 1970b) andupdated by Louveaux et al (1978) The latter includesmany standard methods including the acetolysismethod A more recent publication on pollen analysiswas published in von der Ohe et al (2004)

These methodologies are used today For the firsttechnique the pollen grain content is not removedmaking it difficult to observe some of the structuresHowever more of the elements occurring in honeysediments are preserved The acetolysis technique


enables a better visualization of pollen grain structureswhen only the outer pollen grain wall (exine) is pre-served but there is a significant loss of no resistant ele-ments such as oils fungi hyphae yeast insect fragmentsand organic matter (Haidamus et al 2019) A detailedanalysis of the technique established by Louveaux et al(1978) was presented also by von der Ohe et al (2004)considering quantitative and qualitative pollen counting

212 Other structured elements

Honey sediments may contain several types of particlesbesides pollen grains ( Barth 1989 Zander amp Maurizio1975) These structured elements not nectar indicativeshow what happens during honey sampling and storageand reflect honey quality Some adulterants include a varietyof fungal spores yeasts colorless molds and air dispersedspores green algae bacteria insect bristles and scales andpathogenic mites as well as starch and synthetic fibers

22 Methodology to prepare honey sediment followingLouveaux et al (1970a)

(1) Weigh 10 g of honey into a beaker(2) Add 20mL of distilled water and dissolve well by

stirring (10ndash15min)(3) If the honey is crystallized heat the mixture for a

few seconds in a microwave oven to complete thedissolution process of the sugar crystals

(4) Divide the solution eventually between two conicalcentrifuge tubes of 15mL capacity

(5) Centrifuge at least 252 RCF (relative centrifugalforce or G) that according to a centrifuge modelmay comprise on average 1500 rpm (revolutionsper minute) for 15min

(6) Discard the supernatant and rapidly invert thetubes for approximately 30 s before returning tonormal position

(7) Add 10mL of distilled water to the pellet(8) Centrifuge at 1500 rpm for 15min(9) Discard the supernatant and rapidly invert the cen-

trifuge tubes for approximately 30 s before return-ing to normal position

(10) Add 5mL of glycerin water (solution 1 1 with dis-tilled water and glycerin) to the pellet

(11) Wait for 30min and then centrifuge at least1500 rpm for 15min

(12) Discard the supernatant and rapidly invert the tubeonto absorbent paper (never return to the normalposition) to allow all the liquid to drain

(13) Prepare two microscope slides(14) Put a little piece of glycerin jelly (05mm3) onto a

needle tip and collect part of the sediment into thecentrifuge tube

(15) Place the sediment on the center of the micro-scope slide heat gently on a hot plate only to dis-solve the glycerin jelly

(16) Cover with a coverslip and seal with paraffin

(17) The pollen grain identification is based on the ref-erence pollen slides library from regionalcountryvegetation and with the aid of reference catalogs(eg Roubik amp Moreno 1991)

(18) After the identification and palynological analysisthe prepared slides were stored in a pollen library

23 The acetolysis technique

Acetolysis (Erdtman 1952) is an artificial fossilizationmethod to prepare pollen grains in order to get thebest transparency of the grain envelope




(4) Divide the solution evenly between two centrifugetubes of 15mL capacity

(5) Centrifuge at least 1500 rpm for 15min(6) Discard the supernatant and rapidly invert the

tubes for approximately 30 s before returning tonormal position

(7) Add 5mL of acetic acid (32) centrifuge at least1500 rpm for 15min and discard the supernatant

(8) Add the acetolysis mixture (45mL of acetic anhyd-ride (98) and 05mL of sulfuric acid (99))

(9) Heat in a water bath just to 80 C for 3min(10) Centrifuge at least 1500 rpm for 15min(11) Discard the supernatant and add 20mL of dis-

tilled water(12) Wash twice(13) Discard the supernatant and rapidly invert the


(14) Add 5mL of glycerin water (solution 1 1 with dis-tilled water and glycerin) to the pellet Waitfor 30min

(15) Centrifuge at least 1500 rpm for 15min(16) Discard the supernatant and rapidly invert the tube

onto absorbent paper (never return to the normalposition) to allow all the liquid to drain


needle tip and collect part of the sediment in thecentrifuge tube



24 Additional techniques

A number of other techniques to prepare honey sampleswere purposed and published In general they are complexto execute and require a broad assortment of equipment


Filtration (Lutier amp Vaissiere 1993) ethanol dilution(Bryant amp Jones 2001) mid-infrared spectroscopy (Ruoffet al 2006) multivariate analysis Principal ComponentAnalysis (PCA) (Aronne amp de Micco 2010) ultravioletspectroscopy (Roshan et al 2013) isotopes (Wu et al2015) and pollen grain DNA metabarcoding (Hawkinset al 2015) have all been used to determine honey origin

25 Honey sample evaluation

Since the standard work of Louveaux et al (1978) thequalitative and quantitative evaluation of honey samples(Maurizio 1977) has been a subject of many investiga-tions until the data assembly in von der Ohe et al(2004) The reliability of pollen analysis in honey sam-ples was discussed in detail by Behm et al (1996)Pollen coefficients were discussed by the R-values ofhoney pollen coefficients by Bryant and Jones (2001)

251 Super and subrepresentation of pollen types

For each region of honey production there are plantsthat are significant nectar orand pollen producers Thepollen grains present in the honey sediments can revealthe bee foraging behavior In general the number ofpollen grains present on one microscope slide providesinformation to use to assign a frequency class indicatingif the pollen types observed in the honey samples arepredominant (PP) accessory (AP) important minor pol-len (IMP) or minor pollen (MP) (Louveaux et al1970a 1970b)

Few pollen grains found in a honey sample suggeststhat the bees visited plant species of a low pollen pro-duction but that have a high nectar disponibility likeCitrus spp and Croton sp (Table 1 Figure 1) A highquantity of pollen grains from polliniferous plants (egPoaceae and Melastomataceae) can indicate a super

Table 1 Evaluation of a Croton monofloral honey sample a strong nectariferous and subrepresented plant taxonTotal number of pollen grains counted 962 frac14 100 of the pollenTotal of anemophylous pollen counted 842 frac14 875 of the pollenTotal of polliniferous pollen counted 52 frac14 54 of the pollenTotal of nectariferous pollen counted (frac14100) 68 frac14 71 of the pollenCroton 18 frac14 265 of nectariferous pollen a subrepresented taxonSolanum 18 frac14 265 of nectariferous pollenMyrcia 10 frac14 10 of nectariferous pollenFinal evaluation Monofloral honey of Croton (Euphorbiaceae)

Figure 1 (A) Anemophylous pollen of Piper (B) Polliniferous pollen of Melastomataceae (C) Citrus monofloral honey with honeydewcontribution (organic material and spores) (D) Group of algae cells and a broken pollen grain of Croton of a honeydew honey


representation since these species produce a lot of pol-len but little or no nectar (Louveaux et al 1978)

252 Monofloral and heterofloral honeys

(1) Monofloralunifloral honeys contain predominantlypollen grains from an unique plant species (45 ofall nectariferous pollen grains counted) denominateda (ldquopredominant pollen typerdquo PP)

(2) Bifloral honeys contain pollen grains from two plantspecies with a frequency of 15ndash45 per nectarifer-ous species (ldquoaccessory pollen typesrdquo AP)

(3) Heterofloralplurifloral honeys contain pollen grainsfrom three or more nectariferous plant species withfrequencies in the 3ndash15 (ldquoimportant minor pollentypesrdquo IMP) or lt3 (ldquominor pollen typesrdquo MP)(Barth 1989 Louveaux et al 1978) (Figure 2)

253 Honeydew

Bees can use the excreta of aphids (Aphidae) that feedon plant xylem to create honeydew This bee productcan be difficult to classify and presents a high electricalconductivity and mineral salt content Generally pollengrain occurrence is low and a moderate number of fun-gal spores can be observed (Barth 1971 Maurizio1959) (Figure 1)

26 Geographical origin

Pollen grain analysis allows one to determine the nectarorigin of honey The assembly of plant species identifiedby pollen analysis provides information about honeyrsquoslocal or regional origin A reference pollen slide collec-tion (pollen library) must be available in order to com-pare the morphology of pollen grains obtained directlyfrom flowers with the ones obtained in the honey sedi-ment collection The scope of this collection dependsupon the study area and the radius of action of thebees Plant assemblies recognized in the honey sedimentby means of pollen grain morphology inform about thenectariferous and non-nectariferous local and regionalpotential of honey production (Aronne amp de Micco

2010 Consonni amp Cagliani 2008 Persano Oddo ampBogdanov 2004 Salgado-Laurenti et al 2017)

Unfortunately some honeys do not contain much pollenIn these cases the honey origin is difficult to determine Itmay derive from strong nectar secreting plant species suchas those from the Asclepiadaceae Sometimes certain flow-ers secrete abundant nectar that will drip from the flowerinflorescences making it unlikely to contain pollen Thishappens with Dombeya (Sterculiaceae a shrub) and withsome plants that grow in very humid environments

27 Entomological origin

For purposes of this manuscript we are discussing honeyproduced by bees of the family Apidae genus Apis speciesA mellifera There are several subspeciesraces of A melli-fera occurring in different parts of the world (Carreck etal 2013 Meixner et al 2013) Humans have cultivatedthis bee since antiquity given that it has provided a sourceof sugar There are other types of honey producing beesCollectively called stingless bees (also Apidae) these beesoccur mainly in the tribes Meliponini and Trigonini Beesfrom both tribes produce a different type of honey thanthat produced by A mellifera

Stingless bee honey contains less sugar and morewater than the honey produced by Apis spp These beesare very important pollinators in their native habitatsbeing responsible for maintenance of the local vegeta-tion mainly the forests and for seed production Thereare circa 500ndash800 species occurring exclusively in thetropical and sub-tropical Neotropical regions Apis sppand Meliponini spp forage on some of the same plantsbut many different plants as well Species specific prefer-ences for various forage resources depend upon thebee speciesrsquo affinity as well as the flowering vegetationstrata and environmental conditions (Barth et al 2013)

3 Standard honey physicochemical analysis fornutritional composition chemical compositionand antioxidant activity

In this section we present the main physicochemicalanalyses for nutritional composition chemical

Figure 2 (A) Monofloral Myrcia honey (B) Heterofloral honey


composition and antioxidant activity of honey in step-by-step format Review articles on the composition andproperties of honey from A mellifera colonies are pre-sent in the literature (De-Melo et al 2017 Pascual-Mate et al 2018)

We recommend reading of the primary reference onthis topic Codex Standard for Honey (2001)

31 Water content determination (moisture)

Water content of honey is related to the botanical andgeographical origin of the nectar edaphic and climaticconditions season of harvesting manipulation by bee-keepers processingstorage conditions etc (De-Meloet al 2017) It is an important physicochemical param-eter for honey shelf life (Bogdanov 2011) and it nor-mally ranges between 13 and 25 (Uran et al 2017)with the average being about 17 (Fallico et al 2009)Honeys with high moisture content (gt18) have ahigher probability of fermenting upon storage(Bogdanov 2011) while those with a low moisture con-tent (lt15) are likely to granulate

The Council Directive 2001110EC (EuropeanCommission 2001) as well as the Brazilian legislation(Ministerio da Agricultura Pecuaria e Abastecimento2000) recommend the limit of 20 for moisture inhoney samples (Thrasyvoulou et al 2018)

The standard method for honey moisture determinationindicated by Codex Standard for Honey (2001) uses therefractometric method which is simple and reproducible Itis possible to use an Abbe refractometer or digital refrac-tometers for this purpose (AOAC 1992 Bogdanov 20092011) The refractometric method is described here as areference method (AOAC 1990 Bogdanov 2009)

311 Principle

Water content or moisture determined by the refracto-metric method is based on the fact that the refractiveindex increases with solid content In honey the refrac-tion index can be converted in moisture content usingthe Chataway Table (Bogdanov et al 1997)

312 Apparatus and reagents

Water bath or incubator Honey sample Refractometer

313 Procedure

3131 Samples and standards preparation anddetermination(1) If honey is granulated place it in an airtight closed

flask and dissolve crystals in a water bath or incuba-tor at 50 C

(2) Cool the solution to room temperature and stir

(3) Ensure that the prism of the refractometer is cleanand dry

(4) Cover the surface of the prism from the refractom-eter with the sample and read the refractive index

(5) Read the corresponding moisture content from theChataway Table (Table 2) making the temperaturecorrection if necessary

Other methods can be used to determine water contentof honey including Karl Fischer method and nonstandar-dized method Fourier transform infrared spectroscopy(FTIR) (Almeida-Muradian et al 2013 2014 Almeida-Muradian amp Gallmann 2012 Bogdanov 2009)

32 Sugar content determination (HPLC method)

Honey is mainly composed of sugars (60 85 of thetotal volume) Monosaccharides (fructose and glucose)represent about 75 of the sugars found in honeyDisaccharides (sucrose maltose turanose isomaltosemaltulose trehalose nigerose and kojibiose) composeabout 10ndash15 of the sugars The rest is composed of asmall amount of other sugars including trisaccharides(mainly maltotriose and melezitose)

Due to the wide variety of botanical sources thesugar profile presents great variation however it hasbeen demonstrated that among the same species ofplants the profile of sugars and other physicochemicalcomponents can be used as fingerprint for the identifi-cation of the botanical origin of monofloral honeys (ElSohaimy et al 2015 Ruoff et al 2005 2006)

According to the publication of the HarmonizedMethods of the International Honey Commission (2009)the methodologies for sugar determination are grouped asspecific and nonspecific Liquid chromatography - refractiveindex detector (specific methodology) and a methodologyproposed by Lane-Eynon (nonspecific methodology) areamong the most used methodologies for the quantificationof sugars presented in honeys (Bogdanov 2009)

There are other methods described in the literaturefor the determination of sugars These include (1)enzymatic determination (Gomez-Dıaz et al 2012Huidobro amp Simal 1984) (2) high performance liquidchromatography with pulsed amperometric detectorand anion exchange column (HPLC-PAD) (Bogdanov2009 Cano et al 2006 Nascimento et al 2018Ouchemoukh et al 2010) (3) ultra-performance LCwith and evaporative light scattering detector (UPLC-ELSD) (Zhou et al 2014) (4) gas chromatography(GC) (Bogdanov 2009 Ruiz-Matute et al 2007) (5)capillary electrophoresis (CE) (Dominguez et al 2016)(6) electrochemical determination (EL Alami EL Hassaniet al 2018) (7) FTIR (Almeida-Muradian et al 2012Almeida-Muradian Sousa et al 2014 Almeida-Muradian Stramm et al 2014 Anjos et al 2015) and(8) Raman spectroscopy (RAMAN) ( euroOzbalci et al2013) High-Performance Liquid Chromatography withRefractive Index Detection (HPLC-RI) (Bogdanov 2009)is described herein



Apparatus

HPLC system consisting of pump sample applicatortemperature-regulated RI-detector (30 C) and tem-perature-regulated column oven (30 C)

Ultrasonic bath Micro-membrane 045 mm Volumetric flask (100mL) Syringe (1mL) Beaker (50 and 100mL) Pipette (25mL) Sample vials (15mL)

Reagents

Water (HPLC grade) Methanol (HPLC grade) Acetonitrile (HPLC grade) Analytical stainless-steel column containing amine-

modified silica gel with 5 mm particle size 250mm inlength 46mm in diameter

322 Procedure

3221 Sample preparation and determination(1) Weigh 5 g of honey into a beaker and dissolve in

40mL distilled water

(2) Pipette 25mL of methanol into a 100mL volumetricflask and transfer the honey solution quantitativelyto the flask

(3) Complete the volumetric flask with water until 100mL(4) Filter the solution through a 045 lm membrane and

collect the filtered solution into sample vials(5) Store for four weeks in the refrigerator (4 C) or

for six months at freezer (18 C)

3222 Chromatographic conditions Flow rate 13mLmin Mobile phase acetonitrile water (8020 vv) Column and detector temperature 30 C Sample volume 10 mL

3223 Calculation and expression of results Honeysugars can be identified and quantified by comparison oftheir retention times and the peak areas with those ofthe standard sugars (external standard method)

The mass percentage of the sugars (W) is calculatedaccording to the following formula

W frac14 A1 V1 m1 100= A2 V2 m0

W frac14 Mass percentage of sugars (g 100 g)A1 frac14 Peak areas or peak heights of the given sugar

compound in the sample solution (expressed as units ofarea length or integration)

Table 2 Chataway tablemdashrefractive index and moisture content () 20 C (source International Honey Commission 2009)

Water content (g100 g) Refractive index (20 C) Water content (g100 g) Refractive index (20 C)130 15044 190 14890132 15038 192 14885134 15033 194 14880136 15028 196 14875138 15023 198 14870140 15018 200 14865142 15012 202 14860144 15007 204 14855146 15002 206 14850148 14997 208 14845150 14992 210 14840152 14987 212 14835154 14982 214 14830156 14976 216 14825158 14971 218 14820160 14966 220 14815162 14961 222 14810164 14956 224 14805166 14951 226 14800168 14946 228 14795170 14940 230 14790172 14935 232 14785174 14930 234 14780176 14925 236 14775178 14920 238 14770180 14915 240 14765182 14910 242 14760184 14905 244 14755186 14900 246 14750188 14895 248 14745

250 14740


A2 frac14 Peak areas or peak heights of the given sugarcompound in the standard solution (expressed as unitsof area length or integration)

V1 frac14 Total volume of the sample solution (mL)V2 frac14 Total volume of the standard solution (mL)m1 frac14 Mass amount of the sugar in grams in the total

volume of the standard (V2)m0 frac14 sample weight (g)

33 Reducing sugars

Apparent reducing sugars and apparent sucrose contentof honey are most commonly determined for honeyquality control purposes The Lane and Eynon method(Codex Alimentarius Standard 1969) is used for thedetermination of sugars Apparent reducing sugars aredefined as those sugars that reduce a Fehlings reagentunder the conditions specified (Bogdanov et al 1997Bogdanov amp Martin 2002 Bogdanov 2009 Granato ampNunes 2016)


Apparatus

Burette (25mL) Volumetric pipette (2mL) Volumetric flasks (25 and 100mL) Erlenmeyer flasks (250mL) Beakers (50 and 100mL) Pipette (25mL) Heating plate

Reagents

Copper (II) sulfate pentahydrate (CuSO4 5H2O) Potassium sodium tartrate tetrahydrate (C4H4KNaO6

4H2O) Sodium hydroxide (NaOH) Glucose

332 Procedure

3321 Solutions preparationFehlings solution A

(1) Weigh 6928 g of copper sulfate pentahydrate(CuSO45H2O) in a beaker and solubilize by stirringwith distilled water

(2) Transfer to a 1 L volumetric flask

Fehlingrsquos solution B

(1) Weigh 346 g of sodium and potassium double tartrate(C4H4KNaO64H2O) in 300mL of distilled water

(2) Separately dissolve 100 g of sodium hydroxide(NaOH) into 200mL of distilled water in a plasticbeaker and ice bath in the hood

(3) Combine the solutions of sodium and potassium tar-trate and sodium hydroxide in a 1 L volumetric flask

(4) Homogenize by stirring(5) Raise the volume of the flask to full with dis-

tilled water

3322 Standardization of Fehling solutions It isnecessary to standardize Fehling solutions to obtain thecorrection factor

(1) Prepare a 05 (wv) glucose solution(2) Weigh 05 g of glucose in a beaker add distilled

water dissolve and transfer to a 100mL flask(3) Fill the burette with the glucose solution and holder

as described (in section 3324)(4) For each standardization make a minimum of four

titrations Apply the average of these values in theformula to obtain the correction factor (F) of thesolutions of Fehling

F frac14 ethof glucoseTHORN ethaverage of volumes spentTHORN 001

The correction factor corresponds to the amountof sugar required to reduce 10mL of the Fehlingsolutions

3323 Preparation of the sample(1) Dissolve 50 g of honey in distilled water up to

25mL in a volumetric flask (Solution 15)(2) From this homogenized solution transfer 2mL to a

100mL volumetric flask(3) Make up to volume with distilled water and homogenize

(final dilution 1250) The initial diluted solution 15 shouldbe reserved for the analysis of apparent sucrose

3324 Titration The titration is done with a 25mLburette containing the diluted honey (solution 15) andan Erlenmeyer flask with 5mL of Fehling A solution5mL of Fehling B and 40mL of distilled water

(1) Heat the Erlenmeyer to a hot plate using a tripodand a screen

(2) Heat the solution with the heating plate until boil thenthe titration is initiated releasing in one go 5mL of thesugar solution of the burette With the restart of theboiling the Fehling solution becomes reddish but stillwith a lot of blue color (Cu2thorn ions)

(3) The titration should be restarted this time drop-wise under stirring and the color modification beingobserved The reaction is over when the solutionagainst a fluorescent light does not present anyshade or blue reflection being colored an intensered Titration should not exceed 3min

3325 CalculationThe calculation of the percentage of reducing sugars isgiven by the following formula


AR frac14 ethdilution of honey solution frac14 250THORN F 100average of the volumes spent in the titration

ARfrac14 reducing sugarsF frac14 correction factor obtained from the standardiza-

tion of Fehlingrsquos solutions

34 Apparent sucrose

Apparent sucrose content is determined indirectly bycalculating difference in total reducing sugar before andafter inversion of sugars in honey The determination ofapparent sucrose requires the use of acid hydrolysis tobreak the glycoside bonds of the disaccharides thusreleasing reducing sugars such as glucose and fructose(Bogdanov et al 1997 Granato amp Nunes 2016)


Apparatus

100mL glass beaker 100 and 500mL volumetric flask 1 and 2mL volumetric pipettes 25mL burette 250mL Erlenmeyer flask Hot plate

Reagents

Concentrated hydrochloric acid (HCl) Fehling A solution Fehling B solution Sodium hydroxide (NaOH)

342 Procedure

3421 Sodium hydroxide solution (5 molL)(1) Weigh 100 g of NaOH and dissolve in distilled water(2) Increase the volume in a volumetric flask to 500mL

with distilled water(3) Condition the solution in a plastic container

3422 Sample preparation(1) From the 15 diluted honey solution prepared in the

reducing sugars analysis transfer 2mL to a 100mLglass beaker and add 40mL of distilled water and1mL of concentrated HCl

(2) This solution must be boiled cooled neutralized byadding 5molL NaOH until a pH of 7 plusmn 02

(3) Add distilled water up to 100mL in a volumetricflask and make the titration according to the sameprocedure as ldquoReducing sugarsrdquo (3324) The resultis expressed as percentage of total sugars ( AT)

AT frac14 ethdilution of honey solution frac14 250THORN F 100average of the volumes spent in the titration

ATfrac14 total sugars

F frac14 correction factor obtained from the standardiza-tion of Fehlingrsquos solutions

Heating to boiling under strongly acid conditionsensures that the sucrose is hydrolyzed having as prod-ucts the separate molecules of glucose and fructoseThe molecular mass of sucrose (MM frac14 342molL) is95 of the molecular mass of glucose and fructosetogether (MM frac14 360molL) taking into account that5 of the mass refers to water (MM frac14 18molL) whichwas used in the hydrolysis Thus the percentage ofapparent sucrose in a sample is calculated using the fol-lowing formula

apparent sucrose frac14 eth total sugars ndash reducing sugarsTHORN 095

35 Proteins (proline)

The presence of protein in honey is low and variesdepending on the species of honey bee (A mellifera02ndash16 and A cerana 01ndash33) (Lee et al 1998) andthe contact of these with flower pollen honeydew andnectar (Davies 1975)

In the last decades the use of more sensitive androbust methodologies has enabled the identification of alarger quantity of proteins and amino acids in honeysamples from several countries (De-Melo et al 2017)Among the currently used methodologies the mostpreferred is HPLC (Hermosın et al 2003) associatedwith identification by mass spectrometry (Chuaet al 2013)

The classical method to determine protein uses thetotal nitrogen content that is usually calculated by multi-plying the total nitrogen content from the Kjeldahlmethod by a factor of 625 (AOAC 1990) Howeverabout 40ndash80 of the total honeyrsquos nitrogen comes fromthe protein fraction and most of the remainder residesin the free amino acids (Chua et al 2013)

According to the published update HarmonizedMethods of the International Honey Commission in 2009the proline content is recommended for quality control(being considered a criterion of honey ripeness) and it canbe an indicator of sugar adulteration (Bogdanov 2009)Since most of the amino acids are present in trace quanti-ties only proline content is included for the quality param-eters of honey No other methods for proteinquantification are included in the methods referencedabove or in the Codex Standard for Honey (2001)

351 Principle

Proline and ninhydrin form a colored complex Afteradding 2-propanol color extinction of the sample solu-tion and a reference solution at a wavelength maximumis determined The proline content is determined fromthe ratio of both and it is expressed in mgkg Themethod is based on the original method ofOugh (1969)



Apparatus

Spectrophotometer measuring in the range of 500ndash520nm Cells1 cm 20mL tubes with screw cap or stopper 100mL measuring flask and 100mL beaker Water bath

Reagents

Distilled Water Formic acid (HCOOH) 98ndash100 Solution of 3 ninhydrin in ethylene glycol monomethy-

lether (methyl-cellosolve) Proline reference solution Prepare an aqueous proline

stock solution containing 40mg proline to 50mL dis-tilled water (volumetric flask) Dilute 1mL to 25mL(volumetric flask) with distilled water (solution contain-ing 08 mg25mL)

2-propanol solution 50 by volume in water

Proline reference solution

(1) Prepare an aqueous proline stock solution containing40mg proline to 50mL distilled water

(2) Dilute 1mL to 25mL with distilled water (solutioncontaining 08mg25mL)

(3) Prepare a 2-propanol solution 50 by volumein water

353 Procedure

3531 Preparation of the sample solution(1) Weigh to the nearest milligram about 5 g honey

into a beaker and dissolve in 50 mL dis-tilled water

(2) After dissolving transfer all the solution to a 100mLvolumetric flask

(3) Complete the volume with distilled water andshake well

3532 Determination Note that the coefficient ofextinction is not constant Therefore for each series ofmeasurements the average of the extinction coefficientof the proline standard solution must be determined atleast in triplicate

(1) Pipette 05mL of the sample solution in one tube05mL of distilled water (blank test) into a secondtube and 05mL of proline standard solution intoeach of three additional tubes

(2) Add 1mL of formic acid and 1mL of ninhydrin solu-tion to each tube

(3) Cap the tubes carefully and shake vigorously using avortex machine for 15min

(4) Place tubes in a boiling water bath for 15minimmersing the tubes below the level of the solution

(5) Transfer to a water bath at 70 C for 10min(6) Add 5mL of the 2-propanol-water-solution to each

tube and cap immediately(7) Leave to cool and determine the absorbance using a

spectrophotometer for 45min after removing fromthe 70 C water bath at 510 nm using 1 cm cells

Note Adherence to the above times is critical

3533 Calculation and expression of results Prolinein mgkg honey at one decimal place is calculatedaccording to following equation

Proline ethmg=kgTHORN frac14 EsEa

E1E2

80

Es frac14 Absorbance of the sample solutionEa frac14 Absorbance of the proline standard solution

(average of two readings)E1 frac14 mg proline taken for the standard solutionE2 frac14 Weight of honey in grams80frac14Dilution factorIn Germany a honey with lt 180mg prolinekg

honey is considered either unripe or adulterated(Bogdanov 2009)

36 Vitamins (HPLC)

The presence of vitamins in honey is quite variable and isrelated to the botanical origin of the honey Water sol-uble vitamins (vitamins C and B-complex) occur in higherlevels in honey than do lipid-soluble vitamins (A D Eand K) due to the low presence of lipids in honey Ingeneral this foodstuff cannot be considered as animportant source of vitamins due to the fact that theyare in very low quantities As mentioned by Leon-Ruizet al (2013) the identification of vitamins in differenthoneys allows their characterization to botanical typeand is a valuable approach of honey quality control butno official methods have been described (De-Meloet al 2017)

There are few studies that identified and quantifiedvitamins in honey Some authors such as Ciulu et al(2011) and Leon-Ruiz et al (2013) published worksaiming to investigate the presence of vitamins in honeyusing HPLC The method outlined by Leon-Ruiz et al(2013) is described herein


Apparatus

HPLC system consisting of pump sample applicatorUV-vis variable wavelength detector

Micro-membrane 045 mm Volumetric flask (25mL) Syringe (1mL)


Beaker (50 and 100mL) Pipette (25mL) Sample vials (15mL)

Reagents

Trifluoroacetic acid (0025 vv) (HPLC grade) Acetonitrile (HPLC grade) Analytical column (C18 column 250mm 46mm 5

mm particle size)

362 Procedure

3621 Sample preparation(1) Weigh 10 g of honey into a 50mL beaker and dis-

solve in 10mL of ultra-pure water(2) Add 1 mL of NaOH 2M to complete

solubilization(3) Add 125mL of phosphate buffer 1M (pH frac14 55)(4) Raise the volume to 25mL using ultra-pure water(5) Filter the solution through a 045 lm membrane and

collect in sample vials(6) Store in the refrigerator if it is necessary

3622 Standards preparation The stock standardsolution is prepared by weighing in a 100mL volumetricflask 100mg of vitamin B2 250mg of vitamin B5 and100mg of vitamin B9

(1) Add 40mL of ultra-pure water(2) Add 4mL of NaOH to complete dissolution(3) Add 50mL of phosphate buffer 1M (pH frac14 55)

100mg of vitamin B3 then 100mg of vitamin C(4) Fill the flask to 100mL using distilled water(5) Store the standard solution in the dark at 4 C(6) Prepare the solution fresh daily as needed

3623 Chromatographic analysis The vitamins areidentified and quantified by comparison of the retentiontimes and the peak area of the pure standards The elutionprogram in the RP-HPLC gradient elution of water-solublevitamins in honey is described in Table 3

37 Minerals

The mineral content of honey samples has been receiv-ing significant attention from many scientists globally In

recent years the mineral profile of honey became animportant indicator of quality control and environmentalcontamination by agrochemicals (Almeida-Silva et al2011 Kacaniova et al 2009) In the same way someauthors proposed that mineral profile can be used as anauthentication analysis for botanical identification ordesignation of geographical origin (Anklam 1998Louppis et al 2017)

Many factors can contribute to the mineral compos-ition of honey including the botanical origin of nectarthe harvesting treatment and material of storage sea-sonal climatic variations and geographical origin(Almeida-Silva et al 2011 Anklam 1998) Other miner-als in honey may derive from those present in the airwater and soil Furthermore bees can be exposed tominerals while visiting flowers contacting branches andleaves drinking water from pools and while flying(Kacaniova et al 2009)

The mineral content in honey ranges from 004 inlight honeys to 02 in dark honeys (Alqarni et al2014) Potassium is the most abundant mineral inhoney composing generally up to 13 of the total min-eral content (Alqarni et al 2014 Yeuroucel amp Sultanoglu2013) In smaller quantities honey also contains sodiumiron copper silicon manganese calcium and magne-sium Macro elements (such as potassium calcium andsodium) and trace minerals (such as iron copper zincand manganese) perform a fundamental function in bio-logical systems maintaining normal physiologicalresponses inducing the overall metabolism influencingthe circulatory system and reproduction and acting ascatalysts in various biochemical reactions (Alqarni et al2014 da Silva et al 2016)

Honey should be free from heavy metals in amountsthat may represent a hazard to human health Howevermaximum residue levels of these potentially toxic ele-ments in honey have not been established The WorldHealth Organization (WHO 1996) has proposedacceptable levels of 15lgkg for arsenic 25lgkg forlead 5 lgkg for mercury and 7lgkg for cadmium

As described by Bogdanov et al (2007) the charac-terization of trace elements by inductively coupled sec-tor-field plasma mass spectrometry (ICP-SFMS) issignificantly improved by virtue of enhanced sensitivityand separation of polyatomic interferences in a highresolution mode In contrast the option of inductivelycoupled plasma with mass spectroscopy detector (ICP-

Table 3 Elution program in the RP-HPLC gradient elution of water-soluble vitamins in honey

Time (min) Solvent A (TFA aqueous solution 0025 vv) () Solvent B (acetonitrile) ()0 a 100 05 a 100 0

11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0

Note flow rate 10mLmin amdashOperative wavelength 254 nm bmdashOperative wavelength 210 nm


MS) is widely used by researchers and yields accurateresults Bilandzic et al (2017) published a work withmineral composition of 24 honey samples using an ICP-MS system with a great percentual recovery in thedeterminations This work shows detailed parametersof analysis validation and we chose this method as thestandard analysis for determining mineral content


Apparatus

High-pressure microwave oven Inductively coupled plasma instrument with

mass detector Teflon dishes Volumetric flasks (10 and 50mL)

Reagents

Nitric acid (HNO3) Certified standards consisting of Ag Al As Ba Be Ca

Cd Co Cr Cu Fe K Mg Mn Mo Na Ni Pb Se SbTh U V and Zn

Internal Standard Multi-Element Mix consisting of Li ScGa Y In Tb and Bi

Ultrapure water

372 Procedure

3721 Sample extraction(1) Weigh 05 g of honey samples into a Teflon dish(2) Add 3mL ultra-pure water(3) Add 25mL HNO3 (65)

(4) Perform a wet digestion of the solution using amicrowave oven set at a digestion program consist-ing of three potency steps first step at 500 W for25min second step at 1000 W for 20min and thethird step at 1200 W for 30min

(5) Following the wet digestion cool the samples toroom temperature

(6) Transfer the digested clear solution to a 50mL volu-metric flask and fill it using ultra-pure water

(7) All solutions should be spiked with the internalstandard to a final concentration of 10 mgL

3722 Calculation and expression of results(1) Perform a quantitative analysis using the calibration

curve method(2) Calibration curves should be constructed with a min-

imum of five concentrations of standards per element(3) The limits of detection (LODs) should be calculated

as three times the standard deviation of 10 consecu-tive measurements of the reagent blank multipliedby the dilution factor used for sample preparation(Table 4)

38 Calories

Honey calories can be determined indirectly using calcu-lation criteria according to the Brazilian FoodComposition Table (Food Research Center 2017) and(WHOFAO 2002) which uses the Atwater general fac-tors system

The calculation is done by multiplying the content ofproteins and available carbohydrates by the factorsdescribed as follows

Table 4 Instrumental working parameters for mineral analysis of honey

Torch injector QuartzSpray chamber Peltier cooled cyclonicSample uptake 03 rps (rounds per second)Nebulizer type MicroMistInterface Pt-conesRF power 1550WAr gas flow rate (Lmin) Plasma 15 auxiliary 09Nebulizer pump 01 rpsHe gas flow rate 003mLminIon lenses model X-lensLens voltage 107 VOmega bias 90VOmega lens 102 VAcquisition mode SpectrumPeak Pattern 1 pointIntegration time 2000msReplicate 3Sweepsreplicate 100Tune mode No gas 0 s 01 s(Stabilization time Integ Timemass) He 5 s 05 s HEHe 5 s 1 sICP-MS (standard mode) No gas Be9 Na23 Mg24 Al29 K39 As75Mo95Ag107Sb121Ba137 Tl205Pb208U23

He mode Ca 43V51Cr52Mn55 Co59Ni60Cu63Zn66Cd 111 HEHe Se78Fe56

Internal standards 209Bi 115In 45Sc


Proteins 170 kJg or 40 kcalg Available carbohydrates 160 kJg or 40 kcalg

Observation In the case of lipids dietary fiber and alco-hol it is zero for honey

39 Hydroxymethylfurfural

Hydroxymethylfurfural (HMF) is a compound producedby sugar degradation (dehydration of hexoses in anacidic medium) (Bogdanov 2011) HMF is a freshnessparameter of honey and in fresh honeys it is absent oroccurs in trace amounts High values of HMF are natur-ally present in honeys from areas with warm climates(Sodre et al 2011)

HMF is enhanced with honey processing and heattreatment Adulteration with commercial sugars andlong storage can also enhance HMF (Bogdanov 2011)

There is more than one method found in literatureto determine HMF content (HPLC and spectrophoto-metric method) We have chosen to summarize thespectrophotometric method of Bogdanov et al (1997)

391 Principle

The spectrophotometric method is based on the prin-ciple that HMF absorbs at 284 nm


Apparatus

Spectrophotometer able to measure 284 and 336 nm Quartz cells 1 cm (two cells) Vortex mixer Beaker (50mL) Volumetric flask (50mL) Filter paper Pipettes (05 and 5mL) Test tubes (18 x 150mm) Carrez solution I Carrez solution II Sodium bisulphite solution 02 Sample solution (initial honey solution frac14 50mL)

Reagents

Distilled water Carrez solution I Carrez solution II Sodium bisulphite solution 02 Sample solution (initial honey solution frac14 50 mL)

393 Procedure

The HMF spectrophotometric procedure can be foundin AOAC (1990) and Bogdanov et al (1997)

3931 Samples standards preparation and deter-mination

Carrez I SolutionmdashDissolve 15 g of ferrocyanide inwater and fill to 100mL in volumetric flask

Carrez II SolutionmdashDissolve 30 g of zinc acetate inwater and fill to 100mL in volumetric flask

Sodium Bisulfite Solution 02mdashDissolve 020 g of sodiumbisulfite in water and dilute to 100mL in volumetric flask

(1) Weigh approximately 5 g of honey into a 50mL beaker

(2) Dissolve the sample in approximately 25mL of dis-tilled water and transfer quantitatively into a 50mLvolumetric flask

(3) Add 05mL of Carrez solution I to the 50mL volu-metric flask and mix with a vortex mixer

(4) Add 05mL of Carrez solution II to the 50mL volu-metric flask mix with a vortex mixer and fill to50mL total volume with distilled water

(5) Filter the liquid through filter paper (rejecting thefirst 10mL of the filtrate)

(6) Pipette 50mL of the filtered liquid into each oftwo test tubes (18 150mm)

(7) Add 50mL of distilled water to one of the testtubes and mix with the vortex (sample solution)

(8) Add 50mL of sodium bisulphite solution 02 to thesecond test tube and mix with a vortex (refer-ence solution)

(9) To the sample solution (initial honey solution frac1450mL) add 50mL of distilled water

(10) To the reference solution (initial honey solution frac1450mL) add 50mL of 02 sodium bisulphite solution

(11) Determine the absorbance of the sample solutionagainst the reference solution at 284 and 336 nm in10mm quartz cells within 1 h

(12) If the absorbance at 284 nm exceeds a value of about06 dilute the sample solution with distilled waterand the reference solution with sodium bisulphitesolution (as described before) in order to obtain asample absorbance low enough for accuracy

3932 Calculations Results are calculated using theformula

HMF inmg=kg frac14 ethA284 A336THORN 1497 5 D=W

WhereA284 frac14 absorbance at 284 nmA336 frac14 absorbance at 336 nm

1497 frac14 126 1000 100016830 10 5

frac14 Constant

126frac14molecular weight of HMF16830frac14molar absorptivity e of HMF at kfrac14 284 nm1000frac14 conversion g into mg10frac14 conversion 5 into 50mL1000frac14 conversion g of honey into kg5frac14 theoretical nominal sample weight


D frac14 dilution factor (in case dilution is necessary)W frac14 Weight in g of the honey sampleExpress the results in mgkg

310 Ashes

Ashes as a quality parameter is reported for Brazilian regula-tion but this method will probably be replaced by the con-ductivity measurement Ash content is related to honeyorigin (eg blossom honeys have lower ash content com-pared with honeydew honey) (Bogdanov 2009)The proced-ure for ash determination is described in Bogdanov (2009)

3101 Principle

Samples are ashed using a temperature lt 660 C andthe residue is weighted

3102 Apparatus

Crucible Electric furnace (can run a temperature of 600 C) Desiccator with dry agent Analytical balance

3103 Procedure

31031 Samples standards preparation anddetermination(1) Prepare the ash dish by heating it in the electrical

furnace (ashing temperature 600 C at least 1 h)(2) Cool the ash dish in a desiccator to room tempera-

ture and weigh to 0001g (m2)(3) Weigh 5ndash10 g of the honey sample to the nearest

0001 g into an ash dish that has been previously pre-pared in (1)

(4) Cool the ash dish in the desiccator (2 h) and weigh(5) Continue the ashing procedure (steps 4 and 5) until

a constant weight is reached (m1)

31032 Calculations Make the calculation using thefollowing formula

WA frac14 ethm1 m2THORN100m0

WhereWA is expressed in g100 gm0 frac14 weight of honeym1 frac14 weight of dishthorn ashm2 frac14 weight of dish

311 Free acidity

Free acidity is determined using the method of recom-mended by the AOAC (1990) The International HoneyCommission has proposed 50 milliequivalents as the max-imum permitted acidity in honey (Bogdanov 2009)Furthermore the Council Directive 2001110EC(European Commission 2001) mentions free acidity as a

measured honey quality Finally the Brazilian regulationestablished the maximum of 50 milliequivalentskg of honey

3111 Principle

A honey sample is dissolved in distilled water and freeacidity is measured by titration with 01M sodiumhydroxide solution at pH 80


Apparatus

pH meter Magnetic stirrer Burette or automatic titrator Beaker (250mL)Reagents

Distilled water (carbon dioide free) Buffer solutions (pH 40 and 90) 01M sodium hydroxide solution

3113 Procedure

The procedure for free acidity is found inBogdanov (2009)

31131 Samples and standards preparation(1) The pH meter should be calibrated(2) Dissolve 10 g of the honey sample in 75mL of car-

bon dioxide-free water in a 250mL beaker(3) Stir with the magnetic stirrer(4) Immerse the pH electrodes in the solution and record

the pH(5) Titrate with 01M NaOH to a pH of 830 (complete

the titration within 2min)(6) Record the reading to the nearest 02mL when

using a 10mL burette and to 001mL if the auto-matic titrator has enough precision

31132 Calculations Free acidity is calculated usingthe formula

Free acidity ethmilliequivalents ormillimoles acid=kg honeyTHORNfrac14 mLof 01MNaOH 10

312 Insoluble solids

Honey insoluble solids include pollen honey-comb deb-ris bees and filth particles and is considered a criterionof honey cleanness It is possible to determine insolublesolid content by filtering honey diluted with distilledwater at 80 C in porous plate crucibles (Bogdanov2009 Bogdanov et al 1997 Codex 2001)


Drying oven at 105 C Drying oven


Pump vacuum Thermometer Desiccator with silica gel Metal spatula Beakers (50 and 250mL) Porous crucible 3 1000mL kitassato flask

3122 Procedure

(1) Weigh about 20 g of honey into a 250mL Beaker(2) Dilute with hot distilled water (80 C) until the sam-

ple is dissolved(3) Transfer to a porous crucible 3 coupled in the

kitassato flask (the crucible must be pre-dried at105 C for 12 h cooled in desiccator and weighed)

(4) Filter under vacuum and wash the honey sample inthe crucible with distilled water at 80 C until thevolume of each filtrate reaches 1 L

(5) Dry the crucible again at 105 C for 12 h(6) Cool the crucible in a desiccator and weigh it

3123 Calculation

The percentage of insoluble solids is calculated by thefollowing ratio

Original samplemass frac14 100 Mass of solids

ethfiltered and dried crucible dried crucibleTHORN frac14 x

313 Diastase activity

Diastases (a- and b-amylases) are enzymes naturallypresent in honey Diastase content depends on the flo-ral and geographical origins of the honey Their functionis to digest the starch molecule in a mixture of maltose(disaccharide) and maltotriose (trisaccharide) Diastaseis sensitive to heat (thermolabile) and consequently areable to indicate overheating of the product and thedegree of preservation (Ahmed 2013 da Silva et al2016 Granato amp Nunes 2016)

For this analysis the method recommended by TheInternational Honey Commission (2009) and CodexAlimentarius (2001) is based on the ldquodiastase activityrdquothat corresponds to the activity of the enzyme presentin 1 g of honey which can hydrolyze 001 g of starch in1 h at 40 C expressed as the diastase number inGeuroothe units (Bogdanov et al 1997) In this step-by-stepmethod the modifications proposed by Santos et al(2003) were included to give improvements in the solu-tions preparation and spectrophotometer procedures


Apparatus

UVVIS Spectrophotometer Bucket 1 cm

Analytical balance pH meter Thermometer Stopwatch Water bath 250mL Erlenmeyer flask Volumetric flasks of 50 100 and 500mL 1 L dark volumetric flask 50mL Beaker 100mL test tube 20mL volumetric pipettes Micropipette with a variable volume of 100ndash1000 lL

Reagents

Sodium acetate (CH3COONa) Glacial acetic acid (CH3COOH) Sodium hydroxide (NaOH) Soluble starch Anhydrous sodium chloride Twice sublimated Iodine Potassium iodide (KI) Destilated water

3132 Standard preparation

Acetate buffer solution 01molL pH 53

(1) Pipette 057 mL acetic acid into a 50 mL volumetricflask and fill with distilled water (Solution A)

(2) Weigh 164 g of sodium acetate and dissolve it into80mL of distilled water Adjust the volume to100mL in a volumetric flask (Solution B)

(3) Mix 105mL of Solution A with 395mL of Solution B(4) Check the pH value using the pH meter and adjust if

necessary to 53 with sodium acetate if the pH value isless than 53 or with acetic acid if the pH value isabove 53

(5) Transfer the solution to a 100mL volumetric flaskand fill with distilled water Keep refrigerated

Sodium chloride solution 01molL

(1) Weigh 0585 g of sodium chloride into a50mL Beaker

(2) Dissolve in distilled water(3) Transfer to a 100mL volumetric flask and fill up to

volume with distilled water

Iodine solution 002molL

(1) Weigh 4 g of KI into a 100mL test tube(2) Transfer with about 40mL of distilled water to a

dark volumetric flask glass stopper with a capacityof 1 L

(3) Weigh 254 g of twice sublimated iodine and transferto the KI-containing flask Fill up to volume with dis-tilled water


(4) Shake until complete dissolution of iodine(5) Transfer to a 500mL amber milled flask(6) Store the solution at room temperature with

no light

Starch solution 1 (mv)

(1) Weigh 10 g of soluble starch into a 250mLErlenmeyer flask

(2) Mix with 70mL of distilled water(3) Heat to boiling under constant stirring and keep for

3min Transfer the final volume of 100mL in a volu-metric flask and cool down to room temperature inrunning water The solution should be prepared atthe time of use do not store to avoidcontamination

Sodium hydroxide solution 01molL

(1) Weigh 20 g of NaOH and dissolve in distilled waterby filling the volume in a volumetric flask to 500mL

(2) Store the solution in a plastic container

3133 Experimental procedure

(1) Weigh 50 g of honey into flask(2) Add 20mL of distilled water to the flask(3) Correct the pH of this solution to a value of 53 by

adding 01molL NaOH until the correct pHis obtained

(4) Fill the flask to 50mL with distilled water(5) Reaction system Add 5mL of the honey solution

to a test tube(6) Add 500 lL of the acetate buffer 01 molL pH 53

to the tube and mix It is essential that the honeysolution is buffered before contact with sodiumchloride because at pH below 4 diastase activityis inhibited

(7) Add 500lL of the sodium chloride solution01molL to the tube

(8) Add 150mL of the solution of 002molL iodineand 96mL distilled water to the tube and mix

(9) Keep the tube and its contents in a water bath at40 C plusmn 1 C

(10) Add 250 lL of the 1 starch solution (mv)(11) Start the stopwatch shaking the solution until com-

plete homogenization occurs(12) Transfer a part of the volume of the reaction sys-

tem to complete a 1 cm cell and measure theabsorbance of the solution in the spectrophotom-eter at 660 nm against a water blank This firstreading is the initial absorbance value (Absi) Thetube with the solution should remain in the waterbath at 40 C

(13) Make periodic readings of absorbance alwaysreturning the tube to the water bath when notbeing read until a value between 024 and 020is reached

(14) When this value is reached stop the timer andrecord the elapsed time value

(15) The last recorded absorbance value is consideredthe final absorbance (Absf)

(16) Calculate the diastase index using the following formula

Diastase index frac14 ethAbsi AbsfTHORN 03TethhTHORN V 0016

03frac14 absorbance constant frac14 03mg1 (previouslydetermined by honey-free assay given by method)

T (h) frac14 time (in hours) between Absi and Absfmeasurements

V frac14 volume of the 10 honey solution in the testtube (mL)

0016frac14 total volume in liters of the solution in thetest tube (16mL)

314 Electrical conductivity

This method is easy quick and involves inexpensive instru-mentation Electrical conductivity determination dependson the ash and acid contents of honey (eg the higher ashand acids the higher the result for electrical conductivity)This parameter has been used as criterion of the botanicalorigin of honey (Bogdanov 2009)

According to the European regulation (EuropeanCommission 2001) electrical conductivity of blossomhoney must be lt 08mScm of EC while electrical con-ductivity of honeydew honey and chestnut honey mustbe gt 08mScm Exceptions are honeys from ArbutusBanksia Erica Leptospermum Melaleuca Eucalyptus Tiliaand blends (De-Melo et al 2017)

3141 Principle

Electrical conductivity uses the measurement of theelectrical resistance


Conductimeter lower range 107 S Conductivity cell (platinized double electrodemdashimmer-

sion electrode) Thermometer Water bath Volumetric flasks (100mL and 1000mL) Beakers Distilled water Potassium chloride

3143 Procedure

The standard method for measuring honey electricalconductivity was outlined by Bogdanov (2009)

31431 Samples and standards preparation(1) If the cell constant of the conductivity cell is not

known transfer 40mL of the potassium chloridesolution (01 M) to a beaker


(2) Potassium chloride solution (01 M) preparation dissolve74557 g of potassium chloride (KCl) dried at130 C in freshly distilled water and fill up to 1000mL volumetric flask Prepare fresh on the dayof use

(3) Connect the conductivity cell to the conductiv-ity meter

(4) Rinse the cell thoroughly with the potassium chlor-ide solution and immerse the cell in the solutiontogether with a thermometer

(5) Read the electrical conductance of this solutionin mS after the temperature has equilibratedto 20 C

(6) Calculate the cell constant K using the formulaKfrac14 11691 1G where K frac14 cell constant cm1G frac14 Electrical conductance (mS) measured withthe conductivity cell 11691 frac14 the sum of themean value of the electrical conductivity of freshlydistilled water in mScmndash1 and the electrical con-ductivity of a 01 M potassium chloride solutionat 20 C)

(7) Rinse the electrode with distilled water after thedetermination of the cell constant

(8) Dissolve an amount of honey equivalent to 20 g ofdry matter of honey in distilled water

31432 Determination(1) Transfer 20mL of the solution created in section

ldquoSamples and standards preparationrdquo to a 100 mLvolumetric flask and fill up to 100 mL with dis-tilled water

(2) Pour 40mL of the sample solution into a beaker andplace the beaker in a thermostated water bathat 20 C

(3) Rinse the conductivity cell thoroughly with theremaining part of the sample solution (Step 1)

(4) Immerse the conductivity cell in the sample solutionand read the conductance in mS after temperatureequilibrium has been reached

(5) If the determination is carried out at a differenttemperature because of a lack of thermostatedcell a correction factor can be used for calculat-ing the value at 20 C For temperatures above20 C subtract 32 of the value per C Fortemperatures below 20 C add 32 of the valueper C

31433 Calculations Calculate the electrical conduct-ivity of the honey solution using the following formula

SH frac14 K G

Where SH frac14 electrical conductivity of the honeysolution in mScm1 K frac14 cell constant in cm1 G frac14conductance in mS Express the result to the near-est 001mScmndash1

Also FTIR could be used in order to determinehoney electrical conductivity (Almeida-Muradian et al2012 2013)

315 Trolox equivalent antioxidantcapacity (TEAC)

Determining the scavenging ability of honey to the radicalcation of ABTS [220-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid)] using trolox a water-soluble vitamin E ana-log as the standard for the calibration curve (Miller et al1993 Re et al 1999 Sancho et al 2016) is the methodwe propose for measuring antioxidant capacity Thedescribed method is a modification of Re et al (1999)procedure optimized by Sancho et al (2016)


Spectrophotometer (Visible) to measure absorbance (A)at 734nm

Glass or Plastic cells 1 cm Ultrasonic bath Shaker Stopwatch 100 mL volumetric flask 100 mL beakers Pipettes Distilled water ABTS K2S2O8

Trolox

3152 Solutions

31521 Preparing the solutions(1) 7mM ABTS aqueous solution Dissolve 03841 g 220

ndash azino-bis (3-ethylbenzothiazoline)-6-sulfonic aciddiammonium salt (ABTS) in distilled H2O stir anddilute to 100 mL with distilled H2O

(2) 245 mM K2S2O8 aqueous solution Dissolve 00662 gpotassium persulfate (di-potassium peroxidisulfate) indistilled H2O stir and dilute to 100 mL with dis-tilled H2O

(3) 5mM trolox solution Dissolve 01251 g (plusmn)-6-hydroxy-2578-tetramethylchromane-2ndashcarboxylicacid (trolox) into a mixture of absolute ethanol-dis-tilled H2O (11) The use of an ultrasonic bath for 5min aids dissolution Dilute to 100 mL with dis-tilled H2O

31522 Generation and dilution of ABTS radical cat-ion (ABTSthorn)(1) Undiluted ABTSthorn solution mix 7mM ABTS aqueous

solution and 245mM K2S2O8 aqueous solution inequal amounts (11 vv) and allow them to react inthe dark for 16 h at room temperature UndilutedABTSthorn solution is stable two days stored in thedark at room temperature


(2) Working ABTSthorn solution Dilute the undilutedABTSthorn solution with distilled water to obtain anabsorbance between 070 and 080 at 734nm Theworking ABTSthorn solution must be prepared fresh daily

31523 Samples and standards preparationSample preparation (H-sample)

(1) Weigh 10 g honey into a beaker(2) Dissolve in distilled water(3) Transfer to a 100mL volumetric flask(4) Further dilute to 100mL with distilled water

Trolox calibration curve (standard)

(1) In six volumetric flasks dilute the 5mM trolox solu-tion with distilled water to obtain the following con-centrations 025mM 050mM 075mM 100mM125mM and 150mM

31524 Determination(1) Using a pipette transfer 990 mL of the diluted

ABTSthorn working solution (see ldquoGeneration and dilu-tion of ABTS radical cation (ABTSthorn)rdquo section) intoeach cell One cell corresponds to distilled water(no antioxidant) six cells correspond to the calibra-tion curve (with trolox standard solutions of sectionldquoSamples and standards preparationrdquo) and anothercell corresponds to the sample (of section ldquoSamplesand standards preparationrdquo)

(2) Start the reaction by adding 10 mL of distilled water(DW) trolox standard solution (STANDARD) or samplesolution (H-sample) to the corresponding cell

(3) Mix immediately by turning the cell upside downDetermine the absorbance (A0)

(4) Determine the absorbance (A) after 6min (A6) at734 nm in the cell against a blank of distilled water

This procedure is performed in triplicate

31525 CalculationsPercentage of inhibition

inhibition frac14 ethA0 A6THORN 100A0

A0 is the absorbance at initial time of distilled water(DW) standard solution (STANDARD) or sample solution(H-Sample) referred to section ldquoDeterminationrdquo (3)

A6 is the absorbance measured at 6min of distilledwater (DW) standard solution (STANDARD) or sample

solution (H-Sample) referred to sectionldquoDeterminationrdquo (4)

Linear regression a standard trolox calibration curveis drawn by representing trolox concentrations (mM)on the x-axis and inhibition on the y-axis

inhibition frac14 ax thorn bx frac14 concentration trolox (mM)a frac14 slopeb frac14 interceptTEAC value of the honey sample (mmol troloxg

honey) TEAC antioxidant activity of honey is calculatedas follows

a frac14 slopeb frac14 interceptWhere 100 (of the formula) frac14 1000 (mmolmmol)

100 (mL honey solution)1000 (mLL)

316 Color of honey by the CIELab system

The colorimeter measures the reflected color of thehoney samples both liquid and crystallized of the wholevisible spectrum in a wavelength interval between 380and 740 nm Other methods for measuring a honeysamples color require honey dilution before measuringits color by spectrophotometry at a given wavelengthOr it is necessary to dissolve any crystals in the honeyto obtain a completely liquid honey that is transparentBoth procedures modify the honey color The advan-tage of the colorimeter method is that it is the onlyinstrument that gives the samplesrsquo color the way thehuman eye does whether the sample is liquid or crys-tallized Moreover honey remains suitable for use inother analysis (Sancho et al 2016)

3161 Apparatus

Lab Colorimeter glass Ultrasonic bath

3162 Procedure

(1) Fill the colorimeter glass completely with honeyBubbles in the honey can interfere with honey colorThey can be removed from the honey if it is placedin an ultrasonic bath at room temperature for a fewminutes before color determination (avoidinghoney heating)

(2) Determine the color parameters L (lightness 100for white and 0 for black) and the chromaticitycoordinates a (positive values for redness and

TEAC lmol trolox=g honeyeth THORN frac14 inhibitionH-samples ndash beth THORNa

100honeyweight ethgTHORN


negative values for greenness) and b (positive valuesfor yellowness and negative values for blueness)using illuminant D65 10 observation angle and 450 geometry illumination

(3) Measure the color in triplicate (Sancho et al 2016)(4) Other interesting values for honey comparison or

characterization calculated from a and b are thecoordinates C (chroma saturation vividness orpurity of a color) and h (hue angle or tone)(Tuberoso et al 2014)

C frac14ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffia2 thorn b2

p

h frac14 arctg b=a

4 Investigating honey production with diagnosticradioentomology

Diagnostic radioentomology is a technique based on themeasurement of density from component materialsusing X-rays The density measured by computer tom-ography (CT) scanning reflects the degree to which theenergy of the X-ray beam is reduced when penetratinga certain material This technique allows for the quantifi-cation and visualization of differences in density withminimal disturbance of the colony since the hive doesnot need to be opened to introduce light into the darknest and since no sample need be collected Thus itbecomes possible to investigate noninvasively phenom-ena that are affected by other common research meth-ods that require destructive sampling

Honey originates from nectar and honeydew andundergoes a process of concentration (ie dehydra-tion) The increase in sugar concentration affects honeydensity This can be tracked using radioentomology

Despite the importance of honey for beekeeping theprocesses that lead to its production within the honeybee nest have rarely been investigated (Eyer Grecoet al 2016 Eyer Neumann et al 2016) Diagnosticradioentomology was recently used to shed light onnectar storage strategies of bees by allowing for themeasure of sugar concentration of the content of alarge number of storage cells (Eyer et al 2016 Grecoet al 2013) By following the evolution of the sugarconcentration in individual cells over time it is also pos-sible to monitor several stages of honey production bythe workers from initial deposition of nectar or honey-dew in cells to the capping of mature honey (Eyeret al 2016)

This technique could be used further to determinethe influence of environmental (eg high or low nectarflow diverse or monotonous nectar flow varying wea-ther conditions) and internal factors (eg hive designair circulation colony demography) on honey ripeningand storage A better knowledge of the factors affectinghoney production processes by honey bee workerscould not only lead to a better knowledge of how thehoney bee superorganism manages its food stock but

could also lead to the improvement of beekeeping man-agement practices by favoring a rapid ripening of honeyor by increasing productivity A further application ofthis technique is to track the deposition and spread ofsubstances applied into hives (Rademacher et al 2013)that might contaminate honey stores The concentrationof crop content of live yet motionless honey bees canalso be measured with this technique Rapidly movingindividuals will appear blurred in the scan and thus hin-der precise measurements In the following sections wedescribe how to perform measurements of sugar con-centration of the content of carbohydrate reservesstored in wax combs with a CT scanner

41 Experimental conditions and material requiredto conduct diagnostic radioentomology trials

411 Hive to scanner size ratio

The most commonly available CT-scanners are thoseused for human or veterinary medicine Their dimen-sions allow for scanning hives of most standard sizeswithout honey supers Larger scanners exist for veterin-ary applications and can be used to study hives withsupers When high resolution is required it can beobtained by reducing the size of the experimental hive(eg using Miniplus (R) hives 30 30 34 cm) Thefocalization of the X-ray beam on a smaller areaincreases scan resolution Micro-CT scanners are alsoavailable to study small items (eg individual motionlessworkers a few cells) with high resolution

412 Material to be scanned

Combs built from foundation sheets in moveable frameshave a regular and planar shape which facilitates scan-ning and data analysis However diagnostic radioento-mology can be used to study honey production incombs of any shape (eg in wild nests in logs) thanksto 3D reconstruction tools Depending on the researchquestion combs already containing stores can be usedas starting material When the starting point of thestudy is an empty cell a drawn empty comb can beinserted in the test colony Single combs without work-ers workers alone or entire hives with workers can bescanned Single combs can be gathered in an empty hiveor super box and all scanned at once to decrease thenumber of scans required (Figure 3)

When hives containing honey bees are to bescanned safety of scanning facility staff and of bystand-ers needs to be considered Hive entrances should beclosed early on the day of scanning before foragers starttheir activity and tight hives should be used to preventhoney bees from escaping Ventilation (eg through ascreened bottom) should nevertheless be possible forthe workers to be able to adjust hive temperatureCombs and hives have to be labeled in a way that theycan be recognized on the scan images Notches cut inthe wood or thin metallic letters or numbers can be


used Metallic parts like nails and wires deflect the X-ray beam and produce glares that make data analysis dif-ficult It is thus necessary to remove all metallic partsfrom the material to be scanned or to use plastic orpolystyrol hive material

413 Monitoring the storage of artificial diets

Nectar and honeydew collected by foragers are highlydiverse in their composition Consequently it can beuseful to use artificial diets to simplify the study ofhoney production The storing of artificial diets (egcommercial feeding solutions) can also be the purposeof the experiment Commercial feeding solutions or asolution of controlled sugar concentration can be pro-vided to the colonies in feeders within the hive or out-side of the hive at a close distance which reflects amore natural foraging situation

To prevent the foragers from collecting other sour-ces of carbohydrates than those experimentally pro-vided test colonies can be placed in tunnel cages (seeMedrzycki et al (2013) section ldquoScanning settingsrdquo)When tunnel cages are not available field colonies canbe used during a period without natural nectar flow Toconfirm absence of incoming nectar the concentrationof returning foragersrsquo crop content can be measured atregular interval using the following method

(1) Collect returning foragers that have no pollen bas-kets on their legs at the hive entrance with the helpof forceps

(2) Gently squeeze their abdomens between your fin-gers to force them to regurgitate a small droplet ofcrop content

(3) Place this droplet onto a refractometer adapted tothe range of expected concentrations (see Humanet al (2013) section 4731)

(4) Read the sugar concentration through therefractometer

(5) Repeat this procedure daily for 10ndash50 foragers pertest-colony

If the concentration of crop content does not cor-respond to that of the diet provided the foragers arecollecting nectar from other sources If this input can-not be accounted for in the analysis the experimentshould be performed when naturally available resourcesdo not interfere with the aim of the study

414 Studying the storage of natural diets

When the aim of the experiment is to study naturaldiets field colonies with freely flying honey bees canbe used Choose a period with the nectar or honey-dew flow of interest for your research question Therange of sugar concentrations collected by foragerscan be verified by squeezing the abdomen of returningforagers (see section ldquoMonitoring the storage of artifi-cial dietsrdquo)

415 Labeling of the diet to increase contrast

When the storage of qualitatively different solutions ofsimilar densities needs to be studied it is possible todiscriminate between these solutions by labeling one ofthem with a contrast agent (Eyer et al 2016) Oneexample of a suitable contrast agent is Visipaque whichis harmless to honey bee workers at a concentration of10 Visipaque (iodixanol Injection) is a contrast mediasolution with multiple indications in human medicine(aortography venography urography etc) for use in X-ray scans Other contrast agents may be used but theirtoxicity for honey bees need to be determined(Medrzycki et al 2013)

If a contrast agent is used whether it mixes with thesolution or sediments in the cell needs to be deter-mined prior to the experiment (Eyer et al 2016) Thisverification is required to exclude possible bias fromthe contrast agent when monitoring density patterns ormeasuring density of cell or crop content

416 Calibration

The density of cell content is measured in Hounsfieldunits This unit is used as a proxy for sugar concen-tration A regression of sugar concentrations ondensity of the artificial carbohydrate solutions or nat-ural sources is thus required to convert Hounsfieldunits in sugar concentration (Eyer et al 2016) To

Figure 3 Experimental combs are placed in hive bodies for aCT scan


generate a calibration curve the following stepsare conducted

4161 Artificial diet(1) Prepare vials (20ndash50mL) with carbohydrate solu-

tions (mixing granulated sugar with water) coveringthe range of concentrations required for the experi-ment (eg 30 50 60 70 and 80 sucrose)

(2) Vortex to homogenize solution(3) Scan the vials (see section ldquoComputer tomography

scanningrdquo below)(4) Measure density of 10 randomly selected points in

each vial (using the point measure tool of eFilm forexample) to consider putative density variationswithin the vial

(5) Average these measures(6) Plot the averages for each vial with density meas-

ured on the x-axis and sugar concentration on they-axis

(7) Add a linear trend line to the chart(8) Obtain the equation describing the trend line in

order to determine the slope (m) and the constant(t) of the calibration line

(9) Calculate the sugar concentration (y) by multiplyingthe density measured by the slope value and by add-ing the constant (t) (y frac14 (mx) thorn t) Repeat this cal-culation for each density value measured

4162 Natural diet(1) Sample aliquot of nectar cell or crop content(2) Place aliquot on a refractometer adapted to the

expected sugar concentration (see Human et al(2013) section 4731)

(3) Read the sugar concentration through the refractometer(4) Repeat this procedure until a wide enough range of

concentrations is obtained to perform calibration(5) Perform steps (5ndash9) as indicated in ldquoArtificial

dietrdquo section

4163 Labeled diet A specific calibration curve has to begenerated for the calculations of the sugar concentrations inlabeled diets Repeat steps described in Section ldquoMonitoring

the storage of artificial dietsrdquowith labeled solutions of varioussugar concentrations Densities of labeled and nonlabeleddiets should not overlap at any point of the concentra-tion range

42 Computer tomography scanning

421 Handling and transporting of the combs

Ideally a portable CT-scanner should be used to avoidinterfering with the biological processes in the colonyHowever until the use of portable devices in the fieldbecomes more practical and affordable and since the mostfrequently accessible devices are fixed it is recommendedto minimize transport of the test hives or combs by con-ducting the field component of the experiments near theCT-facility In cases when transportation cannot beavoided its effects can be minimized by careful handlingavoidance of shaking and shocking the hives or combs tobe tested If combs are scanned outside of the hive notusing smoke during comb collection will limit methodo-logical bias by not provoking cell content take up by work-ers Workers can be removed from the combs by gentlybrushing them off taking care that the bristles do not con-tact the cell content Shaking the combs to get rid of work-ers is not recommended due to possible effects on cellcontent especially if storage of nectar of low sugar con-centration is investigated In the absence of honey beesfurther biases such as cell content evaporation can be mini-mized by scanning the comb immediately after collection

422 Scanning settings

The scans can be performed with a Philips Brilliance CT16-slice apparatus (eg Philips Healthcare 5680DABest The Netherlands) using 1200 kVp and 1830mAas settings For other models detailed device settingshould be discussed with scanner operator according tothe output required

423 Performing a scan

Constant conditions during scanning should be main-tained holding the temperature in the scanning room at

Figure 4 Movement of the scanner bed is programed and the hive box is positioned using the scannerrsquos laser beams


18ndash20 C If required by the study question scans canbe repeated at defined time intervals Adhere to safetymeasures as instructed by scanner operator to avoidthe danger of exposure to X-rays

(1) Place the hive or combs to be scanned on the scan-ner bed

(2) Adjust bed movement range and position of materialto be scanned using the devicersquos positioning laserbeams (Figure 4)

(3) Place yourself in the designated protected area per-sonal other than operators leave the room

(4) Press scan and survey the scan-procedure onthe computer

(5) Wait until scan is complete (usually indicated by theextinction of an acoustic signal) before leaving thedesignated area

(6) Save the scan output to a specific location on a ser-ver or a hard drive

43 Data analysis

431 Analysis of density patterns in individual cell

For the visualization of cell content density patternsCT-images can be analyzed with specific 3D renderingsoftware that permits the visualization of differences indensity with a color gradient (Figure 5) This featureenables visual monitoring of density measured followingthis procedure

(1) Load file(2) Choose parameter settings Detailed device settings for

the windowing feature should be discussed with thescanner operator according to the output required Adark color represents low density content whereas lightcolor represents high density content Image analysissoftware also has several coloration presets (eg Figure5) that can be tried to render the best image output forthe analysis There is no empirical manner to determinethe best color settings The appearance of the imagesgenerated will determine which is the most informative

(3) Apply and record the chosen settings(4) Inspect cells from sagittal transverse and coronal

perspectives for specific cell content patterns (Figures5 and 6)

432 Measuring density of cell content

Density quantified in Hounsfield Units (HU) can bemeasured using the software eFilm for example (30-daytest-version is available under httpsestoremergecomnaindexaspx) Density of individual cell content can bemeasured with the ellipse tool as described below Ifrequired more precise measurements of cell contentcan be obtained with the point measurement tool

(1) Load image file(2) NavigateScroll vision plane to the cells of interest

Figure 5 Screenshot of a 3D imaging software with 3D reconstruction panel (top left) and three section planes (sagittal top righttransverse bottom left coronal bottom right)


(3) Use the ellipse tool to draw a circle over the con-tent of the cell three scanning frames away from itsbottom end

(4) Repeat the measurement of the same cell threeframes below the surface of the cell content

Steps 3 and 4 are necessary to take into accountwithin cells variation of density (see Eyer et al 2016)Scanning a few frames away from cell bottoms andopenings helps avoiding the inclusion of air volumes orwax in the circles which would strongly bias themeasurement

(5) Average the values of these measurements

433 Measuring cell filling status

The filling status of the cells can be estimated as thenumber of scanning frames for which the content fillsthe whole cell diameter

(1) Load image file(2) Place vision plane at cell bottom(3) Scroll through each frame with full content

while counting

434 Analyzing content of individual cells over time

(1) Take a picture of each test comb or load the imagefile with eFilm

Figure 6 Density patterns in individual cells (a) low density homogeneous pattern (b) ring of high density content along the cellwalls (c) inhomogeneous pattern with high density speckles (d) homogeneous high density cell content Modified from images pub-lished in Eyer et al 2016 under CC BY 40


(2) Select and mark 10 individual cells (on picturescreenshots (Figure 7) or printouts) for eachtest comb

(3) Define and use specific landmarks (eg particularshapes on the wax combs or marks on the framesthat are not likely to change over time) on theimage filespictures to easily identify individual cellsin subsequent scans

(4) Analyze content of individual cells with eFilm orother dedicated software on subsequent scan timesThis allows the investigation of their filling and ripen-ing dynamics over time

In case the whole process of honey production is to bemonitored but not all cells are capped at the end of theexperiment the selection of cells measured can bedone a posteriori once cells have been capped

435 Spatial analyses at comb level

The following method can be used to investigate spatialpatterns in nectar processing (Eyer et al 2016)

(1) Select combs that contain a sufficient number offilled cells (eg Figure 7)

(2) Select a single vertical scan frame perpendicular tothe cellsrsquo long axes and parallel to the comb midribfor each comb Choose this frame in order to maxi-mize the number of cells showing content

(3) Measure the density of content of a predefined num-ber of cell (eg using the ellipse or point tool ofeFilm see section ldquoMeasuring density ofcell contentrdquo)

(4) Determine the projected X and Y coordinates ofeach cell (eg with the help of the probe toolof eFilm)

(5) Enter coordinates in a spreadsheet in which thedensity values will be recorded

(6) Analyze spatial patterns (eg using the spatial auto-correlation Moranrsquos I test statistic in Arc GIS 102)

(7) Enter feature locations (projected coordinates) andattribute values (sugar concentration) to calculatethe spatial autocorrelation (I index)

(8) Chose the fixed distance model with the mean dis-tance between the centers of two neighboring cellsThis distance can be measured with the line toolof eFilm

(9) Run the analysis and consider the I-index obtainedI-indices close to zero indicate random patternwhereas positive indices indicate a tendency towardclustering Negative indices indicate a tendencytoward uniformity (Eyer et al 2016) P-values indi-cate whether the distribution patterns are significant

44 Pros and cons

Pros The use of CT-scanning permits nondestructiveobservations and measurements within the dark hiveRepeating the observations and measurements in time isthus possible without disrupting the phenomenon underscrutiny Three-dimension imaging is possible

Cons Different materials of similar density cannot bedistinguished based on their Hounsfield value Unless aportable device is available the material to be scannedhas to be transported to the scanning facility Onlysnapshots and hence low frequency time lapse imagescan be captured filming is not possible to observe thebehavior of workers for example Metallic parts createglare and need to be removed from thescanned material

45 Perspectives

Diagnostic radioentomology is a powerful tool to moni-tor otherwise difficult to observe processes Howeverthis recently developed method could be further devel-oped to increase its usefulness Scans performed imme-diately after behavioral observations in hives withtransparent sides can help relate density patterns withthe behavior that generated them Better labeling tech-niques (eg avoiding sedimentation of contrast agent)of cell content would provide further opportunities tomonitor processes involved in nectar storage and honeyproduction with an even higher resolution Scanning ofstorage combs should also be performed more

Figure 7 A coronal scan through a partly filled comb Colors reflect differences in density with black for low density to yellow forhigh density Cell content appear in green-yellow shades wax and wood in blue air in black


frequently as done to date aiming at investigating theprocesses of honey production with higher time reso-lution Further generating a database of densities ofnectar and honey of specific origins (with various sugarcompositions) or at different ripening stages couldimprove the sets of tools available and might relieve theneed for calibration before each experiment Scanninghoney bee workers to investigate crop content forexample is limited by the relatively low scanning speedThis is especially the case with older devices and whenworkers are moving However the motors of new gen-erations of scanners are spinning the X-ray and detec-tion units at high speeds and will allow for the freezingof a greater proportion of the workerrsquos movementOther imaging techniques (eg radioactive labeling andlaser scanning microscopy) could also be employed forstudying honey production by workers

5 Pesticide residues in honey

The term ldquopesticidesrdquo represents many different sub-stances used in various crop protection products totreat plants against pests and in veterinary drugs usedagainst animal pestsparasites (including those used inhoney bee colonies to control bee pestsparasites) inaround structures to protect against structuralpests etc

Pesticides applied on crops can contaminate plantssoil water and air and honey bees may be exposed tothem via contact with these matrices The bees collectand transport contaminated products (nectar pollenand water) into the hive There is also a risk of findingpesticide residues in bee products following treatmentof the hive

Honey samples can be screened for pesticide resi-dues for food safety purposes (ldquoCommission Regulation(EU) No 372010rdquo European Union Commission 2009ldquoRegulation (EU) No 3962005rdquo 2005) The EuropeanUnion (EU) requires that honey be tested for pesticideresidues within the framework of the monitoring pro-gram covered by the Council Directive 9623EC(European Communities 1996) Maximum residue limits(MRLs) are defined for pesticides in honey (EUPesticides Database) which mostly are between 001and 005mgkg For acaricides used in beekeeping twoMRLs were defined in the Commission Regulation (EU)No 372010 (2009) for amitraz (and its metabolite 24-dimethylaniline) and coumaphos 02 and 01mgkgrespectively

51 Chemical families of pesticides

The most common use of pesticides is as plant protec-tion products in agriculture Pesticides are classified inthree main groups according to the nature of the ldquopestrdquoto be controlled herbicides fungicides and insecticides(including acaricides) In each group there are differentchemical families of pesticides

511 Herbicides

Herbicides are used to eliminate weeds which disturbthe growth of the crops They are widely used in agri-culture to kill plants or to inhibit their growth or devel-opment Residues are found in air water and onvarious plant parts There are multiple chemical classesof herbicides carbamates triazines triazoles and ureasto name a few

512 Fungicides

Fungicides are used to kill fungi in plants stored productsor soil or to inhibit their development Fungicides caneither be contact translaminar or systemic The mainchemical families of fungicides used are benzimidazolesdicarboximides triazoles chloronitriles and carbamates

513 Insecticides and acaricides

Insecticides and acaricides are used to kill or disruptthe growthdevelopment of insects or mitesInsecticides are applied on crops to protect themagainst pests They can be classified into two groups (i)Systemic insecticides are incorporated into the tissuesof treated plants Insects ingest the insecticide whilefeeding on the plants (ii) Contact insecticides are toxicby contact to insects Acaricides are also used againsthoney bee parasites such as Varroa in hives (Dietemannet al 2013) The main chemical insecticide families areorganochlorines organophosphorus pyrethroids neoni-cotinoids carbamates and phenylpyrazoles

52 Analytical methods

For protecting the health of consumers the analyticalchallenge is to achieve limits of quantification at orbelow the MRL specified for pesticides under EU orother similar legislation The laboratories conductingthe residue analyses usually develop and validate a mul-tiresidue method Within the framework of official con-trols on pesticide residues laboratories follow therequirements specified in the guidance document onanalytical quality control and validation procedures forpesticide residues analysis in food and feed (ldquoDocumentSANTE118132017rdquo European Commission 2018)

The development of residue analysis methodsdepends on the properties of both the matrices (honeywax pollen etc) and pesticides Residue analysisinvolves several steps There are many extraction andclean-up procedures used by different authors to deter-mine the amount of pesticides in honey (Barganska ampNamiesnik 2010) Solvent extraction and solid-phaseextraction (SPE) are the techniques most commonlyused for the extraction of pesticides from honey

There are two general analytical approaches used todetermine residues in food and environmental samples (i)specific methods where a single pesticide and its metabo-lites are quantitatively determined in the sample and (ii)


multiresidue methods which are analytical methodologiesfor the simultaneous analysis of trace amounts of a largenumber of analytes The number of pesticides tested in thesample can be limited in order to get reliable resultshigher recoveries and lower quantification limits

Several analytical methods have been used to separ-ate and detect pesticides in honey Gas chromatography(GC) and liquid chromatograpy (LC) are used for thedetection and quantification of pesticide residues (SouzaTette et al 2016) The choice of the separation tech-nique depends mostly on the characteristics of the pes-ticides of interest The volatile semi-volatile andthermally stable compounds can be determined by GCwhereas nonvolatile andor thermally unstable onesshould be determined by LC

At least three steps are required for the analysis ofpesticides among them extraction separation anddetection Each one of these steps will be described inthe sections that follow

521 Sampling

The samples to be analyzed must be representative ofthe entire honey batch in question The different stepsfor the sampling of honey are as follow

(1) If the honey sample contains impurities (eg wax)the sample should be filtered through a stainless-steel sieve If needed the honey can be gentlepressed through the sieve with a spatula

(2) If a honeycomb is sampled the honeycomb isdrained through a 05mm sieve without heating inorder to separate honey from the comb

(3) For crystallized honey the sample is homogenizedwith a spatula and an analytical test portion is col-lected by coring of the honey

(4) Honey must be homogenized before analysis(5) According to the protocol of sample preparation

applied for extraction of pesticide residues in honeycollect 1ndash20 g honey into a centrifuge tube or a bea-ker for sample preparation

522 General requirements for pesticide residue analyses

(1) All analyses should include negative control honey(honey free from pesticide residuesmdashthe ldquoblankrdquo)and matrix calibration standards prepared by addingpesticides to blank honey before pretreatment ofthe samples

(2) The sample spiked with pesticides at the level corre-sponding to the limit of quantification (LOQ) for

Table 5 Analysis of pesticide residues in honey using a liquidndashliquid extraction

Pesticide analyzed Extraction protocol ReferencesOrganochlorines (a- b- and

Ccedil-hexachlorocyclohexane(HCH) hexachlorobenzene(HCB) aldrin pprsquo-DDE pprsquo-DDD oprsquo-DDT andpprsquo-DDT)

1 Dissolve 5 g of honey with 50mL 4 aqueous solution ofsodium sulfate in a centrifuge tube

2 Add 20mL of ethyl acetate to the sample Repeat the liquid-liquid extraction two times with 15mL of ethyl acetate

3 Centrifuge at 3000 rpm for 10min if emulsion is formed4 Filtrate the organic phase through anhydrous sodium sulfate5 Evaporate the organic phase under a stream of nitrogen to

25mL for analysis in graduated centrifuge tube6 Analyze by GC-ECD and Gas ChromatographyMass

Spectrometry (GC-MS) for confirmatory analysis followingauthorsrsquo protocols

Blasco Linoet al (2004)

Coumaphos bromopropylateamitraz and tau-fluvalinate

1 Mix 20 g of honey in an Ultra-Turrax blender with a mixtureof n-hexane (60mL) propanol-2 (30mL) and 028 ofammonia The pH of this mixture is 8

2 Filter the solution through a filter paper3 Repeat steps 1 and 2 with the mixture of n-hexane (60mL)

propanol-2 (30mL) and 028 of ammonia4 Rinse the Ultra-Turrax with 40mL of n-hexane and filter this

washing solution on the same filter paper as in step 25 Transfer the combined extracts from steps 2 3 and 4 to a

separating funnel (500mL)6 Add 50mL of distilled water and 028 of ammonia (pH 10)7 Shake the separating funnel vigorously8 Allow the filtrate to separate into two phases9 Discard the aqueous phase (lower)

10 Repeat steps 6 7 8 and 9 twice11 Filter the n-hexane phase through a layer of anhydrous sodium

sulfate placed in a funnel plugged with a filter paper12 Concentrate the extract by evaporation to dryness under

reduced pressure in rotary evaporator using a 35ndash40 Cwater bath

13 Recover the residue obtained with 1mL of acetonebefore analysis

14 Analyze by HPLC-DAD following authorsrsquo protocol

Martel andZeggane (2002)


Table 6 Analysis of pesticide residues in honey using a solid-phase extraction (SPE)

Pesticide analyzed Extraction protocol References450 pesticides 1 Dilute 15 g of the test sample in a 250mL glass jar with 30mL of water

2 Shake for 15min at 40 C in a shaking water bath3 Add 10mL of acetone to the jar4 Transfer the jar contents to a 250mL separating funnel5 Rinse the jar with 40mL of dichloromethane and transfer this rinse to the

separating funnel for partitioning6 Shake the funnel eight times and pass the bottom layer through a funnel

containing anhydrous sodium sulfate into a 200mL pear-shaped flask7 Add 5mL of acetone and 40mL of dichloromethane into the

separating funnel8 Repeat steps 6 and 7 twice9 Evaporate the organic phase to about 1mL with a rotary evaporator at

40 C for clean-up10 Add sodium sulfate into a graphitized carbon black cartridge to

about 2 cm11 Connect the cartridge to the top of the aminopropyl cartridge in series12 Condition the cartridges with 4mL acetonitriletoluene 31 (vv)13 Add 1mL of the sample14 Rinse the pear-shaped flask with 3 2mL acetonitriletoluene 31 (vv)

and decant it into the cartridges15 Elute the pesticides with 25mL acetonitriletoluene 31 (vv)16 Evaporate the eluate to about 05mL using a rotary evaporator at 40 C17 Analyze by GC-MS or LC-MSMS following authorsrsquo protocols according

to the group of pesticides

Panget al (2006)

Organochlorines (a- b- andCcedil-hexachlorocyclohexane(HCH) alachlorheptachlor aldrinendosulfan II 440-DDEdieldrin endrin and 440-DDD) and primiphos-ethyl (internal standard)

1 Add 10 g of honey in a jar and heat the honey at 35 C for 15min2 Add 50mL of distilled water to dissolve honey3 Extract with 3 30mL portions of a binary mixture of petroleum ether

ethyl acetate 8020 (vv) in a separating funnel4 Dry the combined organic extract over anhydrous sodium sulfate5 Evaporate to 2mL with rotary evaporator and transfer into a 5mL glass

tube concentrator6 Evaporate to dryness under a stream of nitrogen7 Dissolve the dried residue with 04mL of n-hexane8 Condition the florisil cartridge with 10mL of n-hexane9 Load the concentrated extract obtained in step 7 onto the cartridge10 Elute the pesticides with 25mL of 20 (vv) of diethyl ether in n-hexane11 Evaporate the eluate to dryness under a stream of nitrogen12 Dissolve the dried residue with 04mL of n-hexane containing 1 mgmL of

internal standard13 Analyze by GC-MSMS following authorsrsquo protocol

Tahboubet al (2006)

15 organophosphorus (OP)17 organochlorines (OC)8 pyrethroids (PYR) 12N-methyl-carbamate(NMC) bromopropylateand the internalstandards 4-bromo-35-dimethylphenyl-Nmethylcarbamate (4-Br-NMC)triphenylphosphate (TPP)and polychlorinatedbiphenyl (PCB) 209

1 Dissolve 10 g of honey in a 60mL glass tube with 10mL of water and10mL of acetone

2 Homogenize the mixture with Ultra Turrax for 2min3 Wash the Ultra Turrax with about 2mL of acetone and collect the

washings into the glass tube4 Load the solution obtained in step 3 into an EXtrelutVRNT 20 column5 Allow to drain for 10min to obtain an even distribution into the

filling material6 Elute pesticides with 5 20mL of dichloromethane using the first aliquot

to wash the glass tube7 Collect the eluate into a 150mL Erlenmeyer flask8 Concentrate to nearly 1mL with a rotary evaporator at 40 C (reduced

pressure) and by drying manually by rotating the flask9 Dissolve the residue in 2mL of n-hexane10 Divide the sample solution obtained in step 9 in two portions11 Transfer one portion of the sample (1mL) into a 25mL Erlenmeyer flask

and dry by manually rotating the flask12 Dissolve the residue in 05mL wateracetonitrile 5050 (vv)13 Add the appropriate amount of 4-Br-NMC as an internal standard14 Inject the sample into the LCDDFl (liquid chromatography-double

derivatization coupled with spectrofluorimetric detector) for thedetermination of NMC pesticides following authorrsquos protocol

15 Add the appropriate internal standards (PCB 209 and TPP) in the secondportion of the original sample left in the Erlenmeyer flask (1mL)

16 Inject this portion in GC-MSD and in GC-FPD following authorsrsquoprotocols in order to analyze bromopropylate OC PYR and OP residuesrespectively

Amendolaet al (2010)


each target analyte must be prepared with othersamples to control the sensitivity of the equipment

(3) A supplementary test (named ldquotest samplerdquo) is con-ducted to measure the recovery of all tar-get analytes

(4) For each analytical sequence the extracts areinjected into the analytical instrument in the follow-ing order blank solvent negative control honey(blank sample) samples spiked with pesticides forcalibration (the ldquomatrix calibration standardsrdquo) blanksample unknown samples (samples to quantify) testsample (to calculate the recovery for each pesticide)and again a spiked sample from the calibration tocontrol any variation during the sequence andblank sample

(5) At the end of the sequence the sample spiked atlevel 1 (corresponding to the LOQ) is injected toverify the ability of the equipment to detect theLOQ Validation of the analytical method should beperformed according to the Guidance document onanalytical quality control and method validation pro-cedures for pesticides residues analysis in food andfeed (Document SANTE118132017 2018)

523 Sample preparation (extraction and clean-up)

The purpose of the extraction is to extract the sub-stance from the sample with minimum co-extractivesmatrix interferences The choice of the solvent isimportant Acetone acetonitrile ethyl acetate and hex-ane are commonly used in this step The analytes areextracted from the matrix and then through a clean-upprocess the co-extractives are removed

The most frequent technique used for sample extrac-tion is via the homogenization of the honey sample withan organic solvent or water followed by clean-up with aliquidndashliquid extraction (LLE) or a solid phase extraction(SPE) on column Relatively new techniques like the dis-persive solid phase extraction (dSPE) and solid phasemicroextraction (SPME) are used Main parameters ofmethodologies mentioned in the literature for theextraction of pesticides from honey are reported byRial-Otero et al (2007) and Souza Tette et al (2016)Different techniques are presented below with corre-sponding protocols The main criterion is to find onemethod that gives acceptable recoveries for all analyteswith one protocol

5231 Liquid-liquid extraction (LLE) In multiresiduemethods an important step is the extraction procedureespecially for complex matrices such as honey whichcontains high sugar content LLE is the most commonextraction and purification technique used in the deter-mination of pesticides in honey (Souza Tette et al2016) However LLE usually employs large sample sizesand toxic organic solvents It is also characterized bythe use of multiple sample handling steps which makes

it susceptible to error and contamination Furthermoreit usually enables the extraction of analytes belonging toonly one chemical class Despite the disadvantagesdescribed above LLE continues to be used in the ana-lysis of pesticides in honey Barganska and Namiesnik(2010) reviewed in their paper the techniques alreadyused to extract pesticides from samples of honey Thegeneral protocol involves dissolving honey in water andapplying a LLE with various solvents (such as petroleumether hexane dichloromethane ethyl acetate or mix-tures of petroleum etherethyl acetate (Tahboub et al2006) acetonitrileacetoneethyl acetatedichlorome-thane (Rissato et al 2007) acetonedichloromethane(Pang et al 2006) hexanepropanol-2 (Martel ampZeggane 2002)) to extract pesticides according to thepesticidersquos polarity The experimental procedures aregiven in Table 5

The extracts obtained before analysis by chromatog-raphy are clean but these methods use a considerableamount of solvent Furthermore these methods arelimited to the extraction of a few pesticides due totheir different solubility

5232 Solid phase extraction (SPE) and dispersivesolid phase extraction (dSPE) Sometimes after a LLEa clean-up step on a SPE cartridge with differentadsorbents may be necessary before quantification ofpesticide residues in honey Protocols using SPE aregiven in Table 6 for the main classes of pesticides

The SPE technique (Barganska amp Namiesnik 2010Rial-Otero et al 2007 Singh et al 2014 Souza Tetteet al 2016) combines in a single step the extractionand the clean-up procedures based on the separationof LC where the solubility and functional group inter-actions of sample solvent and sorbent are optimizedto affect retention and elution In SPE the sample ispassed through a cartridge or a packed column filledwith a solid sorbent where the pesticides are adsorbedand then eluted with an organic solvent Moderatelypolar to polar analytes are extracted from nonpolarsolvents on polar sorbents Nonpolar to moderatelypolar analytes are extracted from polar solvents onnonpolar sorbents Most multiresidue methods includea clean-up step using adsorption columns on polarsorbents (florisil alumina silica gel) for normal-phaseSPE and on nonpolar sorbent like C18 for reversed-phase SPE The reversed-phase C18 cartridge is by farthe most common choice used by researchers for theextraction of insecticides acaricides fungicides herbi-cides organochlorines and organophosphorus pesti-cides from honey (Rial-Otero et al 2007) As florisilretains some lipids preferentially (25 g florisil with 3water retains 1 g of fat) it is particularly well suitedfor the clean-up of fatty foods When a florisil columnis eluted with solvent mixtures of low polarity nonpo-lar residues are recovered almost quantitatively (Singhet al 2014) Florisil sorbent has been used for


pyrethroids organochlorines and organophosphoruspesticides Lores et al (1987) used a silica gel clean-upmethod for organophosphorus pesticide analysis

Other SPE clean-up approaches include the combin-ation of GCB (graphitized carbon black) and PSA (pri-mary secondary amine) columns the combination ofC18 GCB and aminopropyl and the combination ofGCB PSA and SAX (strong anion-exchange sorbent)columns GCB is such a sorbent being nonspecific andgenerally of hydrophobic nature Contrary to sorbentsbased on SiO2 these may be used without the pH ofthe treated solutions being considered Because of diffi-culties with elution of certain planar or aromatic pesti-cides from GCB only PSA is used for very efficientclean-up of acetonitrile extracts The dSPE with PSA iseffectively used to remove many polar matrix compo-nents such as organic acids certain polar pigments andsugars Thus the PSA clean-up method is selected asthe most efficient for cleaning honey samples

The choice of one sorbent or another depends onthe analyte polarity and on the possible co-extractedinterferences Sample pH can be critical to obtain highyields of pesticide retention in the sorbent Thus insome cases sample pH modification can be necessary inorder to stabilize the pesticides and increase theirabsorption in the solid phase Once the pesticides havebeen retained in the SPE cartridges they are theneluted with an organic solvent such as acetonedichloromethane ethyl acetate hexane methanol tetra-hydrofurane or mixtures of hexaneethyl acetate hex-anedichloromethane and methanolwater or methanolethyl acetatedichloromethane

There is also a method based on SPE the on-col-umn liquid-liquid extraction (OCLLE) or liquid-liquidextraction on a solid support (SLE) a technique basedon classical LLE principle but assisted by inert solidsupport (Pirard et al 2007) This inert matrix consistsof diatomaceous earth well-known for its high poros-ity its high dispersing capacities and its high capacityfor aqueous adsorption Pesticides in honey werestudied by Amendola et al (2010) using EXtrelutVRNT20 columns packed with a specially processed widepore kieselguhr with a high pore volume as supportfor the repartition process Honey is dissolved in amixture of water and acetone and is loaded into anEXtrelutVRNT 20 The majority of the co-extractivecompounds are retained on the adsorbent material ofthe column while the pesticides are eluted bydichloromethane (Amendola et al 2010) Other col-umns can be used such as ChemElut 5mL cartridges(Kujawski et al 2014)

5233 Quick Easy Cheap Effective Rugged and Safe(QuEChERS) The QuEChERS method consists in salt-ing-out LLE using acetonitrile MgSO4 and NaCl saltsand a dSPE step based on primary and secondary aminebonded silica (PSA) to remove co-extractive impurities

(US EPA 2013 Barganska amp Namiesnik 2010) TheQuEChERS method is particularly applied for the deter-mination of polar middle polar and nonpolar pesticideresidues in various matrices This method is combinedwith sensitive analytical techniques such as LC-MSMSand GC-MSMS Tomasini et al (2012) demonstratedthat the matrix effect depends on the floral origin ofhoney samples and that quantification by the standardaddition method in blank matrix is needed

With this approach the sample should be gt75water Then an initial dissolution of the honey sampleis required Acetonitrile is used as the water-miscibleextraction solvent and the separation phase is achievedby the addition of MgSO4 The heat produced by thewater binding process promotes extraction to aceto-nitrile The addition of NaCl also increases the extrac-tion efficiency (Kujawski et al 2014) The supernatantis further extracted and cleaned using a dSPE techniqueThe dSPE centrifuge tube format (available in 2mL and15mL sizes) contains magnesium sulfate (to removeresidual water) and PSA sorbent (to remove sugars andfatty acids) These tubes are available with or withoutGCB (to remove pigments and sterols) andor C18-EC(endcapped) packing (to remove nonpolar interferencessuch as lipids) Blasco et al (2011) showed that theQuEChERS method presented the highest recoveries(mean recovery 9167) followed by the SPE (meanrecovery 9025) whereas the solid-phase microextrac-tion (SPME) showed the lowest recovery (mean recov-ery of 4975) for the pesticides studied Thus theQuEChERS method was the most adapted method witharound 58 of recoveries gt90

The QuEChERS multiresidue procedure replacesmany complicated analytical steps commonly employedin traditional methods with easier ones The QuEChERSmethod has been the most commonly used method forthe analysis of pesticides in honey However one limita-tion is that the sample should be gt75 water thus aninitial dissolution of the honey sample is required whichleads to lower concentration of the sample comparedto other sample preparation techniques In order toovercome this limitation Wiest et al (2011) added asample concentration step by evaporation which wassatisfactory for extraction of organohalogens organo-phosphorus pyrethroids and insect growth regulatorsin honey According to these authors evaporation maybe necessary when the MRL is lower than the methodLOQ A general protocol is given in the Table 7

5234 Solid phase microextraction (SPME) SPME is arapid and simple procedure of extraction that can beeasily automated and does not need an organic solventThis technique consists of two separate steps anextraction step and a desorption step Both steps mustbe optimized for the procedure to be successful (Singhet al 2014)


Table 7 Analysis of pesticide residues in honey using the QuEChERS methodology

Pesticide analyzed Extraction protocol References12 organophosphorus and carbamates

insecticides (bromophos-ethylchlorpyrifos-methyl chlorpyrifos-ethyldiazinon fenoxycarb fonofosphenthoate phosalone pirimiphos-methyl profenofos pyrazophosand temephos)

1 Weigh 15 g of honey into a 50mLpolypropylene centrifuge tube

2 Add 3mL of hot water and vortex untildissolution

3 Add 3mL of acetonitrile to the sample andshake the tube vigorously by hand for 30 s

4 Pour the sample and extract into theappropriate tube containing 6 g of MgSO4 and15 g of NaCl

5 Shake the tube vigorously by hand for 1min(avoiding formation of oversized MgSO4agglomerates)

6 Centrifuge the tube at 3000 rpm for 2min7 Transfer 1mL of acetonitrile extract (upper

layer) to the dispersive-SPE tube containing150mg anhydrous of MgSO4 and 50mg of PSA

8 Vortex the dSPE tube for 30 s and centrifuge at3000 rpm for 2min

9 Transfer 05mL of the final extract into thelabeled autosampler vial

10 Analyze by LC-MSMS followingauthorsrsquo protocol

Blasco et al (2011)

80 environmental contaminants 1 Weigh 5 g of honey in a 50mL centrifuge tube2 Add 10mL of water in the centrifuge tube3 Shake the tube to dissolve honey4 When the mixture is homogeneous add 10mL

of acetonitrile (ACN) 4 g of anhydrous MgSO41 g of sodium chloride 1 g of sodium citratedihydrate and 500mg of disodium citratesesquihydrate

5 Shake the tube immediately by hand6 Vortex one minute and then centrifuge for

2min at 5000 rpm7 Transfer 6mL of supernatant in a pre-prepared

15mL PSA tube (900mg of anhydrous MgSO4150mg of PSA bonded silica)

8 Then shake the tube immediately by hand9 Vortex 10 s and centrifuge for 2min

at 5000 rpm10 Evaporate 4mL of the extract in a 10mL glass

cone-ended centrifuge tube until 50 mL are left11 Kept the remaining extract at 18 C until

analysis by LC-MSMS or GC-MS followingauthorsrsquo protocols according to the groupof pesticides

Wiest et al (2011)

Chlorothalonil heptachlor captana-endosulfan b-endosulfan endosulfansulfate and dieldrin

1 Place 10 g of honey sample into apolypropylene tube (50mL) of conical base

2 Homogenize with 10mL of high purity water3 Add 15mL of 1 acetic acid in ethyl acetate

extraction solvent also containing 6 g ofMgSO4 and 15 g of CH3COONa anhydrous tothe tube

4 Shake by hand vigorously for 1min5 Centrifuge at 5000 rpm for 5min6 Transfer an aliquot of 1mL of the supernatant

to a 2mL polypropylene tube containing 50mgof PSA and 150mg of MgSO4

7 Shake by hand vigorously for 30 s andcentrifuge at 5000 rpm for 5min

8 Put 500 lL of the extract obtained in step 7into the 15mL vial and complete with 500lLof ethyl acetate

Vilca et al (2012)

(Continued)


A fused silica fiber coated with a polymeric film isimmersed into an aqueous sample for a given amount oftime The analyte enrichment is by partitioning betweenthe polymer and the aqueous phase according to theirdistribution constant Factors influencing the extractionstep include fiber type extraction time ionic strengthsample pH extraction temperature and sample agita-tion The pesticides are adsorbed into the stationaryphase and later thermically desorbed into the injectionport of a gas chromatograph (Rial-Otero et al 2007)Variables affecting the desorption step include tempera-ture desorption time focusing oven temperature thesolvent used and its volume Most pesticides have beenextracted with polydimethylsiloxane (PDMS100 mm) fiber

The PDMS has the following advantages (i) enhancedreproducibility (ii) lower detection limits (iii) extendedlinearity (iv) improved correlation coefficients (v) lowextraction time and (vi) better chromatograms Theuse of a sol-gel CROWN ETHERVR fiber (40mm) wasalso proposed by Yu et al (2004) to remove 11 organo-phosphorus pesticides from honey with good relativerecoveries (74ndash105) and low detection limits(lt0001mgkg) Blasco et al (2011) employed silicafibers coating with 50 mm carbowaxtemplate resins

(CWTPR) to analyze fenoxycarb penthoate temephosfonofos diazinon pyrazophos phosalone profenofospirimiphos-ethyl bromophos-ethyl chlorpyrifos-methyland chlorpyrifos-ethyl in honey The SPME is accurateas a monitoring method for the extraction of theselected pesticides from honey but cannot be imple-mented as currently applied as a quantification methoddue to its low recovery for pyrazophos chlorpyrifos-methyl temephos and bromophos-ethyl The applica-tion of the internal standard should be considered

5235 Stir bar sorptive extraction (SBSE) SBSE is atechnique theoretically similar to SPME It has beenused with success for the extraction of organic com-pounds from aqueous food biological and environmen-tal samples (Rial-Otero et al 2007)

For SBSE a stir bar is coated with a sorbent andimmersed in the sample to extract the analyte fromsolution The sample is stirred for a given time until theanalyte reaches equilibrium between the polymer andthe aqueous phase according to their distribution con-stant Then the analytes are desorbed by high tempera-tures into the injector port of the GC or by liquidremoval for liquid chromatography-tandem mass spec-trometry (LC-MS) analysis Blasco et al (2004) have

Table 7 (Continued)

Pesticide analyzed Extraction protocol References

9 Analyze by GC-lECD followingauthorsrsquo protocol

200 pesticides 1 Weigh 5 g of the honey sample into a 50mLpolypropylene centrifuge tube

2 Add 10mL of deionized water3 Vortex and incubate in a water bath at 40 C

until complete homogeneity is obtained4 Add 10mL of acetonitrile into the tube5 Shake the content for 1min using a

mechanical shaker6 Add the QuEChERS salt kit (containing 4 g of

anhydrous magnesium sulfate 1 g of sodiumchloride 1 g of sodium citrate and 050 g ofsodium hydrogen citrate sesquihydrate) andimmediately shake for further 1min

7 Centrifuge at 15000 rpm at 4ndash8 C for 5min8 Transfer the whole acetonitrile fraction into a

15mL dSPE polypropylene tube (containing150mg of anhydrous magnesium sulfate and25mg of PSA)

9 Shake the tube for 1min and centrifuge for2min at 15000 rpm using a cooling centrifuge

10 Transfer 2mL of the supernatant into 50mLround bottom glass flask

11 Evaporate under vacuum at 40 C tillcomplete dryness

12 Reconstitute the residue into 2mL of hexaneacetone 91 (vv)

13 Ultra-sonicate and filter the sample through adisposable 045 mm PTFE membrane filter intoan amber glass vial

14 Analyze by GC-MSMS following theauthorsrsquo protocol

Shendy et al (2016)


applied this technique for the extraction of six organo-phosphorus pesticides from honey These authors alsocompared the use of SBSE with SPME and concludedthat although linearity and precision obtained by bothtechniques are similar SBSE is more accurate and sensi-tive and the effect of honey matrix in the quantificationis lower than that of SPME

The most important advantages of SBSE are thesame as those for SPME however higher recoveriesare obtained with SBSE (Blasco et al 2004) because ofthe thicker polydimethylsiloxane (PDMS) coating Theprocedure is presented in Table 8 Nevertheless recov-eries are less than those obtained using other techni-ques (LLE SPE QuEChERS)

5236 Dispersive liquid-liquid microextraction(DLLME) In 2006 the use of DLLME was developed inthe field of separation science for the preconcentrationof organic and inorganic analytes from aqueous matri-ces The basic principle of DLLME is the dispersion ofan extraction solvent (immiscible with water) and a dis-perser solvent (miscible in water and extraction solv-ent) in an aqueous solution that provides a largecontact area between the aqueous phase and theextraction solvent In DLLME extraction and dispersivesolvents are simultaneously and rapidly injected into theaqueous sample using a syringe The main advantages ofDLLME over conventional techniques are simplicity ofoperation rapidity low cost easy handling low con-sumption of organic solvents high recovery high factorenrichment and compatibility with chromatographictechniques such as liquid chromatography (LC) and gaschromatography (GC) However the QuEChERSmethod is demonstrated to be more robust and moresuitable for the determination of pesticides in complexsamples (Tomasini et al 2011) A simple DLLME proto-col for the determination of 15 organochlorine pesti-cides residues in honey is proposed by Zacharis et al(2012) and is described in Table 9 The final DLLMEprotocol involves the addition of 750 lL acetonitrile(disperser) and 50 lL chloroform (extraction solvent)into a 5mL aqueous honey solution followed by centri-fugation The sedimented organic phase (chloroform) isanalyzed directly by GC-ITMS (gas chromatography-ion

trap mass spectrometry) or evaporated and reconsti-tuted in acetonitrile prior to the GC-ECD analysis

524 Chromatographic detection

The quantification is performed by gas or liquid chroma-tography (Barganska amp Namiesnik 2010 Rial-Oteroet al 2007) according to the characteristic of the ana-lyte (i) Gas chromatography (GC) with different detec-tors electron capture (ECD) nitrogen-phosphorus(NPD) and mass spectrometry (GC-MS or GC-MSMS)(ii) liquid chromatography coupled with mass spectrom-etry (LC-MSMS)

Traditionally nonpolar and middle polar pesticides areanalyzed with GC This is the case for organochlorinespyrethroids and organophosphorus pesticides With theemergence of new pesticides (eg neonicotinoids) anddue to their physicochemical properties the LC is used

For the multiresidue analysis of pesticides the mostconvenient detector would be a mass spectrometer (MSMS MSTOF) coupled with either GC or LC dependingon the type of pesticides of interest Volatile semi-vola-tile and thermally stable ones can be determined by GCwhereas nonvolatile andor thermally unstable onesshould be determined by LC When there is a positiveresult it has to be confirmed using mass spectrometrycoupled with chromatography (GC-MSMS and LC-MSMS) Mass spectrometry coupled to a chromatographicseparation method is a very powerful combination foridentification of an analyte in the sample extract

The choice of the GC column is a very important taskin pesticide analysis The stationary phase should beselected as a function of the polarity of the pesticidesNonpolar columns (5 phenyl 95 dimethylpolysiloxane)are the most commonly used for pesticide analysis inhoney (eg DB-5 HP-5MS and DB-XLB) Furthermoreother column parameters such as length inner diameteror film thickness can be optimized as a function of thenumber of pesticides to be determined simultaneouslyUsually the parameters of the column are30m 025mm 025mm (length inner diameter filmthickness) In LC a C18 column (eg 46 and 21mm id)is almost consensus for the separation of pesticides

Table 8 Analysis of pesticide residues in honey using the SBSE protocol (Blasco et al 2004)

Pesticide analyzed Extraction protocol6 organophosphorus insecticides (chlorpyrifos-methyl

diazinon fonofos phenthoate phosalone andpirimiphos-ethyl)

1 Place 25 g of honey into a 50mL glass beaker2 Dilute 110 ratio with water and homogenize over 15min using

a magnetic stirring bar coated with PDMS3 Carry out the sorption for 120min while stirring at 900 rpm4 Remove the stir bar from the aqueous sample with tweezers5 Perform the desorption of the analytes into 2mL vial filled with

1mL of methanol6 Perform the desorption of the pesticides by agitating for 15min7 Inject 5 mL of this extract into the LC-MS system following the

authorsrsquo protocol


6 Antibiotics and chemotherapeutics in honey

Honey is generally considered as a natural healthy andresidue-free product However in the early 2000ssome imported honeys were often contaminated withantimicrobial residues even with residues of chloram-phenicol a forbidden substance (Reybroeck 2018)Importers of honey started with regular screening ofhoney for residues and food authorities increased thenumber of honey samples at border inspection postsand in national monitoring plans In the beginning themonitoring was focused on streptomycin chlorampheni-col tetracyclines and sulfonamides Later the scopewas enlarged to include other compounds Presentlyhoney is monitored for a large list of antibiotics andchemotherapeutics of interest in apiculture tetracy-clines (oxytetracycline) aminoglycosides (streptomycin)sulfonamides (sulfamethazine sulfathiazole sulfadiazine

sulfamethoxazole sulfamerazine sulfadimethoxine) mac-rolides (tylosin erythromycin) lincosamides (lincomy-cin) amphenicols (chloramphenicol) nitrofurans(furazolidone furaltadone nitrofurazone) nitroimida-zoles (metronidazole) fluoroquinolones (enrofloxacin(ciprofloxacin) norfloxacin) and fumagillin (Reybroecket al 2012)

In general residues of antimicrobials in honey ori-ginate from apicultural use since bee diseases causedby microorganisms such as American (Paenibacillus lar-vae de Graaf et al 2013) and European foulbrood(Melissococcus plutonius Forsgren et al 2013) can becured by anti-infectious agents Also nosemosiscaused by spores of the fungi or fungi-related Nosemaapis or N ceranae (Fischer amp Palmer 2005 Fries et al2013) is sometimes treated with antimicrobials likesulfonamides

Table 9 Analysis of pesticide residues in honey using the DLLME protocol

Pesticide analyzed Extraction protocol References15 pesticides (etridiazole chloroneb

propachlor trifluralinhexachlorobenzene chlorothalonilcyanazine chlorpyrifos DCPA trans-chlordane cis-chlordane trans-nonachlor chlorobenzilate cis-permethrin trans-permethrin)

1 Prepare a solution of honey at 50 gL (dissolve 10 gof honey in a flask with 200mL of water)

2 Leave the sample to equilibrate for at least for15min prior to performing the DLLME extraction

3 Transfer an aliquot of 5mL of the diluted sampleinto a 10mL screw cap glass tube withconical bottom

4 Inject rapidly into the sample solution a mixture of750 mL acetonitrile (disperser) and 50mL chloroform

5 Gently shake the mixture by hand for 1min6 Extract the pesticides and the ISTD (1-bromo

decahexane) from the aqueous matrixphase intothe fine chloroform microdroplets

7 Centrifuge the mixture for 3min at 2500 rpm forphase separation

8 Remove the sedimented chloroform volume using amicrosyringe

9 Transfer the extract into an autosampler vial with50 mL insert and 2 mL of the organic solventdirectly inject this into the GC-ITMS (GC withion-trap mass spectrometer detector) following theauthorsrsquo protocol

10 For GC-ECD analysis evaporate 20 mL ofchloroform extract (obtained in step 8) to drynessby a gentle stream of nitrogen

11 Reconstitute the sample by the same volume (20mL) of acetonitrile

Zacharis et al (2012)

Aldrin endrin lindane 240-DDT 240-DDD 240-DDE 440-DDT 440-DDEand a-endosulfan

1 Dissolve in a centrifuge tube 05 g of a homogenizedhoney sample with 3mL of ultrapure water

2 Prepare and inject rapidly a mixture of 450 mLacetone (disperser solvent) and 100 mL chloroform(extract) into the sample to obtain an emulsion

3 After 20 s (including 5 s of shaking) centrifuge thesample (5min 4000 rpm) A two-phase solutionis obtained

4 The resulting volume of sediment phase is 80 mLDuring the extraction a precipitate formed betweenchloroform and aqueous phase

5 Collect the chloroform phase at the bottom of theconical vial with a microlitre syringe

6 Analyze by GC-MS (2 mL of sample are injected)following the authorsrsquo protocol

Kujawski et al (2012)


High levels (mgkg or ppm) of antimicrobial residuesof veterinary drugs applied to hives can be found inhoney especially the first week after dosing (Reybroecket al 2012) Afterward the concentration of residuesin honey decreases via a dilution effect by incoming nec-tar and consumption of contaminated honey by thebees Some compounds (oxytetracycline tylosin furazo-lidone) are also degraded by metabolization In contrastto other food producing animals honey bees do notmetabolize the drugs so residues could remain in thehoney for more than a year (Reybroeck et al 2012)see Table 10

There are some cases reported of residues of antimi-crobials in the honey due to agricultural practices bee-keeping practices and environmental or fraud issues(Reybroeck 2014) contaminated nectar from fruit treestreated with streptomycin against fireblight natural pro-duction of streptomycin by certain Streptomyces bac-teria robbery by bees of contaminated honey feedingbees contaminated honey mixing clean honey withhoney containing residues the collection by bees ofmedicated drinking water from farms or surface waterfrom fields where antibiotic-containing manure has beenspread contamination of nectar with sulfanilamide as adegradation product of the herbicide asulam migrationof residues from polluted wax foundation semicarbazideformed from azodicarbonamide (ADC a blowing agentused in the manufacturing of plastic gaskets in metallids) and finally semicarbazide in heather honey formedfrom elevated arginine levels

For residue analysis in honey it is worth notingthat some pharmacologically active compoundsmetabolize or degrade in honey Thus it is importantto look for the suitable marker residue For examplehoney should be screened for both tylosin A and des-mycosin (tylosin B) (Thompson et al 2007) An over-view of the most suitable marker residues for someantimicrobials of interest in beekeeping is given inTable 11 Reybroeck (2018) published a review aboutresidues of antibiotics and chemotherapeutics inhoney This review can be a basis to decide whichcompounds to analyze in honey

61 Legislation regarding residues of veterinarydrugs in honey

MRLs were established for residues of veterinary medi-cinal products in foodstuffs of animal origin in order toprotect public health No MRLs have been established sofar for antibiotics and sulfonamides in honey(ldquoCommission Regulation (EU) No 372010 of 22December 2009rdquo 2009) This leads to the interpretationthat the use of antibiotics in beekeeping is not permittedin the EU However based on the ldquocascaderdquo systemwhich is open to all animal species (including honey bees)antibiotics can be used for the treatment of bee diseases(Anonymous 2007b) on condition that the active sub-stance concerned is registered as an allowed substance in

Table 1 in the Annex of Commission Regulation (EU) No372010 and a withholding period has been specified bythe prescribing veterinarian In this period no honeycould be harvested for human consumption In reality avery long withdrawal period needs to be considered

Some EU Member States (Belgium France) appliedaction limits recommended target concentrationsnonconformity or tolerance levels for antimicrobialresidues in honey (Reybroeck et al 2012) Presentlyin application of the Commission ImplementingRegulation (EU) 2018470 for honey produced withinthe EU the MRL to be considered for control purposeshall be the lowest of all the MRLs established forother target tissues in any animal species Howeversome Member States do not allow treatment with anti-biotics and chemotherapeutics under ldquocascaderdquo in bee-keeping which is making international honey trademore complex

For certain prohibited or unauthorized analytes infood of animal origin the regulatory limit is theMinimum Required Performance Limit (MRPL) or theReference Point for Action (RPA) MRPLs were fixed toharmonize the level of control of those substances andto ensure the same level of consumer protection in theCommunity So far in honey a MRPL of 03 mgkg wasset for chloramphenicol (European CommunitiesCommission 2003) while the MRPL of 1 mgkg fornitrofuran (furazolidone furaltadone nitrofurantoin andnitrofurazone) metabolites in poultry meat and aquacul-ture products generally is considered as also applicablein honey (SANCO 2004)

For the importation of products of animal originfrom third countries the MRPLs should be employedwhere they exist as RPAs to ensure a harmonizedimplementation of Council Directive 9778EC(Commission 2005)

In a guidance paper of the Community ReferenceLaboratories (CRLs) recommended concentrations fortesting were suggested for the harmonization of the per-formance of analytical methods for national residue controlplans for substances without MRLs (Anonymous 2007a)An overview of European regulatory limits and recom-mended concentrations for testing in honey for residues ofantibiotics and chemotherapeutics of interest for use inbeekeeping is given in Table 11

In the USA there are no authorized residue limitsfor antibiotics in honey despite the authorized use ofcertain antibiotic drugs (oxytetracycline tylosin and lin-comycin) in beekeeping (Administration 2017)

In Europe for national residue monitoring plansgroups of substances that need to be monitored inhoney are indicated (ldquoCouncil Directive 9623EC of 29April 1996rdquo 1996) For honey it concerns group B1(antibacterial substances including sulfonamides and qui-nolones) B2c (other veterinary drugs - carbamates andpyrethroids) B3a and B3b (organochlorine and organo-phosphorus compounds) and B3c (chemical elements)


Despite not being indicated honey should also be moni-tored (ldquoessentialrdquo) for prohibited substances (group A6)such as chloramphenicol nitrofurans and nitroimida-zoles (Anonymous 2017)

Sampling rules are given in Annex IV of the CouncilDirective 9623EC The same Directive specifies thefrequencies and level of sampling while the CommissionDecision 97747EC (European CommunitiesCommission 1997) provides levels and frequencies ofsampling The number of honey samples to be takeneach year must equal 10 per 300 t of the annual pro-duction for the first 3000 t of production and onesample for each additional 300 t Rules for official sam-pling procedures and the official treatment of samplesuntil they reach the laboratory responsible for analysisare given in Commission Decision 98179EC(Commission 1998)

62 Determination of residues of antibiotics andchemotherapeutics in honey

621 Sampling

The analyzed honey samples should be representativeof the honey lot

(1) Crystallized honey should be homogenized beforestarting the analysis This can be done by placing thesample in a water bath at maximum 40 C until thehoney is fully liquified and all of the sugar crystalsare dissolved

(2) The honey should be homogenized before startingthe analysis by stirring thoroughly (at least 2min)

(3) In the event the honey contains extraneous matterthe honey should be strained through a stainless-steel sieve with a mesh diameter of 05mm

(4) If needed the honey could be gently pressedthrough the sieve with a spatula

(5) Comb honey needs to be uncapped and drainedthrough a 05mm sieve without heating in order toseparate honey from the comb

622 Sample pretreatment

Some honeys require a special sample pretreatment Inhoney sulfonamides tend to bind sugars via the forma-tion of N-glycosidic bonds through their aniline group(Sheth et al 1990) Therefore the sulfonamides needto be released from the sugar concentrates by

hydrolysis using strong acids (Schwaiger amp Schuch2000) prior to analysis Otherwise it is possible thatthe sulfonamides are missed or underestimated

623 General remarks for honey testing on antimicro-bial residues

It is necessary to integrate at least one negative andone positive control sample in each test run The nega-tive honey is honey free from antimicrobial residuesand by preference a mixture of the different types ofhoney (origin color and texture) all tested as negativein prior analysis The positive control sample is pre-pared by contaminating blank honey with standardmaterial If commercial kits are used follow the kitinstructions set by the kit manufacturer and validate themethod internally before using the method in routinetesting In Europe validation should be performedaccording to Commission Decision 2002657EC(European Union Commission 2002) For screeningmethods the guidelines published by the CommunityReference Laboratories for Residues could also betaken into account (Anonymous 2010)

624 Microbiological screening tests

Microbiological screening is not often used for screeninghoney for antibiotic residues this due to the very lowdetection capabilities (CCb) that need to be reachedbecause of the issue of zero-tolerance for antimicrobialresidues in honey applied in many countries The highsugar content in this special matrix makes the use ofmicrobial inhibitor tests also less evident A broad-spec-trum detection of antimicrobials in honey by Eclipse 50(ZEULAB Zaragosa Spain) and PremiTest (R-BiopharmAG Darmstadt Germany) was suggested by Gaudinet al (2013) In their validation a high false positive rateof 5 and 14 respectively was observed This studyalso showed that the detection capabilities are not inline with the action or reporting limits or recom-mended concentrations for testing for different com-pounds (eg streptomycin) applied in some Europeancountries The extraction and test procedure for theEclipse 50 and PremiTest for honey testing are given inTables 12 and 13 respectively

Noncommercial microbiological methods two forthe detection of tetracyclines and one for the detectionof tylosin were published for honey based on the useof Bacillus cereus (Gordon 1989) Bacillus subtilis

Table 10 Classification of some veterinary drugs based on their stability in honey

Compounds that do not degrade in honey Compounds metabolizing or degrading in honeyStreptomycin Tetracyclines epimerization to 4-epimersSulfa drugs Tylosin degradation to desmycosin (tylosin B)Lincomycin Furazolidone metabolization to AOZChloramphenicolNote AOZ 3-amino-2-oxazolidone


Table 11 Overview of European regulatory limits in honey and recommended concentrations for testing for residues of anti-infec-tious agents of interest for use in beekeeping

Group Substance Marker residue MRL (in mgkg)Recommended concentration

for testing (in mgkg)Amphenicols chloramphenicol chloramphenicol mdasha 03 MRPLNitrofurans group (furazolidone hellip ) AOZ AHD SEM and AMOZ mdasha 1 MRPLNitro-imidazoles ronidazole hydroxy-metabolites mdasha 3

dimetridazole hydroxy-metabolites mdasha 3metronidazole hydroxy-metabolites mdasha 3

Tetracyclines tetracyclines sum of parent drug and its4-epimer

mdashb 100c 20

Sulfonamides (allsubstancesbelonging to thesulfonamidegroup)

sulfonamides the combined total residuesof all substances within thesulfonamide group

mdashb 100c 50

Aminoglycosides streptomycin streptomycin mdashb 200c 40Macrolides erythromycin erythromycin A mdashb 40c 20

tylosin tylosin A mdashb 50c 20Note MRL Maximum Residue Limit Regulation (EC) No 4702009 of the European Parliament and of the Council (The European Parliament amp theCouncil of European Union 2009) and Commission Regulation (EU) No 372010 and amendments as of August 1 2019 (Commission 2010)MRPL Minimum Required Performance Limit Commission Decision 2003181EC (European Communities Commission 2003) Recommendedconcentration for testing CRL guidance paper (Anonymous 2007a) AOZ 3-amino-2-oxazolidone AHD 1-aminohydantoin SEM semicarbazideAMOZ 3-amino-5-morpholinomethyl-2-oxazolidone a prohibited substance Table 2 in Annex of Commission Regulation (EU) No 372010(Commission 2010) b no MRL fixed in honey c MRL based on application of the Commission Implementing Regulation (EU) 2018470 for honeyproduced within the EU

Table 12 Extraction and test protocol for the Eclipse 50 for honey (Gaudin et al 2013)Extraction protocol1 Weigh 2 g of honey in a 15mL centrifuge tube2 Add 5mL of acetonitrileacetone (vv 7030)3 Mix for 30ndash40 s using a vortex4 Incubate at 625 plusmn 25 C for 5min plusmn 30 s to dissolve honey completely5 Centrifuge at 4000 rpm for 15min6 Transfer the supernatant to a clean tube7 Evaporate under a nitrogen stream at 55 C for 15ndash20min8 Dissolve the residue in 250 mL of UHT consumption milk using a vortex for 1minTest protocol1 Transfer 50 mL of extract into an Eclipse 50 microplate well and seal the wells2 Incubate at 65 C until the negative control changes color or when the absorbances for the negative control are between 02

and 04 (absorbancefrac14 difference of measurement at 590 nm (filter 1) and 650 nm (reference filter))3 Interpret the end color

visual reading yellow is negative purple is positive intermediate color is doubtful optical reading (measurement of absorbancefrac14 difference of measurement at 590 nm (filter 1) and 650 nm (reference

filter)) absorbancegt absorbance for negative control thorn 02 positive

Table 13 Extraction and test protocol for the Premitest for honey (Gaudin et al 2013)Extraction protocol1 Weigh 2 g of honey in a 15mL centrifuge tube2 Add 5mL of acetonitrileacetone (7030)3 Mix for 30ndash40 s (vortex)4 Sonicate for 5min5 Vortex for 30ndash40 s6 Centrifuge at 4500 rpm for 10min at 4 C7 Remove the supernatant8 Evaporate under a nitrogen stream at 40ndash45 C9 Resuspend the residue in Lab Lemco broth 8 g per L (Oxoid Cat No CM0015) and mix wellTest protocol1 Transfer 100 mL of this mixture into the PremiTest ampoule2 Incubate at 64 C until the negative control changes color3 Interpret the end color

visual reading yellow is negative purple is positive intermediate color is doubtful instrumental reading with flatbed scanner and Premiscan software z-value lt0 negative z-value 0 positive


ATCC6633 (Khismatoullin et al 2003) and Micrococcusluteus ATCC 9341 (Khismatoullin et al 2004) respect-ively However the preparation of the test medium andthe testing procedures themselves are not fully andclearly described

63 Immunological and receptor assays

631 Lateral flow devices

For use at the apiary on-site honey tests in the formatof lateral flow devices based on the technology ofColloidal Gold Immune Chromatographic Strip Assay(GICA) are commercially available for the detection ofresidues of several veterinary drugs in honey fromNankai Biotech Co Ltd (Binjiang District HangzhouZhejiang China) These test kits are individually packedand contain all the reagents needed Different testdevices are available for ChloramphenicolStreptomycin Tetracyclines Sulfadiazine SulfaguanidineSulfamethazine Sulfathiazole Sulfonamides FurazolidoneMetabolite (AOZ) Furaltadone Metabolite (AMOZ)Furacilin Metabolite (AHD) Nitrofurans MetaboliteFluoroquinolones Quinolones Gentamicin Kanamycinand Tylosin Residue Rapid Test Device The followingSmarKT kits are available from the same companyGentamicin Kanamycin Penicillin StreptomycinSulfadiazine Sulfamethazine Sulfathiazole SulfonamidesFuraltadone Metabolite (AMOZ) Nitrofurazone (SEM)Furantoin (AHD) Furazolidone (AOZ) Nitrofurans 4-in-1 and Fluoroquinolones Rapid Test Kit AnotherChinese company (Shenzhen Bioeasy Biotechnology CoLtd Shenzhen Guangdong China) is producing theChloramphenicol and Tetracycline Rapid Test Kitfor Honey

The tests are utilizing gold conjugated antibodies assignal reagents and a drug protein conjugate as a solidphase capture reagent As the sample flows through theabsorbent sample pad the liquid reconstitutes the driedmonoclonal gold conjugate The drug in the sample willbind to the conjugate antibody and will migrate further

up the membrane to the test line If there is no drug inthe sample the free antibody conjugate will bind to thetest line giving a negative result In case the sample con-tains drug residues the antibody conjugate will not bindto the test line giving a positive result There is a shortsample pretreatment for honey but without the need ofinstrumentation for most of the tests With all thesetest devices a result is obtained within 3ndash5min and vis-ual interpretation of the result is possible As anexample the test protocol of the ChloramphenicolResidue Rapid Test Device (Nankai Biotech Co Ltd) isgiven in Table 14

The TetraSensor Honey KIT008 (25 tests)KIT009(100 tests) (Unisensor sa Liege Belgium) sensitively(lt10mgkg) detects the four most important tetracy-clines in honey in 30min without any special equip-ment making analysis at the production site possible(Alfredsson et al 2005 Reybroeck et al 2007 Gaudinet al 2013) The test procedure of the TetraSensorHoney is given in Table 15 During this first incubationperiod tetracyclines possibly present in the honey bindwith the specific receptor forming a stable complexAfterward during the second incubation the liquid isabsorbed by the dipstick and while flowing over thedipstick the liquid passes through the green capturelines In case the honey is free from tetracycline resi-dues the first line captures the remaining active recep-tor and a strong red line will appear The second lineserving as a control line takes a certain amount of theexcess of reagent that passed through the first line Ared control line should always become visible other-wise the test is invalid Results can be read visually orby means of a ReadSensor comparing the color inten-sity of both capture lines

During this first incubation period tetracyclines pos-sibly present in the honey bind with the specific recep-tor Afterward the dipstick is dipped into the vial and asecond incubation at room temperature occurs for15min When the liquid passes through the green

Table 14 Extraction and test protocol for the Chloramphenicol Residue Rapid Test DeviceExtraction procedure1 Weigh 4 g of honey into a 15mL centrifuge tube2 Add 1mL PBS A and 1 mL PBS B orderly shake to dissolve fully3 Add 8mL ethyl acetate cap and mix by inversion for approx 8min4 Keep still for clear separation transfer 5mL of supernatant into a 5mL centrifuge tube5 Dry it at 65 C with a stream of air evaporator or nitrogen evaporator6 Dissolve the dried residue in 160lL of CAP PBST bufferTest protocol1 Use a dropper to collect at least 3 drops (100 mL) of prepared sample hold the dropper vertically and transfer 3 full drops

(around 100 mL) of solution to the specimen well (S) of the test kit2 Start the timer3 Wait for red bands to appear4 Interpret the result result should be read in approximately 3ndash5min

visual reading ndash the test line (T) is the same as or darker than the control line (C) negative the test line (T) is lighter thanthe control line (C) or there is no test line it is positive reference line fails to appear invalid


capture lines a red color appears The first line cap-tures the remaining active receptor and the second linetakes a certain amount of the excess reagent thatpassed through the first line The second line serves asa control line and always has to become visible other-wise the test is invalid Results can be read visually orby means of a ReadSensor comparing the color inten-sity of both capture lines

Only limited laboratory equipment is required to runthe Sulfasensor Honey KIT033 (Unisensor sa) a genericmonoclonal antibody test for the detection of sulfona-mides in honey in 20min A sample pretreatment (acidhydrolysis) by heating a mixture of honey sample andbuffer at 95 C for 5min is needed to release the sulfo-namides that are chemically bound to the sugars(Chabottaux et al 2010 Gaudin et al 2012 Reybroeckamp Ooghe 2010) The kit manufacturer claims a detectionof several sulfa drugs at 25mgkg in honey The extrac-tion procedure and test protocol are shown in Table 16

With the competitive multiplex dipstickBee4Sensor KIT059 (Unisensor sa) the screening fortylosin (fluoro)quinolones sulfonamides and chloram-phenicol in honey is possible (Heinrich et al 2013)The test could be used in two different ways as afield test or as a lab test with better detection capa-bilities The sample extraction and test protocol ofthe field test is described in Table 17 To run the labtest one aliquot (A) is dissolved using acid hydrolysiswhereas the other aliquot (B) is dissolved in waterAfter liquidliquid partitioning of both aliquots withethyl-acetate the organic layers are evaporated untildry under nitrogen After reconstitution in a bufferaliquots A and B are combined and applied to thewell of a Bee4Sensor test kit for 5min at 40 C asone sample extract Afterward a dipstick is then incu-bated in this prepared well for 15min at 40 C Thedipstick can be assessed visually or instrumentally viathe ReadSensor

Table 15 Extraction and test protocol for the TetraSensor Honey KIT008009Sample pretreatment1 Fill the lid of the plastic lsquoHoney Dilution Tubersquo with honey (around 600mg)2 Reclose the plastic lsquoHoney Dilution Tubersquo3 Shake well until all honey is dissolved in the bufferTest protocol1 Add 200 mL of this mixture to the lyophilised receptor in the glass vial2 Mix well3 Incubate at room temperature (20 plusmn 5 C) for 15min4 Bring the dipstick into the glass vial5 Continue incubating at room temperature (20 plusmn 5 C) for 15min6 Interpret the result

visual reading intensity of the test line is stronger compared to the reference line negative result intensity of the test lineis equal or weaker compared to the reference line positive result

instrumental reading (ReadSensor) the instrument is calculating the ratio test linereference line Ratio 110 (cut-off)negative 090 ratiolt 110 low positive ratio lt090 positive reference line fails to appear invalid

Table 16 Extraction and test protocol for the Sulfasensor Honey KIT033Extraction protocol1 Fill the lid of the empty plastic lsquoHoney Tubersquo with honey (around 650mg)2 Add 600 mL of ACID Buffer in the empty tube3 Reclose the tube4 Shake well until complete honey dissolution5 Incubate the tubes 5min in boiling water (90ndash100 C)6 Add 600 mL of NEUTRALIZING Buffer close the tube and vortex7 Add 1800 mL of HONEY-II BufferTest protocol1 Place the reagent microwell in the Heatsensor at 40 C2 Add 200 mL of sample and mix 5ndash10 times until complete reagent dissolution3 Incubate for 5min at 40 C4 Bring the dipstick into the microwell5 Continue incubating for 15min at 40 C6 Remove the dipstick remove the filter pad from the dipstick and interpret the result




632 Enzyme-linked immunosorbent assays (ELISA)

There are many commercial and noncommercialELISArsquos developed for the detection of antibiotic resi-dues in different matrices including honey Table 18summarizes existing ELISA kits (the list is nonexhaus-tive) The sample preparation for honey ranges fromvery simple just a dilution followed by a filtration tomore complex including acidic hydrolysis derivatizationsolvent extraction or purification on a SPE column Thetechnical brochures of the different kit manufacturersshould be followed Validation studies of ELISA kitshave been published regarding the detection of chloram-phenicol (Scortichini et al 2005) tylosin and tilmicosin(Peng et al 2012) nitrofuran metabolites (Elizabetaet al 2012) and tylosin en streptomycin (Gaudin et al2013) (Figure 8)

Competitive enzyme immunoassays are used inmost cases for the detection of small molecular weightcomponents such as antimicrobial residues Such assaysuse either an enzyme-linked antibody or an enzyme-linked analyte to detect a particular antigen (drug) Asan example the sample preparation and test procedurefor the detection of chloramphenicol in honey bymeans of the Chloramphenicol ELISA of EuroProxima(Arnhem The Netherlands) is shown in Table 19 Inthis kit the walls of the wells of the microtiterplateare precoated with sheep antibodies to rabbit IgG Aspecific antibody (rabbit anti-CAP) enzyme labeledCAP (enzyme conjugate) and CAP standard or sampleare added to the precoated wells followed by a singleincubation step The specific antibodies are bound bythe immobilized antibodies At the same time freeCAP (present in the standard solution or sample) and

enzyme conjugated CAP compete for the CAP anti-body binding sites After an incubation time of 1 h thenonbound (enzyme labeled) reagents are removed in awashing step The amount of CAP enzyme conjugate isvisualized by the addition of a chromogen substrateThe bound enzyme conjugate elicits a chromogenic sig-nal The substrate reaction is stopped to prevent even-tual saturation of the signal The color intensity ismeasured photometrically at 450 nm The optical dens-ity is inversely proportional to the CAP concentrationin the sample

In some laboratories noncommercial ELISA methodsare used to monitor honey for the presence of anti-microbial residues (Heering et al 1998 Jeon amp RheePaeng 2008) A microplate and magneto iELISA wasdeveloped for the detection of sulfonamides in honeyusing magnetic beads to reduce nonspecific matrix inter-ferences (Muriano et al 2015)

633 Enzyme-linked aptamer assays (ELAA)

Assays were developed using DNA or RNA aptamersto get specific binding to streptomycin (Zhou et al2013) or tetracyclines (Wang et al 2014) in honey

634 Radio-labeled receptorantibody techniques (CharmII tests)

The Charm II (Charm Sciences Inc Lawrence MA) isa scintillation-based detection system for chemical fam-ilies of drug residues utilizing class-specific receptorsor an antibody in immune-binding assay format Thesample preparation is mostly just a dilution of thehoney with an extraction buffer supplied as part of

Table 17 Extraction and test protocol (field test) for the Bee4Sensor KIT059Extraction protocol1a Fill the lid of an empty plastic lsquoHoney Tubersquo (A) with honey (around 650mg)

2a Add 300 mL of ACID Buffer (Solution 1) in the empty tube A3a Reclose the tube4a Shake well until complete honey dissolution5a Incubate the tube 5min in boiling water (90ndash100 C)6a Add 300 mL of NEUTRALIZING Buffer (Solution 3)(preheated) close the tube and mix by inversion7a Let the tube cool down8a Add Dipstick Buffer (Solution 2) to a final extract volume of 35mL and mix well by inversion

1b Fill the lid of an empty plastic lsquoHoney Tubersquo (B) with honey (around 650mg)2b Add 2mL of Dipstick Buffer (Solution 2) in the empty tube B3b Reclose the tube4b Shake well until complete honey dissolution5b Incubate the tube 5min in boiling water (90ndash100 C)6b Let the tube cool down7b Add Dipstick Buffer (Solution 2) to a final extract volume of 35mL and mix well by inversion

Test protocol1 Mix Aamp B in 11 ratio2 Take a reagent microwell and open the microwell3 Add 200 mL of mixture of sample extract (AampB) and mix 5ndash10 times until complete reagent dissolution4 Incubate for 5min at room temperature5 Bring the dipstick into the microwell6 Continue incubating for 20min at 40 C7 Remove the dipstick remove the filter pad from the dipstick and interpret the result visually


Table 18 Commercial and non-commercial ELISA kits for the detection of antibiotic residues in honey (the list is non-exhaustive)

Drug (family) ELISA Kit Kit manufacturer or referenceAHD (1-aminohydantoin) Nitrofuran (AHD) ELISA Test Kit Unibiotest CoLtd (Wuhan China)

MaxSignal Nitrofurantoin (AHD) ELISA Kit Bioo Scientific (Austin TX)AMOZ (3-amino-5-morpholinomethyl-2-oxazolidone)

Furaltadone (AMOZ) ELISA Abraxis (Warminster PA)Irsquoscreen AMOZ v2 Tecna srl (Trieste IT)MaxSignal Furaltadone (AMOZ) ELISA Test Kit Bioo Scientific (Austin TX)Nitrofuran ( AMOZ) ELISA Kit Unibiotest CoLtd (Wuhan China)

AOZ (3-amino-2-oxazolidone) Furazolidone (AOZ) ELISA Abraxis (Warminster PA)Nitrofuran ( AOZ ) ELISA Kit Unibiotest CoLtd (Wuhan China)

Chloramphenicol Chloramphenicol (CAP) ELISA Kit Unibiotest CoLtd (Wuhan China)Chloramphenicol ELISA Abraxis (Warminster PA)Chloramphenicol ELISA EuroProxima bv (Arnhem NL)Chloramphenicol ELISA Randox (Crumlin UK)Irsquoscreen CAP Tecna srl (Trieste IT)Irsquoscreen CAP v2 Tecna srl (Trieste IT)MaxSignal Chloramphenicol (CAP) ELISA Test Kit Bioo Scientific (Austin TX)RIDASCREEN Chloramphenicol r-Biopharm (Darmstadt DE)

Doxycycline MaxSignal Doxycycline ELISA Kit Bioo Scientific (Austin TX)Enrofloxacin Enrofloxacin ELISA EuroProxima bv (Arnhem NL)

MaxSignal Enrofloxacin ELISA Test Kit Bioo Scientific (Austin TX)Erythromycin Erythromycin ELISA EuroProxima bv (Arnhem NL)

Erythromycin ELISA Test Kit Unibiotest CoLtd (Wuhan China)Flumequine Flumequine ELISA Randox (Crumlin UK)Fluoroquinolones Fluoroquinolones (Generic) ELISA EuroProxima bv (Arnhem NL)

Fluoroquinolones II ELISA EuroProxima bv (Arnhem NL)MaxSignal Fluoroquinolone ELISA Kit Bioo Scientific (Austin TX)QUINOLONES ELISA KIT Tecna srl (Trieste IT)Quinolones (QNS) ELISA Kit Unibiotest CoLtd (Wuhan China)

Lincomycin MaxSignal Lincomycin ELISA Test Kit Bioo Scientific (Austin TX)Norfloxacin MaxSignal Norfloxacin ELISA Test Kit Bioo Scientific (Austin TX)Oxytetracycline MaxSignal Oxytetracycline ELISA Kit Bioo Scientific (Austin TX)

Oxytetracycline ELISA Randox (Crumlin UK)SEM (semicarbazide) Nitrofuran (SEM) ELISA Test Kit Unibiotest CoLtd (Wuhan China)Streptomycin Irsquoscreen STREPTO Tecna srl (Trieste IT)

MaxSignal Streptomycin ELISA Test Kit Bioo Scientific (Austin TX)MaxSignal Streptomycin ELISA Test Kit For

Honey SamplesBioo Scientific (Austin TX)

RIDASCREEN Streptomycin r-Biopharm (Darmstadt DE)Streptomycin ELISA EuroProxima bv (Arnhem NL)Streptomycin ELISA Randox (Crumlin UK)

Sulfadiazine MaxSignal Sulfadiazine ELISA Test Kit Bioo Scientific (Austin TX)Sulfamethazine MaxSignal Sulfamethazine ELISA Test Kit Bioo Scientific (Austin TX)Sulfamethoxazole MaxSignal Sulfamethoxazole ELISA Test Kit Bioo Scientific (Austin TX)Sulfaquinoxaline MaxSignal Sulfaquinoxaline (SQX) ELISA Test Kit Bioo Scientific (Austin TX)Sulfonamides B ZERO SULFA Tecna srl (Trieste IT)

Irsquoscreen SULFA Tecna srl (Trieste IT)Irsquoscreen SULFA QL Tecna srl (Trieste IT)MaxSignal Sulfonamide ELISA Test Kit Bioo Scientific (Austin TX)Total Sulfonamides ELISA kit Unibiotest CoLtd (Wuhan China)

Tetracyclines B ZERO TETRA HS Tecna srl (Trieste IT)MaxSignal Tetracycline (TET) ELISA Test Kit Bioo Scientific (Austin TX)RIDASCREEN Tetracycline r-Biopharm (Darmstadt DE)SuperScreen TETRA Tecna srl (Trieste IT)SuperScreen Tetra HS Tecna srl (Trieste IT)Tetracycline ELISA EuroProxima bv (Arnhem NL)Tetracyclines ELISA Abraxis (Warminster PA)Tetracyclines ELISA Randox (Crumlin UK)Tetracyclines(TCs) ELISA Test Kit Unibiotest CoLtd (Wuhan China)

Tilmicosin MaxSignal Tilmicosin ELISA Test Kit Bioo Scientific (Austin TX)Tylosin Irsquoscreen TYLOSIN Tecna srl (Trieste IT)

MaxSignal Tylosin ELISA Test Kit Bioo Scientific (Austin TX)Tylosin Plate Kit Abraxis (Warminster PA)

Note AOZ 3-amino-2-oxazolidone AHD 1-aminohydantoin SEM semicarbazide AMOZ 3-amino-5-morpholinomethyl-2-oxazolidone


the kit The assay itself takes lt 30min to completeCharm II kits are available for the detection of sulfo-namides tetracyclines macrolides (and lincosamides)aminoglycosides ((dihydro)streptomycin) amphenicols(chloramphenicol) nitrofuran AOZ metabolite andbeta-lactams Some Charm II assays were improved oradapted by the integration of solid phase extraction asextract clean-up to limit false positive results due tomatrix quenching effects (McMullen et al 2004) Asan example the procedure of the Charm II MacrolideTest for Honey is given in Table 20 This screeningtest detects macrolides and lincosamides in honey

635 Biochip-based methods

Some biochip-based methods such as Biacore (GEHealthcare Europe GmbH Freiburg DE) and AntiMicrobial Arrays (Randox Laboratories LimitedCrumlin UK) allow the detection of multiple drug resi-dues in honey (McAleer et al 2010)

The Biacore biosensor system is based on surfaceplasmon resonance Analytical Qflex Kits are offered foruse with the Biacore Q-instrument for screening forthe antibiotics chloramphenicol streptomycin sulfona-mides and tylosin (Caldow et al 2005) The samplepreparation includes dissolving the honey in an aqueousbuffer and filtering Analysis for chloramphenicolrequires an extra LLE For sulfonamides a hydrolysisstep is needed to release the sulfonamides bound tosugars With this system a high throughput and a rapid(around 5min) multi-analyte screening in honey is pos-sible (Weigel et al 2005) However the instrumentcosts are high

Randox Laboratories Limited offers a multi-analytequantitative testing platform the Evidence Investigatorusing Biochip Array Technology for the monitoring ofhoney for antimicrobials (Daniela et al 2012 Gaudinet al 2014 OrsquoMahony et al 2010 Popa et al 2012)An overview of available reagents for the monitoring ofhoney on antimicrobials is given in Table 21 The samplepreparation typically takes about 20min for dilutionThis does not include the Antimicrobial Array III which

requires 4 h due to the derivation step or 50min ifCAP only due to extraction step The incubation andassay times take 2 h for all arrays Arrays for the detec-tion of nitroimidazoles in honey are in development

636 Methods using MIPs and other immunotechniques

Molecularly imprinted polymers (MIP) with specific rec-ognitions sites are sometimes used as a preconcentra-tion step as for the determination of chloramphenicol inhoney (Thongchai et al 2010) MIP sensors based onelectropolymerized oligothioaniline-functionalized goldnanoparticles are prepared and applied to the detectionof tetracycline in honey (Bougrini et al 2016)Furthermore there are publications on the develop-ment of a immunosensor for the detection of residuesof sulfonamides in honey (Muriano et al 2013 Valeraet al 2013)

64 Physico-chemical methods(chromatographic techniques)

Methods for the detection of residues of veterinarydrugs in honey based on chromatographic techniqueshave been described in several publications (Benettiet al 2004 2006 Carrasco-Pancorbo et al 2008Dubreil-Cheneau et al 2014 Edder et al 1998Kaufmann et al 2002 Kivrak et al 2016 Maudenset al 2004 Nozal et al 2006 Sporns et al 1986 VanBruijnsvoort et al 2004 Verzegnassi et al 2002) andin a review article (Barganska et al 2011) In the 1990sthe use of HPLC was popular but today confirmationof antibiotic residues in honey is performed by LC-MSmainly LC-MS2 (tandem mass spectrometry) (Blascoet al 2007) When mass fragments are measured usingtechniques other than full scan the system of identifica-tion points (IP) is applied Confirmatory methods mustfulfil the criteria listed in Commission Decision 2002657EC (European Union Commission 2002) and mustbe based on molecular spectrometry providing directinformation concerning the molecular structure of the

Figure 8 Format of a competitive ELISA Residue Xfrac14 target molecule HRPfrac14 horseradish peroxidase enzyme


analyte under examination such as GC-MS and LC-MS(De Brabander et al 2009)

Some compounds such as nitroimidazoles (Polzeret al 2010) fumagillin (Daeseleire amp Reybroeck 2012Kanda et al 2011 Tarbin et al 2010) and nitrofuranresidues (Khong et al 2004 Lopez et al 2007 Tribalatet al 2006) are mostly directly screened in honey using

LC-MSMS detection since no or only few immuno-chemical methods for the detection in honey have beendeveloped By the development of multiresidue meth-ods LC-MS is used more and more for multiclassscreening for antimicrobial residues in honey (Azzouz ampBallesteros 2015 Galarini et al 2015 Hammel et al2008 Lopez et al 2008)

Table 19 Extraction and test protocol (laboratory test) for the Chloramphenicol ELISA (EuroProxima BV)Extraction protocol1 Bring 10 plusmn 01 g of honey into a centrifugation tube of 50mL fill also a centrifugation tube with the same amount of blank

honey honey doped with 03 mgkg of chloramphenicol and honey doped with 01 mgkg of chloramphenicol respectively2 Add to each sample 5mL of distilled water3 Vortex the samples until all honey is dissolved4 Add 5mL of ethyl acetate5 Mix (head over head) for 15min6 After centrifugation (5min at 2600 g) pipette 2mL of the upper layer (ethyl acetate) into a glass test tube7 Evaporate at 45 plusmn 5 C under a mild stream of nitrogen8 Dissolve the residue in 2mL of n-hexane for defatting purposes and vortex9 Add 1mL of reconstitution buffer and vortex for 20 s10 After centrifugation (10min at 2600 g) pipette the layer underneath (reconstitution buffer plusmn 800 mL) into a short glass

test tube11 Add again 1mL of n-hexane and vortex for 20 s12 Add 1mL of reconstitution buffer and vortex for 20 s13 After centrifugation (10min at 2600 g) pipette the layer underneath (reconstitution buffer plusmn 800 mL) into a short glass

test tube14 Use the extract in the ELISA (extract could be stored for one day at 4 plusmn 2 C)

Test protocol1 Before starting the test the reagents should be brought up to ambient temperature by taking them out of the refrigerator

20min before use Keep the substrate away from light After analysis store the remaining reagents as soon as possible in therefrigerator

2 Identify the wells of the microtiterstrip upon the plate configuration sheet3 Pipette 100 mL of reconstitutionzero standard buffer into well A1 (blank)4 Pipette 50 mL of reconstitutionzero standard buffer into well A2 (zero standard)5 Pipette 50 mL of chloramphenicol-free extract buffer into wells B1 and B2 (blank control sample)6 Pipette 50 mL of extract of samples doped at 01 mgkg into wells C1 and C2 (positive control sample 01 ppb)7 Pipette 50 mL of extract of samples doped at 03 mgkg into wells C1 and C2 (positive control sample 03 ppb)8 Pipette 50 mL of each sample extract in duplicate into the remaining wells of the microtiter plate9 Add 25 of conjugate (CAP-HRPO) into all wells except in well A110 Add 25 of antibody solution into all wells except in well A111 Cover the plate with aluminum foil and shake the plate for 1min12 Incubate for 1 h in the dark (refrigerator) at 2ndash8 C13 Discard the solution from the microtiter plate and wash three times with rinsing buffer Fill all the wells each time with

rinsing buffer to the rim Place the inverted plate on absorbent paper and tap the plate firmly to remove residual washingsolution Take care that none of the wells dry out before the next reagent is dispensed

14 Shake the substrate solution before use Pipette 100 mL of substrate solution into each well Cover the plate and shake theplate slowly

15 Incubate for 30min in the dark at room temperature (20ndash25 C)16 Add 10 mL of stop solution into each well17 Read the absorbance values (OD) immediately at 450 nm in a spectrophotometer

Interpretation of results1 Subtract the optical density (OD) value of the blank well (A1) from the individual OD of the other wells2 Calculate the Cut-off by adding 3xSD of repeatability for the positive control sample spiked at 03 ppb (value calculated out

of the validation data) to the mean of both corrected OD values for the two positive control samples spiked at 03 ppb3 Interpret the control samples the negative control sample should test negative and the positive control sample spiked at

01 ppb should give an OD below the OD of the negative control and higher than the OD of the positive control sample at03 ppb The run is invalid if the control samples are not giving correct results

4 Compare the corrected value for each sample to the cut-off value If the corrected OD of the sample is equal to or belowthe cut-off the sample is considered as suspect for the presence of chloramphenicol at 03 mgkg if the corrected OD ofthe sample is higher than the cut-off the sample is free from residues of chloramphenicol at 03 mgkg


Table 20 Extraction and test protocol for Charm II Macrolide Honey Test (Charm Sciences Inc)Honey sample preparation1 Label a 50mL conical centrifuge tube for each sample2 Add 20 grams of honey to an appropriately labeled centrifuge tube Also prepare two negative control samples by using

blank honey and one positive control sample of blank honey spiked with 20 mgkg of erythromycin A3 Add 30mL MSU Extraction Buffer to each tube Mix well until honey is completely dissolved by putting the samples for

30min on a shaker or by vortexing the tubes4 Add 9ndash10 drops of M2-buffer Check pH with pH indicator strips pH of extract should be equivalent to 75 (80) on

pH strip5 If the pH is still too low add M2 Buffer dropwise mix and retest pH until the desired pH is reached

Note If pH is high add 03mL (300 mL) 01 M HCl mix and retest If pH is still high add 01 M HCl drop-wise mixand retest

6 The extract solutions can be kept for 1 day at 4 plusmn 2 C

Sample filtration1 Using the syringe assembly filter the entire sample through a glass fiber filter (eg Millipore-Millex AP prefilter of 25mm)2 Collect the entire sample into a clean container (50mL conical tube or beaker)3 After the sample has been pushed through detach the filter holder Rinse syringe and bivalve with 10mL deionized water

Clean-up over C18 cartridge1 Prepare C18 cartridge by attaching the C18 cartridge to adapter attaching the adapter to the bivalve in the syringe assembly2 Activate the C18 cartridge by pushing through 50mL of methanol The cartridge should be used within 10min of activation3 Wash cartridge with 50mL of deionized or distilled water4 Repeat the washing step with 50mL of water5 Perform the extraction by adding the filtered honey solution to the syringe Push the solution slowly through the

preactivated C18 cartridge one drop at a time The sample may be thick and difficult to push through the cartridge Holdcartridge with two hands to prevent cartridge from popping off due to backpressure Discard liquid that flows throughthe cartridge

6 Wash the cartridge with 50mL distilled water and discard the flow through7 Remove the C18 cartridge from the assembly and add 30mL methanol directly into the cartridge Attach cartridge to the

assembly and bring labeled test tube in position8 Slowly push methanol one drop at a time through the cartridge and collect the eluate in a labeled test tube9 Dry eluate for each sample Dry under nitrogen or air in a 40ndash45 C heat block or water bath and remove from the heat

block or water bath when methanol is completely evaporated10 Once the methanol is completely evaporated reconstitute the dried eluate with 5mL of Zero Control Standard (ZCS) and

vortex extensively11 Cool the diluted samples on ice for 10min prior to running the Charm II Macrolide Test Procedure

Charm II Macrolide test procedure1 Label test tubes and scintillation vials Let Charm II reagents reach room temperature2 Add the white tablet to the empty test tube3 Add 300 plusmn 100 lL water Mix 10 s to break up the tablet Take additional time if required to be sure the tablet is broken up4 Add 5plusmn 025mL diluted sample or control Use a new tip for each sample

Immediately mix by swirling sample up and down 10 times for 10 s5 Incubate at 65 plusmn 2 C for 2min6 Add green tablet (lt 019kBq C14 labeled erythromycin) Immediately mix by swirling the sample up and down 10 times for

15 s The tablet addition and mixing of all samples should be completed within 40 s7 Incubate at 65 plusmn 2 C for 2min8 Centrifuge for 5min at 1750G9 Immediately pour off liquid completely While draining remove fat ring and wipe dry with swabs Do not disturb the pellet10 Add 300 plusmn 100lL water Mix thoroughly to break up the pellet The pellet must be suspended in water before adding

scintillation fluid11 Add 30 plusmn 05mL of scintillation fluid Cap and invert (or shake) until mixture has a uniform cloudy appearance12 Count in liquid scintillation counter for 60 s Read cpm (counts per minute) on [14C] channel Count within 10min of adding

of scintillation fluid13 Calculate the Control Point by taking the highest negative control CPM 3514 Recount if greater than and within 50 cpm of the Control Point

Interpretation of results1 The cpm for the positive control sample should be below the Control Point otherwise the run is not valid2 If the sample cpm is greater than the Control Point the sample is negative Report as ldquoNot Foundrdquo If the sample cpm is less

than or equal to the Control Point the sample is positive The presence of macrolideslincosamides could be confirmed by aconfirmatory method (LC-MSMS)


Table 21 Overview of antimicrobial arrays (Randox Laboratories Limited) for the monitoring for antimicrobials in honey

Antimicrobial Array I Plus Antimicrobial Array II

Assay Compound(s) Assay Compound(s)Sulfadimethoxine Sulfadimethoxine Quinolones Norfloxacin

PefloxacinEnrofloxacinCiprofloxacinOfloxacin

Pipemidic AcidFleroxacinLevfloxacinNadifloxacinOrbifloxacinDanofloxacinMarbofloxacinOxolinic AcidDifloxacinPazufloxacinSarafloxacinEnoxacin

Sulfadiazine Sulfadiazine Ceftiofur CeftiofurDesfuroyceftiofur

Sulfadoxine Sulfadoxine Thiamphenicol FlorphenicolThiamphenicol

Sulfamethizole SulfamethizoleSulfachlorpyridazine

Streptomycin StreptomycinDihydrostreptomycin

Sulfachlorpyridazine Sulfachlorpyridazine Tylosin TylosinTilmicosin

Sulfamethoxypyridazine SulfamethoxypyridazineSulfaethoxypyridazine

Tetracyclines Tetracycline4-epitetracyclineRolitetracycline

4-epioxytetracylcineOxytetracyclineChlortetracyclineDemeclocyclineDoxycycline

4-epichlortetracyclineMethacycline

Sulfamerazine Sulfamerazine Antimicrobial Array IIISulphisoxazole Sulphisoxazole Assay CompoundSulfathiazole Sulfathiazole

SulfadiazineAOZ 4-NP-AOZ

FurazolidoneSulfamethazine Sulfamethazine AMOZ 4-NP-AMOZ

FuraltadoneSulfaquinoxaline Sulfaquinoxaline AHD 4-NP-AHD

NitrofurantoinSulfapyridine Sulfapyridine

SulfasalazineSEM 4-NP-SEM

5-Nitro-2-furaldehydeSemicarbazone

Sulfamethoxazole SulfamethoxazoleSulfamethizoleSulfachlorpyridazine

Chloramphenicol ChloramphenicolChloramphenicol glucuronide

Sulfamonomethoxine Sulfamonomethoxine Antimicrobial Array III (CAP only)Trimethoprim Trimethoprim Assay Compound

Chloramphenicol ChloramphenicolChloramphenicolGlucuronide

Antimicrobial Array IV (continued) Antimicrobial Array IV (continued)Assay Compound Assay CompoundSpiramycinJosamycin Spiramycin

KitasamycinSpiramycin 1

AcetylspiramycinJosamycin

Spectinomycin Spectinomycin

(Continued)


7 Standard method for honey sensory analysis

Sensory analysis of honey describes the organolepticprofile and may be used to assess the quality as well asthe botanical and geographical origin of a honey sampleFurthermore it may be used to assess the pleasantnessto the end-consumers and to monitor the quality ofhoney during harvesting packaging and storage

In order to evaluate uniflorality ldquohoney may be desig-nated according to floral or plant source if it comeswholly or mainly from that particular source and has theorganoleptic physicochemical and microscopic proper-ties corresponding with that originrdquo (Codex 2001) Itshould be noted that some pollen is either under orover-represented in a honey sample and the percentageof one specific pollen does not necessarily correspond tothe amount of nectar from that specific speciesMoreover the pollen entering the nectar during bee col-lection (primary pollen spectrum strictly related tobotanical origin) can be significantly modified from sec-ondary pollen contamination that is pollen grains enter-ing in honey in the hive during transformation fromnectar to honey by bees or as a consequence of bee-keeper operations Using only pollen analysis for deter-mining uniflorality may be misleading and a uniquegeneralized threshold may be too high for under-repre-sented and too low for over-represented pollens (seeeg the descriptive sheets Person Oddo amp Piro 2004)Thus sensory evaluation in addition to physico-chemicaland melissopalinological analyses is essential for deter-mining uniflorality also because organoleptic characteris-tics are the only ones that consumers can identifyand evaluate

Traditional sensory analysis was first applied tohoney by Michel Gonnet who trained specialists toevaluate honey on the basis of their sensory experience(Gonnet et al 1985 Gonnet amp Vache 1979 1992)Modern techniques relying on a panel of assessors and

controlled protocols are applied more regularly now(Piana et al 2004) One selected technique for the sen-sory analysis of honey is presented here while a widerguide is published in Marcazzan et al (2018)

The following proposed standard method will bedeveloped by referring to specific articles(Marcazzan et al 2014 Mucignat-Caretta et al2012 Piana et al 2004 Sabatini et al 2007) or toISO standards (ISO 5492 2008 ISO 6658 2017)For detailed information regarding testing rooms andthe selection and training of assessors or techniciansone can refer to the specific ISO standards (ISO8586 2012 ISO 8589 2007) For a complete list ofISO standards on sensory analysis see the ISO cata-log (httpwwwisoorg)

71 Methods

Many methods for the sensory analysis of honey areavailable The general requirements and tasting proce-dures are common to all methods The descriptivesemiquantitative method will be described in detail as itrequires less training and has a wider use the othermethods are described in Marcazzan et al (2018) andreferences herein

72 General requirements

721 Test room

Evaluations should be conducted under known and con-trolled conditions with minimal distractions Individualtesting booths should be used (Figure 9) and located inrooms with controlled lighting and temperature withoutinterfering noises and odors The general guidance forthe design of test rooms is reported in ISO 8589 (2007)

Table 21 (Continued)

Antimicrobial Array IV (continued) Antimicrobial Array IV (continued)Apramycin Apramycin AmikacinKanamycin Amikacin

Kanamycin AKanamycin B

Bacitracin Bacitracin Lincosamides LincomycinClindamycinPirlimycin

NeomycinParomomycin NeomycinParomomycin

Erythromycin ErythromycinClarithromycinRoxithromycinN-Demethyl

Erythromycin ATobramycin Tobramycin

Kanamycin BStreptomycin

DihydrostreptomycinStreptomycin

DihydrostreptomycinTylosin BTilmicosin Tylosin B

TilmicosinTylvalosinTylosin A

Virginiamycin Virginiamycin



722 Selection and training of assessors and choice of panel

The guidelines for the selection training and monitoringof assessors are reported in ISO 8586 (2012) Panel ses-sions involve at least five specialized expert assessorsworking as part of a panel managed by a panel leader (ISO13300 2006a 2006b) who oversees the selection trainingmonitoring and performance of the assessors The panelleader is responsible for organizing and coordinating thetests as well as collecting and processing the results

723 Product storage

Recommended temperature for honey sample storageshould be lt20 C to maintain the stability of honeyAvoid high temperatures (gt35 C) and exposureto sunlight

724 Preparation and presentation of the samples

The sensory analysis is performed in containers asdetailed below It is useful to separate the visual fromthe olfactory-gustatory assessment general aspect andcertain defects should be assessed in the original con-tainer if possible In any case the container should nothave any sort of identification except a codeOtherwise honey samples must be presented in thecontainers mentioned below

7241 Sampling glassesThe same type of glasses or containers should be usedwithin each tasting session The glassescontainersshould meet the following requirements

Containers should be odorless Present samples in a homogeneous and anonymous way

(identical containers with no distinguishing marks apartfrom the identification code)

Cover samples to prevent the contamination and dis-persion of the honey odors

Use a 150200mL capacity balloon stemmed wine glassfor samples (Figure 10) or other types of glasses if theysatisfy the requirements mentioned above maintainingthe samplevolume ratio near 15

Use colored or opaque containers if it is necessary tomask the color of the honey For example oil samplingbeakers defined in the COIT20Doc No 5 norm(International Olive Oil Council 2007) and ISO stand-ard (ISO 16657 2006) meet these requirements Thecolor of honey may be masked by using a red light

7242 Preparation of the samples(1) Assign each sample a random three-digit code(2) Pour at least 30 g of the sample (suggested for a

150mL glass one for each assessor) into the container(3) Cover samples with a suitable lid (eg petri dish

aluminum foil cling film)(4) Use minimal manipulation during transferring of sam-

ples to ensure that the subsamples look the same(5) To guarantee anonymity the technician preparing

the samples should not perform the testing(6) Maintain the sample at room temperature preferably

between 20 and 25 C(7) The evaluation should ideally occur within 24 h after

sample preparation

Figure 9 Assessors in testing booths Figure 10 Balloon stemmed glasses proposed for sensoryhoney evaluation

Table 22 Guidelines for assessors1 In the day of assessment the assessors should avoid perfumed toiletries and avoid anything else that may introduce

odors into the tasting room2 For 1 h before the evaluation the assessors should avoid smoking and avoid eating3 During the session the assessors should work alone follow the instructions of the panel leader and abstain from expressing any comment


73 Tasting procedures

731 Guidelines

Each assessor will be given instructions and materialsincluding evaluation forms and computers necessary toconduct the sensory analysis The assessors must have attheir disposal neutral spoons (without any odor andortaste metallic material should not be used) to stir thehoney during the olfactory assessment and to take theproduct to be tasted Water and a juicy slightly acidicapple are suggested to cleanse the mouth between tast-ings Also rose-hip tea or low-salt bread may be usedThe assessors must follow simple but important rules(ISO 6658 2017 Piana et al 2004) see Table 22

7311 Session protocol(1) Limit the number of samples according to the type

of honey to prevent oversaturation and fatigue ofthe senses

(2) The evaluation should occur when sensory sensitiv-ity is at its optimum at least 2 h after a meal

(3) Rest at least 30min between sessions(4) Randomly present the order of the samples to

each assessor

732 Evaluation of the visual characteristics

(1) The evaluation is carried out under day light in a wellilluminated room or using an artificial illumination aslights with a correlated color temperature of 6 500 K

(2) Evaluate the visual cleanliness and quality of the crys-tallization of a honey in its original container

(3) Evaluate all other characteristics by assessing thesample in a separate glass

733 Evaluation of the olfactory characteristics

Evaluate olfactory characteristics before tasting Avoidreceptor desensitization by taking short sniffs of the sample

(1) Spread the honey around the inside of the glassusing the spoon This increases the surface area andreleases the volatiles

(2) Take first sniff to evaluate odor immediately(3) Wait 15ndash20 s before taking a second sniff so that

the full intensity of the odor is perceived(4) Leave part of crystallized honey untouched for tact-

ile evaluation

734 Evaluation of the olfactory-gustatory and tactilecharacteristics

(1) Bring to the mouth 12 g of honey with a spoon(2) Allow the honey sample to dissolve slowly in the

mouth before swallowing(3) Evaluate olfactory-gustatory characteristics

immediately(4) Evaluate the taste (sweet salty sour and bitter)

(5) Evaluate the retro-nasal odor aroma intensity andquality persistence after-taste

(6) Evaluate other mouth sensations (viscosity texturetype of crystals)

(7) Concentrate on the chemical aspects of the mouthsensations apart from the tactile characteristics

(8) Wait 1 or 2min before taking a second taste lon-ger in the case of persistent honeys Eat a slice ofapple or bread or cleanse the mouth with water orrose-hip tea if necessary

(9) Evaluate tactile characteristics simultaneously Weencourage doing so in a second taste Press the samplebetween tongue and palate in order to perceive andassess the viscosity texture and crystals characteristics

(10) Wait a few minutes between samples eat a slice ofapple or bread or cleanse the mouth with water orrose-hip tea

74 Descriptive semiquantitative analysis

All numbered lists correspond to a scale while bulletedlists correspond to a nonexhaustive list of descriptorsAn example of descriptive card is presented inFigure 11

741 Appearance and visual descriptors

The principal visual descriptors for honey are

(1) Physical state liquid or crystallized(2) Color intensity 1 very light 2 light 3 medium 4

dark 5 very dark(3) Color tone normal honey color bright yellow gray-

ish reddish with orange tone fluorescent greendull bright

(4) Presentation depending on the aim of the evaluation(eg competition or checking botanical declarationetc) the importance of the assessment of some orall of these elements may vary The following charac-teristics can be assessed clarity (for liquid honey)impurities air bubbles or foam

(5) Defects can be assessed as marbling or white spotsincomplete crystallization phase separation

Viscosity cohesion and type of crystals are observedin both visual and tactile evaluation but assessed only inthe latter (see Section ldquoTexture and other tactiledescriptorsrdquo for description)

742 Olfactory descriptors

(1) The intensity of the odor may be defined as 0mdashabsent 1mdashweak 2mdashmedium 3mdashstrong

(2) The description of the odors may be classified infamilies and subfamilies These are shown in Table23 The examples used in the table to describe thefamilies should be considered useful butnot exhaustive


Figure 11 Example of descriptive card


743 Flavor descriptors

Flavor is the ldquocomplex combination of the olfactorygustatory and trigeminal sensations perceived duringtasting It may be influenced by tactile thermal painfulandor kinaesthesic effectsrdquo (ISO 5492 2008)

(1) The intensity of the taste sensation sweetness sour-ness salty and bitterness (Table 24) is quantifiedaccording to the scale 0 absent 1 weak 2medium 3 strong The taste of umami is not consid-ered if perceived it is reported as present

(2) The odor perceived via retro-nasal pathways (aroma)is described as reported in olfactory description

(3) Other sensations we can perceive in the mouthinclude (i) piquant an aggressive and burning sensa-tion perceived by the mucous membranes andor in

the throat (ii) metallic a feeling similar to a slighttingling sensation due to the presence of traces ofiron (or perhaps other metals) in ionic form in somehoneys In honey it indicates a defect

(4) Persistence can be evaluated Persistence refers tothe duration of the aroma sensations after swallow-ing Less than 30 s is considered ldquoshortrdquo persistenceand longer than 5min ldquolongrdquo persistencePersistence is evaluated according to the scale 0absent 1 short 2 medium 3 long

(5) Aftertaste indicates the presence of aroma sensa-tions in the mouth that remain after swallowing theproduct and are different from those initially per-ceived The quality of the sensations can bedescribed with the same terminology used to explainthe olfactory sensations (Table 23)

Table 23 Attributes for honey olfactory description

Attribute Definition Reference1 Floral Odor reminiscent of flowers orange flower (distilled) water b-ionone (subfamily ldquofinerdquo)

rose ylang ylang phenylacetaldehyde (subfamily ldquoheadyrdquo)2 Fruity Odor reminiscent of fruit fruit aroma (strawberry pear apple raspberry) (subfamily ldquofresh fruitrdquo)

isoamyl acetate c-decalactone d-decalactone (subfamily ldquotropical fruitrdquo)grape sugar (concentrated rectified must) (subfamily ldquosyruprdquo)apricot fruit juice peeled tomatoes (subfamily ldquoprocessed fruitrdquo)white wine (subfamily ldquofermented fruitrdquo)

3 Warm Odor reminiscent of foodcharacterized by sweet taste

vanillin benzoin beeswax (subfamilyldquofinerdquo)concentrated sugared milk diacetyl (subfamily ldquolacticrdquo)cane sugar furanone (subfamily ldquocandiedrdquo)toasted hazel nuts toasted almonds toasted peanuts (subfamily ldquotoastedrdquo)barley malt syrup rice syrup (subfamily ldquomaltedrdquo)toasted barley toasted bread (charred part) (subfamily ldquoburntrdquo)

4 Aromatic Odor reminiscent ofsomething fresh balsamiccosmetic not necessarilyrelated to food products

cloves nutmeg (subfamily ldquospicyrdquo)mugo pine incense (subfamily ldquoresinousrdquo)oak musk (subfamily ldquowoodyrdquo)peppermint anise eucalyptus (subfamily ldquobalsamicrdquo)lemon sweet orange (subfamily ldquocitrus fruitrdquo)coumarin (subfamily ldquobitter almondrdquo)

5 Chemical Odor generally not related tofood products characterizedby a certain degree ofaggressiveness

phenol vegetal tar (subfamily ldquophenolicrdquo)castile soap (not perfumed) (subfamily ldquosoaprdquo)jute smoke cigarette butt extract (subfamily ldquosmokyrdquo)white wine vinegar (subfamily ldquovinegarrdquo)ammonia (subfamily ldquoammoniardquo)

6 Vegetal Odor reminiscent of non-aromatic plants

fresh raw beans cut grass fresh ivy leaves (subfamily ldquogreenrdquo)raw champignon mushrooms lightly boiled spinach (subfamily ldquodamprdquo)green tea hay (subfamily ldquodryrdquo)

7 Animal Odor reminiscent of animalworld or decomposition

hardboiled egg dimethyl sulphide methionine (subfamily ldquosulfurizedrdquo)dried ceps mushrooms stock cube (subfamily ldquoproteicrdquo)isovalerianic acid butyric acid hexanoic acid (subfamily ldquovalerianicrdquo)cassis (subfamily ldquocassisrdquo)

Table 24 Attributes for honey taste description

Attribute Definition Reference1 Sweetness Basic taste produced by dilute aqueous solutions of natural or

artificial substances such as sucrose or aspartameSucrose in water 12 gL

2 Sourness Basic taste produced by dilute aqueous solutions of mostlyacid substances (eg citric acid and tartaric acid)

Citric acid in water 06 gL

3 Saltiness Basic taste produced by dilute aqueous solutions of varioussubstances such as sodium chloride

Sodium chloride in water 2 gL

4 Bitterness Basic taste produced by dilute aqueous solutions of varioussubstances such as quinine or caffeine

Caffeine in water 027 gL


744 Texture and other tactile descriptors

Tactile characteristics refer to those perceived in themouth although the visual inspection and handling ofthe sample contribute to its assessment

(1) Viscosity is evaluated in liquid honey according tothe scale 1 very fluid 2 fluid 3 normal viscosity 4viscous 5 very viscous

(2) Texture of the mass in crystallized honeys is eval-uated and can be described as 1 deliquescent 2soft 3 pasty 4 firm 5 compact

(3) Crystals can be differentiated according to their size0 not perceptible 1 fine 2 medium 3 large

(4) Crystals can be differentiated depending on theshape and can differ according to solubility that isto say the tendency to melt in the mouth more orless easily The crystals can be described as sharpgritty or roundish

A further tactile descriptor for honey is astringencya complex sensation due to substances such as the tan-nins of persimmon or unripe fruit which precipitate theproteins in the saliva responsible for lubricating themucous membranes in the mouth The astringency isperceived as a lack of lubrication and dryness of mucousmembranes it is often associated with a bitter taste

In finely crystallized honey it is common to experi-ence a refreshing sensation This is not due to the tem-perature of the product but it is linked to the solubilityof some type of crystals (glucose)

AcknowledgmentsThe following authors were responsible for the respectivemanuscript sections

Ligia Bicudo de Almeida-Muradian (Introduction) OrtrudMonika Barth and Alex da Silva de Freitas (Honey authenti-citymdashbotanical geographical and entomological origin) LigiaBicudo de Almeida-Muradian Maria Teresa Sancho AnaPascual-Mate Jose Augusto Gasparotto Sattler (Honey physi-cochemical analysis for nutritional chemical composition andantioxidant activity) Michael Eyer and Vincent Dietemann(Investigating honey production with diagnostic radioentomol-ogy) Anne-Claire Martel (Pesticide residues in honey) WimReybroeck (Antibiotics and chemotherapeutics in honey) GianLuigi Marcazzan Carla Mucignat Carla Marina Marchese(Sensory analysis) The authors thanks Lucia Piana for partici-pation in the Sensory Analysis section Ana Pascual-Mate andMaria Teresa Sancho-Ortiz are very grateful to CiprianoRamos and Monica Pascual for their valuable help Ligia Bicudode Almeida-Muradian thanks Stefan Bogdanov and PeterGallmann for their technical support and also AdrianeAlexandre Machado de Melo We further thank LeilaAparecida Bonadio for helping with the refer-ence management

The COLOSS (Prevention of honey bee COlony LOSSes)Association aims to explain and prevent massive honey beecolony losses It was funded through the COST ActionFA0803 COST (European Cooperation in Science andTechnology) is a unique means for European researchers tojointly develop their own ideas and new initiatives across allscientific disciplines through trans-European networking ofnationally funded research activities Based on a pan-European

intergovernmental framework for cooperation in science andtechnology COST has contributed since its creation morethan 40 years ago to closing the gap between science policymakers and society throughout Europe and beyond COST issupported by the EU Seventh Framework Program forresearch technological development and demonstration activ-ities (Official Journal L 412 30 December 2006) TheEuropean Science Foundation as implementing agent of COSTprovides the COST Office through an EC Grant AgreementThe Council of the European Union provides the COSTSecretariat The COLOSS network is now supported by theRicola FoundationmdashNature amp Culture

We would also like to express our gratitude to ProfessorJamie Ellis for his kind support all the way and for providinghelpful and constructive suggestions

Disclosure statementNo potential conflict of interest was reported bythe author(s)

ORCIDLigia Bicudo de Almeida-Muradian httporcidorg0000-0001-5322-1358Ortrud Monika Barth httporcidorg0000-0002-2418-8605Vincent Dietemann httporcidorg0000-0002-0791-9939Michael Eyer httporcidorg0000-0003-1047-1714Alex da Silva de Freitas httporcidorg0000-0002-8665-7649Anne-Claire Martel httporcidorg0000-0002-1179-4286Gian Luigi Marcazzan httporcidorg0000-0002-1695-9713Carla Marina Marchese httporcidorg0000-0002-7495-0688Carla Mucignat-Caretta httporcidorg0000-0001-5307-0414Wim Reybroeck httporcidorg0000-0002-2269-6534M Teresa Sancho httporcidorg0000-0002-9128-9422Jose Augusto Gasparotto Sattler httporcidorg0000-0002-7760-0501

References

Administration U S F amp D (2017) Title 21 of the US Code ofFederal Regulations Part 556 Subpart B Specific tolerances forresidues of new animal drugs Section 556360 LincomycinSection 556500 Oxytetracycline Section 556740 Tylosin httpwwwecfrgovcgi-bintext-idxSID=635d19511f68344e66597151f2534107ampmc=trueampnode=pt216556amprgn=div5sp216556b

Ahmed M (2013) The relationship between bioactive com-pounds with diastase activity and antibacterial synergy ofhoney and potato starch combinations against Klebsiellapneumonia Clinical Microbiology Open Access 2(3) 2ndash5 doi1041722327-50731000131

Alfredsson G Branzell C Granelli K amp Lundstreuroom Aring(2005) Simple and rapid screening and confirmation of tet-racyclines in honey and egg by a dipstick test and LCndashMSMS Analytica Chimica Acta 529(1ndash2) 47ndash51 doi101016jaca200408050

Almeida-Muradian L B amp Gallmann P (2012)Generalizability of PLS calibrations with FT-IR ATR spec-trometry for the prediction of some physicochemical meas-urands of honey ALP Science 541 1ndash20

Almeida-Muradian L B Luginbeurouhl W Badertscher R ampGallmann P (2012) Generalizability of PLS calibrationswith FT-IR ATR spectrometry for the prediction of some


physicochemical measurands of honey ALP Science 4411ndash20

Almeida-Muradian L B amp Penteado M V (2015) VigilanciaSanitaria Topicos sobre Legislac~ao e Analise de Alimentos (2nded) Guanabara Koogan

Almeida-Muradian L B Sousa R J Barth O M ampGallmann P (2014) Preliminary data on brazilian monoflo-ral honey from the northeast region using ft-ir atr spectro-scopic palynological and color analysis Quimica Nova37(4) 716ndash719 1059350100-404220140115

Almeida-Muradian L B Stramm K M amp Estevinho L M(2014) Efficiency of the FT-IR ATR spectrometry for theprediction of the physicochemical characteristics ofMelipona subnitida honey and study of the temperaturersquoseffect on those properties International Journal of FoodScience amp Technology 49(1) 188ndash195 doi101111ijfs12297

Almeida-Muradian L B Stramm K M Horita A BarthO M Freitas A S amp Estevinho L M (2013)Comparative study of the physicochemical and palynologicalcharacteristics of honey from Melipona subnitida and Apismellifera International Journal of Food Science amp Technology48(8) 1698ndash1706 doi101111ijfs12140

Almeida-Silva M Canha N Galinha C Dung H M FreitasM C amp Sitoe T (2011) Trace elements in wild andorchard honeys Applied Radiation and Isotopes 69(11)1592ndash1595 doi101016japradiso201101013

Alqarni A S Owayss A A Mahmoud A A amp Hannan M A(2014) Mineral content and physical properties of local andimported honeys in Saudi Arabia Journal of Saudi ChemicalSociety 18(5) 618ndash625 doi101016jjscs201211009

Amendola G Pelosi P amp Dommarco R (2010) Solid-phaseextraction for multi-residue analysis of pesticides in honeyJournal of Environmental Science and Health Part B 46(1)24ndash34 doi101080036012342010515170

Anjos O Campos M G Ruiz P C amp Antunes P (2015)Application of FTIR-ATR spectroscopy to the quantificationof sugar in honey Food Chemistry 169 218ndash223 doi101016jfoodchem201407138

Anklam E (1998) A review of the analytical methods todetermine the geographical and botanical origin of honeyFood Chemistry 63(4) 549ndash562(98)00057-0 doi101016S0308-8146(98)00057-0

Anonymous (2007a) CRLs view on state of the art analytical methodsfor national residue control plans CRL Guidance Paper of 7thDecember httpseceuropaeufoodsitesfoodfilessafetydocscs_vet-med-residues_guideline_validation_screening_enpdf

Anonymous (2007b) Note of the Commission dated 24 July2007 to the members and observers of the VeterinaryPharmaceutical Committee (GG) In Summary record of theStanding Committee on the Food Chain and Animal Health held on18 September in Brussels httpseceuropaeufoodsitesfoodfilessafetydocsreg-com_biosec_sum_18092007_enpdf

Anonymous (2010) Guidelines for the validation of screening meth-ods for residues of veterinary medicines (initial validation and trans-fer) httpseceuropaeufoodsitesfoodfilessafetydocscs_vet-med-residues_guideline_validation_screening_enpdf

Anonymous (2017) Table 2 Substances or Group of substances (1)to be monitored for in the relevant commodity E frac14 ldquoessentialrdquo HDfrac14 ldquohighly desirablerdquo httpseceuropaeufoodsitesfoodfilessafetydocscs_vet-med-residues_animal-imports-non-eu_resi-due-monitoring-progs_required-table2pdf

AOAC (Association of Official Agricultural Chemists) (1990)AOAC official methods of analysis (15th ed Vol 1 pp136ndash138)

AOAC (1992) AOAC 96938B MAFF validated method V21for moisture in honey Journal of the Association of PublicAnalysts 28 183ndash187

Aronne G amp DE Micco V (2010) Traditional melissopaly-nology integrated by multivariate analysis and sampling

methods to improve botanical and geographical character-isation of honeys Plant Biosystems - an International JournalDealing with All Aspects of Plant Biology 144(4) 833ndash840 doi101080112635042010514125

Azzouz A amp Ballesteros E (2015) Multiresidue method forthe determination of pharmacologically active substances inegg and honey using a continuous solid-phase extractionsystem and gas chromatography-mass spectrometry FoodChemistry 178 63ndash69 doi101016jfoodchem201501044

Barganska Z amp Namiesnik J (2010) Pesticide analysis of beeand bee product samples Critical Reviews in AnalyticalChemistry 40(3) 159ndash171 doi101080104083472010490484

Barganska Z Namiesnik J amp Slebioda M (2011)Determination of antibiotic residues in honey TrAC Trendsin Analytical Chemistry 30(7) 1035ndash1041 doi101016jtrac201102014

Barth O M (1989) O polen no mel brasileiro Online httpscholargooglecomscholarhl=enampbtnG=Searchampq=intitleO+Polenthornno+Mel+Brasileiro0

Barth O M (1971) Mikroskopische Bestandteile brasilia-nischer Honigtau-Honigen Apidologie 2(2) 157ndash167 doi101051apido19710202

Barth O M Freitas A S Sousa G L amp Almeida-MuradianL B (2013) Pollen and physicochemical analysis of Apisand Tetragonisca (Apidae) honey Interciencia 38(4)280ndash285 httpwwwredalycorghtml33933926985012

Behm F von der Ohe K amp Henrich W (1996)Zuverleuroassigkeit der Pollenanalyse von Honig DeutscheLebensmittel-Rundschau 192 183ndash188

Benetti C Dainese N Biancotto G Piro R amp Mutinelli F(2004) Unauthorised antibiotic treatments in beekeepingDevelopment and validation of a method to quantify and con-firm tylosin residues in honey using liquid chromatography-tandem mass spectrometric detection Analytica Chimica Acta520(1ndash2) 87ndash92 doi101016jaca200404070

Benetti C Piro R Binato G Angeletti R amp Biancotto G(2006) Simultaneous determination of lincomycin and fivemacrolide antibiotic residues in honey by liquid chromatog-raphy coupled to electrospray ionization mass spectrom-etry (LC-MSMS) Food Additives and Contaminants 23(11)1099ndash1108 doi10108002652030600699338

Bilandzic N Tlak Gajger I Kosanovic M Calopek BSedak M Solomun Kolanovic B Varenina I Luburic-D B Varga I amp -Dokic M (2017) Essential and toxicelement concentrations in monofloral honeys from south-ern Croatia Food Chemistry 234 245ndash253 doi101016jfoodchem201704180

Blasco C Fernandez M Pico Y amp Font G (2004)Comparison of solid-phase microextraction and stir barsorptive extraction for determining six organophosphorusinsecticides in honey by liquid chromatography-mass spec-trometry Journal of Chromatography A 1030(1ndash2) 77ndash85doi101016jchroma200311037

Blasco C Lino C M Pico Y Pena A Font G amp SilveiraM I N (2004) Determination of organochlorine pesticideresidues in honey from the central zone of Portugal andthe Valencian community of Spain Journal of ChromatographyA 1049(1ndash2) 155ndash160 doi101016jchroma200407049

Blasco C Pico Y amp Torres C M (2007) Progress in ana-lysis of residual antibacterials in food TrAC Trends inAnalytical Chemistry 26(9) 895ndash913 doi101016jtrac200708001

Blasco C Vazquez-Roig P Onghena M Masia A amp PicoY (2011) Analysis of insecticides in honey by liquid chro-matography-ion trap-mass spectrometry Comparison ofdifferent extraction procedures Journal of ChromatographyA 1218(30) 4892ndash4901 doi101016jchroma201102045


Bogdanov S (2009) Harmonised methods of the internationalhoney commission International Honey CommissionRetrieved from httpwwwihc-platformnetihcme-thods2009pdf

Bogdanov S (2011) The honey book httpwwwbee-hexa-gonnethoney

Bogdanov S amp Martin P (2002) Honey authenticity A reviewMitteilungen Aus Dem Gebiete Der LebensmitteluntersuchungUnd Hygiene 6 1ndash20 httpwwwbee-hexagonnetfilesfilefileEHoneyAuthenticityRevue_Internetpdf

Bogdanov S Haldimann M Luginbeurouhl W amp Gallmann P(2007) Minerals in honey Environmental geographical andbotanical aspects Journal of Apicultural Research 46(4)269ndash275 doi10108000218839200711101407

Bogdanov S Martin P amp Leuroullmann C (1997) Harmonizedmethods of the European Honey Commission ApidologieExtra Issue 1ndash59

Bougrini M Florea A Cristea C Sandulescu R Vocanson FErrachid A Bouchikhi B El Bari N amp Jaffrezic-Renault N(2016) Development of a novel sensitive molecularlyimprinted polymer sensor based on electropolymerization of amicroporous-metal-organic framework for tetracycline detec-tion in honey Food Control 59 424ndash429 httpsdoiorghttpsdoiorg101016jfoodcont201506002 doi101016jfoodcont201506002

Bryant V M amp Jones G D (2001) The R-values of honeyPollen coefficients Palynology 25(1) 11ndash28 doi1010800191612220019989554

Caldow M Stead S L Day J Sharman M Situ C ampElliott C (2005) Development and validation of an opticalSPR biosensor assay for tylosin residues in honey Journal ofAgricultural and Food Chemistry 53(19) 7367ndash7370 doi101021jf050725s

Cano C B Felsner M L Bruns R E Matos J R ampAlmeida-Muradian L B (2006) Optimization of mobilephase for separation of carbohydrates in honey by high per-formance liquid chromatography using a mixture designJournal of the Brazilian Chemical Society 17(3) 588ndash593 doi101590S0103-50532006000300024

Carrasco-Pancorbo A Casado-Terrones S Segura-Carretero A amp Fernandez-Gutierrez A (2008) Reversed-phase high-performance liquid chromatography coupled toultraviolet and electrospray time-of-flight mass spectrom-etry on-line detection for the separation of eight tetracy-clines in honey samples Journal of Chromatography A1195(1ndash2) 107ndash116 doi101016jchroma200805003

Carreck N L Andree M Brent C S Cox-Foster DDade H A Ellis J D Hatjina F amp vanEngelsdorp D(2013) Standard methods for Apis mellifera anatomy anddissection Journal of Apicultural Research 52(4) 1ndash39

Chabottaux V Bonhomme C Muriano A Stead SDiserens J M Marco M P amp Granier B (2010) Effect ofacidic hydrolysis on sulfamides detection in honey with newgeneric antibody-based dipstick assay In Abstract book ofthe 6th International Symposium on Hormone andVeterinary Drug Residue Analysis (Vol 63 pp 1ndash4) httpwwwconffidenceeuwp-contentuploads201408enews_2_VDRA_abstract_CHABOTTAUX_SULFASENSORpdf

Chua L S Lee J Y amp Chan G F (2013) Honey proteinextraction and determination by mass spectrometryAnalytical and Bioanalytical Chemistry 405(10) 3063ndash3074doi101007s00216-012-6630-2

Ciulu M Solinas S Floris I Panzanelli A Pilo M I PiuP C Spano N amp Sanna G (2011) RP-HPLC determin-ation of water-soluble vitamins in honey Talanta 83(3)924ndash929 doi101016jtalanta201010059

Codex (2001) Codex Alimentarius standard for honey 12-1981 Revised Codex standard for honey Standards and

standard methods (Vol 11) Retrieved April 2020 fromhttpwwwcodexalimentariusnet

Codex Alimentarius Commission (1969) RecommendedEuropean regional standard for honey (CACRS 12-1969)

Codex Standard for Honey (2001) CODEX STAN 12-1981(pp 1ndash8)

Commission E (1998) Commission Decision 98179EC of 23February 1998 laying down detailed rules on official samplingfor the monitoring of certain substances and residues thereof inlive animals and animal products Official Journal of the EuropeanCommunities L65 31ndash34 httpeur-lexeuropaeulegal-con-tentENTXTPDFuri=CELEX31998D0179ampfrom=EN

Commission E (2005) Commission Decision 200534EC of11 January 2005 laying down harmonized standards for thetesting for certain residues in products of animal originimported from third countries Official Journal of theEuropean Union L16 61ndash63 httpeur-lexeuropaeulegal-contentENTXTPDFuri=CELEX32005D0034ampfrom=EN

Commission E (2010) Commission Regulation (EU) No 372010 of 22 December 2009 on pharmacologically active sub-stances and their classification regarding maximum residue lim-its in foodstuffs of animal origin Official Journal of the EuropeanUnion L15 1ndash72 httpseceuropaeuhealthsiteshealthfilesfileseudralexvol-5reg_2010_37reg_2010_37_enpdf

Commission E (2018) Commission Implementing Regulation (EU)2018470 of 21 March 2018 on detailed rules on the maximumresidue limit to be considered for control purposes for food-stuffs derived from animals which have been treated in the EUunder Article 11 of Directive 200182EC Official Journal of theEuropean Union L79 16ndash18 httpseur-lexeuropaeulegal-con-tentENTXTqid=1561736258606ampuri=CELEX32018R0470

Consonni R amp Cagliani L R (2008) Geographical character-ization of polyfloral and Acacia honeys by nuclear magneticresonance and chemometrics Journal of Agricultural and FoodChemistry 56(16) 6873ndash6880 doi101021jf801332r

Council Directive (1996) Council Directive 9623EC of 29 April1996 on measures to monitor certain substances and residuesthereof in live animals and animal products and repealingDirectives 85358EEC and 86469EEC and Decisions 89187EEC and 91664EEC Official Journal of the European CommunitiesL125 10ndash32 httpeur-lexeuropaeulegal-contentENTXTPDFuri=CELEX31996L0023ampfrom=EN

da Silva P M Gauche C Gonzaga L V Costa A C O ampFett R (2016) Honey Chemical composition stability andauthenticity Food Chemistry 196 309ndash323 doi101016jfoodchem201509051

Daeseleire E amp Reybroeck W (2012) Development and val-idation of a liquid chromatographic-tandem mass spectro-metric method for the detection of fumagillin in honey Usein a stability study In I International Symposium on BeeProducts (pp 9ndash12) Braganca Portugal

Daniela I Morariu P Schiriac E Matiut S amp CuciureanuR (2012) Method validation for simultaneous determin-ation of 12 sulfonamides in honey using biochip array tech-nology Farmacia 60 143ndash154 httpspdfssemanticscholarorg96c0944ef59d8e1a03fc7c966532a528bb232c43pdf

Davies A M C (1975) Amino acid analysis of honeys fromeleven countries Journal of Apicultural Research 14(1)29ndash39 doi10108000218839197511099798

De Brabander H F Noppe H Verheyden K VandenBussche J Wille K Okerman L Vanhaecke LReybroeck W Ooghe S amp Croubels S (2009) Residueanalysis Future trends from a historical perspective Journalof Chromatography A 1216(46) 7964ndash7976 doi101016jchroma200902027

de Graaf D Alippi A Antunez K Aronstein K Budge GDe Koker D De Smet L Dingman D Evans J Foster LFeurounfhaus A Garcia-Gonzalez E Gregore A Human HMurray K Nguyen B Poppinga L Spivak M van


Engelsdorp D Wilkins S amp Genersch E (2013) Standardmethods for American foulbrood research Journal ofApicultural Research 52(1) 1ndash28 httpsdoiorg103896IBRA152111

De-Melo A A M Almeida-Muradian L B Sancho M T ampPascual-Mate A (2017) Composition and properties ofApis mellifera honey A review Journal of Apicultural Research57(1) 5ndash37 doi1010800021883920171338444

De-Melo A A M Almeida-Muradian L B Sancho M T ampPascual-Mate A (2017) Composition and properties ofApis mellifera honey A review Journal of Apicultural Research8839(June) 1ndash33 doi1010800021883920171338444

Dietemann V Nazzi F J Martin S L Anderson D LockeB S Delaplane K Wauquiez Q Tannahill C Frey EZiegelmann B Rosenkranz P D amp Ellis J (2013)Standard methods for varroa research In V Dietemann JD Ellis amp P Neumann (Eds) The COLOSS BEEBOOK VolumeII standard methods for Apis mellifera pest and pathogenresearch Journal of Apicultural Research 52(1) doi103896IBRA152109

DOCUMENT SANTE118132017 (2018) Guidance documenton analytical quality control and method validation proceduresfor pesticides residues analysis in food and feed EuropeanCommission


Dominguez M A Jacksen J Emmer Aring amp Centurion M E(2016) Capillary electrophoresis method for the simultan-eous determination of carbohydrates and proline in honeysamples Microchemical Journal 129 1ndash4 doi101016jmicroc201605017

Dubreil-Cheneau E Pirotais Y Verdon E amp Hurtaud-Pessel D (2014) Confirmation of 13 sulfonamides in honeyby liquid chromatography-tandem mass spectrometry formonitoring plans Validation according to European UnionDecision 2002657EC Journal of Chromatography A1339(37) 128ndash136 doi101016jchroma201403003

Edder P Cominoli A amp Corvi C (1998) Dosage de residusde streptomycine dans le miel par HPLC avec postderivati-sation et detection fluorimetrique Mitteilungen Aus DemGebiete Der Lebensmitteluntersuchung Und Hygiene 89(3)369ndash382

EL Alami EL Hassani N Tahri K Llobet E Bouchikhi BErrachid A Zine N amp EL Bari N (2018) Emergingapproach for analytical characterization and geographicalclassification of Moroccan and French honeys by means of avoltammetric electronic tongue Food Chemistry 243 36ndash42doi101016jfoodchem201709067

EL Sohaimy S A Masry S H D amp Shehata M G (2015)Physicochemical characteristics of honey from different ori-gins Annals of Agricultural Sciences 60(2) 279ndash287 doi101016jaoas201510015

Elizabeta D S Gordana A Zehra H M Biljana S D Risto UAleksandra T amp Goran S (2012) In-house validation and qualitycontrol of commercial enzyme-linked immunosorbnet assays forscreening of nitrofuran metabolites in food of animal originMacedonian Veterinary Review 35(1) 13ndash21 httpswwwresearchgatenetprofileRisto_Uzunovpublication260184219_In-house_validation_and_quality_control_of_commercial_enzyme-linked_immunosorbnet_assays_for_screening_of_nitrofuran_metabolites_in_food_of_animal_originlinks0a85e52fea989dc8bc00000

Erdtman G (1952) Pollen morphology and plant taxonomy -Angiosperms AlmqvistampWiksell

EU PESTICIDES DATABASE httpeceuropaeufoodplantpes-ticideseu-pesticides-databasepublicevent=productresultatamplanguage=ENampselectedID=375

European Commission (2001) Council Directive 2001110ECof 20 December 2001 relating to honey Retrieved fromhttpwwwihc-platformnethoneydirective2001pdf

European Communities Commission (1997) CommissionDecision 97747EC of 27 October 1997 fixing the levels andfrequencies of sampling provided for by Council Directive 9623EC for the monitoring of certain substances and residuesthereof in certain animal products Official Journal of the EuropeanCommunities L303 12ndash15 httpeur-lexeuropaeulegal-con-tentENTXTPDFuri=CELEX31997D0747ampfrom=EN

European Communities Commission (2003) CommissionDecision 2003181EC of 13 March 2003 as regards the set-ting of minimum required performance limits (MRPLs) forcertain residues in food of animal origin Official Journal of theEuropean Union L71 17ndash18 httpeur-lexeuropaeulegal-contentENTXTPDFuri=CELEX32003D0181ampfrom=EN

European Union Commission (2002) Commission Decision2002657EC of 12 August 2002 implementing CouncilDirective 9623EC concerning the performance of analyticalmethods and the interpretation of results Official Journal ofthe European Communities L221 8ndash36 httpeur-lexeuropaeulegal-contentENTXTPDFuri=CELEX32002D0657ampfrom=EN

Eyer M Greco M K Lang J Neumann P amp Dietemann V(2016) No spatial patterns for early nectar storage inhoney bee colonies Insectes Sociaux 63(1) 51ndash59 doi101007s00040-015-0432-4

Eyer M Neumann P amp Dietemann V (2016) A look intothe cell Honey storage in honey bees Apis mellifera PLoSOne 11(8) e0161059ndash20 doi101371journalpone0161059

Fallico B Arena E amp Zappala M (2009) Prediction ofhoney shelf life Journal of Food Quality 32(3) 352ndash368 doi101111j1745-4557200900253x

Ferreira I C F R Aires E Barreira J C M amp EstevinhoL M (2009) Antioxidant activity of Portuguese honey sam-ples Different contributions of the entire honey and phen-olic extrac Food Chemistry 114(4) 1438ndash1443 doi101016jfoodchem200811028

Fischer W M amp Palmer J D (2005) Evidence from small-subunit ribosomal RNA sequences for a fungal origin ofMicrosporidia Molecular Phylogenetics and Evolution 36(3)606ndash622 doi101016jympev200503031

Food Research Center (2017) Tabela Brasileira de Composic~aode Alimentos (TBCA)

Forsgren E Budge G Charriere JD amp Hornitzky M(2013) Standard methods for European foulbrood researchJournal of Apicultural Research 52(1) 1ndash14 httpsdoiorg103896IBRA152112

Fries I Chauzat MP Chen YP Doublet V Genersch EGisder S Higes M McMahon D Martın-Hernandez RNatsopoulou M Paxton R Tanner G Webster T ampWilliams G (2013) Standard methods for Nosemaresearch Journal of Apicultural Research 52(1) 1ndash28 httpsdoiorg103896IBRA152114I

Galarini R Saluti G Giusepponi D Rossi R amp Moretti S(2015) Multiclass determination of 27 antibiotics in honeyFood Control 48 12ndash24 doi101016jfoodcont201403048

Gaudin V DE Courville A Hedou C Rault A DiomandeS E Creff-Froger C amp Verdon E (2013) Evaluation andvalidation of two microbiological tests for screening anti-biotic residues in honey according to the European guide-line for the validation of screening methods Food Additivesamp Contaminants Part A 30(2) 234ndash243 doi101080194400492012738367

Gaudin V Hedou C amp Verdon E (2013) Validation of twoELISA kits for the screening of tylosin and streptomycin inhoney according to the European decision 2002657ECFood Additives amp Contaminants Part A 30(1) 93ndash109 doi101080194400492012722696


Gaudin V Hedou C Soumet C amp Verdon E (2014)Evaluation and validation of biochip multi-array technologyfor the screening of six families of antibiotics in honeyaccording to the European guideline for the validation ofscreening methods for residues of veterinary medicinesFood Additives amp Contaminants Part A 31(10) 1699ndash1711doi101080194400492014952784

Gaudin V Rault A amp Verdon E (2012) Validation of acommercial receptor kit SulfasensorVR Honey for thescreening of sulfonamides in honey according toCommission Decision 2002657EC Food Additives ampContaminants Part A 29(6) 942ndash950 doi101080194400492012668718

Gaudin V Rault A amp Verdon E (2013) Validation of acommercial receptor kit for tetracycline residues in honeyaccording to the European guideline for screening methodsFood and Agricultural Immunology 24(1) 111ndash128 doi101080095401052011651446

Gomes S Dias L G Moreira L L Rodrigues P ampEstevinho L (2010) Physicochemical microbiological andantimicrobial properties of commercial honeys fromPortugal Food and Chemical Toxicology 48(2) 544ndash548 doi101016jfct200911029

Gomez-Dıaz D Navaza J M amp Quintans-Riveiro L C(2012) Physicochemical characterization of Galician honeysInternational Journal of Food Properties 15(2) 292ndash300 doi101080109429122010483616

Gonnet M amp Vache G (1979) Technique de degustationdes miels et recherche drsquoun systeme de notation et de clas-sification objectif pour apprecier leur qualite par lrsquoanalysesensorielle In 27th Apimondia International ApiculturalCongress (pp 499ndash506) Athenes

Gonnet M amp Vache G (1992) The taste of honeyApimondia

Gonnet M Vache G amp Jean-Prost P (1985) Le gout du mielUNAF

Gordon L (1989) Quantitative determination of oxytetracyc-line in honey by cylinder plate microbioassay AustralianJournal of Agricultural Research 40(4) 933ndash940 doi101071AR9890933

Granato D amp Nunes D S (2016) Analises quımicas proprie-dades funcionais e controle de qualidade de alimentos e bebi-das uma abordagem teorico-pratica Elsevier Brasil

Greco M K Lang J Gallmann P Priest N Feil E ampCrailsheim K (2013) Sugar concentration influences deci-sion making in Apis mellifera L workers during early-stagehoney storage behaviour Open Journal of Animal Sciences3(3) 210ndash218 doi104236ojas201333031

Haidamus SL Lorenzon MCA amp Barth OM (2019)Biological elements and residues in Brazilian honeys GreenerJournal of Biological Sciences 9(1) 8ndash14 httpsdoiorg1015580GJBS20191022119038

Hammel Y A Mohamed R Gremaud E Lebreton M H ampGuy P A (2008) Multi-screening approach to monitor andquantify 42 antibiotic residues in honey by liquid chromatog-raphyndashtandem mass spectrometry Journal of ChromatographyA 1177(1) 58ndash76 httpreindiaenvironmentportalorginfilesMulti screening approach to monitorpdf

Hawkins J de Vere N Griffith A Ford C RAllainguillaume J Hegarty M J Baillie L amp Adams-Groom B (2015) Using DNA metabarcoding to identifythe floral composition of honey A new tool for investigat-ing honey bee foraging preferences PLoS One 10(8)e0134735ndash20 doi101371journalpone0134735

Heering W Usleber E Dietrich R amp Meuroartlbauer E (1998)Immunochemical screening for antimicrobial drug residuesin commercial honey The Analyst 123(12) 2759ndash2762 doi101039a805047c

Heinrich K Macarthur R VON Holst C amp Sharman M(2013) An inter-laboratory validation of a multiplex dipstickassay for four classes of antibiotics in honey RapidDetection in Food and Feed Analytical and BioanalyticalChemistry 405(24) 7875ndash7884 doi101007s00216-013-7070-3

Hermosın I Chicon R M amp amp Dolores Cabezudo M(2003) Free amino acid composition and botanical origin ofhoney Food Chemistry 83(2) 263ndash268 doi101016S0308-8146(03)00089-X

Huidobro J F amp Simal J (1984) Contribution to the sugarsdetermination on honey Anales de Bromatologıa 36247ndash264

Human H Brodschneider R Dietemann V Dively G EllisJ D Forsgren E Fries I Hatjina F Hu F-L Jaffe RJensen A B Keuroohler A Magyar J P euroOzkyrym A PirkC W W Rose R Strauss U Tanner G Tarpy D Rhellip Zheng H-Q (2013) Miscellaneous standard methodsfor Apis mellifera research In V Dietemann J D Ellis amp PNeumann (Eds) The COLOSS BEEBOOK Volume I standardmethods for Apis mellifera research Journal of ApiculturalResearch 52(4) 1ndash53 doi103896IBRA152410

International Olive Oil Council (2007) Sensory analysis of oliveoil Standard Glass for oil tasting COIT20Doc No 5Rev1Retrieved from httpswwwinternationaloliveoilorg

ISO 16657 (2006) Sensory analysis Apparatus ndash Olive oiltasting glass International Organization for StandardizationRetrieved from https wwwisoorg

ISO 13300 (2006a) Sensory analysis General guidance for thestaff of a sensory evaluation laboratory - Part 1 Staff responsi-bilities International Organization for Standardizationhttpswwwisoorg

ISO 13300 (2006b) Sensory analysis General guidance for thestaff of a sensory evaluation laboratory - Part 2 Recruitmentand training of panel leaders International Organization forStandardization httpswwwisoorg

ISO 5492 (2008) Sensory analysis Vocabulary InternationalOrganization for Standardization httpswwwisoorg

ISO 6658 (2017) Sensory analysis Methodology - general guid-ance International Organization for Standardization httpswwwisoorg

ISO 8586 (2012) Sensory analysis General guidelines for theselection training and monitoring of selected assessors andexpert sensory assessors International Organization forStandardization httpswwwisoorg

ISO 8589 (2007) Sensory analysis General guidance for thedesign of test rooms International Organization forStandardization httpswwwisoorg

Jeon M amp Rhee Paeng I (2008) Quantitative detection oftetracycline residues in honey by a simple sensitiveimmunoassay Analytica Chimica Acta 626(2) 180ndash185 doi101016jaca200808003

Kacaniova M Knazovicka V Melich M Fikselova MMassanyi P Stawarz R amp Putała A (2009) Environmentalconcentration of selected elements and relation to physico-chemical parameters in honey Journal of EnvironmentalScience and Health - Part A ToxicHazardous Substances andEnvironmental Engineering 44(4) 414ndash422 doi10108010934520802659802

Kamal M A amp Klein P (2011) Determination of sugars inhoney by liquid chromatography Saudi Journal of BiologicalSciences 18(1) 17ndash21 doi101016jsjbs201009003

Kanda M Sasamoto T Takeba K Hayashi H Kusano TMatsushima Y Nakajima T Kanai S amp Takano I (2011)Rapid determination of fumagillin residues in honey byliquid chromatographyndashtandem mass spectrometry usingthe QuEChERS method Journal of AOAC International 94(3)878ndash899 httpwwwingentaconnectcomcontentaoac


jaoac20110000009400000003art00023 doi101093jaoac943878

Kaufmann A Roth S Ryser B Widmer M amp Guggisberg D(2002) Quantitative LCMS-MS determination of sulfonamidesand some other antibiotics in honey Journal of AOACInternational 85(4) 853ndash860 httpwwwingentaconnectcomcontentoneaoacjaoac20020000008500000004art00009-crawler=trueampmimetype=applicationpdfampcasa_token=cI5pUDQmtlQAAAAAA3SyqI7aIZndvjCZA0bR2IuaZyBleUKUQ-U1YQT5p8t6vqxxk30JLPlxd2xbeYzIwMQb9-lQl

Khismatoullin R G Lyapunov Y E Kuzyaev R ZYelovikova E A Legotkina G I amp Zakharov A V(2004) Bacteriological detection of tylosin residues inhoney [Paper presentation] Proceedings of the First EuropeanConference of Apidology (pp 19ndash23)

Khismatoullin R Kuzyaev R Lyapunov Y amp Elovikova E(2003) Modification of microbiological detection method oftetracycline in honey Apiacta 38 246ndash248 httpswwwapimondiacomapiactaarticles2003khismatoullin_1pdf

Khong S P Gremaud E Richoz J Delatour T Guy P AStadler R H amp Mottier P (2004) Analysis of matrix-bound nitrofuran residues in worldwide-originated honeysby isotope dilution high-performance liquid chromatog-raphy-tandem mass spectrometry Journal of Agricultural andFood Chemistry 52(17) 5309ndash5315 doi101021jf0401118

Kivrak _I Kivrak S amp Harmandar M (2016) Developmentof a rapid method for the determination of antibiotic resi-dues in honey using UPLC-ESI-MSMS Food Science andTechnology 36(1) 90ndash96 doi1015901678-457X0037

Kujawski M W Barganska Z Marciniak K MiedzianowskaE Kujawski J K Slebioda M amp Namiesnik J (2014)Determining pesticide contamination in honey by LC-ESI-MSMS - Comparison of pesticide recoveries of two liquid-liquid extraction based approaches LWT - Food Science andTechnology 56(2) 517ndash523 doi101016jlwt201311024

Kujawski M W Pinteaux E amp Namiesnik J (2012)Application of dispersive liquid-liquid microextraction forthe determination of selected organochlorine pesticides inhoney by gas chromatography-mass spectrometry EuropeanFood Research and Technology 234(2) 223ndash230 doi101007s00217-011-1635-1

Lazarevic J S Palic R M Radulovic N S Ristic N R ampStojanovic G S (2010) Chemical composition and screen-ing of the antimicrobial and antioxidative activity of extractsof Stachys species Journal of the Serbian Chemical Society75 1347ndash1359 doi102298JSC100601117L

Lee D-C Lee S-Y Cha S-H Choi Y-S amp Rhee H-I(1998) Discrimination of native bee-honey and foreign bee-honey by SDS-PAGE Korean Journal of Food Science andTechnology 30(1) 1ndash5

Leon-Ruiz V Vera S Gonzalez-Porto A V amp San AndresM P (2013) Analysis of water-soluble vitamins in honey byisocratic RP-HPLC Food Analytical Methods 6(2) 488ndash496doi101007s12161-012-9477-4

Lopez M I Feldlaufer M Williams A amp Chu P-S (2007)Determination and confirmation of nitrofuran residues inhoney using LC-MS MS Journal of Agricultural and FoodChemistry 55(4) 1103ndash1108 doi101021jf0625712

Lopez M I Pettis J S Smith I B amp Chu P-S (2008)Multiclass determination and confirmation of antibiotic resi-dues in honey using LC-MSMS Journal of Agricultural andFood Chemistry 56(5) 1553ndash1559 doi101021jf073236w

Lores E M Moore J C amp Moody P (1987) Improved silica gelcleanup method for organophosphorus pesticides Chemosphere16(5) 1065ndash1069 doi1010160045-6535(87)90043-9

Louppis A P Karabagias I K Kontakos S KontominasM G amp Papastephanou C (2017) Botanical discriminationof Greek unifloral honeys based on mineral content in com-bination with physicochemical parameter analysis using a

validated chemometric approach Microchemical Journal 135180ndash189 doi101016jmicroc201709004

Louveaux J Maurizio A amp Vorwohl G (1970a)Internationale Kommission Feurour Bienenbotanik Der I U BS Methodik Der Melissopalynologie Apidologie 1(2)193ndash209 doi101051apido19700205

Louveaux J Maurizio A amp Vorwohl G (1970b) Methods ofmelissopalynology Bee World 51(3) 125ndash138 doi1010800005772X197011097312

Louveaux J Maurizio A amp Vorwohl G (1978) Methods ofmelissopalynology Bee World 59(4) 139ndash157 doi1010800005772X197811097714

Lutier P M amp Vaissiere B E (1993) An improved methodfor pollen analysis of honey Review of Palaeobotany andPalynology 78(1ndash2) 129ndash144(93)90019-Q doi1010160034-6667(93)90019-Q

Marcazzan G L Magli M Piana L Savino A amp StefanoM A (2014) Sensory profile research on the main Italiantypologies of monofloral honey Possible developments andapplications Journal of Apicultural Research 53(4) 426ndash437doi103896IBRA153409

Marcazzan G L Mucignat-Caretta C Marchese C M ampPiana M L (2018) A review of methods for honey sensoryanalysis Journal of Apicultural Research 57(1) 75ndash87 doi1010800021883920171357940

Martel A C amp Zeggane S (2002) Determination of acari-cides in honey by high-performance liquid chromatographywith photodiode array detection Journal of ChromatographyA 954(1ndash2) 173ndash180(02)00126-7 doi101016S0021-9673(02)00126-7

Maudens K E Zhang G F amp Lambert W E (2004)Quantitative analysis of twelve sulfonamides in honey afteracidic hydrolysis by high-performance liquid chromatog-raphy with post-column derivatization and fluorescencedetection Journal of Chromatography A 1047(1) 85ndash92 doi101016jchroma200407007

Maurizio A (1959) Zur Frage der Mikroskopie von Honigtau-Honigen Annales de Labeille 2 145ndash157 doi101051apido19590203

Maurizio A (1977) Honig-Mikroskopie In H Freud (Ed)Handbuch der Mikroskopie in der Technik (vol VIII pp627ndash652) Umschau Verlag

McAleer D Mcconnell R I Tohill A amp Fitzgerald S P(2010) Biochip arrays for multi-analytical screening of anti-biotics in honey [Paper presentation] Abstract Book of the6th International Symposium on Hormone and Veterinary DrugResidue Analysis (Vol 129 pp 1ndash4) Ghent

McMullen S E Lansden J A amp Schenck F J (2004)Modifications and adaptations of the Charm II rapid antibodyassay for chloramphenicol in honey Journal of Food Protection67(7) 1533ndash1536 doi1043150362-028X-6771533

Medrzycki P Giffard H Aupinel P Belzunces L PChauzat M-P Claszligen C Colin M E Dupont TGirolami V Johnson R Le Conte Y Leurouckmann JMarzaro M Pistorius J Porrini C Schur A SgolastraF Delso N S van der Steen J J M hellip Vidau C (2013)Standard methods for toxicology research in Apis melliferaIn V Dietemann J D Ellis amp P Neumann (Eds) The COLOSSBEEBOOK Volume I standard methods for Apis melliferaresearch Journal of Apicultural Research 52(4) 1ndash60 doi103896IBRA152414

Meixner MD Pinto MA Bouga M Kryger P Ivanova Eamp Stefan Fuchs S (2013) Standard methods for character-ising subspecies and ecotypes of Apis mellifera Journal ofApicultural Research 52(4) 1ndash27

Miller N J Rice-Evans C Davies M J Gopinathan V ampMilner A (1993) A novel method for measuring antioxi-dant capacity and its application to monitoring the


antioxidant status in premature neonates Clinical Science84(4) 407ndash412 doi101042cs0840407

MINISTERIO DA AGRICULTURA PECUARIA EABASTECIMENTO B (2000) Instruc~ao Normativa n 11 de20 de outubro 2000

Mucignat-Caretta C Marcazzan G L amp Piana M L (2012)Il Miele In Atlante sensoriale dei prodotti alimentari (pp286ndash295) Techniche Nuove

Muriano A Chabottaux V Diserens J M Granier BSanchez-Baeza F amp Marco M P (2015) Rapid immuno-chemical analysis of the sulfonamide-sugar conjugated fractionof antibiotic contaminated honey samples Food Chemistry178 156ndash163 doi101016jfoodchem201501037

Muriano A Pinacho D-G Chabottaux V Diserens J-MGranier B Stead S Sanchez Baeza F Pividori M I ampMarco M-P (2013) A portable electrochemical magneto-immunosensor for detection of sulfonamide antimicrobialsin honey Rapid Detection in Food and Feed Analytical andBioanalytical Chemistry 405(24) 7885ndash7895 doi101007s00216-013-7219-0

Nascimento K S D Gasparotto Sattler J A Lauer MacedoL F Serna Gonzalez C V Pereira de Melo I L da SilvaAraujo E Granato D Sattler A amp de Almeida-MuradianL B (2018 January) Phenolic compounds antioxidant cap-acity and physicochemical properties of Brazilian Apis melli-fera honeys LWT 91 85ndash94 doi101016jlwt201801016

Nozal M J Bernal J L Gomez M T Jimenez J J BernalJ amp Higes M (2006) Trace analysis of antibacterial tylosinA B C and D in honey by liquid chromatography-electro-spray ionization-mass spectrometry Journal of SeparationScience 29(3) 405ndash413 101002jssc200500423

OrsquoMahony J Moloney M Mcconnell I Benchikh E LowryP amp Danaher M (2010) Biochip array detection of nitro-furan metabolites in honey [Paper presentation] AbstractBook of the 6th International Symposium on Hormone andVeterinary Drug Residue Analysis (Vol 176 pp 1ndash4) Ghent

Ouchemoukh S Louaileche H amp Schweitzer P (2007)Physicochemical characteristics and pollen spectrum ofsome Algerian honeys Food Control 18(1) 52ndash58 doi101016jfoodcont200508007

Ouchemoukh S Schweitzer P Bachir Bey M Djoudad-Kadji H amp Louaileche H (2010) HPLC sugar profiles ofAlgerian honeys Food Chemistry 121(2) 561ndash568 doi101016jfoodchem200912047

Ough C (1969) Rapid determination of proline in grapes andwines Journal of Food Science 34(3) 228ndash230 doi101111j1365-26211969tb10327x

euroOzbalci B Boyaci I H Topcu A Kadilar C amp Tamer U(2013) Rapid analysis of sugars in honey by processingRaman spectrum using chemometric methods and artificialneural networks Food Chemistry 136(3ndash4) 1444ndash1452 doi101016jfoodchem201209064

Pang G-F Fan C-L Liu Y-M Cao Y-Z Zhang J-J FuB-L Li X-M Li Z-Y amp Wu Y-P (2006) Multi-residuemethod for the determination of 450 pesticide residues inhoney fruit juice and wine by double-cartridge solid-phaseextraction gas chromatography-mass spectrometry andliquid chromatography-tandem mass spectrometry FoodAdditives and Contaminants 23(8) 777ndash810 doi10108002652030600657997

Pascual-Mate A Oses S M Fernandez-Mui~no M A ampSancho M T (2018) Methods of analysis of honey Journalof Apicultural Research 57(1) 38ndash74 doi1010800021883920171411178

Peng D Ye S Wang Y Chen D Tao Y Huang L LiuZ Dai M Wang X amp Yuan Z (2012) Development andvalidation of an indirect competitive enzyme-linkedimmunosorbent assay for the screening of tylosin and tilmi-cosin in muscle liver milk honey and eggs Journal of

Agricultural and Food Chemistry 60(1) 44ndash51 doi101021jf2037449

Persano Oddo L amp Bogdanov S (2004) Determination ofhoney botanical origin Problems and issues Apidologie35(Suppl 1) S2ndashS32004044 doi101051apido2004044

Piana M L Persano Oddo L Bentabol A Bruneau EBogdanov S amp Guyot Declerck C (2004) Sensory ana-lysis applied to honey State of the art Apidologie 35(Suppl1) S26ndashS372004048 doi101051apido2004048

Pirard C Widart J Nguyen B K Deleuze C Heudt LHaubruge E Haubruge E DE Pauw E amp Focant J F(2007) Development and validation of a multi-residuemethod for pesticide determination in honey using on-col-umn liquid ndash liquid extraction and liquid chromatography ndashtandem mass spectrometry Journal of Chromatography A1152(1ndash2) 116ndash123 doi101016jchroma200703035

Polzer J Kindt K Radeck W amp Gowik P (2010Determination of nitro-imidazoles in honey Method devel-opment validation and relevance in practice [Paper presen-tation] Abstract Book of the 6th International Symposium onHormone and Veterinary Drug Residue Analysis (Vol 185 pp1ndash4) Ghent

Popa I D Schiriac E C amp Cuciureanu R (2012) Multi-ana-lytic detection of antibiotic residues in honey using a multi-plexing biochip assay Revista Medico-Chirurgicala a Societatiide Medici Si Naturalisti Din Iasi 116(1) 324ndash329

Quintana-Rizzo J Salter R S amp Saul S J (2005) Solid phaseextraction procedures for validation of Charm II sulfonamidestreptomycin and chloramphenicol positives and detection oftylosin in honey Apiacta 40 55ndash62 httpwwwapimondiacomapiactaarticles2005quintana-rizzo_1pdf

Rademacher E Fahlberg A Raddatz M Schneider S ampVoigt K (2013) Galenics Studies of the toxicity and distri-bution of sugar substitutes on Apis mellifera Apidologie44(2) 222ndash233 doi101007s13592-012-0174-5

Re R Pellegrini N Proteggente A Pannala A Yang M ampRice-Evans C (1999) Antioxidant activity applying animproved ABTS radical cation decolorization assay FreeRadical Biology and Medicine 26(9ndash10) 1231ndash1237(98)00315-3 doi101016S0891-5849(98)00315-3

Regulation E (2005) Regulation (EU) No 3962005 on max-imum residue levels of pesticides in or on food and feed ofplant and animal origin and amending Council Directive 91414EEC Official Journal of the European Union L70 1ndash16httpseur-lexeuropaeulegal-contentENTXTPDFuri=CELEX32005R0396ampfrom=en

Reybroeck W (2014) Quality control of honey and beeproducts In Beekeeping for poverty alleviation and livelihoodsecurity (pp 481ndash206) Springe 101007978-94-017-9199-1

Reybroeck W (2018) Residues of antibiotics and chemother-apeutics in honey Journal of Apicultural Research 57(1)97ndash112 doi1010800021883920171338129

Reybroeck W amp Ooghe S (2010) Detection of sulfa drugsin honey by Sulfa-Sensor Honey and Charm II Sulfa TestHoney A comparison [Paper presentation] Proceedings ofthe 4th European Conference of Apidology (p 148) Kence M

Reybroeck W Daeseleire E de Brabander H F amp Herman L(2012) Antimicrobials in beekeeping Veterinary Microbiology158(1ndash2) 1ndash11 doi101016jvetmic201201012

Reybroeck W Ooghe S DE Brabander H amp Daeseleire E(2007) Validation of the tetrasensor honey test kit for thescreening of tetracyclines in honey Journal of Agricultural andFood Chemistry 55(21) 8359ndash8366 doi101021jf071922p

Rial-Otero R Gaspar E M Moura I amp Capelo J L (2007)Chromatographic-based methods for pesticide determin-ation in honey An overview Talanta 71(2) 503ndash514 doi101016jtalanta200605033

Rissato S R Galhiane M S DE Almeida M V GerenuttiM amp Apon B M (2007) Multiresidue determination of


pesticides in honey samples by gas chromatography-massspectrometry and application in environmental contamin-ation Food Chemistry 101(4) 1719ndash1726 doi101016jfoodchem200510034

Roshan A R A Gad H A EL-Ahmady S H KhanbashM S Abou-Shoer M I amp Al-Azizi M M (2013)Authentication of monofloral yemeni sidr honey using ultra-violet spectroscopy and chemometric analysis Journal ofAgricultural and Food Chemistry 61(32) 7722ndash7729 doi101021jf402280y

Roubik DW amp Moreno JE (1991) Pollen and spores of BarroColorado Island Missouri Botanical Garden (pp 268)(Monographs in Systematic Botany n36)

Ruiz-Matute A I Sanz M L amp Martinez-Castro I (2007)Use of gas chromatography-mass spectrometry for identifi-cation of a new disaccharide in honey Journal ofChromatography A 1157(1-2) 480ndash483 doi101016jchroma200705056

Ruoff K Karoui R Dufour E Luginbeurouhl W Bosset J-OBogdanov S amp Amado R (2005) Authentication of thebotanical origin of honey by front-face fluorescence spec-troscopy A preliminary study Journal of Agricultural andFood Chemistry 53(5) 1343ndash1347 doi101021jf048384q

Ruoff K Luginbeurouhl W Keurounzli R Iglesias M T BogdanovS Bosset J O von der Ohe K von der Ohe W ampAmado R (2006) Authentication of the botanical and geo-graphical origin of honey by mid-infrared spectroscopyJournal of Agricultural and Food Chemistry 54(18) 6873ndash6880doi101021jf060838r

Sabatini A G Bortolotti L amp Marcazzan G L (Eds)(2007) Conoscere il miele Avenue Media

Salgado-Laurenti C R Tellerıa M C amp Coronel J M(2017) Botanical and geographical origin of honey from thedry and humid Chaco ecoregions (Argentina) Grana 56(6)450ndash461 doi1010800017313420161276619

Sancho M T Pascual-Mate A Rodrıguez-Morales E GOses S M Escriche I Periche A amp Fernandez-Mui~noM A (2016) Critical assessment of antioxidant-relatedparameters of honey International Journal of Food Science ampTechnology 51(1) 30ndash36 doi101111ijfs12988

SANCO (2004) Summary record of the Standing Committeeon the Food Chain and Animal Health held in Brussels on21 September 2004 (Section Biological Safety of the FoodChain) (Section Controls and Import Conditions) Article16 Statement of the Commission SANCO -E2(04)D521927 httpseceuropaeufoodsitesfoodfilessafetydocsreg-com_cic_summary35_enpdf

Santos K S Malaspina O amp Palma M S (2003 March)Cinetica da diastase em meis de diferentes origens floraisUm novo protocolo experimental Mensagem Doce 70 2ndash4

Schwaiger I amp Schuch R (2000) Bound sulfathiazole residuesin honey ndash need of a hydrolysis step for the analyticaldetermination of total sulfathiazole content in honeyDeutsche Lebensmittel-Rundschauche Lebensmittel-Rundschau96(3) 93ndash98

Scortichini G Annunziata L Haouet M N Benedetti FKrusteva I amp Galarini R (2005) ELISA qualitative screen-ing of chloramphenicol in muscle eggs honey and milkMethod validation according to the Commission Decision2002657EC criteria Analytica Chimica Acta 535(1ndash2)43ndash48 doi101016jaca200412004

Shendy A H Al-Ghobashy M A Mohammed M N GadAlla S A amp Lotfy H M (2016) Simultaneous determin-ation of 200 pesticide residues in honey using gas chroma-tography-tandem mass spectrometry in conjunction withstreamlined quantification approach Journal ofChromatography A 1427 142ndash160 doi101016jchroma201511068

Sheth H B Stiles M E Sporns P Yaylayan V A amp LowN H (1990) Reaction of reducing sugars with sulfathiazoleand importance of this reaction to sulfonamide residue ana-lysis using chromatographic colorimetric microbiologicalor ELISA methods Journal of Agricultural and Food Chemistry38(4) 1125ndash1130 doi101021jf00094a047

Singh S K Dwevedi R Deopa P amp Krishna V (2014) Agradual advancement of the extraction and cleanup techni-ques for residue analysis of pesticides in food matrices andliquid products A review Iosr Journal of EnvironmentalScience Toxicology and Food Technology 8(1) 113ndash118httpspdfssemanticscholarorg2b36bf5f5e0e60d0c972ceb53b71b830ce87779fpdf doi1097902402-0815113118

Sodre G D S Marchini L C Moreti A C D C COtsuk I P amp Carvalho C A L D (2011) Physico-chem-ical characteristics of honey produced by Apis mellifera inthe Picos region state of Piauı Brazil Revista Brasileira deZootecnia 40(8) 1837ndash1843 doi101590S1516-35982011000800030

Souza Tette P A Guidi L R DE Abreu Gloria M B ampFernandes C (2016) Pesticides in honey A review onchromatographic analytical methods Talanta 149 124ndash141doi101016jtalanta201511045

Sporns P Kwan S amp Roth L A (1986) HPLC analysis ofoxytetracycline residues in honey Journal of Food Protection49(5) 383ndash388 doi1043150362-028X-495383

Tahboub Y R Zaater M F amp Barri T A (2006)Simultaneous identification and quantitation of selectedorganochlorine pesticide residues in honey by full-scan gaschromatography-mass spectrometry Analytica Chimica Acta558(1ndash2) 62ndash68 doi101016jaca200511004

Tarbin J A Read W amp Sharman M (2010 Developmentand validation of a rapid method for the determination offumagillin in honey [Paper presentation] Abstract Book ofthe 6th International Symposium on Hormone and VeterinaryDrug Residue Analysis (Vol 217 pp 1ndash4) Ghent

The European Parliament and the Council of European Union(2009) Regulation (EC) No 4702009 of the EuropeanParliament and of the Council of 6 May 2009 laying downCommunity procedures for the establishment of residuelimits of pharmacologically active substances in foodstuffs ofanimal origin repealing Council Regulation (EEC) No 237790 and amending Directive 200182EC of the EuropeanParliament and of the Council and Regulation (EC) No 7262004 of the European Parliament and of the Council layingdown a Community procedure for the establishment ofmaximum residue limits of veterinary medicinal products infoodstuffs of animal origin Official Journal of the EuropeanUnion L152 11ndash22

Thompson T S Pernal S F Noot D K MelathopoulosA P amp VAN DEN Heever J P (2007) Degradation ofincurred tylosin to desmycosin-Implications for residue ana-lysis of honey Analytica Chimica Acta 586(1ndash2) 304ndash311doi101016jaca200609043

Thongchai W Liawruangath B Liawruangrath S ampGreenway G M (2010) A microflow chemiluminescencesystem for determination of chloramphenicol in honey withpreconcentration using a molecularly imprinted polymerTalanta 82(2) 560ndash566 doi101016jtalanta201005007

Thrasyvoulou A Tananaki C Goras G Karazafiris EDimou M Liolios V Kanelis D amp Gounari S (2018)Legislation of honey criteria and standards Journal ofApicultural Research 57(1) 88ndash96 doi1010800021883920171411181

Tomasini D Sampaio M R F Caldas S S Buffon J GDuarte F A amp Primel E G (2012) Simultaneous deter-mination of pesticides and 5-hydroxymethylfurfural in honeyby the modified QuEChERS method and liquid


chromatography coupled to tandem mass spectrometryTalanta 99 380ndash386 doi101016jtalanta201205068

Tomasini D Sampaio M R F Cardoso L V Caldas S Samp Primel E G (2011) Comparison of dispersive liquidndashli-quid microextraction and the modified QuEChERS methodfor the determination of fipronil in honey by high perform-ance liquid chromatography with diode-array detectionAnalytical Methods 3(8) 1893 doi101039c1ay05221g

Tribalat L Paisse O Dessalces G amp Grenier-LoustalotM F (2006) Advantages of LC-MS-MS compared to LC-MSfor the determination of nitrofuran residues in honeyAnalytical and Bioanalytical Chemistry 386(7ndash8) 2161ndash2168doi101007s00216-006-0878-3

Tuberoso C I G Jerkovic I Sarais G Congiu FMarijanovic Z amp Kus P M (2014) Color evaluation ofseventeen European unifloral honey types by means ofspectrophotometrically determined CIEL a -Cab a - Hab achromaticity coordinates Food Chemistry 145 284ndash291doi101016jfoodchem201308032

US Environmental Protection Agency (EPA) (2013 June) Asummary of analytical methods used for the analyses of pesti-cide residues in bees and hive matrices U S EnvironmentalProtection Agency Region 5 Land and Chemicals DivisionChemicals Management Branch pp 1ndash10 httpswwwepagovsitesproductionfiles2013-09documentsbee-analytical-methodspdf

Uran H Aksu F amp Altiner D D (2017) A research on thechemical and microbiological qualities of honeys sold inIstanbul Food Science and Technology 37(suppl 1) 30ndash33(doi1015901678-457x32016

Valera E Muriano A Pividori I Sanchez-Baeza F amp MarcoM P (2013) Development of a Coulombimetric immuno-sensor based on specific antibodies labeled with CdS nano-particles for sulfonamide antibiotic residues analysis and itsapplication to honey samples Biosensors and Bioelectronics43(1) 211ndash217 doi101016jbios201212017

VAN Bruijnsvoort M Ottink S J M Jonker K M amp DEBoer E (2004) Determination of streptomycin and dihy-drostreptomycin in milk and honey by liquid chromatog-raphy with tandem mass spectrometry Journal ofChromatography A 1058(1ndash2) 137ndash142 doi101016jchroma200407101

Verzegnassi L Savoy-Perroud M C amp Stadler R H (2002)Application of liquid chromatography-electrospray ioniza-tion tandem mass spectrometry to the detection of 10 sul-fonamides in honey Journal of Chromatography A 977(1)77ndash87 doi101016S0021-9673(02)01341-9

Vilca F Z Corrreia-Oliveira M E Leal R M P MonteiroS H Zanardi O Z Marchini L C amp Tornisielo V L(2012) Quechers approach for the determination of sevenpesticide residues in Brazilian honey samples using GC-mECD Journal of Food Science and Engineering 2 163ndash169

Vit P Pedro S R amp Roubik D (2018) Pot-pollen in stinglessbee melittology Springer 481p

von der Ohe W Persano Oddo L Piana M L Morlot Mamp Martin P (2004) Harmonized methods of

melissopalynology Apidologie 35(Suppl 1) S18ndashS25 101051apido2004050

Wang S Yong W Liu J Zhang L Chen Q amp Dong Y(2014) Development of an indirect competitive assay-basedaptasensor for highly sensitive detection of tetracyclineresidue in honey Biosensors and Bioelectronics 57 192ndash198doi101016jbios201402032

Weigel S Gatermann R amp Harder W (2005) Screening ofhoney for residues of antibiotics by an optical biosensorApiacta 40 63ndash69 httpwwwapimondiacomapiactaarticles2005weigel_1pdf

WHO (World Health Organization) (1996) Trace elements inhuman nutrition and health World Health Organization pp 1ndash360

WHOFAO (2002) Food energy ndash methods of analysis and conver-sion Fao Food and Nutrition Paper httpsdoiorg0254-4725

Wiest L Bulete A Giroud B Fratta C Amic S LambertO Pouliquen H amp Arnaudguilhem C (2011) Multi-resi-due analysis of 80 environmental contaminants in honeyshoney bees and pollens by one extraction procedure fol-lowed by liquid and gas chromatography coupled with massspectrometric detection Journal of Chromatography A1218(34) 5743ndash5756 doi101016jchroma201106079

Wu Z Chen L Wu L Xue X Zhao J Li Y Ye Z ampLin G (2015) Classification of Chinese honeys accordingto their floral origins using elemental and stable isotopiccompositions Journal of Agricultural and Food Chemistry63(22) 5388ndash5394 doi101021acsjafc5b01576

Yu J Wu C amp Xing J (2004) Development of new solid-phase microextraction fibers by sol-gel technology for thedetermination of organophosphorus pesticide multiresiduesin food Journal of Chromatography A 1036(2) 101ndash111 doi101016jchroma200402081

Yeuroucel Y amp Sultanoglu P (2013) Characterization of honeysfrom Hatay Region by their physicochemical propertiescombined with chemometrics Food Bioscience 1 16ndash25doi101016jfbio201302001

Zacharis C K Rotsias I Zachariadis P G amp Zotos A(2012) Dispersive liquid-liquid microextraction for thedetermination of organochlorine pesticides residues inhoney by gas chromatography-electron capture and iontrap mass spectrometric detection Food Chemistry 134(3)1665ndash1672 doi101016jfoodchem201203073

Zander E amp Maurizio A (1975) Der Honig In A Maurizio(Ed) Handbuch der Bienenkunde (2nd ed pp 212) VerlagEugen Ulmer Stuttgart

Zhou J Qi Y Ritho J Duan L Wu L Diao Q Li Y ampZhao J (2014 February) Analysis of maltooligosaccharidesin honey samples by ultra-performance liquid chromatog-raphy coupled with evaporative light scattering detectionFood Research International 56 260ndash265 doi101016jfoo-dres201401014

Zhou N Wang J Zhang J Li C Tian Y amp Wang J(2013) Selection and identification of streptomycin-specificsingle-stranded DNA aptamers and the application in thedetection of streptomycin in honey Talanta 108 109ndash116doi101016jtalanta201301064


Abstract

Introduction

Honey authenticitymdashbotanical geographical and entomological origin

Botanical origin

Pollen grains

Other structured elements

Methodology to prepare honey sediment following Louveaux et al (1970a)

The acetolysis technique

Additional techniques

Honey sample evaluation

Super and subrepresentation of pollen types

Monofloral and heterofloral honeys

Honeydew

Geographical origin

Entomological origin

Standard honey physicochemical analysis for nutritional composition chemical composition and antioxidant activity

Water content determination (moisture)

Principle

Apparatus and reagents

Procedure

Samples and standards preparation and determination

Sugar content determination (HPLC method)


Procedure

Sample preparation and determination

Chromatographic conditions

Calculation and expression of results

Reducing sugars


Procedure

Solutions preparation

Standardization of Fehling solutions

Preparation of the sample

Titration

Calculation

Apparent sucrose


Procedure

Sodium hydroxide solution (5 molL)

Sample preparation

Proteins (proline)

Principle


Procedure

Preparation of the sample solution

Determination


Vitamins (HPLC)


Procedure

Sample preparation

Standards preparation

Chromatographic analysis

Minerals


Procedure

Sample extraction


Calories

Hydroxymethylfurfural

Principle


Procedure

Samples standards preparation and determination

Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure

Samples and standards preparation

Calculations

Insoluble solids


Procedure

Calculation

Diastase activity


Standard preparation

Experimental procedure

Electrical conductivity

Principle


Procedure


Determination

Calculations

Trolox equivalent antioxidant capacity (TEAC)


Solutions

Preparing the solutions

Generation and dilution of ABTS radical cation (ABTSbull+)


Determination

Calculations

Color of honey by the CIELab system

Apparatus

Procedure

Investigating honey production with diagnostic radioentomology

Experimental conditions and material required to conduct diagnostic radioentomology trials

Hive to scanner size ratio

Material to be scanned

Monitoring the storage of artificial diets

Studying the storage of natural diets

Labeling of the diet to increase contrast

Calibration

Artificial diet

Natural diet

Labeled diet

Computer tomography scanning

Handling and transporting of the combs

Scanning settings

Performing a scan

Data analysis

Analysis of density patterns in individual cell

Measuring density of cell content

Measuring cell filling status

Analyzing content of individual cells over time

Spatial analyses at comb level

Pros and cons

Perspectives

Pesticide residues in honey

Chemical families of pesticides

Herbicides

Fungicides

Insecticides and acaricides

Analytical methods

Sampling

General requirements for pesticide residue analyses

Sample preparation (extraction and clean-up)

Liquid-liquid extraction (LLE)

Solid phase extraction (SPE) and dispersive solid phase extraction (dSPE)

Quick Easy Cheap Effective Rugged and Safe (QuEChERS)

Solid phase microextraction (SPME)

Stir bar sorptive extraction (SBSE)

Dispersive liquid-liquid microextraction (DLLME)

Chromatographic detection

Antibiotics and chemotherapeutics in honey

Legislation regarding residues of veterinary drugs in honey

Determination of residues of antibiotics and chemotherapeutics in honey

Sampling

Sample pretreatment

General remarks for honey testing on antimicrobial residues

Microbiological screening tests

Immunological and receptor assays

Lateral flow devices

Enzyme-linked immunosorbent assays (ELISA)

Enzyme-linked aptamer assays (ELAA)

Radio-labeled receptorantibody techniques (Charm II tests)

Biochip-based methods

Methods using MIPs and other immunotechniques

Physico-chemical methods (chromatographic techniques)

Standard method for honey sensory analysis

Methods

General requirements

Test room

Selection and training of assessors and choice of panel

Product storage

Preparation and presentation of the samples

Sampling glasses

Preparation of the samples

Tasting procedures

Guidelines

Session protocol

Evaluation of the visual characteristics

Evaluation of the olfactory characteristics

Evaluation of the olfactory-gustatory and tactile characteristics

Descriptive semiquantitative analysis

Appearance and visual descriptors

Olfactory descriptors

Flavor descriptors

Texture and other tactile descriptors

Acknowledgments

Disclosure statement

References

REVIEW ARTICLE

Standard methods for Apis mellifera honey research

Ligia Bicudo de Almeida-Muradiana Ortrud Monika Barthb Vincent Dietemannc Michael Eyerc Alex da Silva de Freitasd Anne-Claire Martele Gian Luigi Marcazzanf Carla Marina Marcheseg Carla Mucignat-Carettah Ana Pascual-Mateij Wim Reybroeckk M Teresa Sanchoi andJose Augusto Gasparotto Sattlera

aPharmaceutical Science School University of S~ao Paulo S~ao Paulo Brazil bInstituto Oswaldo Cruz Fiocruz Rio de Janeiro BrazilcAgroscope Swiss Bee Research Centre Bern Switzerland dInstituto de Geociencias Universidade Federal Fluminense Niteroi BrazileSophia Antipolis Laboratory Honeybee Pathology Unit ANSES Sophia France fCREA ndash Council for Agricultural Research and EconomicsResearch Centre for Agriculture and Environment Bologna Italy gRed Bee Honey American Honey Tasting Society Weston FL USAhDepartment of Molecular Medicine University of Padova Padua Italy iDepartamento de Biotecnologıa y Ciencia de los AlimentosNutricion y Bromatologıa Universidad de Burgos Burgos Spain jFacultad de Ciencias de la Salud Universidad Isabel I Spain kResearchInstitute for Agriculture Fisheries and Food Technology and Food Science Unit Melle Belgium

(Received 3 May 2018 accepted 27 February 2020)

Honey is an important food for man and has been used as a natural sweetener since ancient times It is a viscous andaromatic product made by honey bees using the nectar of flowers or honeydew Honey is composed of a complex mix-ture of carbohydrates and other substances such as organic acids amino acids proteins minerals vitamins lipids aromacompounds flavonoids pigments waxes pollen grains several enzymes and other phytochemicals This chapterpresents some properties of Apis mellifera honey as well as the main methods of honey analysis All methods are basedon specialized literature including the Codex Alimentarius AOAC and publications of the International HoneyCommission Herein we describe methods related to honey authenticity botanical origin geographical origin physico-chemical analysis radioentomology pesticide and antibiotic contamination chemotherapeutics and sensory analysis Allmethods are described in a step-by-step model in order to facilitate their use

La miel es un alimento importante para el hombre y se ha utilizado como edulcorante natural desde la antigeurouedad Esun producto viscoso y aromatico hecho por las abejas de la miel usando el nectar de las flores o el rocıo de miel Lamiel esta compuesta por una mezcla compleja de carbohidratos y otras sustancias como acidos organicos aminoacidosproteınas minerales vitaminas lıpidos compuestos aromaticos flavonoides pigmentos ceras granos de polen variasenzimas y otros fitoquımicos En este capıtulo se presentan algunas propiedades de la miel de Apis mellifera ası comolos principales metodos de analisis de la miel Todos los metodos se basan en literatura especializada incluido el CodexAlimentarius AOAC y publicaciones de la Comision Internacional de la Miel A continuacion describimos metodos rela-cionados con la autenticidad de la miel el origen botanico el origen geografico el analisis fisicoquımico la radio-entomologıa la contaminacion por pesticidas y antibioticos la quimioterapia y el analisis sensorial Todos los metodosse describen en un modelo paso a paso para facilitar su uso

蜂蜜是人类重要的食品自古以来就被用作天然甜味剂它是蜜蜂使用花蜜或蜜露酿造而成的粘性芳香产品蜂蜜由碳水化合物和其他物质（例如有机酸氨基酸蛋白质矿物质维生素脂质香气化合物类黄酮色素蜡花粉粒几种酶和其他植物化学物质）组成的复杂混合物本章介绍西方蜜蜂蜂蜜的一些特性以及主要分析方法所有方法均基于专业文献包括食品法典 AOAC和国际蜂蜜委员会的出版物在此我们描述与蜂蜜真实性蜜源植物地理来源理化分析放射昆虫学农药和抗生素污染化学治疗和感官分析有关的方法为了便于使用所有方法都采用分步描述的方式加以介绍

Keywords Apis mellifera honey physicochemical analysis honey authenticity botanical and geographical origindiagnostic radioentomology pesticides sensory antibiotics and chemotherapeutics

Corresponding author Email ligiabiuspbrPlease refer to this paper as de Almeida-Muradian L B Barth O M Dietemann V Eyer M da Silva de Freitas A MartelA-C Marcazzan G L Marchese C M Mucignat-Caretta C Pascual-Mate A Reybroeck W Sancho M Teresa amp Sattler J AG (2020) Standard methods for Apis mellifera honey research In V Dietemann P Neumann N Carreck amp J D Ellis (Eds) TheCOLOSS BEEBOOK ndash Volume III Part 2 Standard methods for Apis mellifera product research Journal of Apicultural Research 59(3) 1ndash62httpsdoiorg1010800021883920201738135

2020 International Bee Research Association

Journal of Apicultural Research 2020Vol 59 No 3 1ndash62 httpsdoiorg1010800021883920201738135

Table of contents

PageNo

1 Introduction 6


6


211 Pollen grains 6



7





8


253 Honeydew 9




9


10

311 Principle 10


313 Procedure 10


10


10


322 Procedure 11






Table of contents

PageNo

332 Procedure 12




3324 Titration 12

3325 Calculation 12



342 Procedure 13




351 Principle 13


353 Procedure 14






362 Procedure 15




37 Minerals 15


372 Procedure 16



Table of contents

PageNo


38 Calories 16


391 Principle 17


393 Procedure 17


17


310 Ashes 18

3101 Principle 18

3102 Apparatus 18

3103 Procedure 18


18


311 Free acidity 18

3111 Principle 18


3113 Procedure 18





3122 Procedure 19

3123 Calculation 19






Table of contents

PageNo

3141 Principle 20


3143 Procedure 20





21


3152 Solutions 21



21





22

3161 Apparatus 22

3162 Procedure 22


23


23






416 Calibration 24






Table of contents

PageNo




43 Data analysis 26


26




27


44 Pros and cons 28

45 Perspectives 28



511 Herbicides 29

512 Fungicides 29



521 Sampling 30


30


32



32


33



Table of contents

PageNo


36



37


38


39

621 Sampling 39



39





43



43



45


45


49

71 Methods 49


721 Test room 49


50



50



Table of contents

PageNo



731 Guidelines 51





51

Table of contents

PageNo


51





Acknowledgments 54

References 54


1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

Table of contents

PageNo

1 Introduction 6


6


211 Pollen grains 6



7





8


253 Honeydew 9




9


10

311 Principle 10


313 Procedure 10


10


10


322 Procedure 11






Table of contents

PageNo

332 Procedure 12




3324 Titration 12

3325 Calculation 12



342 Procedure 13




351 Principle 13


353 Procedure 14






362 Procedure 15




37 Minerals 15


372 Procedure 16



Table of contents

PageNo


38 Calories 16


391 Principle 17


393 Procedure 17


17


310 Ashes 18

3101 Principle 18

3102 Apparatus 18

3103 Procedure 18


18


311 Free acidity 18

3111 Principle 18


3113 Procedure 18





3122 Procedure 19

3123 Calculation 19






Table of contents

PageNo

3141 Principle 20


3143 Procedure 20





21


3152 Solutions 21



21





22

3161 Apparatus 22

3162 Procedure 22


23


23






416 Calibration 24






Table of contents

PageNo




43 Data analysis 26


26




27


44 Pros and cons 28

45 Perspectives 28



511 Herbicides 29

512 Fungicides 29



521 Sampling 30


30


32



32


33



Table of contents

PageNo


36



37


38


39

621 Sampling 39



39





43



43



45


45


49

71 Methods 49


721 Test room 49


50



50



Table of contents

PageNo



731 Guidelines 51





51

Table of contents

PageNo


51





Acknowledgments 54

References 54


1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

Table of contents

PageNo


38 Calories 16


391 Principle 17


393 Procedure 17


17


310 Ashes 18

3101 Principle 18

3102 Apparatus 18

3103 Procedure 18


18


311 Free acidity 18

3111 Principle 18


3113 Procedure 18





3122 Procedure 19

3123 Calculation 19






Table of contents

PageNo

3141 Principle 20


3143 Procedure 20





21


3152 Solutions 21



21





22

3161 Apparatus 22

3162 Procedure 22


23


23






416 Calibration 24






Table of contents

PageNo




43 Data analysis 26


26




27


44 Pros and cons 28

45 Perspectives 28



511 Herbicides 29

512 Fungicides 29



521 Sampling 30


30


32



32


33



Table of contents

PageNo


36



37


38


39

621 Sampling 39



39





43



43



45


45


49

71 Methods 49


721 Test room 49


50



50



Table of contents

PageNo



731 Guidelines 51





51

Table of contents

PageNo


51





Acknowledgments 54

References 54


1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

Table of contents

PageNo




43 Data analysis 26


26




27


44 Pros and cons 28

45 Perspectives 28



511 Herbicides 29

512 Fungicides 29



521 Sampling 30


30


32



32


33



Table of contents

PageNo


36



37


38


39

621 Sampling 39



39





43



43



45


45


49

71 Methods 49


721 Test room 49


50



50



Table of contents

PageNo



731 Guidelines 51





51

Table of contents

PageNo


51





Acknowledgments 54

References 54


1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

Table of contents

PageNo



731 Guidelines 51





51

Table of contents

PageNo


51





Acknowledgments 54

References 54


1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

1 Introduction










21 Botanical origin


211 Pollen grains






























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References



























































253 Honeydew



















311 Principle




313 Procedure















Apparatus



Reagents



322 Procedure










W frac14 A1 V1 m1 100= A2 V2 m0





250 14740





33 Reducing sugars



Apparatus


Reagents



332 Procedure









tilled water





















34 Apparent sucrose



Apparatus


Reagents


342 Procedure

















351 Principle




Apparatus


Reagents









353 Procedure
















E1E2

80




36 Vitamins (HPLC)




Apparatus





Reagents


mm particle size)

362 Procedure









37 Minerals









11 a 75 2511 b 75 2519 b 55 4520 b 60 4022 b 100 0





Apparatus



Reagents




Ultrapure water

372 Procedure










38 Calories














391 Principle



Apparatus


Reagents


393 Procedure





















1497 frac14 126 1000 100016830 10 5

frac14 Constant




310 Ashes


3101 Principle


3102 Apparatus


3103 Procedure










311 Free acidity



3111 Principle



Apparatus



3113 Procedure















3122 Procedure






3123 Calculation








Apparatus



Reagents



















no light
































3141 Principle





3143 Procedure



















SH frac14 K G








Trolox

3152 Solutions































3161 Apparatus


3162 Procedure










p

h frac14 arctg b=a





























416 Calibration



















dietrdquo section



















43 Data analysis



















while counting




















44 Pros and cons



45 Perspectives











511 Herbicides


512 Fungicides












521 Sampling










































Panget al (2006)






Tahboubet al (2006)


















































Blasco et al (2011)










Wiest et al (2011)









Vilca et al (2012)

(Continued)







Table 7 (Continued)
















Shendy et al (2016)































































621 Sampling























mdashb 100c 20



mdashb 100c 50




















































































































































(Continued)








71 Methods



721 Test room


















723 Product storage
















sample preparation






731 Guidelines






each assessor










ile evaluation










































































References



























































































































































































































































Abstract

Introduction


Botanical origin

Pollen grains








Honeydew

Geographical origin




Principle


Procedure




Procedure




Reducing sugars


Procedure




Titration

Calculation

Apparent sucrose


Procedure


Sample preparation

Proteins (proline)

Principle


Procedure


Determination


Vitamins (HPLC)


Procedure

Sample preparation



Minerals


Procedure

Sample extraction


Calories


Principle


Procedure


Calculations

Ashes

Principle

Apparatus

Procedure


Calculations

Free acidity

Principle


Procedure


Calculations

Insoluble solids


Procedure

Calculation

Diastase activity





Principle


Procedure


Determination

Calculations



Solutions




Determination

Calculations


Apparatus

Procedure








Calibration

Artificial diet

Natural diet

Labeled diet



Scanning settings

Performing a scan

Data analysis






Pros and cons

Perspectives



Herbicides

Fungicides


Analytical methods

Sampling













Sampling

Sample pretreatment












Methods


Test room


Product storage


Sampling glasses


Tasting procedures

Guidelines

Session protocol







Flavor descriptors


Acknowledgments


References

Standard methods for Apis mellifera honey research

Documents

Transcript of Standard methods for Apis mellifera honey research