Guidebook Food Product - Shimadzu · 4. 1 Aromatic Components of Alcohols ... 1. 22 Analysis of...

C180-E059B

Analysis of Pesticide Residue in Foods Using GC/MS (2) – GC/MS----------------------723. 7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (1) – GC/MS-----------73

Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (2) – GC/MS-----------743. 8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (1) – GC/MS----------75

Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (2) – GC/MS----------763. 9 Analysis of Pesticides with Specific Threshold Levels in Foods (1) – LC----------------77

Analysis of Pesticides with Specific Threshold Levels in Foods (2) – LC----------------783. 10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (1) – LC------------79

GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (2) – LC------------803. 11 Analysis of Carbamate Pesticides – LC------------------------------------------------------813. 12 Analysis of Imazalil in Oranges - LC----------------------------------------------------------823. 13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (1) – LC/MS--------------83

Analysis of Pesticide Residue in Agricultural Products Using LC/MS (2) – LC/MS--------------843. 14 Analysis of N-methylcarbamate Pesticides Using LC/MS (1) – LC/MS------------------85

Analysis of N-methylcarbamate Pesticides Using LC/MS (2) – LC/MS------------------863. 15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (1) – LC/MS--------87

Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (2) – LC/MS--------883. 16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (1) – LC/MS----------------------89

Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (2) – LC/MS----------------------90

4. 1 Aromatic Components of Alcohols - GC-----------------------------------------------------914. 2 Aromatic Components of Tea - GC-----------------------------------------------------------924. 3 Essential Oil (Headspace Analysis) - GC-----------------------------------------------------934. 4 Essential Oil (Direct Analysis) - GC-----------------------------------------------------------944. 5 Diketones - GC----------------------------------------------------------------------------------954. 6 Fruit Fragrances - GC---------------------------------------------------------------------------964. 7 Vegetable Fragrances - GC--------------------------------------------------------------------974. 8 Flavor of Rice – GC-----------------------------------------------------------------------------984. 9 Flavoring Agent for Food Product - GC------------------------------------------------------994. 10 Analysis of Fishy Smell in Water (1) - GC/MS---------------------------------------------100

Analysis of Fishy Smell in Water (2) - GC/MS---------------------------------------------1014. 11 Analysis of Alcohols (1) - GC/MS-----------------------------------------------------------102

Analysis of Alcohols (2) - GC/MS-----------------------------------------------------------1034. 12 Analysis of Strawberry Fragrances - GCMS------------------------------------------------1044. 13 Analysis of Beverage Odors (1) - GC/MS---------------------------------------------------105

Analysis of Beverage Odors (2) - GC/MS---------------------------------------------------1064. 14 Analysis of Fragrant Material (1) - GC/MS-------------------------------------------------107

Analysis of Fragrant Material (2) - GC/MS-------------------------------------------------108Analysis of Fragrant Material (3) - GC/MS-------------------------------------------------109

5. 1 Analysis of Inorganic Ions in Milk (1) - LC-------------------------------------------------110Analysis of Inorganic Ions in Milk (2) - LC-------------------------------------------------111

5. 2 Analysis of Pb in Milk Using Atomic Absorption Spectrophotometry - AA--------------1125. 3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (1) - AA--------------113

Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (2) - AA--------------1145. 4 Analysis of Cadmium in Rice (1) – AA------------------------------------------------------115

Analysis of Cadmium in Rice (2) – AA------------------------------------------------------1165. 5 Analysis of Inorganic Components in Powdered Milk (1) - ICP-AES-------------------117

Analysis of Inorganic Components in Powdered Milk (2) - ICP-AES-------------------1185. 6 Analysis of Canned Beverage (Green Tea) - ICP-AES ------------------------------------1195. 7 Analysis of Inorganic Components in Processed Food Products - ICP-AES-------------1205. 8 Analysis of Cooking Oil – ICP-AES----------------------------------------------------------1215. 9 Analysis of Pastry – ICP-AES----------------------------------------------------------------1225. 10 Analysis of Nutrition Function Food Products (1) – ICP-AES---------------------------123

Analysis of Nutrition Function Food Products (2) – ICP-AES---------------------------1245. 11 Analysis of Plants – ICP-AES----------------------------------------------------------------1255. 12 Analysis of Powdered Milk Using ICPM-8500 – ICP/MS---------------------------------1265. 13 Analysis of Plants Using ICPM-8500 – ICP/MS-------------------------------------------127

6. 1 Analysis of Organotin Compounds Using Capillary GC-FPD – GC-----------------------1286. 2 Analysis of Organotin in Fish (1) – GC/MS-------------------------------------------------129

Analysis of Organotin in Fish (2) – GC/MS-------------------------------------------------1306. 3 Analysis of Shellfish Toxins (1) - LC--------------------------------------------------------131

Analysis of Shellfish Toxins (2) - LC--------------------------------------------------------1326. 4 Analysis of Oxytetracycline - LC-------------------------------------------------------------1336. 5 Analysis of Closantel - LC--------------------------------------------------------------------1346. 6 Simultaneous Analysis of Synthetic Antibacterial Agent (1) - LC------------------------135

Simultaneous Analysis of Synthetic Antibacterial Agent (2) - LC------------------------1366. 7 Analysis of Enrofloxacin in Broiled Eel (1) - LC--------------------------------------------137

Analysis of Enrofloxacin in Broiled Eel (2) - LC/MS---------------------------------------1386. 8 Analysis of Malachite Green in Farmed Fish – LC-----------------------------------------1396. 9 Analysis of New Type Quinolone Antibacterial Agents in Poultry - LC/MS----------1406. 10 Analysis of Aminoglycoside Antibiotics(1)-LC/MS----------------------------------------141

Analysis of Aminoglycoside Antibiotics(2)-LC/MS----------------------------------------1426. 11 Analysis of Fumonisin in Sweet Corn (1) - LC---------------------------------------------143

Analysis of Fumonisin in Sweet Corn (2) - LC---------------------------------------------1446. 12 Analysis of Aflatoxins (1) – LC--------------------------------------------------------------145

Analysis of Aflatoxins (2) – LC--------------------------------------------------------------146Analysis of Aflatoxins (3) – LC--------------------------------------------------------------147

6. 13 Analysis of Aflatoxins Using LC/MS (1) – LC/MS-----------------------------------------148Analysis of Aflatoxins Using LC/MS (2) – LC/MS-----------------------------------------149

6. 14 Analysis of Patulin Using LC/MS-LC/MS---------------------------------------------------1506. 15 Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (1) – LC/MS------------------151

Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (2) – LC/MS------------------152

1. 1 Analysis of Fatty Acids (1) – GC/MS-----------------------------------------------------------1Analysis of Fatty Acids (2) – GC/MS-----------------------------------------------------------2Analysis of Fatty Acids (3) /Derivatization - Fat Extraction Method------------------------------------------------------3Analysis of Fatty Acids (4) /Derivatization - Preparation of Methyl Fatty Acids----------------------------------------4Analysis of Fatty Acids (5) /Derivatization - Alkali Hydrolysis of Fat------------------------------------------------------5Analysis of Fatty Acids (6) / Derivatization (1)- Preparation of Methyl Ester Derivative----------------------------------------------------6Analysis of Fatty Acids (6) / Derivatization (2)- Methyl Ester Derivative---------------------------------------------------------------------7

1. 2 Fatty Acids (Fish Oil) - GC-----------------------------------------------------------------------81. 3 Triglycerides - GC--------------------------------------------------------------------------------91. 4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (1) - IR---------------10

Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (2) - IR---------------111. 5 Application of the ELSD-LT Low Temperature Evaporative Light Scattering Detector – LC-----------121. 6 Analysis of Decenoic Acid in Royal Jelly - LC-----------------------------------------------131. 7 Analysis of Fatty Acids - LC-------------------------------------------------------------------141. 8 Analysis of Organic Acid in Beer - LC--------------------------------------------------------151. 9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn

Derivatization (1) - LC-----------------------------------------------------------------------16Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (2) - LC-----------------------------------------------------------------------17

1. 10 Analysis of Amino Acids in Fermented Foods – LC-----------------------------------------181. 11 Simultaneous Analysis of D- and L-Amino Acids (1) - LC--------------------------------19

Simultaneous Analysis of D- and L-Amino Acids (2) - LC---------------------------------201. 12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (1) – LC-----------------------------21

Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (2) – LC-----------------------------221. 13 Analysis of Theanine in Green Tea – LC-----------------------------------------------------231. 14 Obligation to Display Nutritive Components in Processed Foods - UV-------------------241. 15 Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products

Using Fluorescence Photometry (1) - RF-------------------------------------------------25Analysis of Trace Amounts of Vitamins B1 and B2 in Food Products Using Fluorescence Photometry (2) - RF-------------------------------------------------26

1. 16 Analysis of Water Soluble Vitamins Using Semi-micro LC System - LC----------------271. 17 Analysis of Vitamin B Group - LC------------------------------------------------------------281. 18 Analysis of Tocopherol in Milk - LC----------------------------------------------------------291. 19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (1) – LC-----------30

Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (2) – LC-----------311. 20 Analysis (Measurement of K Value) of Nucleotide in Tuna Meat - LC--------------------321. 21 Analysis of Oligosaccharide in Beer - LC----------------------------------------------------331. 22 Analysis of Oligosaccharides in Beer Using the ELSD-LT Low Temperature Evaporative Light Scattering Detector – LC----------341. 23 Analysis of Saccharides in Fermented Foods – LC-----------------------------------------351. 24 Analysis of Nonreducing Sugar Using Postcolumn Derivatization with

Fluorescence Detection - LC----------------------------------------------------------------361. 25 Analysis of Sugar in Yogurt - LC--------------------------------------------------------------371. 26 Analysis of Alliin in Garlic - LC----------------------------------------------------------------381. 27 Analysis of Catechins in Green Tea - LC-----------------------------------------------------391. 28 Analysis of Chlorogenic Acid in Coffee - LC-------------------------------------------------401. 29 Analysis of Lycopene and β-Carotene in Tomato - LC-------------------------------------411. 30 Melting of Fats and Oils - TA------------------------------------------------------------------421. 31 Gelatinization of Starch - TA-------------------------------------------------------------------43

2. 1 Propionic Acid in Cookies and Bread - GC---------------------------------------------------442. 2 Saccharine and Sodium Saccharine - GC----------------------------------------------------452. 3 Ethylene Glycols in Wine - GC-----------------------------------------------------------------462. 4 Sorbic Acid, Dehydroacetic Acid and Benzoic Acid - GC-----------------------------------472. 5 Analysis of Preservatives in Food Products with Absorption

Photometry (1) - UV-------------------------------------------------------------------------48Analysis of Preservatives in Food Products with Absorption Photometry (2) - UV-------------------------------------------------------------------------49

2. 6 Color Control of Food Products (1) - UV-----------------------------------------------------50Color Control of Food Products (2) - UV-----------------------------------------------------51

2. 7 Analysis of Sweetener in Soft Drink - LC----------------------------------------------------522. 8 Analysis of Fungicide in Oranges - LC-------------------------------------------------------532. 9 Analysis of Phenol Antioxidant in Foods - LC-----------------------------------------------542. 10 Analysis of L-Ascorbic Acid 2-Glucoside - LC---------------------------------------------552. 11 Analysis of EDTA in Mayonnaise - LC-------------------------------------------------------562. 12 Analysis of Benzoyl Peroxide in Food Product - LC----------------------------------------572. 13 Analysis of p-Hydroxybenzoates in Soy Sauce - LC----------------------------------------582. 14 Analysis of Potassium Bromate in Bread - LC----------------------------------------------592. 15 Simultaneous Analysis of Water-soluble Tar Pigments - LC------------------------------60

3. 1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) – GC------------61Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) – GC------------62

3. 2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) – GC------------63Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) – GC------------64

3. 3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (1) – GC----------65Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (2) – GC-------------66

3. 4 Simultaneous Analysis of Pesticides (1) - GC/MS------------------------------------------67Simultaneous Analysis of Pesticides (2) - GC/MS------------------------------------------68

3. 5 Analysis of Pesticides Using NCI (1) - GC/MS----------------------------------------------69Analysis of Pesticides Using NCI (2) - GC/MS----------------------------------------------70

3. 6 Analysis of Pesticide Residue in Foods Using GC/MS (1) – GC/MS----------------------71

6. Others

IndexC

H O 1. Food Product Components

4. Aromas and Odors

CaMg

Na

5. Inorganic Metals

2. Food Additives

3. Residual Pesticides

1

1.1 Analysis of Fatty Acids (1) - GC/MS

■ ExplanationFatty Acids exist in a great many food products. Andderivatization process is used to measures them. The aims of derivatization process are as follows.1) Weaken the polarity of compounds.2) Lower the boiling point.3) Increase molecular ion peak and ion intensity in high

mass region.In the case of fatty acids, derivatization process is used toachieve item 1). The methyl esterization ortrimethylsilylation can be used but generally methylesterization employing diazomethane is used for thederivatization.

Normally, the molecular ion peak that displays themolecular weight is detected for the Ei mass spectrum'ssaturated fatty acid methyl ester and, as determination ofmolecular weight is easy, a carbon count is possible. However, the molecular ion peak often does not appearwhen the level of unsaturation increases, which meansthat not only molecular weight but also the carbon countand unsaturated level cannot be determined. In such

cases, the Ci mass spectrum is measured. With the Cimass spectrum, the ion denoting the molecular weightappears as an ion (M+1) with added proton in themolecular weight for detection of molecular weight + 1 ion.Measuring the Ei and Ci mass spectra enables qualitativeanalysis of compounds in fatty acid methyl estermeasuring. Also, the columns used in this measuringinclude the slightly polar column DB-1 and polar column DB-WAX. The polarity column produces peaks in thesaturated and unsaturated order while the slightly polarcolumn produces peaks in the reverse order.

■ Analytical Conditions

43

55

74

87

97 111 129143

157 185 199 213 227255

267298(M)

C18:0x 5.0

50 100 150 200 250 300 350 400

Fig. 1.1.1 Ei mass spectrum of C18:0

100 150 200 250 300 350 400

113 131 165 196 219 244 265

C18:0

299

(M+H)+

450

Fig. 1.1.2 Ci mass spectrum of C18:0

Instrument

Column

Column Temp.

Inj. Temp.

I/F Temp.

Carrier Gas

Reagent Gas

: GCMS-QP5000

: DB-WAX

(30m×0.25mmI.D. df=0.25µm)

: 60˚C-10˚C/min-250˚C

: 250˚C

: 250˚C

: He(100kPa)

: Isobutane

C

H O1. Food Product Components

2

271

243

269 26

7

265

263

263 29

929

729

7

295

295

293 29

329

3

291

291

327

325

319

319

317

355

311

345

383

343

2779198

345

369

381

TIC243.00271.00269.00267.00✻ 10.00265.00✻ 10.00263.00✻ 10.00299.00✻ 10.00297.00295.00✻ 10.00293.00✻ 10.00291.00✻ 10.00327.00✻ 10.00325.00✻ 10.00319.00✻ 10.00317.00355.00✻ 10.00331.00✻ 10.00369.00345.00✻ 10.00383.00✻ 10.00343.00✻ 10.00381.00✻ 10.00

1.1 Analysis of Fatty Acids (2) - GC/MS

Fig. 1.1.5 Mass chromatogram of protonized molecules for fatty acid methyl ester

50 100 150 200 250 300 350 400

41

55

67

79

93119

145 159 181 199 252

C20:5x 5.0

Fig. 1.1.3 Ei mass spectrum of C20:5

100

109 135 149 175195 221 235 267

285

C20:5

317

(M+H)+

150 200 250 300 350 400 450

Fig. 1.1.4 Ci mass spectrum of C20:5

3

Food Product ComponentsC

H O

1.1 Analysis of Fatty Acids (3) / Derivatization - Fat Extraction Method

■ Pretreatment for Fatty Acid AnalysisFat must be extracted from the food product andhydrolysis and methylation performed for GC andGC/MS analysis of fatty acids in food products. Here,

This shows an example of fat extracted from a sample.

ReferencesStandard Methods of Analysis for Hygienic Chemists and Notes 1990 Appended supplement (1995) Pharmaceutical Society of Japan Edition, published by Kanehara & Co., Ltd (1995)

several representative pretreatment methods will beintroduced from the numerous methods available.

1. Fat extraction

1. Fat extraction

2. Preparation of methylated fatty acid

3. Alkali hydrolysis of fat 4. Methylation of fatty acid

GC, GC/MS analysis

GC, GC/MS analysis

Alternatively

1. Fat Extraction

Sample 5g

Dehydrate and filter with anhydrous Na2SO4

(suitable amount)

Water Layer

Water Layer

CHCR3Layer

CHCR3Layer

Filtrate

Fat Extract

(Precisely measured)

Add 16mL of H2O

Homogenize

Separate sample into separating funnel with 100mL of CHCR3 : MeOH (2:1)

Shaking extraction for 5 min

Shaking extraction for 100mL of CHCR3 : MeOH (2:1) Two Times

Shaking extraction for 5 min

Rinse with 100mL of 0.5% NaOH

Remove solvent by spraying nitrogen gas at 40˚C or less

Fig. 1.1.6 Fat extraction method

1.1 Analysis of Fatty Acids (4) / Derivatization - Preparation of Methyl Fatty Acids

4

This shows a transmethylation method for extracting fatusing an alkali catalyst that does not require fat extractionof food oils, etc. This easy method just requireshydrolysis and fatty acid extraction so labor is reduced.

ReferenceStandard Methods of Analysis for Hygiene Chemists and Notes 1990 Appended supplement (1995) Pharmaceutical Society of Japan Edition, published by Kanehara & Co., Ltd

2. Preparation of Methylated Fatty Acid

Note, however, that amide-bonded fatty acid and freefatty acid do not methylate.

20mg of Extracted Fat

Water Layer

GC, GC/MS

Water Layer

Hexane Layer

Hexane Layer

Filtrate


Dissolve in 1mL of benzene

Add 2mL of 0.5N sodium methoxide(diluted with anhydrous MeOH) and shake, and leave for 10 min at room temperature

Add 0.5N acetic acid water solution to neutralize

Add 5mL of hexane and perform shaking extraction for 1 min

Re-extract with 5mL of hexane

Add small amounts of anhydrous Na2SO4 + NaHCO3 (2+1), leave for 30 min, and filter

Remove solvent by spraying nitrogen gas at 40˚C or less

Dissolve in 5mL of hexane

Fig. 1.1.7 Preparation of methylated fatty acid

1.1 Analysis of Fatty Acids (5) / Derivatization - Alkali Hydrolysis of Fat

5


H O

3. Alkali Hydrolysis of FatExtracted fat is triacylglycerol which emerges as glyceroland potassium salt's fatty acid (water soluble) usingalkali. Fatty acid hardly separates when acidified, which

ReferenceOrganic Chemistry Testing Guidebook No. 5, Handling Biological materials, Toshio Goto, Tetsuo Shiba, TeruoMatsuura ed, Kagaku-Dojin Publishing Company, INC (1991)

enables extraction with non-polar solvent. Here, anexample of alkali hydrolysis is introduced.

5g of Extracted Fat

Fatty Acid


Water Layer

Add 15mL of 20% (w/w) KOH (in 40% EtOH water solution)

Reflux for approximately 1 hr in 85˚C heated bath

After cooling, separate reaction liquid using separatory funnel, and add 6N-HCr to adjust to pH1.

Perform shaking extraction with diethyl ether (3 extractions: 30mL, 20mL and 20mL)

Add anhydrous Na2SO4, stir, leave for 1 hr, and filter

Combine diethyl ether with separatory funnel, clean 3 times with 20mL of saturated Na2CO3 water solution, and then rinse 3 times with 20mL of water

Diethyl Ether Layer

Remove solvent using rotary evaporator or nitrogen gas spraying

Filtrate

Fig. 1.1.8 Alkali hydrolysis of fat

1.1 Analysis of Fatty Acids (6) / Derivatization (1) - Preparation of Methyl Ester Derivative

6

4. Methyl Ester Derivative Preparation MethodHigh-class fatty acids are generally derived into methyl

(1) Methyl Esterization using BF3-CH3OH

(2) Methyl Esterization Using H2SO4-CH3OH

ester. The currently used methods are introduced here.

20mg of Fatty Acid

GC,GC/MS

(Remove solvent if it is in solution)

Boil approximately 3mL of BF3-CH3OH over a water bath for 2 min

Shake 20mL of n-hexane + 20mL of distilled water

Water Layer n-Hexane Layer

Filtrate

Add anhydrous Na2SO4 (suitable amount), let stand, filter

Remove solvent by spraying nitrogen gas over 50˚C water bath

20mg of Fatty Acid

GC,GC/MS

Boil approximately 20mL of H2SO4-CH3OH over a water bath for 1 hr

Shake 20mL of n-hexane + 20mL of distilled water

Water Layer n-Hexane Layer

Filtrate

• Repeatedly rinse with 10mL batches of distilled water to neutralize

• Add anhydrous Na2SO4

(suitable amount), let stand, filter

Remove solvent

Fig. 1.1.10 Methyl esterization using sulphuric acid-methanol

Fig. 1.1.9 Methyl esterization using boron trifluoride-methanol

1.1 Analysis of Fatty Acids (6) / Derivatization (2) - Methyl Ester Derivative

7


H O

(3) Methyl Esterization Using CH2N2

A diazomethane generator is assembled as shown in thediagram. And ethyl ether (I), 50% potassium hydroxidewater solution (II), 10mg of fatty acid + 2mL of ethylether (III) and acetic acid are sealed in tubes.1. A suitable amount of nitrogen gas is passed through

test tube I.2. Some 0.5 to 1mL of N-methyl-N’-nitroso-p-

toluenesulfonamide with 20% ethyl ether is injectedinto test tube II to create diazomethane.

3. Remove test tube III from diazomethane generatoronce the ethyl ether liquid inside has turned yellow.

4. Leave test tube III to stand for 10 min to enrich theethyl ether, and inject into GC or GCMS.

● Notes and coutions- Handle diazomethane with care, as it is carcinogenic.- For the above reason, only adjust small amounts and be sure to

use a ventilating hood.- Do not use ground glass stoppers because there is a danger of

explosion.- Small amounts of ether solution (100mL or less) can be stored

in a refrigerator for several days.● Several relatively easy-to-handle diazomethane generators are

available in market.

(4) Methyl Esterization Using Dimethylformamide Dialkylacetals (CH3) 2NCH(OR)2

Add 300µL of esterification reagent to some 5 to 50mg offatty acid. Dissolve the sample and inject the resultantreaction liquid into the GC or GCMS. (Normally it is bestto heat this at 60˚C for 10 to 15 min.)

(5) Methyl Esterization Using Phenyltrimethyl Ammonium Hydroxide (PTAH)

Dissolve the fatty acid in acetone, add PTAH/methanolsolution (1 to 1.5M%), thoroughly stir sample andreaction reagent, leave to stand for 30 min, and inductinto GC or GCMS.This methyl esterization using on-column injectionn is amethod where the PTAH/methanol reagent and fatty acidare mixed in advance, injected into the GC and made toreact in a GC injector. Compared to other methodstreatment is quick and simple and there is no volatile lossbecause the reaction is in a GC injector. Furthermore,harmful, dangerous reagents are not required.

Fig. 1.1.11 Methyl esterization using diazomethane

8

1.2 Fatty Acids (Fish Oil) - GC

■ ExplanationAmong high-class fatty acids, unsaturated fatty acids arecurrently in the limelight, for example, much attention isbeing given to the antithrombogenic effect ofeicosapentaenoic acid, etc. From the outset, gaschromatographs have been used to separate and quantifyhigh-class fatty acids.High-class fatty acids have absorptivety and high boilingpoints, which means that derivatization (usually methylesterization) is performed for GC analysis. This exampleintroduces capillary column analysis of fatty acid methylester in fish oil. Fig. 1.2.1 shows constant pressureanalysis at 110kPa and Fig. 1.2.2 shows programmedpressure analysis from 110kPa to 380kPa. Programmedpressure analysis provides quicker analysis withimproved sensitivity because separation hardly changes.

■ PretreatmentMethyl esterization of fatty acids in fish oil is performedin accordance with Fig. 1.1.11 followed by GC analysis.


C16

C18

C161=

C182=

C183=

C184=

C201=

C204=

C205=

C221=

C241=

C226=

C225=

C181=

C14

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

20.0

22.0

24.0

26.0

28.0

30.0

32.0

34.0

36.0

38.0

+

(min)

Pressure 110kPa

Fig. 1.2.1 Analysis of fatty acid methyl ester in fish oil (constant pressure)

(min)

Pressure

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

16.0

18.0

C14

C16

C161=

C181=

C182=

C183=

C183=

C201=

C204=

C205=

C221=

C226=

C18C24

1=

C225=+

110kPa

1min10kPa/min

180kPa 30kPa/min

380kPa(10min)

Fig. 1.2.2 Analysis of fatty acid methyl ester in fish oil (programmed pressure)

Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

: CBP20 (25m×0.22mm I.D. df=0.25µm)

: 210˚C

: 230˚C

: 230˚C(FID)

: He 100kPa

(0.52mL/min at 210˚C)

: Split 1:100

9


H O

C30

C40

C50

C54

min

0 164 8 12 20 24 28 32 36 40 44 48 52 56

Fig. 1.3.1 Analysis of triglyceride in butter

C30

C50

C40

min

0 164 8 12 20 24 28 32 36 40 44

Fig. 1.3.2 Analysis of triglyceride in palm oil

1.3 Triglycerides - GC

■ ExplanationTriglycerides are compounds with a high boiling pointsand strong absorptivity. Separation is poor in analysis ofthese compounds when a short column filled with highlyheat resistant packing is used with the packed-columnGC.In comparison to this kind of column a capillary columnfilled with fused silica offers minimal absorptivity at highseparation and excellent heat resistance. However, evenbetter heat resistance is required for high-boiling-pointcompounds like triglycerides.Stainless steel capillary columns or aluminum coatedones are extremely heat resistant and, as such, are suitablefor analysis of triglycerides. Also, cold on-columninjector suppress discrimination of samples.

■ Analytical ConditionsColumn

Column Temp.

Det. Temp.

Carrier Gas

Injection Method

: CBM65 (25m×0.22mm I.D. df=0.10µm)

: 50˚C(1min)-20˚C/min-240˚C

-6˚C/min-390˚C

: 390˚C(FID)

: He(1.5mL/min)

: Cold on-column

10

1.4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (1) - IR

■ ExplanationFood products are mixtures of various compounds thatrequire liquid chromatography (LC) or gaschromatography (GC) separation procedures forcomponent analysis. However, injections of a largeamount of samples are difficult due to column loadrestrictions in chromatography, so at maximum theamount of component existing in one peak of achromatogram will be only in the µg order. Nevertheless,if a FTIR is used, infrared measuring is possible andcomponents can be quantified.Here, component analysis of food products using apreparative LC-FTIR method will be introduced.

■ PretreatmentRed wine that has been filtered through a membrane filterwas injected into an LC. Fig. 1.4.1 shows achromatogram detected by the UV detector. Theseparated substances in peaks A to C have been collected,but because there are numerous coexisting substances inthe collected substances, the collected substance is re-injected into the LC using a mobile phase of water, andthe chromatogram measured. The separated substances inthe largest peak obtained from this operation is collected,the mobile phase vaporized from within the collectedsubstance, this collected substance is mixed with KBrpowder and measured using a diffuse reflection method.Fig. 1.4.2 shows the infrared spectrum of peak A.Absorption of coexisting substances is overlaid buttartaric acid can be clearly confirmed.

Fig. 1.4.3 shows the infrared spectrum of peak B. Thecarboxylic acid peak can be confirmed in the region of1730cm-1 and, as glucose (a coexisting substance) is equalto the holding time, glucose absorption has mostlybecome infrared spectrum.Fig. 1.4.4 shows the infrared spectrum of peak C. In thiscase there is no interference from other components andthe spectrum is only for succinic acid.

■ Analytical ConditionsInstrument

Column

Mobile Phase

Flow Rate

Column Temp.

Detector

Instrument

Resolution

Accumulation

Appodization

Detector

: LC-VP Series

: Shim-pack SCR-102H

(300mmL.×8.0mm I.D.)

: 5mM Trifluoroacetic Acid Aqueous Solution

: 1mL/min

: 50˚C

: UV-VIS Detector 210nm

: FTIR

: 4cm-1

: 50

: Happ-Genzel

: Pyroelectric Detector

11


H O

1.4 Analysis of Fatty Acids in Red Wine Using Infrared Spectrophotometry (2) - IR

A

C

B

5

-

10

-

15

-

20

-

Fig. 1.4.1 LC chromatogram of red wine

1 0 0 . 0 0

8 8 . 0 0

7 6 . 0 0

6 4 . 0 0

5 2 . 0 0

4 0 . 0 0

1 0 0 . 0 0

8 8 . 0 0

7 6 . 0 0

6 4 . 0 0

5 2 . 0 0

4 0 . 0 0

%

Fig. 1.4.2 Infrared spectrum of peak A (T: tartaric acid peak)

1 0 0 . 0 0

8 6 . 0 0

7 2 . 0 0

5 8 . 0 0

4 4 . 0 0

3 0 . 0 0

1 0 0 . 0 0

8 6 . 0 0

7 2 . 0 0

5 8 . 0 0

4 4 . 0 0

3 0 . 0 0

G

G

G

G

G

G

%

Fig. 1.4.3 Infrared spectrum of peak B (G: glucose)

1 0 0 . 0 0

8 6 . 0 0

7 2 . 0 0

5 8 . 0 0

4 4 . 0 0

3 0 . 0 0

1 0 0 . 0 0

8 6 . 0 0

7 2 . 0 0

5 8 . 0 0

4 4 . 0 0

3 0 . 0 0

%

Fig. 1.4.4 Infrared spectrum of peak C (succinic acid)

12

min0 20 40 60

mV

0

20

40

Fig. 1.5.2 Chromatogram of safflower oil

1.5 Application of the ELSD-LT Low Temperature Evaporative Light Scattering Detector - LC- Analysis of Triglyceride in Cooking Oil -

■ ExplanationThe ELSD-LT Low Temperature Evaporative LightScattering Detector is an HPLC general-purpose detectorthat converts the target components into minute particlesby evaporating the mobile phase and makes light-scattering measurements of the particles.The ELSD-LT can, in principle, detect nearly allnonvolatile compounds and so it is ideal for the analysisof compounds such as sugars, oils, and surfactants, whichhave a low absorbency and are difficult to detect with UVdetectors. It can also be used for gradient elution, whichis not possible using a differential refractive indexdetector (RID), allowing application in a wide variety offields.Application of the ELSD-LT to the analysis oftriglyceride in cooking oil is described here as anexample.


Column

Mobile Phase

Flow Rate

Temperature

Detection

: Shim-pack VP-ODS (250mmL.×4.6mm I.D.)

: A : Acetonitrile

B : Acetone

Linear Gradient B 50% → 70% (10 to 40min)

: 1.0mL/min

: 30˚C

: ELSD-LT

Temperature : 35˚C

Gain : 5

Nebulizer Gas : Air

Gas Pressure : 350kPa

mV

min0 20 40 60

0

5

10

15

Fig. 1.5.3 Chromatogram of sesame oil

Mobile phase

Nebulization

Photomultiplier

Detection

Light

Gas

Evaporation

Fig. 1.5.1 Operating principle of ELSD-LT

■ Analysis of Triglyceride in Cooking OilCooking oil contains many types of triglyceride, whichdiffer according to the acyl group. Quality control ofcooking oils is also carried out by performing patternanalyses of these triglycerides.Fig. 1.5.2 and 1.5.3 show the results obtained bypreparing 2.0g/L solutions of commercial safflower oil

and sesame oil (solvent: acetonitrile/acetone = 1/1, v/v)and injecting 20µL of these solutions.In general, elution is faster for triglycerides with smaller acylcarbon numbers or with a larger numbers of doublebonds.

1.6 Analysis of Decenoic Acid in Royal Jelly - LC

13


H O

0 1 0 m i n

Inte

rna

l s

tan

da

rd s

ub

sta

nc

e

10

-HD

A

Fig. 1.6.1 Analysis of decenoic acid in raw royal jelly

■ ExplanationRoyal jelly is widely known as a food product and herbalmedicine, and its peculiar component is 10-hydroxy-δ-decenoic acid (10-HAD). The amount of this and theinvestigation method are vital points in compositionstandards for royal jelly. The following is an analysisexample.

ReferenceStudy group text related to royal jelly compositionstandard testing method provided by Japan Royal JellyFair Trade Council

■ PretreatmentDistilled water is added to a specific amount of sample,dissolved through mixing, a specific amount of internalstandard (benzoic acid) was added and the mixturefiltered through a disposable 0.45µm filter.


Mobile Phase

Flow Rate

Temperature

Detection

: STR ODS-2(150mmL.×4.6mm Ι.D)

: 10mM Sodium Phosphate Buffer

(pH2.6)/Methanol=55/45 (v/v)

: 1.0mL/min

: 40˚C


1.7 Analysis of Fatty Acids - LC

14

■ ExplanationFatty acid can be detected using carboxyl groupabsorbent (210nm) in the same way as organic acid, etc.However, this kind of short wavelength is susceptible toimpurities and some samples are difficult to analyze.Here, a prelabel agent is derived into a fluorescentsubstance and detected using a fluorescent detector. Thecompound labeling agent ADAM (9-Anthryl-diazomethane) possessing the carboxyl group is aprelabel agent that targets the methylating agent(diazomethane) reaction.Here, direct analysis using UV absorption detection andprelabel derivatization detection using ADAM agent willbe introduced.

ReferenceShimadzu HPLC Food Analysis Applications Data Book(C190-E047)


Mobile Phase

Flow Rate

Temperature

Detection

: Shim-pack CLC-ODS (150mmL.×6.0mm I.D.)

: Acetonitrile/Water = 95/5 (v/v)

: 1.0mL/min

: 45˚C

: Fluorescence Detector

Ex : 365nm Em : 415nm

0

1

2

3

4

65

5 1 0 1 5 ( m i n )

■ Peaks

1. Lauryl acid

2. Myristic acid

3. Linoleic acid

4. Palmitic acid

5. Oleic acid

6. Stearic acid

Fig. 1.7.1 Analysis of fatty acid using UV absorption detection

0

1

2

3

4

1 0 2 0 3 0 4 0 5 0 6 0 ( m i n )

■ Peaks

1. Lauryl acid

2. Myristic acid

3. Palmitic acid

4. Stearic acid

Fig. 1.7.2 Analysis of high-class fatty acid using precolumnderivatization method with ADAM

CHN2

HOCOR-N2

CHN2OCOR

ADAM (9-Anthryldiazomethane) (Ex365nm Em412nm)

+

Fig. 1.7.3 Reaction equation for ADAM and fatty acid

15


H O

1.8 Analysis of Organic Acid in Beer - LC

0

1

2

3

4

5

67

8

9

5 1 0 1 5 2 0 ( m i n )

■ Peaks

1. Phosphoric acid 2. Citric acid 3. Pyruvic acid 4. Malic acid 5. Succinic acid 6. Lactic acid 7. Formic acid 8. Acetic acid 9. Levulinic acid

Fig. 1.8.1 Analysis of beer

■ ExplanationIn the case of analysis of organic acid usingabsorptiometry, carboxyl group absorption at 200 to210nm is used, but some samples are difficult to analyzebecause of poor selectivity and impurity interference atthis wavelength.In such cases, a conductivity detector that detects ionizedsubstances at selectively high sensitivity is used.

ReferencesHayashi, Shimadzu Review 49 (1), 59 (1992)Shimadzu LC Application Report No. 18Shimadzu HPLC Food Analysis Applications Data Book(C190-E047)

■ PretreatmentBeer is injected in without any pretreatment.


Mobile Phase

Flow Rate

Temperature

Reaction Reagent

Reaction Reagent Flow Rate

Cell Temperature

Detection

: Shim-pack SCR-102H×2

(300mmL.×8.0mm I.D.)

: 5mM p-Toluenesulfonic Acid

: 0.8mL/min

: 45˚C

: 5mM p-Toluenesulfonic Acid

20mM Bis-Tris

100µM EDTA

: 0.8mL/min

: 48˚C

: Conductivity Detector

Mobile phase

Pump

Injector

Analysis column Mixer

Column oven

Reaction agent

Conductivity detector

Fig. 1.8.2 Flowchart of organic acid analysis system

16

1.9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (1) - LC

■ ExplanationA separation method (precolumn derivatization method)exists using reversed phase chromatography with aderivatization reaction performed on the samplepretreatment stage. Here, the analysis example shows anOPA (o-phthalaldehyde) precolumn derivatizationmethod.

■ PretreatmentSee next page for details.

Column

Precolumn

Mobile Phase

Flow RateTemperatureDetection

: Shim-pack CLC-ODS(150mmL.×6.0mm I.D.)with Guard Column

: Shim-pack GRD-ODS(250mmL.×4.0mm I.D.)

: (A)10mM Sodium Phosphate Buffer (pH 6.8)(B)A/Acetonitrile = 2/1(C)80% Acetonitrile Water SolutionGradient Method

: 1.0mL/min: 45˚C: Fluorescence Detector

Ex : 350nm Em : 460nm (Primary Amino Acid)Ex : 485nm Em : 530nm (Secondary Amino Acid)

( m i n )

AS

PG

LU

PR

O

AS

NS

ER

PE

AG

LN

GL

YT

HR

AL

A

TY

R

LE

U

VA

Ln

––V

AL

(I.

S.)

ILE

OR

N LY

S

PH

E

ME

TH

IS

TA

U

α––A

BA

AR

G

Fig. 1.9.1 Analysis of cooking vinegar using prelabel amino acid analysis method


Ex485nm

Em530nm

Ex350nm

Em460nm

17


H O

1.9 Analysis of Amino Acid in Cooking Vinegar Using Precolumn Derivatization (2) - LC

PretreatmentSample 10~20µL

-Mercapt reagent

OPA reagent

NBD-Cl reagent

Mixing

Mixing and Wait ing

Inject ion

1)

2)

3)

-Mercapt propionic acid

100mM Borate buffer (pH 9.0)

100mM Borate buffer (pH 9.0)

NBD-Cl

OPA

Acetoni t r i le

Acetoni t r i le

to HPLC

200µL

200µL2)

1)

200µL

10µL

10µL

10mL

20mg

3mL

10mL

100mg

10mL

3)

Fig. 1.9.2 Pretreatment conditions

4 . 0

0 . 0 1

3 9 . 0 1

4 8 . 0 1

5 4 . 0 15 4 . 0 2

8 . 0

1 6 . 0

1 8 . 0

2 5 . 0

3 0 . 0

3 9 . 0

4 2 . 0

4 8 . 0

4 9 . 0

5 3 . 0

5 4 . 0

B . C O N C

B . C O N C

B . C O N C

B . C O N C

B . C O N CS T O P

B . C O N C

B . C O N C

B . C O N C

B . C O N C

B . C O N C

B . C O N C

B . C O N C

B . C O N C

S V

S V

B . C O N C

1 5

5

7 0

1 0 0

0

2 0

2 7

3 0

4 5

5 0

6 5

7 5

8 0

1

0

1 0 0

Fig. 1.9.3 Gradient conditions

18

1.10 Analysis of Amino Acids in Fermented Foods - LC

Abbreviation Amino acid Abbreviation Amino acid

P-SER o-Phosphoserine MET

TAU Taurine ILU

P-ET-AMINE o-PhosphoethanolamineCYSTATHIO

NINELEU

TYR

PHE

β-ALA

β-A-I-B-A

γ-A-B-A

ASP

OH-PRO

THR

SER

ASN

GLU

GLN

SAR

α-A-A-A

PRO

GLY

ALA

CTRULINE

α-A-B-A

VAL

CYS

L-Aspartic Acid

Hydroxy-L-proline

L-Threonine

L-Serine

L-Asparagine

Glutamic Acid

L-Glutamine

Sarcosine

α-Aminoadipic Acid

L-Proline

Glycine

L-Alanine

L-Citrulline

DL-α-Amino-n-butyric Acid

L-Valine

L-Cystine

TRP

HIS

3-ME-HIS

1-ME-HIS

CARNOSINE

ANSERINE

OH-LYS

ORNITHINE

LYS

ARG

L-Methionine

L-Isoleucine

L-Cystathionine

L-Leucine

L-Tyrosine

L-Phenylalanine

β-Alanine

DL-β-Aminoisobutyric Acid

γ-Aminobutyric Acid

L-Tryptophan

L-Histidine

L-3-Methylhistidine

L-1-Methylhistidine

L-Carnosine

L-Anserine

Hydroxylysine

L-Ornithine

L-Lysine

L-Arginine

Table 1.10.1

■ ExplanationThe detection method is an important factor to improvesensitivity and selectivity in the analysis of amino acidsusing HPLC. For this reason, a wide variety of pre- andpost-column derivatization methods have beendeveloped. Of these methods, post-column fluorescentderivatization using o-phthalaldehyde as the reagentoffers significant advantages in terms of detectionsensitivity, selectivity, and operational ease and is used ina number of fields, including foods.

Shimadzu's Amino Acid Analysis System incorporatesthe Na type method, which enables protein hydrolysis andamino-acid analysis, and the Li type method, whichenables the analysis of free amino acids. The analysis ofamino acids in fermented foods using the lithium methodis described here as an example.


Mobile Phase

Flow Rate

Temperature

Reaction Reagent

Flow Rate of Reaction Reagent

ReactionTemperature

Detection

: Shim-pack Amino-Li (100mmL. × 6.0mm I.D.)

: AminoAcid Mobile-Phase Kit (Lithium type)

Gradient Elution method

: 0.6mL/min

: 39˚C

: AminoAcid Reaction Reagent Kit

: 0.3mL/min

: 39˚C

: RF-10AXL

Ex : 350nm Em : 450nm

min0 20 40 60 80 100 120 140

P-S

ER

(TA

U)

(P-E

T-A

MIN

E)

AS

P

(OH

-PR

O) TH

RS

ER

(AS

N)

GLU

(GLN

)

(SA

R)

(α-A

-A-A

)P

RO

GLY

ALA

(CT

RU

LIN

E)

(α-A

-B-A

)V

AL

(CY

S)

ME

TIL

E(C

YS

TA

TH

ION

INE

) LEU

TY

RP

HE

β-A

LA(β

-A-I

-B-A

)

γ-A

-B-A

TR

P

HIS

(3-M

E-H

IS)

(1-M

E-H

IS)

(CA

RN

OS

INE

)(A

NS

ER

INE

)

OH

-LY

S(O

RN

ITH

INE

)

LYS

NH

4,E

-AM

INE

AR

G

Fig. 1.10.1 Analysis of soy sauce

min

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

(P-S

ER

) TA

U(P

-ET

-AM

INE

)

AS

P

OH

-PR

OH

TH

RS

ER

AS

NG

LUG

LN

(SA

R)

(α-A

-A-A

)P

RO

GLY A

LA(C

TR

ULI

NE

)(α

-A-B

-A)

VA

L

(CY

S)

ME

T

ILU

(CY

ST

AT

HIO

NIN

E)

LEU

TY

RP

HE

(β-A

LA)

( β-A

-I-B

-A)

γ-A

-B-A

TR

P

(HIS

)3

-ME

-HIS

(1-M

E-H

IS)

(CA

RN

OS

INE

)(A

NS

ER

INE

)

OH

-LY

S(O

RN

ITIH

NE

)

LYS

NH

4,E

-AM

INE AR

G

Fig. 1.10.2 Analysis of sweet sake

■ Analysis of Soy Sauce and Sweet SakeThe soy sauce was diluted by a factor of 200 in citric-acid(lithium) buffer solution (for sample dilution) and thesweet sake was diluted by a factor of 10. After filtrationwith a membrane filter, 10µL of each solution wasinjected.

19


H O

1.11 Simultaneous Analysis of D- and L-Amino Acids (1) - LC

■ ExplanationMeasurement of optical purity in the food product field isvital. In the case of amino acid, optical separation ofconfigured amino acid is necessary because, in particular,optical purity greatly affects synthetic peptide and itsphysiological activity in derivatives.Optical isomer separation methods in LC are broadlydivided among the Chiral column solid phase method,Chiral mobile phase method and the Chiral derivatizationmethod. This explanation introduces the Chiralderivatization method.OPA/N-acetylcysteine agent was used as thederivatization agent.

ReferencesN. Nimura and T. Kinoshita, J. Chromatogr., 352, 169(1986)Murakita, et al Clinical Chemistry, Supplement No. 2,pp71b, 21 (1992)Murakita, et al Summary of Symposium on SeparationScience and Related Techniques, pp101 (1993)Shimadzu Application News No. L235(C190-E063)


Precolumn

Mobile Phase

Flow Rate

Temperature

Detection

: Develosil ODS-UG-5

(200mmL.×6.0mm I.D.)

with Guard Column

: Shim-pack GRD-ODS

(250mmL.×4.0mm I.D.)

: (A) 50mM Sodium Acetate

(B) Methanol

(A)→(B)Gradient Method

: 1.2mL/min

: 35˚C


Ex : 350nm Em : 450nm

0

1

9

8

7

6

5

10

11

12

1314

15 18 20

21 23

24

22

191715

4

2

3

20 60 min40

1 . D-ASP■ Peaks

each component c.a. 200pmol

2 . L-ASP 3 . L-GLU 4 . D-GLU 5 . D-SER 6 . L-SER

7 . D-THR 8 . L-THR 9 . L-ARG10. D-ARG11. D-ALA12. L-ALA

13. L-TYR14. D-TYR15. L-VAL16. D-MET17. L-MET18. D-VAL

19. D-PHE20. L-PHE21. L-ILE22. D-ILE23. D-LEU24. L-LEU

Fig. 1.11.1 Analysis of D-, L-amino acid standard solution

20

1.11 Simultaneous Analysis of D- and L-Amino Acids (2) - LC

CHO

OPA

COOH

C

S CH2

H

COOH

CN

R

H

CH3COHN

COOH

C

S CH2

H

H

CN

R

COOH

CH3COHN

CHOHS-CH2CHCOOH

NHCOCH3

R-CH-COOH

N-acetyl-L-cysteine

D,L-amino acid

NH2

Fig. 1.11.2 Chiral derivatization reaction

standard solution 300µL

buffer solution 200µL

reagent A 100µL

*1

*2

*1

reagent B 100µL*3

mix

mix

mix and wait 3min

inject 20µL

0.1N sodium tetraborate

*2 2% N-acetyl - L-cysteine(0.1N sodium tetraborate solution)

*3 1.6% o-Phthalaldehyde(methanol solution)

Fig. 1.11.4 Derivatization conditions

TIME

1624295059

59.0164

64.0165

FUNCTION

BCONCBCONCBCONCBCONCBCONCBCONCBCONCBCONCSTOP

VALUE

242440406780800

Fig. 1.11.3 Gradient conditions

21


H O

1.12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (1) - LC

Injection into HPLC

5g of cooked rice

←25mL of 80% ethanol solution

Mixing, homogenizing, Mixing, homogenizing, and centrifugationMixing, homogenizing, and centrifugation

Supernatant ResidueSupernatant Residue

Supernatant ResidueSupernatant Residue

←20mL of 80% ethanol solution

Mixing, homogenizing, Mixing, homogenizing, and centrifugationMixing, homogenizing, and centrifugation

Volume increased to 50mL with 80% ethanol solution→1mL sample collected

Evaporation to drynessEvaporation to dryness

1mL of mobile phase→

■ Explanation4-amino-n butyric acid (gamma-aminobutric acid,GABA) is a type of amino acid that is common inanimals and plants. It stimulates blood circulation inanimals and supports the metabolic function. A largeamount of this substance is present in rice, particularlyunpolished rice, and so it is an important considerationwhen considering the role of unpolished rice as a healthfood.

GABA can be analyzed simply with HPLC by combiningseparation using reverse-phase ion-pair mode anddetection using post-column derivatization. The batchanalysis of other amino acids as well as GABA is alsopossible using Shimadzu's Amino Acid Analysis System.

Simple analysis of GABA using the reverse-phase ion-pair mode and batch analysis of 18 amino acids using thecation exchange mode are described here as examples.


Mobile Phase

Flow Rate

Temperature

Reaction Reagent


Reaction Temperature

Reaction Coil

Detection

: Shim-pack VP-ODS (150mmL. × 4.6mm I.D.)

: 20mM Sodium Phosphate Buffer Solution containing

10mM Sodium 1-Hexane Sulfonate (pH 2.5)

: 0.8mL/min

: 45˚C

: Amino Acid Reaction Reagent Kit, Solution B

: 0.4mL/min

: 45˚C

: SUS, 2m × 0.8mm I.D.


Ex : 350nm Em : 450nm

■ Analysis by reverse-phase ion-pairChromatography with post-column derivatization

Fig.1.12.1 shows a chromatogram obtained by thismethod after GABA is extracted by 80% ethanol solutionfrom polished and unpolished rice then replaced bysolvent.the analyzed unpolished and polished rice (cooked withrice-cooker) contained GABA 2.7mg and 0.3.mgrespectively in 100g.

■ Pretreatment

■ Peak 1.GABA

15

10

mV

5

0

0 5 10min

15 20

1

Polished rice

Unpolished rice

Fig. 1.12.1 Chromatograms of polished and unpolished rice

22

1.12 Analysis of 4-Amino-n-Butyric Acid (GABA) in Rice (2) - LC

■ Peaks1. Asp 7. Ala2. Thr 8. Val3.Ser 9. Phe4. Glu 10. GABA5. Pro 11. Arg6. Gly

60

mV

0 20 40min

Polished rice

Unpolished rice

1

40

0

202 3

4

56

7

8 9

10

11

Fig. 1.12.2 Chromatograms of polished and unpolished rice

■ ExplanationBatch Analysis of GABA and Other Amino Acids UsingShimadzu's Amino Acid Analysis System

As an example, Fig. 1.12.2 shows the results obtainedwhen GABA and other amino acids in polished andunpolished rice were measured with Shimadzu's AminoAcid Analysis System using the sodium method.

Using this system, it was possible to separate 18 aminoacids obtained by protein hydrolysis and GABA. Thequantitative results are shown in Table 1.12.1.

The overlapping of peaks was observed, however, withsome of the amino acids analyzed in this batch, such asthreonine. To perform the batch separation andquantitative analysis of these acids, use the lithiummethod, which enables the separation of even moreamino acids.


Column

Ammonia Trap Column

Mobile Phase

Flow Rate

Temperature

Reaction Reagent



Detection

: LC-VP Amino Acid Analysis System

: Shim-pack Amino-Na (150mmL. × 6.0mm I.D.)

: Shim-pack ISC-30/S0504 (50mmL. × 4.0mm I.D.)

: Amino Acid Mobile Phase Kit (Na type)

Gradient Elution Method

: 0.4mL/min

: 60˚C

: Amino Acid Reaction Reagent Kit (Solutions A and B)

: 0.3mL/min

: 60˚C


Ex : 350nm Em : 450nm

Aspartic acidThreonine SerineGlutamic acidProlineGlycineAlanineValinePhenylalanine4-amino-n-butyric acidArginine

Polished rice1.71.20.61.9n.d.0.20.80.1n.d.0.4n.d.

Unpolished rice3.41.71.34.50.80.62.90.40.32.81.0

n.d. : not detected

Table 1.12.1 Results of Quantitative Analysis (mg/100g)

23


H O

1.13 Analysis of Theanine in Green Tea - LC

■ ExplanationTheanine is the predominant amino acid in green tea, andis known as the component responsible for the good,sweet taste of green tea. Regarding the efficacy oftheanine, various types of research have been conducted,and these researches have reported its efficacy to soothethe excited nerves induced by caffeine and othersubstances, and to lower the blood pressure. Theanineanalysis can be conducted simultaneously with otheramino acids by the post-column derivatization methodwith fluorescence detection using OPA (o-phthalaldehyde) as the reaction reagent. Here weintroduce the simultaneous analysis of theanine togetherwith other amino acids in a commercially sold green teabeverage using the Shimadzu amino acid analysis system.

Column

Mobile Phase

Mobile Phase Flow Rate

Column Temperature

Reaction Reagent

Reaction Reagent

Flow Rate


Detection

Injection Volume

: Shim-pack Amino-Li

(100 mmL. × 6.0 mm I.D.)

: Amino Acid Mobile Phase Kit

(Li type)

Gradient Elution method

: 0.6 mL/min

: 39˚C

: Amino Acid Reagent Kit

: 0.3 mL/min

: 39˚C

: RF-10AXL

Ex : 350 nm Em : 450 nm

: 20 µL


Fig. 1.13.2 Chromatogram of green tea

0

1

2

3

4

5

6 ■ Peaks1.ASP2.THR3.SER4.GLU5.GLN

6. THEANINE7. ALA8. VAL9. ILU

10. LEU

11.TYR12.PHE13.β-ALA14.γ-A-B-A15.TRP16.ARG

7

8 9 10111214

13 15

16

10 20 30 40 50 60 70 80 90 100 110 120 130 140

0

1

2

3

4

mV

■ Pretreatment

Sample 5 mL

Filter using disposable filter (0.45 µm)

2 mL

Make the volume 10 mL with mobile phase A

Inject 20 µL

CH

COOH

NH2

CH2CH2 CH2 CH3

O

C NH

Fig. 1.13.1 L-theanine structural formula

24

1.14 Obligation to Display Nutritive Components in Processed Foods - UV

■ ExplanationJapanese government national health policy since 1986dictates that processed food must display nutritivecomponents. Within the regulations governing thispolicy, energy, proteins, lipid, saccharine and table saltcan be displayed.The above policy also includes directives for nutritivecomponent analysis method standards and analyzers.Here, analysis of vitamin C using a spectrophotometer forultraviolet and visible region will be introduced.

■ PretreatmentReducing vitamin C is converted into oxidized vitamin C,and red osazone created through reaction of 2,4-Dinitrophenylhydrazine. This osazone is dissolved in85% sulfuric acid and measured using aspectrophotometer.Here, vitamin C in a nutritious candy was dissolved usingmetaphosphoric acid solution and measured.


Sample

Solvent

Cell

Range

Slit

: UV Spectrophotometer

: Candy

: Metaphosphoric Acid Solution

: 10mm

: 0∼ 0.5ABS

: 2nm

Vitamine C

1.5mg/dL

1.0mg/dL

0.5mg/dL

0.2500.5

Abs.

0 450.0

500.0

550.0

650.00.250

0.50

Fig. 1.14.1 Absorption spectrum of vitamin C

ABS.

CONC.

0.270

0

0

K5.7000

B-0.0009

R**20.9997

1.500

Fig. 1.14.2 Calibration curve for vitamin C

STD. NO.01 0 002 0.5000 0.087003 1.0000 0.178004

K

NO.0102

ABS.0.19400.1640

CONC.C=C=

1.10480.9338

Balance FoodCandy

B R✽✽ 2

5.7000 -0.0009 0.9997

1.5000 0.2620

CONC. ABS.

CONC.=K✽ ABS.+B

Fig. 1.14.3 Quantitative results for vitamin C

25


H O

1.15 Analysis of Trace Amounts of Vitamins B1 and B2 in FoodProducts Using Fluorescence Photometry (1) - RF

■ ExplanationVitamins are one valuable form of nutrition. They help tocondition physiological function in minute amounts andhave been much used in physiology and pharmacologyfrom ancient times.Vitamin analysis differs for the characteristics (watersoluble, fat soluble) and types. And the JapanesePharmacopoeia and Standard Methods of Analysis forHygienic Chemists state that on the whole analysisshould be conducted using chromatography,absorptiometry and fluorescence photometry. The latterbeing often used where the vitamin is chemicallyprocessed to increase its unique fluorescence formeasuring. Here, a measuring example using afluorescence photometry will be introduced.

■ PretreatmentVitamin B2 - or riboflavin as it is commonly known - iscopiously contained in milk, eggs and grains andpromotes growth in animals. A riboflavin deficiencyleads to various inflammations such as oral ulcers andvision impairment.Water-solution riboflavin is lime green and shows a greenfluorescence. And when it is in an alkali solution, and anultraviolet is irradiated onto that solution, it becomes alumiflavin with strong fluorescent properties uniquelyinactive.

Instrument

Sample

Solvent

Excitation

Slit

: RF Spectrofluorophotometer

: Vitamin B2 in Soya bean

: Chloroform

: 469nm

: Ex : 10nm Em : 10nm

Fig. 1.15.1 shows the creation process for lumiflavin.And measurement of lumiflavin provides a good way forquantifying vitamin B2, which also has been adopted forthe Standard Methods of Analysis for Hygienic Chemists.Here, vitamin B2 copiously found in Soya beans waspretreated in accordance with the Standard Methods ofAnalysis for Hygienic Chemists and measured.Photolysis was performed in an alkali solution on thevitamin B2 that had been hot-water extracted. And afteroxidation, the liquid extracted with chloroform wasmeasured. Vitamin B2 itself is fluorescent and thatexcitation and fluorescent spectrum is shown in Fig.1.15.2. Fig. 1.15.3 shows the spectrum after pretreatment.Fig. 1.15.4 shows the data for processed and measuredSoya bean. A comparison with the standard productshows that 2 µg of vitamin B2 exist in 1g of Soya bean.


H3C N

CH2(CHOH)3CH2OH

O

NH

N

O

Riboflavin

Lumiflavin

Photolysis

NH3C

H3C N

CH3

O

NH

N

O

NH3C

Fig. 1.15.1 Creation process of lumiflavin

400 450 500 550 600nm

Excitation spectrum Fluorescent spectrumRelative fluorescent intensity

Fig. 1.15.2 Excitation and fluorescent spectrum of vitamin B2 (riboflavin)

26

1.15 Analysis of Trace Amounts of Vitamins B1 and B2 in FoodProducts Using Fluorescence Photometry (2) - RF

400 450 500 550 600nm

Excitation spectrum Fluorescent spectrum

Relative fluorescent intensity

Fig. 1.15.3 Excitation and fluorescent spectra of lumiflavin created from photolysis

450 500 550 600nm

A

B

C

A: Sample solution + standard sampleB: Sample solution onlyC: Sample blank

Relative fluorescent intensity

Fig. 1.15.4 Measurement of vitamin B2 in soya bean

Soya bean

Homogenize

Hot-water extraction

Test solution + standard solution

Measuring solution A Measuring solution B Measuring solution C

Test solution + purified water

Add 1N NaOH

Photolysis

Test solution + purified water

Dark-location storage

Add acetic acid, 4% KMnO4

Chloroform extraction

Fig. 1.15.5 Pretreatment for vitamin B2 analysis

27


H O

1.16 Analysis of Water Soluble Vitamins Using Semi-micro LC System - LC

■ ExplanationA column with an inner diameter of 4 to 6mm is usuallyused in HPLC analysis, but in recent years semi-microscale columns are being employed in this area and willundoubtedly become the mainstream column for thefollowing reasons.(1) Mass sensitivity (sensitivity based on mass) is

increased.(2) The amounts of mobile phase and sample used are

reduced.Fig. 1.16.1 shows a semi-micro LC analysis example ofthe vitamin B group and caffeine in a vitamin drink.Some 2µL of sample was injected.

ReferenceShimadzu Application News No. L239 (C190-E065)

■ PretreatmentA 0.45 µm membrane filter was used for filtration.


Mobile Phase

Flow Rate

Temperature

Detection

: STR ODS-II (150mmL. × 2.0mmI.D.)

: 10mM Phosphate Buffer (pH 2.6)

containing 5mM Sodium Hexanesulfonate Acid

/Acetonitrile = 9/1 (v/v)

: 0.2mL/min

: 25˚C


0 1 2 3 4 5 6 7 8 9 10 11 12min

1

2

3

4

5

Fig. 1.16.1 Analysis of vitamin B group and caffeine in vitamin drink

28

0 5 15(min)10

■ PeaksNicotinic acidNicotinamidePantothenic acidPyridoxineRiboflavin phosphateThiamineCaffeineFolic acidBiotinRiboflavin

1.2.3.4.5.6.7.8.9.10.

0 5 15(min)10

■ Peaks Nicotinic acid Nicotinamide Pyridoxine Riboflavin phosphate Thiamine Caffeine Folic acid Riboflavin

1.2.4.5.6.7.8.10.

1.17 Analysis of Vitamin B Group - LC

■ ExplanationQuantification methods for vitamins have shifted frombiological methods to chemical methods.GC and HPLC incorporated methods are almost alwaysused for fat-soluble Vitamins whereas GC analysis ofwater-soluble vitamins is complicated to the point that itis impractical thus the HPLC analysis method is the mostfavored. Ion conversion and normal-phase partitionchromatography are used for separation but, from thepoint of view of column durability and analysis stability,reversed phase chromatography has become themainstream method.There are individual test methods for each vitamin, andchromatography simultaneous analysis capabilities forsamples with comparatively few impurities and largeamounts of target components are often found in medicalproducts and drink materials. Here, the conditions forsimultaneous analysis and the analysis example itself areshown for the vitamin B group.

ReferenceShimadzu HPLC Application Report No. 14(C196-E035)


Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack CLC-ODS(150mmL. × 6.0mm I.D.)


(pH 2.1) containing 0.8mM Sodium Octanesulfonate

/Acetonitrile = 9:1 (v/v)

: 40˚C

: 1.5mL/min

: UV-VIS Detector 210nm or 270nm

Fig. 1.17.1 Analysis example (210nm) of vitamin B group Fig. 1.17.2 Analysis example (270nm) of vitamin B group

29


H O

1.18 Analysis of Tocopherol in Milk - LC

■ ExplanationLC vitamin analysis is broadly separated into water-soluble vitamin analysis and fat-soluble vitamin analysis.Use of HPLC enables simultaneous analysis of thecomponents, which has made it a popular form ofanalysis from the outset.Here, analysis of fat-soluble vitamin tocopherol isintroduced.

ReferencesShimadzu HPLC Food Analysis Applications Data Book(C190-E047)Shimadzu HPLC Application Data book (C190-E001)

■ Pretreatment1. Add chloroform to sample for extraction.2. After vaporizing and dry hardening the chloroform

layer, the sample is dissolved in a small amount ofhexane and then concentrated.

3. The dissolved liquid sample is injected.


Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack CLC-NH2

(150mmL.×6.0mm I.D.)

: n-Hexane/Isopropyl Alcohol

= 100/4 (v/v)

: 40˚C

: 1.5mL/min


0 5 15(min)10

1

2

4

3

Peaks

1. α-tocopherol

2. β-tocopherol

3. γ-tocopherol

4. δ-tocopherol

Fig. 1.18.1 Analysis of tocopherol types in milk

30

1.19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (1) - LC■ ExplanationIn April 2002, the Food with Health Claims System wasestablished in Japan. The aim of this system is to regulatethe wide variety of so-called "health foods" byrecognizing the health claims of foods that satisfy certainrequirements.Foods with health claims are divided into two categories:food for specific health use and food with nutrientfunction claims. Foods with nutrient function claims aresubject to specific standards. The content of 12 vitaminsand 2 minerals must lie between the lower and upperlimits for the recommended daily intake.The analysis of the water-soluble vitamins that arespecified nutrients for food with nutrient function claimsis described here as an example.

■ Analytical Conditions[Fig. 1.19.1]

[Fig. 1.19.2]

[Fig. 1.19.3]

Column

Mobile Phase

Flow Rate

Temperature

Detection


: A: 100mM Sodium Phosphate Buffer Solution

containing 0.8mM Sodium 1-octanesulfonate (pH 2.1)

B: Acetonitrile

A/B = 10/1 (v/v)

: 1.2mL/min

: 40˚C

: UV (LC-2010) 210nm

Sample

Add 1mM NaOH 10mL

Pound in a mortar

(dosage per day) (10 times of dosage per day) (dosage per day)

Mixing

Add each buffer 100mL

Mixing Filtration Inj. 20µL

Fig.1.19.1 Fig.1.19.2 Fig.1.19.3

■ Analysis of Food with Nutrient ClaimsFig. 1.19.1, 1.19.2, and 1.19.3 show analysis examplesfor commercial tablet-shaped sweets (sample A) and formultivitamin tablets (sample B). The vitamin B groupincludes vitamins that only dissolve in dilute alkalis andso the following pretreatment was carried out. 20mL ofeach sample solution was injected.

■ Pretreatment

Column

Mobile Phase

Flow Rate

Temperature

Detection


: A: 100mM Sodium Phosphate Buffer Solution (pH 2.1)

B: Acetonitrile

A/B = 8/1 (v/v)

: 1.2mL/min

: 40˚C

: SPD-10AVVP 550nm

Column

Mobile Phase

Flow Rate

Temperature

Detection

: Asahipak NH2P-50 4E (250mmL. × 4.6mm I.D.)

: A: 100mM Phosphate (Triethanolammonium)

Buffer Solution (pH 2.2)

B: Acetonitrile

A/B = 1/4 (v/v)

: 1.0mL/min

: 40ºC

: SPD-10AVVP 240nm

31


H O

1.19 Analysis of Water-soluble Vitamins in Foods with Nutrient Function Claims (2) - LC

■ Peak 1. Vitamin B12

min0 2 4 6 8 10

mA

U

0.05

0.1

0.15

0.2

1

Fig. 1.19.2 Chromatogram of sample B

■ Peaks 1. Nicotinamide 2. Ca pantothenate 3. Vitamin B6

4. Vitamin B1

5. Folic acid 6. Vitamin B2

min0

mA

U

30

20

10

1 2 3

4

5

6

5 10 15

0

min

mA

U

40

30

20

10

0

3

4

5

6

21

151050

Sample A Sample B

Fig. 1.19.1 Chromatograms of commercial foods with nutrient claims

■ Peak 1. Vitamin C

min0 2 4 6 8 10

mA

U

0

10

20

30

40

1

Fig. 1.19.3 Chromatogram of sample B

32

1.20 Analysis (Measurement of K Value) of Nucleotide in Tuna Meat - LC

■ ExplanationNucleic acid base and nucleotide are usually analyzedusing reversed phase chromatography as they can besimultaneously analyzed.Here, a separation example using reversed phasechromatography for 8 adenine derivative components isshown.This form of analysis is applied to measuring of fishfreshness indicated by the K value (freshness constant)because the 4 kinds of nucleotides, hypoxanthine andinosine can be individually quantified.


■ Pretreatment1. Add 25mL of 1M perchloric acid to 10g of tuna meat

and homogenize.2. Centrifugally separated (3000 rpm for 5 min).3. Skim off top layer, and add 1M potassium bicarbonate

solution to adjust sample to pH 6.5.4. Remove the created potassium perchlorate sediment,

and filter top layer through membrane filter.5. Inject 5µL of filtered solution.


Mobile Phase

Temperature

Flow Rate

Detection

: STR ODS-2 (150mmL×.4.6mm I.D.)

: A : 100mM

Phosphate(Triethylammonium)Buffer(pH 6.8)

B : Acetonitrile

A / B = 100/1 (v/v)

: 40˚C

: 1.0mL/min


Hyp+Ino

Hyp+Ino+IMP+AMP+ADP+ATPK=

Fig. 1.20.1 Analysis of adenine derivative components Fig. 1.20.2 Analysis of tuna meat

■ Peaks

1.Hyp

2.IMP

3.Adenine

4.Ino

5.AMP

6.ADP

7.Adenosine

8.ATP

■ Peaks

1.Hyp

2.IMP

3.Adenine

4.Ino

5.AMP

6.ADP

7.Adenosine

8.ATP

33


H O

1.21 Analysis of Oligosaccharide in Beer - LC

■ ExplanationIn the case of analysis of sugars using the partitionmethod, the mobile phase is a mixture of water andacetonitrile used with an aminopropyl column. Theelation position can be adjusted by changing the water toacetonitrile ratio.Fig. 1.21.1 shows an analysis example of monosaccharideand oligosaccharide standard solutions and Fig. 1.21.2shows an analysis example of oligosaccharide in beer.

ReferencesShimadzu HPLC Food Analysis Applications Data Book(C190-E047)Mikami, Egi, Shimadzu Review, Nos. 44 (3), 47 (1987)Shimadzu HPLC Application Report No. 11 (C196-E036)


Column

Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack CLC-NH2 (150mmL.×6.0mm I.D.): Acetonitrile/Water = 60/40 (v/v)

: 25˚C

: 1.0mL/min

: Refractive Index Detector

0 5 15(min)10

1

2

3

4

5

67

■ Peaks

1. Glucose

2. Maltose

3. Maltotriose

4. Tetraose (L-DP-4)

5. Pentaose (L-DP-5)

6. Hexaose (L-DP-6)

7. Heptaose (L-DP-7)

L : Straight-chain structure

B : Branching structure

DP: Sugar number

Fig. 1.21.1 Analysis of sugar and oligosaccharide standard samples

0 5 10 15 20

12

3

4

5

678

9 10 11 1213

■ Peaks

1. Fructose

2. Glucose

3. Maltose

4. Maltotriose

5.L-DP-4

6.L-DP-5

7.B-DP-5

8.L-DP-6

9.B-DP-6

10.B-DP-7

11.B-DP-8

12.B-DP-9

13.B-DP-10

(min)

Fig. 1.21.2 Analysis of oligosaccharides in beer

34

1.22 Analysis of Oligosaccharides in Beer Using the ELSD-LT Low Temperature Evaporative Light Scattering Detector - LC

■ ExplanationCombining the GE method with the ELSD-LT enablesefficient separation when analyzing oligosaccharides.

Fig. 1.22.1 shows the results of analyzing theoligosaccharides in beer using the isocratic elutionmethod and the GE method. 10µL of beer was injectedafter filtering with a membrane filter. There are branchedoligosaccharides (1 -> 6 glycosidic linkage), linearoligosaccharides (1 -> 4 glycosidic linkage), and othertypes of oligosaccharide. In general, the different types ofoligosaccharide are mixed together when eluted. Theelution times for monosaccharides as well as lineardisaccharides, trisaccharides, and heptasaccharides areindicated in the chromatogram. It can be seen that the GEmethod enables the efficient separation and detection ofoligosaccharides up to 20-mer. The results of analyzingcommercial beers under the same GE conditions areshown in Fig. 1.22.2 and 1.22.3.


Mobile Phase

Flow Rate

Temperature

Detection

: NH2P-50 (250mmL. × 4.6mm I.D.)

: (1)Acetonitrile/Water = 6/4 (v/v)

Fig. 1.22.1

(2)A : Acetonitrile

B : Water

Linear Gradient B 30% → 60%

Fig. 1.22.2 and 1.22.3

: 1.0mL/min

: 40˚C

: ELSD-LT

Temperature : 35˚C

Gain : 7

Nebulizer Gas : N2

Gas Pressure : 350kPa

0 28

mV

0

200

400

600

800

1000

1200

min2 4 6 8 10 12 14 16 18 20 22 24 26

Fig. 1.22.2 Chromatogram of beer A

min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

mV

-500

0

500

1000

1500

2000

2500

1

4321

2 3 4

■ Elution Times1.Glucose2.Maltose3.Maltotriose4.Maltoheptaose

Gradient elution method

Isocratic elution method

Fig. 1.22.1 Analysis of oligosaccharides in beer

min0 2 4 6 8 10 12 14 16 18 20 22 24 26 28

mV

0

200

400

600

800

1000

1200

Fig. 1.22.3 Chromatogram of beer B

35


H O

1.23 Analysis of Saccharides in Fermented Foods - LC

2mL of water added to 200mg of miso or sake lees

Mixing

Centrifugation (3,000rpm, 5min)

Supernatant

Filtration

Diluted by factor of 10 with water

10µL injected

■ ExplanationFoods created by the fermentation process are attracting agreat deal of interest because of the many benefits theyoffer. It is often the case, however, that foods with manybenefits contain a large number of constituents, and,especially when the target constituent is only present in avery small amount, analysis can be difficult. In order tominimize the influence of impurities when analyzing thiskind of sample, a selective and highly sensitive detectionmethod is required.

In terms of selectivity and sensitivity, post-columnfluorescent derivatization is suited to the analysis ofsaccharides in fermented foods. The batch analysis ofsaccharides in miso and sake lees with Shimadzu'sReducing Sugar Analysis System, which uses an originalarginine reagent, is described here as an example.


Guard Column

Mobile phase

Flow Rate

Temperature

Reaction Reagent



Detection

: Shim-pack ISA-07/S2504 (250mmL. × 6.0mm I.D.)

: Guard column ISA (50mmL. × 4.0mm I.D.)

: A: 0.1M Borate (Potassium) Buffer Solution

(pH8)

B: 0.4M Borate (Potassium) Buffer Solution

(pH9)

A → B/Linear Gradient Elution Method

: 0.6mL/min

: 65˚C

: 1% L-Arginine, 3% Boric Acid Solution

: 0.5mL/min

: 150˚C

: RF-10AXL

Ex : 320nm Em : 430nm

■ Analysis of Miso and Sake LeesMiso and sake lees were pretreated in the way shown onthe right and 10µL of each were injected.

■ Pretreatment

1

23

4

5mV10

0

■ Peaks1. Ribose2. Fructose3. Galactose4. iso-Maltose5. Glucose

20 40 60 80min

0

5

Fig. 1.23.1 Analysis of miso

3

4

21

mV10

5

■ Peaks1. Ribose2. Mannose3. iso-Maltose4. Glucose

806040200 min

0

Fig. 1.23.2 Analysis of sake lees

36

4

3

2

1 5

8

9

7

6

1011

12

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

mobile

pump

injecto

colum

colum

reage

pump

reactio

coolin

detect

coil

waste

Fig. 1.24.2 Flowchart diagram of nonreducing sugar analysis system

1.24 Analysis of Nonreducing Sugar Using Postcolumn Derivatizationwith Fluorescence Detection - LC

■ ExplanationNonreducing sugars such as sucrose, raffinose andstachyose can be analyzed at high sensitivity and highselectivity by adding taurocyamine (as a fluorescentreaction agent for postcolumn fluorescence detection) toreducing sugar.Fig. 1.24.1 shows an analysis example for mixed standardsolutions of sucrose, raffinose and stachyose. Some500pmol of each component was injected.

ReferenceT. Kinoshita, et al, J. Liquid Chromatogr., No. 14 (10),1929 (1991)


Mobile Phase

Temperature

Flow Rate

Reaction Reagent

Reaction Reagent Flow Rate


Detection

: Asahipak NH2P-50 (250mmL.×4.6mm I.D.)

: Acetonitrile/Water = 65/35 (v/v)

: 40˚C

: 1.0mL/mi

: 0.1M Potassium Tertraborate Solution

containing 20mM Taurocyamine,

1mM Sodium Periodate (adjust to pH

10.5 using 10MKOH solution)

: 1.0mL/min

: 150˚C


Ex : 320mm Em : 450nm

0 5 10 (min)

1

2

3■ Peaks

1. Sucrose2. Raffinose3. Stachyose

Fig. 1.24.1 Analysis of nonreducing oligosaccharide

■ Peaks1. Sucrose2. Raffinose3. Stachyose

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

mobile phase

pump

injector

column

column oven

reagent

pump

reaction oven

cooling coil

detector

coil

waste

37


H O

1.25 Analysis of Sugar in Yogurt - LC■ ExplanationThe ligand conversion chromatography column SCR-101series consists of the 101N, 101C and 101P types withends made respectively of Na, Ca and Pb. And theretaining behavior of sugars differs with each one. Inparticular, in the case of sugar alcohol analysis, 101C or101P is recommended. Also, glucose and galactoseseparation is possible with the 101C type. Fig. 1.25.1shows an analysis example of a Japanese pickle liquidand Fig. 1.25.2 shows an analysis example of sugar inyogurt.

ReferencesShimadzu LC Application Report No. 11 (C196-E036)Shimadzu HPLC Food Analysis Applications Data Book(C190-E047)

■ Pretreatment[Analysis of Japanese pickle liquid]1. Filter the pickle liquid through a membrane filter.2. Inject 10 µL of filtered liquid.

[Analysis of yogurt]1. Add perchloric acid to the yogurt, and mix to

deproteinize.2. Centrifugally separate, and filter upper layer through a

membrane filter.3. Inject 10 µL of filtered liquid.

■ Analytical Conditions[Pickle liquid]

[Yogurt]

Column

Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack SCR-101C

(300mmL.×7.9mm I.D.): Water

: 80

: 0.8mL/min


Column

Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack SCR-101C

(300mmL.×7.9mm I.D.): Water

: 85

: 1.0mL/min


g Peaks

1. Sucrose2. Glucose3. Fructose4. Sorbitol

0 10 (min)20

1

2

4

3

Fig. 1.245.1 Analysis of pickle liquid

0 5 15 (min)10

1 2

4

3

■ Peaks

1. Lactose2. Glucose3. Galactose4. Fructose

Fig. 1.25.2 Analysis of yogurt

38

1.26 Analysis of Alliin in Garlic - LC

Column

Mobile Phase

Mobile Phase Flow Rate

Column Temperature

Reaction Reagent

Reaction Reagent

Flow Rate


Detection

Injection Volume

: Shim-pack Amino-Na

(100 mmL. × 6.0 mm I.D.)

: A: 0.1M Sodium Citrate Buffer

Solution (pH 3.2)

B: 0.2M Sodium HydroxideAqueous

Solution

Step Gradient Elution Method

0 – 23 (min) A 100%

23 – 33 (min) B 100%

33 – 50 (min) A 100%

: 0.4 mL/min

: 60˚C

: Amino Acid Reagent Kit (Solution B)

: 0.4 mL/min

: 60˚C

: RF-10AXL Ex : 350 nm Em : 450 nm

: 10 µL

■ ExplanationThe active ingredient alliin in garlic quickly changes toallicin by the action of the enzyme alliinase. As theefficacy of these substances has been elucidated, they areoften marketed as health food products. Since alliin is atype of amino acid, it can be analyzed by the post-columnderivatization method with fluorescence detection usingOPA (o-phthalaldehyde) as the reaction reagent. Here weintroduce the analysis of alliine in garlic using theShimadzu amino acid analysis system.


Fig. 1.26.2 Analysis of garlic

min0 10

1

20 30

■ Peak1.Alliin

Fig. 1.26.3 Analysis of processed garlic (tablet)

Fig. 1.26.1 Alliin structural formula

min0 10

1

20 30

■ Peak1.Alliin

CH

COOH

NH2

CH2 CH2 CH2

O

S CH

■ Pretreatment

Grated garlic 700 mg

or

processed garlic 100 mg

5% trifluoro acetic acid

aqueous solution 50 mL

Mix together

Filter

Inject 10 µL

39


H O

1.27 Analysis of Catechins in Green Tea - LC■ ExplanationThe efficacy of catechins, including antioxidative andcancer inhibition effects have been reported in variousliterature, and today catechins are receiving attentionbecause several commercially sold beverages are nowusing them. Five types of catechins are known, and theyare epigallocatechin, catechin, epigallocatechin gallate,epicatechin and epicatechin gallate. These componentscan be easily analyzed by HPLC.

(Gradient Program)Time(min) Func Value(%)

0 B.Conc 76 B.Conc 7

20 B.Conc 2020.01 B.Conc 5025 B.Conc 5025.01 B.Conc 735 STOP


Fig. 1.27.2 Chromatogram of green tea beverage

min0 10 20

mAU

0

5

10

15

12

■ Peaks1.EGC2.C3.Caffeine4.EC5.EGCG6.ECG

3

4

5

6

Fig. 1.27.3 Chromatogram of oolong tea beverage

Fig. 1.27.1 Structural formulas of catechins

min0 10 20

mAU

0

5

10

15

2

3

5 61 4

HO

OHOH

OH

OH

O

(+)-C

HO

OHOH

OH

OH

O

(-)-EC

HO

OHOH

OH

OH

OH

O

(-)-EGC

HO

OHO

O

OH

OH

OH

OH

OH

O

(-)-ECG

HO

OHO

O

OH

OH

OH

OH

OH

OOH

(-)-EGCG

Column

Mobile Phase

Flow Rate

Column Temperature

Detection

Injection Volume

: Shim-pack FC-ODS (75mmL. × 4.6mmI.D.)

: A: 10 mM Sodium Phosphate Buffer

Solution (pH 2.6)

B: Acetonitrile

Gradient Elution Method

: 1.0 mL/min

: 40˚C

: UV-VIS Detector 270 nm

: 5 µL

40

1.28 Analysis of Chlorogenic Acid in Coffee - LC■ ExplanationChlorogenic acid (3-caffeoylquinic acid) is a type ofpolyphenol compound widely distributed in higher plants,and is found in large quantities in coffee, potatoes andsweet potatoes. The antioxidative effects of chlorogenicacid are now receiving attention, and various types ofresearch on the efficacy of chlorogenic acid are beingadvanced. We present here the analysis of threecomponents of processed coffee. They are chlorogenicacid, caffeine and caffeic acid (3,4- dihydroxycinnaminicacid) which is a structural component of chlorogenic acid.

* It is sometimes referred to as 5-caffeoylquinic acid. However, here weuse the term 3-caffeoylquinic acid as used in “The Merck Index 13thEdition”.


Fig. 1.28.3 Chromatograms of canned coffee

at 325nm

at 270nm

0 5 10 15min

0

100

200

mA

U

min

mA

U

0 5 10 15

0

50

100

3

1

1

2 ■ Peaks1. Chlorogenic Acid2. Caffeine

■ Peaks1. Chlorogenic Acid3. Caffeic Acid

Fig. 1.28.4 Chromatograms of coffee beans

Fig.1.28.2 Spectra of chlorogenic acid

at 325nm

at 270nm

0 5 10 15min

min

0

50

100

mA

Um

AU

0 5 10 15

0

5

10

15

2

1

1

3

■ Peaks1. Chlorogenic Acid2. Caffeine

■ Peaks1. Chlorogenic Acid3. Caffeic Acid

0

50

100

mA

U

200 300 nm

Spectrum of Standard

Spectrum of Sample(Canned Coffee)

Fig. 1.28.1 Structural formula of chlorogenic acid

HOOC OH

OH

OH

OH

HO OC

C C

O

(Gradient Program)Time(min) B Conc(%)

0 105 10

15 3015.01 7018 7018.01 1025 STOP

Column

Mobile Phase

Flow Rate

Column Temperature

Detection

: Shim-pack VP-ODS (150 mmL. × 4.6 mm I.D.)

: A:10 mM Sodium Phosphate Buffer

Solution (pH 2.6)

B: Acetonitrile

Low Pressure Gradient Elution Method

: 1.0 mL/min

: 40˚C

: SPD-M10AVP 270 nm 325 nm

41


H O

1.29 Analysis of Lycopene and ß-Carotene in Tomato - LC■ ExplanationLycopene is a type of carotenoid, and is found in largequantities as a red pigment in red tomatoes, etc. Theantioxidative effects of lycopene are said to be 100 timesstronger than that of vitamin E and more than twicestronger than that of ß-carotene, and lycopene is receivingattention for its effects to prevent lifestyle diseases suchas cancer and arteriosclerosis, and to slow aging. Thoughlycopene and ß-carotene have similar structures, they canbe easily separated by reversed phase chromatographyusing a non-aqueous mobile phase.


Fig. 1.29.3 Spectra of lycopene

TomatoStandard

Fig.1.29.4 3-D Chromatogram of tomato

200

300

400

500

600 0

5

10

15min

0

20

40

60

80

100

mAU

nm

Tomato 5 g

Chloroform 40 mL

Homogenization 1 min

Shaking 5 min

Aqueous phase (upper layer)

Discard

Organic phase (lower layer)

Evaporative drying

Chloroform 5 mL

Inject 5 µL

min

mAU

0 5 10 15 20

0

25

50

75

1001

2

1:Lycopene■ Peaks

2: -Caroteneβ

Fig. 1.29.2 Chromatogram of tomato

Fig. 1.29.1 Structural formulas for lycopene and ß-carotene

Lycopene -Caroteneβ

CH3 CH3 CH3

CH3

CH3

CH3 CH3

H3C

H3C

CH3

CH3 CH3 CH3

CH3CH3 CH3

H3CH3C

CH3H3C

■ Pretreatment

Column

Mobile Phase

Flow Rate

Column Temperature

Detection

Cell Temperature

Injection Volume


: Acetonitrile / Ethanol = 4/1 (v/v)

: 1.0 mL/min

: 50˚C

: SPD-M20A 450 nm

Slit width: 8 nm, Bandwidth: 8 nm

: 50˚C

: 5 µL

42

1.30 Melting of Fats and Oils - TA■ ExplanationThe melting process of various edible fats and oils ismeasured by DSC. Six types of crystals exist in cocoa oil,a component of chocolate. Of these, V type crystal is saidto possess good thermal stability. Since V type crystalmelts at about 34˚C, DSC measurement can be used toknow the condition of V type crystal contained in aparticular sample of chocolate. Figure 1.30.1 shows aDSC curve of the chocolate sample heated at 3˚C/min.Figure 1.30.2 shows a DSC curve of the same chocolatesample, reheated after cooling the melted sample to -50˚Cto harden it. It is evident that the peak at 30.4˚C hascompletely disappeared.


0.00 50.00Temp [˚C]

–6.00

–4.00

–2.00

0.00

DSCmW

30.44˚C

13.99˚C3.39˚C

–36.25J/g

Fig. 1.30.1 Chocolate measurement (1st time)

0.00 50.00Temp [˚C]

–6.00

–4.00

–2.00

0.00

2.00

DSCmW

–23.45J/g

–11.71˚C

19.60˚C

16.30˚C

7.92˚C

Fig. 1.30.2 Chocolate measurement (2nd time)

Instrument

Sample

Sample Amount

Atmospheric Gas

Gas Flow Rate

Heating Rate

: DSC-60

: Chocolate

: 22.87 mg

: Nitrogen

: 30 mL/min

: 3˚C/min

[Temperature Program]

1.31 Gelatinization of Starch - TA

43


H O

■ ExplanationStarches gelatinize when heated with water. Thegelatinization reaction can be analyzed by DSC because itis accompanied by endothermic reaction. Here weconducted measurement of flour starch (17.4%).

Fig. 1.31.1 Gelatinization temperature of flour (17.4%)

20.00 40.00 60.00 80.00 100.00Temp [˚C]

–1.00

0.00

1.00

DSCmW

59.10˚C

Fig. 1.31.2 Gelatinization temperature of corn (19.9%)

● Flour

● Corn

40.00 60.00 80.00Temp [˚C]

–1.00

0.00

1.00

DSCmW

68.90˚C

■ ExplanationHere we conducted measurement of corn starch (19.9%).It is known that when sucrose and salt are added tostarch, the gelatinization temperature changes.


Sample

Sample Amount

Atmospheric Gas

Gas Flow Rate

Heating Rate

: DSC-60

: Flour

: 4.21 mg

: Nitrogen

: 30 mL/min

: 5˚C/min



Sample

Sample Amount

Atmospheric Gas

Gas Flow Rate

Heating Rate

: DSC-60

: Corn

: 4.97 mg

: Nitrogen

: 30 mL/min

: 5˚C/min


44

2.1 Propionic Acid in Cookies and Bread - GC■ ExplanationPropionic acid is one of the components that form flavorand fragrance, included in fermented products such asmiso, soy sauce and cheese as a microbial metabolite. Itis also used as a preservative in cookies and breadbecause of its low toxicity and minimal effect on breadyeast.When propionic acid is analyzed using GC with FID, thetotal calculation of the natural propionic acid, which isinherently included in the food, and the added propionicacid is obtained as the quantitative value.

References1) Standard Methods of Analysis for Hygienic Chemists

(annotation) 455 (1990), edited by the PharmaceuticalSociety of Japan

2) Ministry of Health and Welfare (currently Ministry ofHealth, Labour and Welfare), Environmental HealthBureau, Food Sanitation Testing Policy, 33-35 (1989)

■ PretreatmentPropionic acid was extracted using steam distillationmethod.


2

1

S 10min

1. Propionic acid2. Crotonic acid (I.S.)

Peaks■

Column

Col. Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

: 10% PEG6000 on shimalite TPA

1m × 3mm I.D.(glass)

: 150°C

: 230°C

: 200°C(FID)

: N2

2. Food Additives

Fig. 2.1.1 Analysis of propionic acid

45

Food Additives

2

1

5minS

1. Saccharine2. (IS) trans-stilbene

Peaks■

2.2 Saccharine and Sodium Saccharine - GC

Fig. 2.2.1 Analysis of saccharine

■ ExplanationSaccharine and sodium saccharine are used as artificialsweeteners. Saccharine is only used in chewing gumbecause it does not dissolve easily in water whereassodium saccharine does and is widely used in pickles andjams.Saccharine and sodium saccharine are extracted fromfood products and refined, and after being methylated,they are analyzed by GC with FID or FPD. Here, a GCwith FID analysis example will be introduced.

ReferenceStandard Methods of Analysis for Hygienic Chemists(annotation) 493 to 495 (1990), edited by thePharmaceutical Society of Japan.

■ Pretreatment1. Extract and refine sample by dialysis extraction or

direct extraction.2. Produce a derivative (methylate) of saccharine using

diazomethane, etc.3. Dissolve in ethyl acetate, etc. and use this liquid as the

sample.


Col. Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

: SE-30 5% on chromosorb W

1.5m × 3mm I.D. (glass)

: 190°C

: 250°C

: 230°C (FID)

: N2

46

2.3 Ethylene Glycols in Wine - GC

■ ExplanationNormally wine does not contain ethylene glycol but therehave been reports of temporary errors where diethyleneglycol was mixed into wine.Here, ethylene glycol and diethylene glycol have beenadded to wine and directly analyzed by GC. Analysis waspossible without any interference from impurities in thewine.

ReferenceShimadzu Application News No. G110

■ PretreatmentEthylene glycol and diethylene glycol were added to ashop-sold wine for direct analysis.


min8642START

3

21

4

1.Propylene glycol

2.Ethylene glycol

3.Dipropylene glycol

4.Diethylene glycol

Peaks■

Fig. 2.3.1 Analysis of glycols (standard products)

min8642START

2

1

1.Ethylene glycol

2.Diethylene glycol

Peaks■

Fig. 2.3.2 Analysis of shop-sold wine with glycols added

Column

Col. Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection

: ULBON HR-20M

(25m × 0.25mmI.D. df = 0.25µm)

: 150°C

: 200°C

: 200°C (FID)

: He 2mL/min

: Split 1:30

47

Food Additives

ether. Reversely extract the ether layer using sodiumhydrogen carbonate solution, re-extract using ethylether, and concentrate. GC analyze the final liquid asan acetone.

2. Steam distillationPulverize the sample, add water, and neutralize pH.Add tartaric acid solution and salt and perform steamdistillation. Extract residue using ethyl ether aspreviously described.


5 5 10min

Sor

bic

Aci

d

Deh

ydro

acet

ic A

cid

Ben

zoic

Aci

d

tran

s-S

tilbe

ne(I

.S.)

Fig. 2.4.1 Analysis of preservatives

2.4 Sorbic Acid, Dehydroacetic Acid and Benzoic Acid - GC

■ ExplanationThe preservatives sorbic acid, dehydroacetic acid andbenzoic acid are analyzed by UV absorption spectrummethod or GC method. The UV method is fast andefficient but can be affected by coexisting substancessuch as fragrances, whereas GC has the advantage ofbeing able to easily separate out such substances.Here, these preservatives were extracted from a foodproduct by direct extraction or steam distillation andrefined to be analyzed by GC with FID.

ReferenceStandard Methods of Analysis for Hygienic Chemists(annotation) 445 to 451 (1990), edited by thePharmaceutical Society of Japan

■ Pretreatment1. Direct extraction

Add saturated saline solution and sulfuric acid,homogenize with strong acidity and extract with ethyl

Column

Col. Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

: 5% DEGS+1%H3PO4 on chromosorb W

2m × 3mm I.D.(glass)

: 185°C

: 230°C

: 250°C (FID)

: N2

48

2.5 Analysis of Preservatives in Food Products with Absorption Photometry (1) - UV

■ ExplanationVarious preservatives are added to preservative andprocessed foods to prevent putrefaction and to keepfreshness. The use of these food additives is strictlygoverned by the Food Sanitation Law to ensure thatconcentrations do not exceed the permitted safeconcentrations for human consumption.Here, preservatives in food products regulated by theFood Sanitation Law were analyzed with a Shimadzudouble-beam spectrophotometer after pretreatment inaccordance with the law.

■ Pretreatment- Sodium nitrite in a food product

The sodium nitrite preservative in meat was separatedby distillation, and sulfamic acid was diazotized usingnitrite acid under acidity of hydrochloric acid, andcolored with naphthylethylenediamine for measurement.

- Benzoic acid in a food product

The benzoic acid preservative was separated andextracted from soy sauce using steam distillation inreadiness for UV absorption measurement.

- Sorbic acid in a food productThe sorbic acid preservative was separated andextracted from boiled fish paste using steam distillationin readiness for UV absorption measurement.

- Dehydroacetic acid in a food productThe dehydroacetic acid preservative was separated andextracted from bean jam using steam distillation inreadiness for UV absorption measurement.


Reference

Solvent

Cell

Range

: UV Spectrophotometer

: blank

: H2O

: 10mm

: 0~2Abs

0.7

0.6

0.5

0.4

0.3

0.2

0.1

0.5µg

0.4µg

0.3µg

0.2µg

0.1µg

650600550500450

Wavelength (nm)

Sample

Abs

orba

nce

Fig. 2.5.1 Absorption spectrum for sodium nitrite

0.25

µg

0.8

0.6

0.4

0.2

Sample

0 0.50.40.30.20.1

Sodium nitrite concentration(µg/mL)

Abs

orba

nce

Fig. 2.5.2 Calibration curve for sodium nitrite

49

Food Additives

2.5 Analysis of Preservatives in Food Products with Absorption Photometry (2) - UV

Absorption spectrum for dehydroacetic acid standard liquids

200 250 300 350

320µg/20mL

240µg/20mL

160µg/20mL

80µg/20mL

Wavelength (nm)

0.6

0.5

0.4

0.3

0.2

0.1

Abs

orba

nce

0.6

0.5

0.4

0.3

0.2

0.1

Absorption spectrum for sample solutions

Sample No. 2

Sample No. 1

200 250 300 350Wavelength (nm)

Abs

orba

nce

Fig. 2.5.7 Absorption spectrum for dehydroacetic acid

194 21

0

0.6

0.4

0.2

0 320240 16080

Dehydroacetic acid concentration(µg/mL)

No.2No.1

Abs

orba

nce

Fig. 2.5.6 Calibration curve for dehydroacetic acid

0.7

0.6

0.5

0.4

0.3

0.2

0.1

200 250 300 350 250 300 350Wavelength (nm) Wavelength (nm)

Absorption spectrum for sorbic acid in a food product

Absorption spectrum for sorbic acid standard liquids

2.5.2

Abs

orba

nce

Fig. 2.5.5 Absorption spectrum for sorbic acid

7.2µ

g

0.8

0.6

0.4

0.2

0 8642

Sorbic acid concentration(µg/mL)

Sample

Abs

orba

nce

Fig. 2.5.4 Calibration curve for sorbic acid

0.7

0.6

0.5

0.4

0.3

0.2

0.1

Wavelength (nm) Wavelength (nm)

Absorption spectrum for benzoic acid standard liquids

Absorption spectrum for benzoic acid in a food product

200 250 300200 250 300

Abs

orba

nce

Fig. 2.5.3 Absorption spectrum for benzoic acid

50

2.6 Color Control of Food Products (1) - UV

100.00

50.00

0.00

380.0

1

4

3

2

580.00 780.00Wavelength (nm)

1. Vinegar

2. Ketchup

3. Sauce

4. Mayonnaise

Ref

lect

ive

ratio

Fig. 2.6.1 Transmittance and reflectance spectra

■ ExplanationColor control is an important factor in quality control, ascolors have large psychological effect and consumerimage of products largely depends on the color of theircoating resin or paint. Thus, colorimeters, whichdetermine color of objects, are widely used in variousfields.Colorimetry methods are largely divided into two: one isspectral colorimetry in which a spectrophotometer is usedto measure reflectance or transmittance spectrum, and thetristimulus values X, Y and Z are determined bycalculation; the other is the direct reading of thetristimulus values where a photoelectric photometer isused to directly measure the tristimulus values.Here, a measurement example using the colormeasurement software with the spectrophotometer UV-3100PC will be introduced.

Color Measurement of Processed Food Productsavailable in consumer marketThe colors of processed foods can greatly enhance theirappearance for marketing purposes, which makes colorcontrol an important facet of the food industry. Here,color measurement was performed on shop-sold flavoringproducts.1. Vinegar2. Ketchup3. Sauce4. Mayonnaise

Vinegar was analyzed using transmittance measurementand the other products by reflective measurement. Fig.2.6.1 shows the spectra for the products. Next, based onthese spectrum data, the x, y, Y stimulus values and L*,a*, b* values were calculated under the conditions of Cilluminant and 2* field of view. Fig. 2.6.2 shows theprintout of the results. Also, under the same conditions,CIE (xy) and UCS chromaticity diagrams were drawn up(see Fig. 2.6.3 and Fig. 2.6.4). The xy chromaticitydiagram shows chromaticity (hue and saturation) usingthe x and y chromaticity coordinates. The closer to thecenter, the lower the saturation. Color differences can bediscerned at a glance. In the Lab chromaticity diagram,the left-side L* displays brightness between zero and 100while a* and b* on the right denote chromaticity. Plus a*is the red direction, minus a* the green direction, plus b*the yellow direction and minus b* the blue direction. Thecloser to the center, the lower the saturation and thecloser to the edge, the higher the saturation. This is thechromaticity diagram most widely used.


Sample

Reference

Range

: UV-3101PC with color

measurement software

: Vinegar, ketchup, sauce,

and mayonnaise

: MgO

: 0 to 100%

51

Food Additives

2.6 Color Control of Food Products (2) - UV

Measurement results of x, y, Y and L*,a*,b* values

Title : COLOR MEASUREMENT

Comment : UV-3100PC+ISR-3100

Illuminant : C Field of view (degree) : 2

Reference value : 0.00 0.00 0.00 0.00 0.0000 0.0000

Sample ID L* a* b* Y x y

1 97.44 -2.67 12.39 93.51 0.3286 0.3406 Vinegar2 20.59 21.05 17.83 3.14 0.4985 0.3491 Ketchup3 9.46 2.88 3.37 1.06 0.3546 0.3345 Sauce4 75.63 -2.77 20.77 49.29 0.3511 0.3657 Mayonnaise

Fig. 2.6.2 Measurement results

1.000

y 0.500

0.0000.000 0.400

x0.800

4

1 3 2

Fig. 2.6.3 CIE (x,y) chromaticity diagram

100.01

50.0

0.0

L*

a*

b*

60.000

0.000

-60.000-50.000 0.000 60.000

42

31

4

2

3

Fig. 2.6.4 UCS (Lab) chromaticity diagram

52

2.7 Analysis of Sweetener in Soft Drink - LC

■ ExplanationThis is an example of simultaneous analysis of thesweeteners aspartame, saccharine, benzoic acid, sorbicacid and glycyrrhizic acid.


■ PretreatmentA soft drink was directly injected without pretreatment.


Mobile Phase

Temperature

Flow Rate

Detection

■ Peaks

1. Saccharine

2. Aspartame

3. Benzoic acid

4. Sorbic acid

1

3

4

2

0 16 (min)8

Fig. 2.7.1 Analysis of sweetener in soft drink

: STR ODS-M(150mmL. × 4.6mmI.D.)

: 40mM Sodium Acetate Buffer (pH 4.0)/

Methanol = 3/1 (v/v)

: 40˚C

: 1.0mL/min

: UV 250nm

53

Food Additives

2.8 Analysis of Fungicide in Oranges - LC

■ ExplanationIn Japan the use of o-phenylphenol (OPP), thiabendazole(TBZ) and diphenyl is permitted for preventing mold incitrus.Here, the simultaneous analysis of these componentsusing fluorescent detection will be introduced.


■ Pretreatment1. Add 0.5g of anhydrous sodium acetate, 15g of

anhydrous sodium sulfate and 40mL of ethyl acetate to

10g of orange, and homogenize twice.

2. Filter using glass filter.

3. Add 2.5mL of butanol to the acquired ethyl acetate

layer.

4. Concentrate at 40˚C until 2.5mL is obtained.

5. Add methanol to dilute to 10mL and filter through

membrane filter.

6. Inject 5µL of filtrate.


■ Peaks

1. O-phenylphenol (OPP)

2. Thiabendazole (TBZ)

3. Diphenyl (DP)

2

1

0 10 20 (min)

3

Fig. 2.8.1 Analysis of fungicide in orange

Column

Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack CLC-ODS(150mmL.× 6.0mmI.D.)

: Acetonitrile/Methanol/Water

= 30/35/35 (v/v/v)

Prepare it to pH 2.4 with phosphoric acid

containing 10mM sodium dodecyl acetate.

: 40˚C

: 1.0mL/min


Ex : 285nm Em : 325nm

54

2.9 Analysis of Phenol Antioxidant in Foods - LC

■ ExplanationThe exposure of food constituents to oxygen in the airleads to the creation of oxidation products anddeterioration in quality. To prevent this, variousantioxidants are used as food additives. Here, we will belooking at how phenol antioxidants, which are usedparticularly often in oil products, can be analyzed withHPLC.

There are four types of phenol antioxidant that areapproved as food additives in Japan: BHT (butylatedhydroxytoluene), BHA (butylated hydroxyanisole),NDGA (nordihydroguaiaretic acid), and PG (propylgallate). They are authorized for use in oil, fat, and butter,as well as frozen and dried seafood products.

The analysis of nine phenol antioxidants, the fourmentioned above and five that are used in other countries,is described here as an example.

■ Analysis of ButterThe results of analyzing butter after performing thepretreatment described on the right are shown in Fig.2.9.1. The lower line represents the result of analyzingbutter and the upper line represents the result of analyzingbutter after adding 20mg/L of the nine phenolantioxidants at the pretreatment stage.


Mobile Phase

Flow Rate

Temperature

Detection

■ Peaks 1.PG 2.THBP 3.TBHQ 4.NDGA

5.BHA 6.HMBP 7.OG 8.BHT 9.DG

50

mV

0

100 20 min

4

2

3

1

56

7

8

9

Fig. 2.9.1 Analysis of butter

: Shim-pack FC-ODS (75mmL. × 4.6mm I.D.)

: A: 5% Acetic Acid Solution

B: Methanol/Acetonitrile = 1/1 (v/v)

B 40% → 80% /15min

Linear Gradient

: 1.0mL/min

: 40˚C

: SPD-10AVP 280nm

■ Pretreatment

0.5g of butter

Mixing

Left for 1 hour at -20˚C

Centrifugation at 3,000rpm for 5min

Supernatant

Filtration

10µL injected

1g of sodium sulfate5mL of acetonitrile/isopropanol/ethanol = 2/1/1 (v/v/v)

55

Food Additives

2.10 Analysis of L-Ascorbic Acid 2-Glucoside - LC■ ExplanationL-ascorbic acid 2-glucoside (2-o-α-D-glucopyranosyl L-ascorbic acid) is a type of vitamin C derivative that canexist stably when exposed to heat and light and even inwater, and in the body it attains vitamin C activitythrough the action of glucosidase. L-ascorbic acid 2-glucoside is specified as a food additive (January 20,2004, Ministry of Health, Labour and Welfare;Department of Food Safety, No. 0120001), andnotification has been made that its analysis in foodproducts is to be conducted by HPLC (May 13, 2004,Ministry of Health, Labour and Welfare; Department ofFood Safety, No. 0513003). Though L-ascorbic acid(vitamin C) is currently used in various food products onthe market as an antioxidant and nutrition enrichmentagent, it is believed that L-ascorbic acid 2-glucoside willalso be widely used.We introduce here an analysis of L-ascorbic acid 2-glucoside in accordance with the Department of FoodSafety notification, No. 0513003.


Mobile Phase

Flow Rate

Temperature

Detection

: Shim-pack VP-ODS (250 mmL. × 4.6mm I.D.)

: A : Water 800 mL + KH2PO4 1.4 g +

TBAH* (10%) 26 mL →pH=5.2 using Phosphoric Acid →1000 mL using Water

(*Tetrapbutyl Ammonium Hydroxide)

B : Acetonitrile

A/B=9/1 (v/v)

: 0.8mL/min

: 40˚C

: SPD-20A 260 nm

Cell Temperature: 40˚C

■ Pretreatment

■ Peaks1. Nicotinamide2. L-Ascorbic Acid 2-Glucoside3. L-Ascorbic Acid(Vitamin C)4. Riboflavin(Vitamin B2)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5

0

25

50

75

100

125

1

3

min

mA

U 2

4

Fig. 2.10.2 Chromatogram of soft drink beverage (spiked with L-ascorbic acid 2-glucoside)

Soft drink beverage 5 g

Add mobile phase to 50 mL

Filter (0.45 µm)

Inject 10µL

100 mg/L standard product 10 mL

H

HO

HOH

H

H

OH

O

HO

H O

HO

OO

H

OH

H

OH

200 250 300 350nm

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

mA

U

Fig. 2.10.1 Structural formula for L-ascorbic acid 2-glucoside and absorption spectrum

56

2.11 Analysis of EDTA in Mayonnaise - LC■ ExplanationEDTA in mayonnaise was analyzed after chelation of Feion. Reversed-phase ion pair chromatography withtetrabutylammonium ions was used for separation. In thisanalysis, a polymer column (ODP), instead of a silicacolumn (ODS), was used because of the high pH of themobile phase and the basicity of thetetrabutylammonium.The following chromatogram shows the measurement ofmarketed mayonnaise with PDTA (the internal standardsubstance) and EDTA added.

ReferencesShimadzu HPLC Food Analysis Applications Data Book(C190-E047)Shimadzu Application News No. L214 (C190-E050)

■ Pretreatment1. Add chloroform to sample, mix together, and

centrifugally separate (12000 r.p.m for 2 min,twice).

2. Add 0.01M FeCl3 solution to water layer and mixtogether.

3. Inject 20µL of sample.


Mobile Phase

Temperature

Flow Rate

Detection

: Asahipak ODP-50 (150mmL. × 6.0mmI.D.)

: 20mM Sodium Phosphate Buffer (pH 6.9)

containing 10mM Tetrabutylammonium

Hydrogensulfate

(adjust to pH 7.5 with 4M of NaOH)

: 40˚C

: 0.8mL/min

: UV 255nm

■ Peaks

1. EDTA-Fe

2. PDTA-Fe (internal standard substance)2

1

0 8 16 (min)

Fig. 2.11.1 Analysis of EDTA in mayonnaise

57

Food Additives

2.12 Analysis of Benzoyl Peroxide in Food Product - LC

0 5 10 15 20 25min

30 35 40 45

0

1.0

2.0

mA

U

3.0

4.0

5.0

1 ■ Peak1.Benzoyl Peroxide

Fig. 2.12.2 Analysis of flour : (above) spiked with 1.0 mg/L of standard benzoyl peroxide (below) not spiked (each with 20 µL injection)

Fig. 2.12.1 Structural formula for benzoyl peroxide

■ ExplanationIn Japan, benzoyl peroxide is a food additive authorizedfor use only in flour as a processing agent, and its amountfor use as diluted benzoyl peroxide is prescribed to be“0.30 g/kg or less” by the Ministry of Health, Labour andWelfare in “Standards for Foods and Food Additives”. Inaddition, the Ministry of Health, Labour and Welfare(May 13, 2004, Department of Food Safety; No.0513003)revised the testing method so that the analytical methodwas changed from gas chromatography to HPLC. Herewe introduce an analysis of benzoyl peroxide inaccordance with the Department of Food Safetynotification No. 0513003.


Mobile Phase

Flow Rate

Temperature

Detection


: Water/Acetonitrile = 45/55 (v/v)

: 1.0 mL/min

: 40˚C

: UV 235 nm

■ Analysis of FlourFigure 2.12.2 shows the chromatogram of a pretreatedsample of domestically produced flour, and thechromatogram of the same pretreated sample with theadditional 1.0 mg/L (equivalent to flour 5.0 mg/kg) ofbenzoyl peroxide (indicated by the dotted arrow inSample Pretreatment).

■ Pretreatment

Flour 10 g

50 mL acetonitrile added

Mix using stirrer for 15 min

Filter (0.45 µm)

Inject 20 µL

5.0 g/L standard product 10 µL

C O CO

O O

58

2.13 Analysis of p-Hydroxybenzoates in Soy Sauce - LC

■ ExplanationLC is a great force in the analysis of preservatives used infood products. In particular, LC is useful forsimultaneous analysis of such components. Here, ananalysis example for p-hydroxybenzoates added to soysauce will be introduced.


■ Pretreatment1. Add pure water to soy sauce until diluted by 10 fold.

2. Filter through membrane filter.

3. Inject 10µL of filtrate.


Mobile Phase

Temperature

Flow Rate

Detection

: STR ODS-2 (150mmL. × 4.6mm I.D.)


(pH 2.6)/Methanol = 1/1 (v/v)

: 40°C

: 1.5mL/min

: UV 270nm

■ Peaks

1. Methyl p-Hydroxybenzoate

2. Ethyl p-Hydroxybenzoate

3. Isopropyl p-Hydroxybenzoate

4. Propyl p-Hydroxybenzoate

5. Iso butyl p-Hydroxybenzoate

6. Butyl p-Hydroxybenzoate

1

2

3 4

56

0 10 20 (min)

Fig. 2.13.1 Analysis of p-hydroxybenzoates in soy sauce

59

Food Additives

2.14 Analysis of Potassium Bromate in Bread - LC

■ ExplanationThe use of potassium bromate as an additive is allowed inbread production to make the bread-making process moreeffective. However, to ensure safety, it must not remainin the final product. Therefore, it is necessary to verifythat there is no potassium bromate left in the bread.Chemical Hygiene No. 119 issued from the Japanese

50mL of WaterBread 10.0g

Stirred in Room Temperature for 30 min.

Left for 5 min

Centrifugation for 30 min (5˚C, 10000G)

Filtration (Filter Paper No.5A)

Filtration (0.45µm membrane filter)

Filtration (C18(ODS) mini cartridge column)

Filtration (Ion exchange mini cartridge column (Ag form) )

Filtration (Ultarfiltration)

Filtration (Ion exchange mini cartridge column (H form) )

Injection (200µL)

Fig. 2.14.2 Pretreatment

0 4 8 12

1

16 20(min)

■ Peak1. Bromate

Fig. 2.14.1 Analysis of potassium bromate in bread

InstrumentColumnMobile Phase

Flow RateTemperatureInjection VolumeReaction Regent

Reaction UnitTemperatureDetection

:

:

:

:

:

:

:

:

:

:

Shimadzu LC-VP Bromate Analysis System

Shim-pack VP-ODS (250mmL. × 4.6mmI.D.)

100mL of Methanol, 2.0g of Acetic Acid and 19g of Tetrabutylammonium Hydroxide were added to 700mL of Water, and pH

of Solution was adjusted to 6.3 - 6.5. And then, this solution was diluted to 1000 mL with Water.

1.0mL/min

40˚C

200µL

A ; 60mL of Nitric Acid (70%), 10.0g of Potassium Bromide was added to 700mL of Water.

B ; 500mg of o-Dianisidine Dihydrochloride was added to 200mL of Methanol.

Solution A and B were mixed, and diluted to 1000 mL with Water.

Piping Kit for Bromate Analysis

60˚C

UV-VIS Detector (450nm)

Table 2.14.1 Analytical conditions

Ministry of Health, Labour and Welfare on September11, 1997 stipulates the post-column derivatization HPLCmethod using o-dianisidine as a reaction reagent toanalyze potassium bromate in bread. This section shows an example of analyzing potassiumbromate contained in bread.

Fig. 2.14.1 shows the chromatogram obtained byanalyzing commercially sold bread after the pretreatmentprocedure shown in Fig. 2.14.2 that conforms toChemical Hygiene No. 119 (lower chromatogram), andthe chromatogram obtained when 326 µg of potassiumbromate standard (equivalent to 250 µg of bromate ions)was added to 10 grams of bread before pretreatment(upper chromatogram). No bromate ions were detectedwhen analyzing bread alone. When analyzing the breadwith potassium bromate added, 325.4 µg of potassiumbromate (249.4 µg of bromate ions) was quantified in 10grams of bread, demonstrating approximately 100%recovery rate.

60

2.15 Simultaneous Analysis of Water-soluble Tar Pigments - LC■ ExplanationSynthetic and natural compounds are used as foodpigments, and HPLC is a powerful tool for analyzingsuch compounds. The photodiode array analysis, whichallows simultaneous analysis at multiple wavelengths andspectrum display, further facilitates the analysis andidentification of unknown components.Here, a simultaneous analysis example for water-solubletar pigments will be introduced showing multichromatograms for each absorption wavelength using aphotodiode array detector.

ReferenceMasaaki Ishikawa et al; Summary of the 31st AnnualConference of the Japan Hygienic Chemistry Council(1994)

1. Yellow No. 42. Red No. 23. Blue No. 24. Yellow No. 203a5. Red No. 1026. Yellow No. 4037. Yellow No. 58. Yellow No. 203b9. Red No. 227

10. Red No. 4011. Yellow No. 20212. Orange No. 20713. Red No. 50314. Red No. 50415. Green No. 40116. Red No. 23017. Orange No. 40218. Blue No. 119. Red No. 50220. Red No. 321. Black No. 40122. Orange No. 20123. Red No. 10624. Green No. 20125. Red No. 10426. Yellow No. 40727. Yellow No. 40628. Red No. 10529. Red No. 50630. Brown No. 20131. Violet No. 40132. Red No. 40133. Red No. 21334. Yellow No. 402

mAbs100

50

0

100

50

0

100

50

0

100

50

0

100

50

0100

50

0

100

50

0

100

50

0

1

Ch1 250nm

3

1924

31

Ch2 410nm

26 27 34

Ch3 430nm

4 68

Ch5 510nm

510

11 1213 14 29

3022

177

Ch4 470nm

Ch6 520nm

29

1620

24 32

33

282523Ch7 550nm

Ch8 620nm 19 1821

0 20 40 60 (min)

■ Peaks

Fig. 2.15.1 Simultaneous analysis of water-soluble tar pigments


■ Gradient Conditions

Column

Mobile Phase

Temperature

Flow Rate

Detection

: STR ODS-2 (150mmL. × 4.6mm I.D.)

: A: 20mM Ammonium Phosphate Buffer

(pH 6.8)/Isopropanol = 25/1 (v/v)

B: Acetonitrile

Gradient Elution

: 40˚C

: 1.0mL/min

: Photodiode Array Detection from

220nm to 700nm

Time

0.00 min (initial condition)

15:00 min

45.00 min

55.00 min

55.01 min

65.00 min

B concentration

0%

20%

40%

70%

0%

0%

61

3. Residual Pesticides

3.1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) - GCAnalysis Based on Standards for Foods and Additives Specified in Japan's Food Sanitation Law (Notice 370 Issued by Japan's Ministry of Health and Welfare)

■ ExplanationAs a result of the diversification of foods andimprovements in the ways foods are transported andstored, a wide variety of food products are imported fromall over the world and eaten as part of our daily diet.There are, however, many reported cases of excessivelevels of pesticide residue being found in imported foods,particularly imported vegetables, and the safety ofimported vegetables has become an issue of someconcern. The analysis of organophosphorus pesticidebased on the standards for foods and additives specified

Sample

Purification

Quantitativeanalysis

Extraction

Column tube of inner diameter 15mm and length 300mm filled with 5g of silica gel and 5g of Na2SO4

Extract injected

Elution with 100mL mixture of acetone and n-hexane (1:1)

Vacuum concentration of eluate at 40ºC max.; acetone added to residue to make 5mL solution

GC-FTD, FPD

Addition of 100mL of acetone; homogenization for 3min; suction filtration through layer of diatomaceous earthHomogenization of residue and 50mL of acetone; suction filtration; vacuum concentration at 40˚C max.300mL separating funnel; 100mL of saturated NaCl solution

Extracted twice with 1:4-mixture of ethyl acetate and n-hexane solution (first time: 100mL; second time: 50mL)Dehydration of organic solvent layer; vacuum concentration at 40˚C max.

Residue dissolved with 1:1 mixture of acetone and n-hexane (approx. 5mL)

(Fruits, vegetables, green powdered tea, or hops)

Approx. 1kg of sample homogenized

20g of sample weighed out

in Japan's Food Sanitation Law (Japan's Ministry ofHealth,Labour and Welfare: Notice 370, D, item (6)) isdescribed here as an example.

The analysis method varies with the sample; samples arecategorized into three groups that each have differentanalysis methods: fruits, vegetables, green powdered tea,and hops; grain, beans, nuts, and seeds; and teas otherthan green powdered tea. The sample processing methodsfor the first two groups are described below.

■ Pretreatment(Fruits, vegetables, green powdered tea, and hops) (Grain, beans, nuts, and seeds)

Sample

Degreasing

Purification


Extraction

Column tube of inner diameter 15mm and length 300mm filled with 5g of silica gel and 5g of Na2SO4

Extract injected

Elution with 100mL mixture of acetone and n-hexane (1:1)

Vacuum concentration of eluate at 40˚C max.; acetone added to residue to make 5mL solution

GC-FTD, FPD

100mL separating funnel; 30mL of n-hexane saturated with acetonitrile; shaken for 5min; extracted another 2 times (3 times in total)Vacuum concentration of acetonitrile layer at 40˚C max.

Residue dissolved with a 1:1-mixture of acetone and n-hexane (approx. 5mL)

Addition of 100mL of acetone; homogenization for 3min; suction filtration through layer of diatomaceous earthHomogenization of residue and 50mL of acetone; suction filtration; vacuum concentration at 40˚C max.300mL separating funnel; 100mL of saturated NaCl solutionExtracted twice with 1:4 mixture of ethyl acetate and n-hexane solution (first time: 100mL; second time: 50mL)Dehydration of organic solvent layer; removal of organic solvent at 40˚C max.20mL of n-hexane added to residue

(Grain, beans, nuts, or seeds)

Pulverization; 420µm standard sieve

10g; 20mL of water; left for 2 hours

62

3.1 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) - GC

4

5.0 10.0 15.0 20.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0µV (×100,000)

1

2

35 6

8

9

10

7

Fig. 3.1.1 shows a chromatogram of 0.1mg/L standardorganophosphorus pesticide solution. Analysis wasperformed after adding standard organophosphorus

pesticide solution.Fig. 3.1.2 to 3.1.4 show the chromatograms of apples,spinach, and soybeans.

Fig. 3.1.3 Chromatogram of spinach (0.05µg/g standard pesticide solution added)

Instrument

Column

Column Temp.

Carrier Gas

Detector

Inj. Temp.

Det. Temp

Injection Method

Injection Volume

: GC-2010AF, FPD-2010, AOC-20i

: Rtx-1 (15m × 0.53mm I.D., df = 1.5µm)

: 80˚C(1min)-8˚C/min-250˚C(5min)

: He, 46kPa (16.5mL/min, 120cm/s,

constant-velocity mode)

: FPD-2010 (P Filter)

: 230˚C

: 280˚C

: Splitless (1min)

: 1µL

5.0 10.0 15.0 20.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

1

2

3

5 67

8

9

10

µV (×100,000)

4

Fig. 3.1.4 Chromatogram of soybeans (0.1µg/g standard pesticide solution added)

5.0 10.0 15.0 20.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

1

2

3

4

5 67

8 9 10

µV (x100,000)

■ Peaks12345

DDVP Dimethoate Diazinon IBP Methylparathion

:::::

678910

MEPMalathionChlorpyrifosProthiofosEPN

:::::

each 0.1mg/L

Fig. 3.1.1 Chromatogram of standard organophosphoruspesticide solution (0.1mg/L)

5.0 10.0 15.0 20.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0µV (×100,000)

12

3

4

5 67

8

9 10

Fig. 3.1.2 Chromatogram of apples (0.05µg/g standard pesticide solution added)


63

Residual Pesticides

3.2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (1) - GCAnalysis Based on the Rapid Analysis Method Specified in Japan's Food Sanitation Law (Notices43, 44, and 45 Issued by the Environmental Health Bureau's Food Chemistry Division in 1997)

GC-FTD, FPD (organophosphates, organonitrogens)

GC-ECD (organochlorines, pyrethroids)

1mL of extract A collected; solvent removed; dissolved in 1mL of methanol; HPLC measurement (pirimicarb)Dilute hydrochloric acid added to 0.3mL of remaining sample to make solution of 3mL; filtration; HPLC measurement (N-methylcarbamate)

HPLC (carbamates)

2mL of extract A injected into silica-gel mini-column

Eluted with 20mL of acetone and hexane (1:1)

Solvent removed; 3:17 mixture of ether and hexane added to residue to make 4mL solution (extract B)

Vegetables, fruits, green powdered tea, hops: 20gGrain, beans, nuts, seeds: 10g of sample passed through 420µm standard sieve; 20mL of water added; left for 2 hours

100mL of acetone added; homogenization and extraction for 3min; suction filtration through layer of diatomaceous earth

Filtrate residue and 50mL of acetone

Concentrated to 20mL max. at 40˚C max.; 6g of NaCl added

Total volume injected into diatomaceous-earth column; left for 10min

Container used for concentration washed with 150mL of ethyl acetate; wash liquid injected into column

Sample

GPC clean-up

Quantitative analysis

Eluted with 1:1 mixture of ethyl acetate and cyclohexaneSeparate out fraction corresponding to elution times between fluvalinate and quinomethionateConcentrated at 40˚C max.; dissolved into a 4mL (1:1) mixture of acetone and hexane (extract A)

2mL of extract injected into GPC clean-up apparatus

2mL of extract B collected; solvent removed; residue dissolved in 2mL (3:17) mixture of ether and hexane2mL of extract B injected into Florisil mini-column; eluted with 18mL (3:17) mixture of ether and hexane; eluted with 15mL (3:17) mixture of acetone and hexane; each outflow liquid concentrated; residue dissolved in hexane to make 2mL (1mL for grain or beans) solution


2mL of extract B collected; solvent removed; residue dissolved in acetone to make 2mL (1mL for grain or beans) solution


Mini-columnpurification

Extraction

■ ExplanationIn 1997, Japan's Ministry of Health, Labour and Welfareissued notification of a pesticide-residue rapid analysismethod as a simple and quick way for screening manypesticide residues. With this method, the same analysismethod can be used for many different agriculturalproducts and pesticides and some of the pretreatmentoperations can be automated using the GPC method. Thepesticides are analyzed in groups, such as chlorine,phosphorus, and nitrogen, using GC-ECD, GC-FPD, andGC-FTD. If, however, pesticide residue with aconcentration exceeding approximately 50% of theregulated value is detected using the rapid analysismethod, quantitative measurement must be carried out asstipulated by the corresponding notification. The analysisof organophosphorus pesticide is described as anexample.

■ PretreatmentExtraction with acetone is carried out on the sample and,after redissolving in ethyl acetate with a diatomaceous-earth column, GPC clean-up is performed and the sampleis purified with a silica-gel mini-column.Organophosphorus pesticides are analyzed with GC-FPDor GC-FTD after concentration. Carbamate pesticides areanalyzed by taking a sample of the solution afterperforming GPC clean-up and analyzing the sample inthis state or after diluting with hydrochloric acid.Organochlorine or pyrethroid pesticides are investigatedby performing analysis after refining first with silica geland then with a Florisil mini-column.

64

3.2 Analysis of Organophosphorus Pesticide Residue in Agricultural Products (2) - GC

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

µV(×100,000)

1

2

3

4

5 6

7

89

10

Fig. 3.2.3 Chromatogram of processed soybean liquid with0.1µg/g standard pesticide solution added

Instrument

Column

Column Temp.

Carrier Gas

Detector

Inj. Temp.

Det. Temp.

Injection Method

Injection Volume

: GC-2010AF, FPD-2010, AOC-20i

: Rtx-1 (15m × 0.53mm I.D., df = 1.5µm)

: 80˚C(1min)-8˚C/min-250˚C(5min)

: He, 46kPa (16.5mL/min, 120cm/s,


: FPD-2010 (P Filter)

: 230˚C

: 280˚C

: Splitless (1min)

: 1µL

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

0.0

2.5

5.0

7.5

µV(×100,000)

1

2

3

4

56

7

8

910

Fig. 3.2.4 Chromatogram of processed rice liquid with0.1µg/g standard pesticide solution added

5.0 10.0 15.0 20.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0µV(x100,000)

1

2

3

4

5 6 7

8 9 10

■ Peaks1 : DDVP2 : Dimethoate3 : Diazinon4 : IBP5 : Methylparathion6 : MEP7 : Malathion8 : Chloropyrifos9 : Prothiofos10 : EPN

each 0.1mg/l

Fig. 3.2.1 Chromatogram of standard organophosphoruspesticides solution (0.1mg/L)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

µV(×100,000)

1

2 34

56

7

8

9 10

Fig. 3.2.2 Chromatogram of processed spinach liquid with0.05µg/g standard pesticide solution added


ReferenceHandbook for Food Sanitation Laws, 2003 Edition,Shinnippon-hoki Publishing Co., Ltd., (2002)

65

Residual Pesticides

3.3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (1) - GC

1mL of extract A collected; solvent removed; dissolved in 1mL of methanol; HPLC measurement (pirimicarb)Dilute hydrochloric acid added to 0.3mL of remaining sample to make solution of 3mL; filtration; HPLC measurement (N-methylcarbamate)

2mL of extract B injected into Florisil mini-column; eluted with 18mL (3:17) mixture of ether and hexane (first fraction); eluted with 15mL (3:17) mixture of acetone and hexane (second fraction); each outflow liquid concentrated; residue dissolved in hexane to make 2mL (1mL for grain or beans) solution

GC-FTD, FPD (organophosphates, organonitrogens)

GC-ECD (organochlorines, pyrethroids)

2mL of extract A injected into silica-gel mini-column

Eluted with 20mL of acetone and hexane (1:1)

After removal of solvent; 3:17 mixture of ether and hexane added to residue to make 4mL solution (extract B)

Vegetables, fruits, green powdered tea, hops: 20gGrain, beans, nuts, seeds: 10g of sample passed through 420µm standard sieve; 20mL of water added; left for 2 hours

100mL of acetone added; homogenization and extraction for 3min; suction filtration through layer of diatomaceous earth

Filtrate residue and 50mL of acetone

Concentrated to 20mL max. at 40˚C max.; 6g of NaCl added

Total volume injected into diatomaceous-earth column; left for 10min

Container used for concentration washed with 150mL of ethyl acetate; wash liquid injected into column

Vacuum concentration at 40˚C max.; dissolved in 4mL (1:1) mixture of ethyl acetate and cyclohexane

2mL of extract injected into GPC clean-up apparatus

Eluted with 1:1 mixture of ethyl acetate and cyclohexaneSeparate out fraction corresponding to elution times between fluvalinate and quinomethionateConcentrated at 40˚C max.; dissolved into a 4mL (1:1) mixture of acetone and hexane (extract A)

HPLC (carbamates)

2mL of extract B collected; solvent removed; residue dissolved in acetone to make 2mL (1mL for grain or beans) solution

Sample

GPC clean-up




Mini-column purification

Extraction

■ ExplanationThe analysis of organonitrogen and pyrethroid pesticidesusing the rapid analysis method is described here. Fig. 3.3.1and 3.3.2 show chromatograms of standardorganophosphorus and standard organonitrogen pesticidesolutions analyzed under the same conditions. Bothorganophosphorus and organonitrogen pesticides can bedetected with GC-FTD. Also, in the pretreatment for therapid analysis method, both pesticides are eluted into thesame fraction. Fig. 3.3.3 shows a chromatogram ofprocessed soy bean liquid with 13 organophosphorus and14 organonitrogen pesticides added.

Fig. 3.3.4 shows a chromatogram of a standard pyrethroidpesticide solution. Separation and quantitative analysiscan be difficult for pyrethroid pesticides as there are oftenmany isomers within a standard product. Consideration isalso required for substances that convert their forms at theGC injector inlet (for example, deltamethrin changes totralomethrin). Fig. 3.3.5 shows a chromatogram ofprocessed spinach liquid with a standard pyrethroidpesticide added.

■ Pretreatment(Pretreatment for Pesticide-residue Rapid Analysis Method)

3.3 Analysis of Organonitrogen and Pyrethroid Pesticide Residue in Agricultural Products (2) - GC

66

P1 DDVP P2 Ethoprophos P3 Dimethoate P4 Diazinon P5 IBP P6 Parathion-methyl P7 Pirimiphos-methyl P8 MEP P9 Malathion P10 Chlorpyrifos P11 Parathion P12 Prothiofos P13 EPN

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0 µV (x100,000)

P1

P2

P3 P4

P5 P6

P7 P8

P9 P10

P11

P12 P13

Fig. 3.3.1 Chromatogram of standard organophosphate pesticidesolution obtained using GC-FTD

Instrument

Column

Column Temp.

Carrier Gas

Detector

Inj. Temp.

Det. Temp.

Injection Method

Injection Volume

: GC-2010AF, FPD-2010, AOC-20i, GCsolution

: BPX5 (30m × 0.25mm I.D., df = 0.25µm)

: 80˚C(1min)-20˚C/min-190˚C-5˚C/min-280˚C(5min)

: He, 143kPa (2.4mL/min, 45cm/s,


: FTD-2010

: 250˚C

: 280˚C

: High-pressure, splitless (300kPa, 1min)

: 1µL

■ Analytical Conditions 1

Instrument

Column

Column Temp.

Carrier Gas

Detector

Inj. Temp.

Det. Temp.

Injection Method

Injection Volume

: GC-17A, ECD-17, AOC-20i, GCsolution

: ZB-1 (30m × 0.25mm I.D., df = 0.25µm)

: 50˚C(1min)-25˚C/min-175˚C-10˚C/min-300˚C(4min)

: He, 150kPa (1.7mL/min, constant-pressure mode)

: ECD-17

: 280˚C

: 310˚C

: High-pressure, splitless (300kPa, 1min)

: 1µL

■ Analytical Conditions 2

N1 Isoprocarb N2 Alachlor N3 Diethofencarb N4 Paclobutrazol N5 Flutolanil N6 Pretilachlor N7 Mepronil N8 Lenacil N9 Thenylchlor N10 Tebufenpyrad N11 Pyriproxyfen N12 Mefenacet N13 Fenarimol N14 Bitertanol 1 N15 Bitertanol 2

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0µV (x10,000)

N1

N2 N3

N4

N5 N6

N7

N8 N9

N10

N11

N12 N13 N14

N15

Fig. 3.3.2 Chromatogram of standard organonitrogen pesticidesolution obtained using GC-FTD

12.5 15.0 20.02.5 5.0 7.5 10.0 17.5 22.5 min

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0 µV (x10,000)

P1

P2

P3

P4

P5

N2

P6

P7

P8

P9

N3+P10

P11

N4 N5

N6+P12

N7

N8

N9

P13

N10 N11

N12

N13 N14

N15

N1

Fig. 3.3.3 Chromatogram of processed soy bean solutionobtained using GC-FTD (13 organophosphates and14 organonitrogens: 0.2 to 0.5µg/g added)

18.012.0 13.0 14.0 15.0 16.0 17.0 min

0.00

0.25

0.50

0.75

1.00

1.25

µV (x1,000,000)

1 2 3 4

5

6

7+8

9+10+11

12

13

1

4

15

1617

18

19

20

21

22

23

24(25)

26(27)

1 Pyrethrine 12 Pyrethrine 23 Pyrethrine 34 Pyrethrine 45 Bifenthrin 6 Pyrethrine 5 7 Cyhalothrin 18 Acrinathrin 1 9 Cyhalothrin 210 Acrinathrin 2 11 Acrinathrin 312 Acrinathrin 4 13 Permethrin 114 Permethrin 215 Cyfruthrin 1 16 Cyfruthrin 2

17 Cypermethrin 118 Cypermethrin 219 Flucythrinate 120 Flucythrinate 221 Fenbalerate 122 Fenbalerate 223 Fluvalinate 24 Tralomethrin 125 Deltamethrin 126 Tralomethrin 227 Deltamethrin 2

Fig. 3.3.4 Chromatogram of standard pyrethroid pesticidesolution obtained using GC-ECD (1mg/L)

9+10+11

26(27)

12.0 13.0 14.0 15.0 16.0 17.0 18.0 min

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

µV (x100,000)

1

2

5

7+8

12

13

14

1516

17

18

19

20

21

22

23

24(25)

3 6

Fig. 3.3.5 Chromatogram of processed spinach liquid obtainedusing GC-ECD (first fraction, 0.1µg/g pesticideadded)

67

Residual Pesticides

3.4 Simultaneous Analysis of Pesticides (1) - GC/MS

■ExplanationResidual Pesticides on vegetables and fruits are a matterof concern. There are various kinds of pesticides used,among which approximately 240 are subjected toregulations in Japan. A good way of analyzing thesepesticides is simultaneous GC/MS measurement.Here, an example of a simultaneous analysis of 86pesticides using GC/MS is shown.

■Analytical ConditionsInstrumentColumnCol.Temp.

Inj.Temp.I/F Temp.Carrier Gas

: GCMS-QP5000: DB-1 30m × 0.25mmI.D. df=0.25µm: 50°C(2min)-20°C/min-130°C

-3°C/min-300°C(7min): 280°C: 280°C: He 120kPa(2min)-2kPa/min-250kPa

Component Molecular weight1 Methamidophos 1412 Dichlorvos 2203 Propamocarb 1884 Acephate 1835 Isoprocarb 1936 Fenobucarb 2077 Ethoprophos 2428 Chlorproham 2139 Bendaiocarb 22310 Dimethipin 21011 α-BHC 28812 Dimethoate 22913 Thiometon 24614 β-BHC 28815 γ-BHC 28816 σ-BHC 28817 Terbufos 28818 Diazinon 30419 Ethiofencarb 22520 Etrimfos 29221 Pirimicarb 23822 Metribuzin 21423 Bentazone 25424 Parathion-methyl 26325 Carbaryl 20126 Heptachlor 37027 Fenitrothion 27728 Methiocarb 22529 Dichlofluanid 33230 Esprocarb 26531 Pirimifos-methyl 30532 Thiobencarb 25733 Malathion 33034 Aldrin 36235 Fenthion 27836 Parathion 29137 Chlorpyrifos 34938 Diethofencarb 26739 Captan 29940 Heptachlor epoxide 38641 Pendimethalin 28142 α-Chlorfenvinphos 35843 Pyrifenox 294

Component Molecular weight44 Chinomethionat 23445 β-Chlorfenvinphos 35846 Quinalphos 29847 Phenthoate 32048 Triadimenol 29549 Vamidothion 28750 Trichlamide 33951 Methoprene 31052 Flutolanil 32353 Dieldrin 37854 Prothiofos 34455 Myclobutanil 28856 p,p'-DDE 31657 Pretilachlor 31158 Endrin 37859 Fensulfothion 30860 Chlorobenzilate 32461 p,p'-DDD 31862 o,p'-DDT 35263 Mepronil 26964 Lenacil 23465 Edifenphos 31066 Captafol 34767 p,p'-DDT 35268 Propiconazole 34169 EPN 32370 Dicofol 37071 Phosalone 36772 Mefenacet 29873 Amitraz 29374 Cyhalothrin 44975 Bitertanol 33776 Pyridaben 36477 Inabenfide 33878 Permethrin 39079 Cyfluthrin 36380 Cypermethrin 41581 Flucythrinate 45182 Fenvalerate 41983 Fluvalinate 50284 Pyrazoxyfen 43785 Deltamethrin 50386 Tralomethrin 661

Table 3.4.1 List of pesticides and molecular weights

68

3.4 Simultaneous Analysis of Pesticides (2) - GC/MS

TIC

10

2

1

3

4

5

6

7

8 9

11

14

15 16

17

18

19

21

22

2425

26

2728

32

34

40

46

48

43

50

51

5354

56

57

5859

61

62

6667

68

697171

72

73 7475

76

78

80

81

81 82

82

83

84

83

85+86

15 20 25 30 35 40 45 50

18466908

Fig. 3.4.1 Analysis of 86 pesticides using DB-1

69

Residual Pesticides

3.5 Analysis of Pesticides Using NCI (1) - GC/MS

■ExplanationTrace analysis is required for the measurement of residualpesticides in vegetables and fruits, but it is difficult toextract only pesticides, even after a cleanup pretreatment.NCI is an effective method for this analysis.Generally, positive ions are detected in massspectrometry, but negative-ion analysis may be useddepending on the compound. The negative ions of suchcompounds allow microanalysis with minimalinterference from the matrix. Trace amount of pesticidesthat cannot be detected using the conventional EI methodcan be detected by this method.

■Analytical ConditionsInstrumentColumnCol.Temp.

Inj.Temp.I/F Temp.Carrier Gas

: GCMS-QP5050A: DB-1 30m × 0.25mmI.D. df=0.25µm: 50°C(2min)-20°C/min-130°C

-3°C/min-300°C(7min): 280°C: 280°C: He 120kPa(2min)-2kPa/min-250kPa

50

0

38

51

7585

96

100

109

121

145

181

200

219

50

0

7135

100 200

Cl

Cl

Cl

Cl

Cl

Cl

14,284

Fig. 3.5.1 α-BHC mass spectrum (upper: EI, lower: NCI)

70

125 15

TIC * 1.0

183.00 * 100.0219.00 * 100.0

3.5 Analysis of Pesticides Using NCI (2) - GC/MS

Component : δ-BHC 11.867

11.863

11.867

11.75 12

183.00 * 1.0

181.00 * 1.0

217.00 * 1.0

Fig. 3.5.2 SIM chromatogram using EI

Component : δ-BHC11.908

13.905

13.5 14

35.00 * 1.0

71.00 * 1.0

Fig. 3.5.3 SIM chromatogram using NCI

Fig. 3.5.4 MC and mass spectrum using EI Fig. 3.5.5 MC and mass spectrum using NCI

10 125

α–BHCβ–BHC γ–BHC

δ–BHC

15

35.00 * 3.0TIC * 1.0

71.00 * 3.0

50

0

35

57

71

73

100

100 126 147160 180

200

197 224 253 280 306

300

318 331 344 372 393

400

Fig. 3.5.6 MC and mass spectrum using EI

50

0

43

69 95

100

113

122 150 167 183

200

221 262273 289

300

318 343

21 22 23 24

TIC * 1.0

163.00 * 100.0

50

0

45

6996

109

100

121143

157

181191

207

200

215 227 245 265

281

288

300

317 344 360373393

Fig. 3.5.7 MC and mass spectrum using NCI

10 20

cypermethrin

TIC* 1.0

205.10* 3.0207.05* 3.0

167.10* 3.0

50

0

35

55 71

106

97

100

112 126137

156

171

197

200

207

223 235 260 278

300

305 318 343 379 392

400

71

Residual Pesticides

213.15 (1.22)600

500

400

300

200

100

21.4 21.5 21.6 21.7 21.8

121.00 (1.00)255.10 (2.22)

3.6 Analysis of Pesticide Residue in Foods Using GC/MS (1) - GC/MS

■ ExplanationThe measurement of pesticide using the NCI (negativechemical ionization) method is described here as anexample. NCI enables detection with a higher degree ofsensitivity than EI (electron ionization) for somechemical compounds. NCI is particularly effective forpesticides that contain chlorine but the analysis ofpesticides not containing chlorine is also described hereas an example. Fig. 3.6.1.1 to 3.6.3.4 show EI and NCImass spectra and 10ppb SIM chromatograms forisofenphos, pyributicarb, and fenvalerate. Isofenphos andpyributicarb do not contain chlorine.


Fig. 3.6.3 EI 10ppb-SIM chromatogram of isofenphos

Instrument

[GC]

Column

Column Temp.

Inj. Temp.

I/F Temp.

Carrier Gas

Injection Method

[MS]

Scan Range

Reaction Gas

: GCMS-QP5050A

: DB-5 (30m × 0.25mm I.D. df = 0.25µm)

: 50˚C(1min)-20˚C/min-100˚C-5˚C/min-300˚C(1.5min)

: 300˚C

: 300˚C

: He 100kPa (2min) – 3kPa/min – 220kPa (3min)

: Splitless (2min)

: EI: m/z 35 to 550; NCI: m/z 10 to 550

: Isobutane

100 200 300 400 500

58

213121

1859643

255138

92

200 286

OCH(CH3)2

O

OP

S

OC2H5

(CH3)2CHNH

0

25000

50000

75000

100000

125000

Fig. 3.6.1 EI mass spectrum of isofenphos

182.05 (1.00)

1000

8000

7000

6000

5000

4000

3000

2000

21.4 21.5 21.6 21.7 21.8

Fig. 3.6.4 NCI 10ppb-SIM chromatogram of isofenphos

100 200 300 400 500

182

95 256 344216136 3020

25000

50000

75000

100000

125000

Fig. 3.6.2 NCI mass spectrum of isofenphos

72

165108

18193

166

41

330

NO N

S

O

25000

50000

75000

100000

125000

0100 200 300 400 500

Fig. 3.6.5 EI mass spectrum of pyributicarb

3.6 Analysis of Pesticide Residue in Foods Using GC/MS (2) - GC/MS

250

500

750

1000

27.5 27.6 27.7 27.8 27.9

181.15 (3.00)165.15 (1.00)

Fig. 3.6.7 EI 10ppb-SIM chromatogram of pyributicarb

100 200 300 400 5000

2500

5000

7500

10000

12500

15000

17500 125

167

152 22577

209

51 115

419

197

103

OO

O CN

Cl

Fig. 3.6.9 EI mass spectrum of fenvalerate

100

150

200

250

300

350

33.25 33.50 33.75 34.00

226.00 (3.00)163.15 (1.00)

Fig. 3.6.11 EI 10ppb-SIM chromatogram of fenvalerate

149

150

181 22342 710e3

100e3

200e3

300e3

400e3

500e3

600e3

700e3

800e3

100 200 300 400 500

Fig. 3.6.6 NCI mass spectrum of pyributicarb

25000

50000

75000

100000

27.6 27.7 27.8 27.9 28.0

149.10 (1.00)

Fig. 3.6.8 NCI 10ppb-SIM chromatogram of pyributicarb

0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

400e3

100 200 300 400 500

167

211

39112773

Fig. 3.6.10 NCI mass spectrum of fenvalerate

5000

10000

15000

20000

25000

30000

35.00 35.25 35.50

211.05 (2.00)167.05 (1.00)

Fig. 3.6.12 NCI 10ppb-SIM chromatogram of fenvalerate

73

Residual Pesticides

3.7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (1) - GC/MS

■ ExplanationAfter extracting Vegetable juice with n-hexane(concentration factor of 10), standard pesticide productswere added to concentrations of 20ng/mL and thenanalyzed with GC/MS. Library searches and quantitativeanalysis were performed on the three representativecomponents for which the elution positions are indicatedwith arrows in Fig. 3.7.2. Clean-up was not performed forthe sample.


Column

Column Temp.

Inj. Temp.

I/F Temp.

Ion Source Temperature

: GCMS-QP2010

: ZB1 30m × 0.32mm I.D. df = 0.25µm)

: 70˚C(1min)-20˚C/min-120˚C/min-

10˚C/min-270˚C(4min)

: 270˚C

: 250˚C

: 200˚C

Chlorpyrifos

Fig. 3.7.2 TIC chromatogram (enlarged) and mass chromatogram of vegetable juice

Sim

azin

e (C

AT)

Phe

nant

hren

e-d1

0

Piri

mic

arb

Chl

orpy

rifos

-met

hyl

Mal

atho

n

Chl

orpy

rifos

Pyr

ene-

d10

α-en

dosu

lfan

Mep

roni

l

Chr

ysen

e-d1

0

Am

itraz

Fen

vale

rate

Fig. 3.7.1 TIC chromatogram of standard pesticides (1ppm each)

74

Library search resultsTargetLine #: 1 Retention time: 8.400 (scan #: 109) Mass peak #: 56 Base peakRaw mode: Single 8.400 (109) Background mode: 8.425 (112)

Hit #: 1 Entry #: 67749 Library: NIST 107.LIBSI: 74 Molecular formula: C6H6Cl6 CAS: 319-84-6 Molecular weight: 288 Retention index: 0Compound:alpha.-Lindane $$ Cyclohexane, 1,2,3,4,5,6-hexachloro-,(1.

3.7 Analysis of Pesticide Residue in Vegetable Juice Using GC/MS (2) - GC/MS

■ Results of Library Searches and Quantitative Analysisα-BHC

TargetLine #: 4 Retention time: 10.450 (scan #: 355) Mass peak #: 46 Base peakRaw mode: Single 10.450 (355) Background mode: 10.425 (352)

Hit #: 1 Entry #: 19366 Library: NIST 21.LIBSI: 66 Molecular formula: C9H11Cl3NO3PS CAS: 2921-88-2 Molecular weight: 349 Retention index: 0 Compound: Chlorpyrifos

Library search results

TargetLine #: 5 Retention time: 12.592 (scan #: 612) Mass peak #: 69 Base peakRaw mode: Average 12.592-12.600 (612-613) Background mode: Average 1

Hit #: 1 Entry #: 84517 Library: NIST 107.LIBSI: 62 Molecular formula: C14H9Cl5 CAS: 50-29-3 Molecular weight: 352 Retention index: 0Compound:Chlorophenothane $$ p,p’-DDT $$ Benzene, 1,1’-(2,2,2-tricl

Library search results

Chlorpyrifos

p,p’-DDT

Compound: a-BHC

Concentration (ppb) Area ratio

m/z: 181.00 Calibration curve: Quadratic Origin: Through origin Internal standard methodf(x)=-0.000000*x^2+0.001749*x+0.000000Correlation coefficient (R) = 0.999982 Contribution (R'2): 1.000000

Compound: a-BHC

ID #: 1 m/z: 181.00Type: TargetRetention time: 8.405Area: 9821Concentration: 26.201ppb

Compound: Chlorpyrifos



Compound: p,p'-DDT



Compound: p,p'-DDT

ID #: 12 m/z: 235.00Type: TargetRetention time: 12.602Area: 14105Concentration: 30.207ppb

75

Residual Pesticides

3.8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (1) - GC/MS

■ExplanationIn recent years, there has been an increase in regulationsregarding residual pesticides in food products and moreagricultural products are subject to such regulations. Inresponse to this trend, there has been an increaseddemand for automating and speeding up the pretreatmentprocedures for the analysis of residual pesticides. TheJapnese Ministry of Health, Labour and Welfare hasissued a notice about the rapid method for analyzingresidual pesticides. This method employs GPC clean-upfor part of pretreatment in order to simultaneouslyanalyze multiple pesticide components (1997 ChemicalHygiene No. 43, 44 and 45).

■Equipment OverviewSamples extracted from food contain large quantities ofoils and pigments that will interfere with pesticideanalysis. The GPC column separates the fat and pigmentsubstances from the pesticides in the extracted samples inaccordance with their molecular size. By switching thevalve, fat and pigment substances which elute morequickly are discharged and the target pesticides are takeninto the trapping loop. The pesticides trapped in the loopare injected into the GC, separated in the GC column andthen detected in the MS section.

■Analytical Conditions

In order to further improve the rapid analysis method,Shimadzu has developed a system that connects theGC/MS and GPC clean-up systems online. By completelyautomating the GPC and GC/MS processes, the OnlineGPC-GC/MS (Prep-Q) system realizes simpler andquicker analysis of residual pesticides. Prep-Q wasdeveloped under the proposal and directives of the OsakaPrefectural Public Health Laboratory. 1) 2)

This section shows an analysis example where pesticideswere added to an actual sample (potato) and analyzed byPrep-Q.

Systems

GPC Column

GC Column

: Prep-QGPC : LC-VP SeriesGC/MS : GCMS-QP5050A

: CLNpak EV-200(Shodex 150mmL. × 2mm I.D.)

: J&Wuncoated : deactivated silica tubing

(5m × 0.53mm I.D.)pre-column : DB-5

(5m × 0.25mm I.D. df=0.25µm)analysis : DB-5

(30m × 0.25mm I.D. df=0.25µm)

■Sample Extraction and PretreatmentSample pretreatment is performed in accordance with therapid analysis method for residual pesticides.

Fig. 3.8.1 Outline of Prep-Q

Fig. 3.8.2 Flow of pretreatment

Table 1.12.1

-GPC-Mobile Phase Flow RateInjection VolumeFraction

-GC-PTVColumn TemperatureCarrier Gas

-MS-Interface TemperatureScan RangeInterval

::::

:::

:::

Fats

GPC Column

Trapping Loop

Pigments

Pesticides (1, 2 and 3)

sampleExhaust

InjectionFats Pigments Pesticides

(1, 2 and 3)

12 3

GCMS-QP5050A

LC-10A

Pesticides (1, 2 and 3)

m/z

Data

Fats Pigments

GC/MS

Homogenize 20 g of sample↓Extract with 100 mL acetone↓Filter extract and wash with 50 mL acetone↓Vacuum concentrate and add 6 g Chart salt

Load to diatom earth column↓Elute with 150 mL ethyl acetate

After vacuum concentrating,dissolve in 10 mL acetone : cyclohexane (3:7)

Inject 20µL sample into Prep-Q

Solvent extraction

Dehydration

Re-extract

GPC-GC/MS

76

3.8 Analysis of Pesticide Residue in Foods Using On-line GPC-GC/MS Prep-Q (2) - GC/MS■Example of Actual Sample AnalysisIn this case, a standard solution of pesticides was added to potato extract and the mixture was analyzed with Prep-Q.The mass chromatograms and mass spectra for four pesticide substances (fenobucarb, BHC, diazinon and permethrin)are provided as an example in Fig. 3.8.3.

■Comparison to Rapid Analysis MethodPrep-Q employs a small GPC column in the clean-up GPCsection in order to reduce the time necessary for the clean-up procedure. In addition, by injecting a large quantity ofsample into the GC, the concentration process of the rapidanalysis method can be eliminated. As a result, the analysistime per sample was reduced to about one half compared tothe conventional rapid analysis method. The amount ofsolvent used for clean-up GPC was also reduced from 200mL to 1 mL per sample. Prep-Q enables environmentallyfriendly and economical analysis of residual pesticides.

■ConclusionThe Prep-Q system, developed specifically for analyzing residual pesticides in food products, fully automates allprocedures from pretreatment and reduces analysis time and solvent consumption. Therefore, residual pesticide analysiscan be accomplished more simply and quickly than the conventional rapid analysis method. In addition, since thesystem is automated, improvements in analytical accuracy and ease of validation (for both equipment and method) canbe expected, enabling even more reliable analysis.

■References1) Study for making the GPC-GC/MS process of analyzing residual pesticides in foods online - large volume injections to GC

Osaka Prefectural Public Health Laboratory: Mikiya Kitagawa, Shinjiro Hori, et al. Food Hygienic Society of Japan,73rd Technical Symposium

2) Analysis of Residual Pesticides in Foods Using Online GPC-GC/MSOsaka Prefectural Public Health Laboratory: Mikiya Kitagawa, Shinjiro Hori, et al. Food Hygienic Society of Japan,77th Technical Symposium

min

Intensity

10 11 12 13 14 0

50000000

100000000

TIC*1.00

121.00*50.00

150.00*50.00

#1 Retention time: 12.033 (scan #: 749)Base peak: 121.20 (1927587)

m/z90 100 110 120 130 140 150

91 107

121

150

Fenobucarb

I )

I )

min

Intensity

12 13 14 0

50000000 TIC*1.00

183.00*150.00181.00*400.00


m/z90 110 130 150 170 190 210 230 250

86

109145 158

181

205

219

252

α-BHC

II)

II)

min

Intensity

13 14 15 16 0

10000000150000002000000025000000300000003500000040000000

TIC*1.00

304.00*300.00179.00*100.00


m/z90 120 150 180 210 240 270 300

88

93

111

124137

152

163

179

183

199

216 248 276 304

Diazinon

III)

III)

min

Intensity

24 25 26 0

50000000

100000000

150000000

TIC*1.00

183.00*150.00184.00*300.00


m/z90 100 110 130 150 170 180

89 115129 147 163

168

183

Permethrin1

IV)

IV)

Permethrin2

I ) Fenobucarb0.05 ppm

II) α-BHC0.025 ppm

III) Diazinon0.05 ppm

IV) Permethrin0.05 ppm

Fig. 3.8.3 Mass Chromatograms and mass spectra for pesticides in potato extract

Rapid Analysis Method

Extraction Re-extraction

Concentration

30min 60min + 30min 40min 170min10min

30min 40min 80min10min

GPC + (concentration) (solid phase column)

Simultaneous multi-substance

GC/MS measurementPesticide A, B, C, D ...

Simultaneous multi-substance

GC/MS measurementPesticide A, B, C, D ...

Can be operated at night.

Dehydration

Extraction Re-extraction GPCDehydration

Online GPC-GC/MS Method

Fig. 3.8.4 Comparison of analysis times for rapid analysis and on-line GPC-GC/MS methods

77

Residual Pesticides

3.9 Analysis of Pesticides with Specific Threshold Levels in Foods (1) - LC

■ ExplanationThe regulations on pesticide residue in foods based on the

Food Sanitation Law have undergone many revisions

since October 1992 and, as of January 2005, regulated

values were specified for 244 of pesticides. The analysis of

some standard pesticides for which HPLC is used is

described here as an example.


Mobile Phase

Flow Rate

Temperature

Detection



: 1.0mL/min

: 40˚C

: SPD-10AVVP 254nm min0 2 4 6 8 10 12

mA

U

0

200

400

600

121:FenpyroximateZ

2:FenpyroximateE

■ Peaks

Fig. 3.9.1 Chromatogram of fenpyroximate

■ Analysis of FenpyroximateThe fenpyroximate content can be obtained from the sum

of fenpyroximate-E and fenpyroximate-Z.


Mobile Phase

Flow Rate

Temperature

Detection

: Shodex Asahipak NH2P-50 4E (250mmL. × 4.6mm I.D.)


: 0.8mL/min

: 40˚C

: SPD-10AVVP 215nmmin

0 2 4 6 8 10 12

mA

U

0

50

1001

1:Cyromazine ■ Peak

Fig. 3.9.2 Chromatogram of cyromazine

■ Analysis of CyromazineBecause cyromazine has a high polarity, there is

insufficient retaining power with an ODS column and so

an aminopropyl column is used.

78

3.9 Analysis of Pesticides with Specific Threshold Levels in Foods (2) - LC


Mobile Phase

Flow Rate

Temperature

Detection


: 50mM KH2PO4/Methanol = 17/3 (v/v)

: 0.8mL/min

: 40˚C

: SPD-10AVVP 270nm

min0 2 4 6 8 10 12 14

mA

U

0

1000

2000 11:Nitenpyram

■ Peak

Fig. 3.9.3 Chromatogram of nitenpyram

■ Analysis of NitenpyramIn the nitenpyram testing method, the nitenpyram content

is obtained by analyzing nitenpyram with HPLC and

analyzing its CPF metabolite with GC.


Mobile Phase

Flow Rate

Temperature

Detection



: 0.8mL/min

: 40˚C

: SPD-10AVVP 250nm min0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0

mA

U

0

50

100

1

2

3

4 5

6 7

1:Diflubenzuron 2:Tebufenozide 3:Hexaflumuron 4:Teflubenzuron 5:Lufenuron 6:Flufenoxuron 7:Chlorfluazuron

■ Peaks

Fig. 3.9.4 Chromatogram of 7 pesticides including chlorfluazuron

■ Analysis of ChlorfluazuronSeven pesticides, including chlorfluazuron, can be

analyzed simultaneously.

79

Residual Pesticides

3.10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (1) - GC/MS

■ ExplanationIn the analysis of pesticide residue in agriculturalproducts, fats and pigments in the sample can causecontamination of the GC or GC/MS injection port andpeaks that interfere with the target components and sothey must be removed as part of the pretreatment process.The conventional solvent extraction method requiresconsiderable time and effort and so difficulties arise whenprocessing large numbers of samples.GPC (gel permeation chromatography) is a technique thatseparates the sample components by molecular size.Using this technique, the pesticide components can beeasily separated from the fats and pigments, which haverelatively large molecular weights, and clean-up can beautomated. For this reason, GPC is adopted as one of theclean-up methods in the pesticide-residue rapid analysismethod prescribed by Japan's Ministry of Health, Labourand Welfare (Notice 43 issued by the EnvironmentalHealth Bureau's Food Chemistry Division on 8 April1997).The principle of the GPC clean-up method and anapplication example of Shimadzu's GPC Clean-upSystem are described here.

[References]1) Committee for Studying and Developing the Pesticide-residue Rapid Analysis Method: Food Hygiene Research,Vol. 47, P35 (1997)2) Isao Saito: LCtalk, Vol. 35, P3 (1995)

Packingmaterial

Fig. 3.10.1 Principle of GPC clean-up method

■ Principle of GPC Clean-up MethodFig. 3.10.1 shows the principle of the GPC clean-upmethod. There are small holes (pores) of a fixed size inthe packing material of the GPC column. Components inthe sample with a small molecular size (e.g., pesticides:gray sections in the figure) can permeate deep into thepores while constituents with a large molecular size (e.g.,fats and pigments: striped sections in the figure) cannot.For this reason, fats and pigments are eluted from thecolumn sooner than pesticides*) and so the sample can bepurified by fractionating this pesticide eluate.

*)In practice, the separation process is not onlyaffected by the molecular size but also by theadsorption onto the packing material.

■ Pretreatment for the Pesticide-residue Rapid Analysis Method

Fig. 3.10.2 shows the different stages in the pesticide-residue rapid analysis method. With the notified method(individual analysis method), fats and pigments wereremoved using liquid-liquid extraction and solid-phaseextraction, whereas with the pesticide-residue rapid

analysis method, they are removed using GPC. Thepesticide-residue rapid analysis method makes it possibleto perform pretreatment for all the pesticides together inalmost the same amount of time required by the notified(individual analysis) method.

Pirimicarb

N-methylcarbamatesHydrochloric acid processing

Acetoneextraction

GPCclean-up

Organophosphates, organonitrogens

Organochlorines, pyrethroids

Florisilcolumn

purification

Silica-gelcolumn

purification

Diatomaceous-earthcolumn solid-phase

extraction

Fig. 3.10.2 Stages of pretreatment using the rapid analysis method

80

3.10 GPC Clean-up Method Used in the Analysis of Pesticide Residue in Foods (2) - LC■ Fractionation ConditionsThe fractionation conditions for extracting a rice samplein accordance with the rapid analysis method andpurifying it with Shimadzu's GPC Clean-up System aregiven in Table 3.10.1. The corresponding chromatogramis shown in Fig. 3.10.3.

Fig. 3.10.3 also shows the chromatogram for twopesticides, fluvalinate and quinomethionate, obtainedwith GPC.

In general, the pesticides that are analyzed with the rapidanalysis method are eluted between fluvalinate andquinomethionat and so fractionation is performed for theinterval between the elution times of these twoconstituents.


Fig. 3.10.3 GPC chromatogram of rice extract

Instrument

Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Detection

Injection Method

: GC-2010

: Rtx-1 (15m × 0.53mm I.D. df = 1.5µm)

: 80˚C(1min)-8˚C/min - 250˚C(10min)

: 230˚C

: 280˚C

: He, 16.5mL/min

: FPD-2010

: Splitless (1min)

■ Analysis Example for Organophosphorus PesticidesFig. 3.10.4 shows the result obtained by purifyingsoybean, to which organophosphorus pesticides are added,using Shimadzu's GPC Clean-up System, purifying with asilica-gel mini-column, redissolving with acetone, andthen analyzing the sample solution using GC.

Fig. 3.10.5 shows the result obtained by processingsoybean, to which organophosphorus pesticides are added,in accordance with the notified (individual analysis)method, and analyzing with GC. It can be seen that almostidentical results are obtained with both methods.

Fig. 3.10.5 Chromatogram of organophosphorus pesticides obtainedusing the notified (individual analysis) method

Instrument

Column

Mobile Phase

Flow Rate

Detection

: Shimadzu GPC Clean-up System

: CLNpak EV-G+CLNpak EV-2000

: A: Ethyl Acetate B: Cyclohexane

A/B = 1/4 (v/v)

: 4.0mL/min

: SPD-10AVP 254nm

Table 1.14.1 Fractionation conditions

Fig. 3.10.4 Chromatogram of organophosphorus pesticides obtainedusing the rapid analysis method

min0 5 10 15 20 25 30 35 40

Fluvalinate

Chinomethionat

Rice sampleStandard sample

FIG.4

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

1.0

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

DD

VP

Dim

etho

ate

Dia

zino

nIB

PP

arat

hion

-met

hyl

ME

P Mal

athi

on

Pro

thio

fos

EP

N

Chl

orpy

rifos

FIG.5

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

0.00

0.25

0.50

0.75

1.00

1.25

DD

VP

Dim

etho

ate

Dia

zino

nIB

P Par

athi

on-m

ethy

lM

EP

Mal

athi

on

Pro

thio

fos

EP

N

Chl

orpy

rifos

81

Residual Pesticides

3.11 Analysis of Carbamate Pesticides - LC

■ Analytical Conditions[Separation]ColumnMobile PhaseFlow RateTemperature[Detection]Reaction Reagent 1Flow RateTemperatureReaction Reagent 2Flow RateTemperatureDetection

: Shim-pack FC-ODS (75mm × 4.6mm I.D.): Water/Methanol (Gradient Elution Method): 1.0mL/min: 50˚C

: 50mM NaOH: 0.5mL/min: 100˚C: OPA Solution: 0.5mL/min: 50˚C

: RF-10AXL Ex: 340nm Em: 445nm

■ ExplanationN-methylcarbamate pesticides are used widely asinsecticides and herbicides. In a publication issued by theJapanese government in 1994 (Ministry of Health,Labour and Welfare, Notice 199), post-columnfluorescent derivatization using HPLC was adopted as themethod for analyzing N-methylcarbamate pesticides.N-methylcarbamate pesticides undergo hydrolysis inalkaline conditions and generate methylamine, which is aprimary amine that can be analyzed by fluorescentdetection after fluorescent derivatization.

min

0 5 10 15 20 25

1. Oxamyl2. Methiocarb-sulfoxide3. Methiocarb-sulfone4. Aldicarb5. Bendiocarb6. Carbaryl7. Ethiofencarb8. Fenobucarb9. Methiocarb

1

23

4

56

78

9

■ Peaks

Fig. 3.11.1 Analysis of standard N-methylcarbamate pesticides (1ppm each, 10µL injected)

min

0 5 10 15 20 25

1. Oxamyl2. Methiocarb-sulfoxide3. Methiocarb-sulfone4. Aldicarb5. Bendiocarb6. Carbaryl7. Ethiofencarb8. Fenobucarb9. Methiocarb

12 3

4

56

7

8 9

■ Peaks

Fig. 3.11.2 Analysis of standard N-methylcarbamate pesticides (5ppm each, 10µL injected)

■ High-sensitivity Analysis Example forN-methylcarbamate Pesticides

Fig. 3.11.2 shows the results of injecting 10µL of asample with a concentration of 5ppb and performing

high-sensitivity analysis.

■ Rapid Analysis of N-methylcarbamate PesticidesFig. 3.11.1 shows the result of analyzing nine standardsubstances, including the N-methylcarbamate pesticidesmentioned in Notice 199 issued by Japan's Ministry ofHealth, Labour and Welfare. By using the high-separationFC-ODS column as the analysis column, and optimizingthe gradient program, methiocarbs, which are the slowest

to elute, can be eluted in about 25 minutes. Because thecolumn is shorter than those employed in conventionalmethods, gradient re-equilibration time and columncleaning time are also reduced. The time for one analysiscycle can be reduced to 32 minutes.

82

3.12 Analysis of Imazalil in Oranges - LC

■ExplanationFungicide imazalil is mostly contained in importedoranges and bananas imported to Japan. Here, analysis ofimported oranges will be introduced. The target component was confirmed by comparison withUV spectrum of standard Sample using a photodiodearray UV-VIS detector.


■PretreatmentPerformed in accordance with Standard Methods ofAnalysis for Hygienic Chemists, annotation(supplement 1995)

■Analytical ConditionsColumnMobile Phase

Flow RateTemperatureDetection

: STR ODS-II (150mmL. × 4.6mm I.D.): 5mM (Sodium) Phosphate Buffer(pH6.9)

/Acetonitrile=45/55 (v/v): 1.0mL/min: 40°C: Photodiode Array Detectionλ=210nm to 300nm

mAbs

10

5

00 5

Ch1 220nm 1

10min

1. imazalil■ Peaks

Fig. 3.12.1 Chromatogram of imazalil in imported orangesample (220nm)

Peak spectrum16

0

mAbs

250Wavelength (nm)

300

Peak spectrum3

mAbs

0250

Wavelength (nm)300

Fig. 3.12.2 Spectra of imazalil (upper: standard sample, lower: sample)

83

Residual Pesticides

3.13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (1) - LC/MS

2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 min

250e3

500e3

750e3

1000e3

1250e3

1500e3

1750e3

2000e3

Int.

1:305.95(1.00)

2:537.80(8.00)2:486.90(8.00)2:458.85(4.00)2:308.90(4.00)2:327.90(40.00)2:347.05(30.00)2:337.00(4.00)2:268.90(4.00)1:422.00(11.36)

1:328.90(2.46)1:345.90(2.59)1:402.90(10.53)1:353.05(2.19)1:268.95(1.00)1:339.90(1.28)1:296.85(1.00)1:333.95(7.24)1:221.90(2.44)1:201.85(1.00)

1:305.95(1.00)

2:537.80(8.00)2:486.90(8.00)2:458.85(4.00)2:308.90(4.00)2:327.90(40.00)2:347.05(30.00)2:337.00(4.00)2:268.90(4.00)1:422.00(11.36)

1:328.90(2.46)1:345.90(2.59)1:402.90(10.53)1:353.05(2.19)1:268.95(1.00)1:339.90(1.28)1:296.85(1.00)1:333.95(7.24)1:221.90(2.44)1:201.85(1.00) 1

2

3

4

56

7

89

10

11

12

201918

171615

14

13

Fig. 3.13.1 Mass chromatogram of pesticides in agricultural products

■ ExplanationUnder Japan's Food Sanitation Law, the levels ofpesticide residue in agricultural products are strictlyregulated, and at present there are 244 pesticides for 262types of agricultural products. Out of the regulatedpesticides, LC is used to analyze non-volatile pesticidesor pesticides that are easily decomposed by heating.Because there are many impurities in food extracts,qualitative determination using LC/MS, which uses amass spectrometer for detection and thereby offersgreater selectivity, is increasingly employed for thepurpose of verification.

The batch analysis of 20 residual pesticides inagricultural products is described here as an example.Fig. 3.13.1 shows a mass chromatogram obtained in scanmode. There is no need to perform optimization for eachof the pesticides and high-sensitivity analysis is possibleunder the conditions set with autotuning. Also, if multi-sequence mode is used, reliable qualitative andquantitative determination is possible with one analysisby performing mass chromatography with positive ions ornegative ions using the mass numbers of each of thepesticides.

■ PeaksESI-Positive mode1. thiabendazole MW 2012. methabenzthiazuron MW 2213. furametpyr MW 3334. imazalil MW 2965. etobenzanid MW 3396. daimuron MW 2687. tebufenozide MW 3528. pyrazoxyfen MW 4029. triflumizole MW 34510. pencycuron MW 32811. buprofezin MW 30512. fenpyroximate MW 421

ESI-Negative mode13. imibenconazole-debenzyl MW 27014. inabenfide MW 33815. myclobutanil MW 28816. iprodione metabolite MW 32917. diflubenzuron MW 31018. hexaflumuron MW 46019. flufenoxuron MW 48820. chlorfluazuron MW 539

84

3.13 Analysis of Pesticide Residue in Agricultural Products Using LC/MS (2) - LC/MSFig. 3.13.2 shows SIM chromatograms and calibrationcurves (n=5) for pencycuron (12.5pg) and hexaflumuron(25pg) and Table 3.13.1 and 3.13.2 give thereproducibility results for each substance. As shown in

this example, constituents with roughly the sameretention times and different measurement modes(positive and negative ions) can be quantified at the sametime.

10 15 min

2500

2750

3000

3250

3500

Int. 328.90(1.00)

14.4

70

0 25 50 75 Conc0e3

100e3

200e3

300e3

400e3

500e3

Area

Cl CH2

N CONH

C19H21ClN2OExact Mass: 328.13

Mol. Wt.: 328.84

Y = (5655.35133)X + (4034.05823) r2=0.99982

10 15 min

1800

1900

2000

2100

2200

2300

2400

Int. 458.85(1.00)

14.1

70

0 50 100 150 200 Conc0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

400e3

450e3Area

F

CONHCONH

F

O

Cl

Cl

CF2CHF2

C16H8Cl2F6N2O3

Exact Mass: 459.98Mol. Wt.: 461.14

Y = (1766.53398)X + (-192.42839) r2=0.99998

Fig. 3.13.2 SIM chromatogram and calibration curves for pencycuron and hexaflumuron

ColumnMobile PhaseGradient Program

Flow RateColumn TemperatureInjection VolumeProbe VoltageCDL TemperatureBlock Heater TemperatureNebulizer Gas Flow RateCDL VoltageQ-array DC VoltageQ-array RFScan Range

: Shim-pack VP-ODS (150mmL. × 2.0mm I.D.): A: Water B: Acetonitrile: 20% B → 60% B (0.03min) → 80% B (20min) → 100% B (20.01-30min) →20% B (30.01-40min)

: 0.2mL/min: 40˚C: 5µL: +4.5kV (ESI-Positive Mode), -3.0kV (ESI-Negative Mode): 200˚C: 200˚C: 4.5L/min: +0V (ESI-Positive Mode), +0V (ESI-Negative Mode): Scan Mode: Scan Mode: m/z 50-650 (1.5sec/scan)


Table 3.13.2 Repeatability for hexaflumuron

Table 3.13.1 Repeatability for pencycuron

12.5pg25pg50pg

125pg250pg500pg

1139173371067238150565289289586968

2145263371068996145253287762581783

3140183024269932

144468270265560675

4139483079366772

152698288482575145

5152513133161325

140439273127551669

Average14332.00 31957.20 66852.60

146684.60 281785.00 571248.00

Standard deviation570.191631645.75323346.12684929.21899281.106

14734.456

CV3.98 % 5.15 %5.01 %3.36 %3.29 %2.58 %

25pg50pg

125pg250pg500pg

1250pg

111153186904288181842174911437000

28984192294372689280177600437627

39859

204734000190536

180001439882

49766

185804484086530

174125434111

59007

177624364691154

172789460184

Average9753.80

18946.80 43018.80 87868.40

175885.20 441760.80

Standard devaition883.074011001.644

1825.95093808.30292894.571310502.585

CV9.05 %5.29 %4.24 %4.33 %1.65 %2.38 %

85

Residual Pesticides

3.14 Analysis of N-methylcarbamate Pesticides Using LC/MS (1) - LC/MS

H3C C

H3CS

NOCONHCH3

OCON

N

N

CH3

H3C

H3C

CH3

CH3

CH3

N

OCONHCH3

CH3

CH3O

O

C

H3CHNOCO

OCONHCH3

CH2SC2H5

OCONHCH3

CH3

CH3SH3C

100 125 150 175 200 225 250 275 m/z0e3

250e3

500e3

750e3

1000e3

Int.239.15

240.30168.30

100 125 150 175 200 225 250 275 m/z0e3

50e3

100e3

150e3

200e3Int.

224.05

183.90167.30 255.65184.65 226.05

100 125 150 175 200 225 250 275 m/z0e3

250e3

500e3

750e3Int.

208.10

240.05152.45

100 125 150 175 200 225 250 275 m/z0e3

100e3

200e3

300e3

400e3

500e3Int. 226.05

258.20168.90 207.95

100 125 150 175 200 225 250 275 m/z0e3

100e3

200e3

300e3

Int.223.05

255.05

239.65166.00 254.00147.85

100 125 150 175 200 225 250 275 m/z0e3

100e3

200e3

300e3Int.

226.05

164.05

106.80258.05

100 125 150 175 200 225 250 275 m/z0e3

50e3

100e3

150e3

200e3

250e3Int. 202.05

144.95

234.35195.05 219.05

100 125 150 175 200 225 250 275 m/z0e3

50e3

100e3

150e3

Int.163.00

106.00

282.75179.20195.15

OCONHCH3

CHC2H5

H3C

CHCS N

CH3

CH3O

NH

CH3O CH3CO

O

1. Aldicarbsulfone M.W. 222 5. Carbaryl M.W. 201

2. MethomylM.W. 162

6. Ethiofencarb M.W. 225

3. Primicarb M.W. 238 7. Fenobucarb M.W. 207

4. Bendiocarb M.W. 223 8. Methiocarb M.W. 225

Fig. 3.14.1 Structures and mass spectra of n-methylcarbamate pesticides

■ ExplanationN-methylcarbamate pesticides are used widely ininsecticides and herbicides and their residue inagricultural products has become an issue of concern. Themethod of separating eight n-methylcarbamate pesticideswith an HPLC column, performing on-line hydrolysis,and applying fluorescent derivatization to the resultingmethylamine is adopted as the testing method in revisionsto the standards for foods and additives made by Japan'sMinistry of Health, Labour and Welfare. Here, however,

the analysis of n-methylcarbamate pesticides directlyusing LC/MS in order to increase the simplicity andsensitivity, without applying derivatization, is describedas an example.

Fig. 3.14.1 shows the structures and mass spectra of n-methylcarbamate pesticides. The protonated moleculescan be confirmed in each case.

86

3.14 Analysis of N-methylcarbamate Pesticides Using LC/MS (2) - LC/MSFig. 3.14.2 shows the chromatograms obtained when 5µL(5ng) of a 1ppm mixture of eight n-methylcarbamatepesticides is injected. The components that do not haveclear peaks in the TIC chromatogram can be qualitativelyanalyzed easily by drawing mass chromatograms basedon the characteristic mass numbers.

Fig. 3.14.3 shows the SIM chromatogram for fenobucarbat 40pg (8ppb, 5µL) and Fig. 3.14.4 shows the calibrationcurve (n = 5) between 40pg and 5ng. The CV values atdifferent concentration are in the range 2% to 6%; highlyprecise results are obtained.

9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 min0e3

500e3

1000e3

1500e3

2000e3

2500e3

3000e3

3500e3

4000e3

4500e3

5000e3

Int.1

/Ald

ica

rbsu

lfon

2/M

eth

om

yl

3/P

rim

ica

rb

4/B

en

dio

carb

5/C

arb

ary

l

6/E

thio

fen

carb

7/F

en

ob

uca

rb8

/Me

thio

carb

TIC223.05163.00239.15224.05202.05226.05208.10

Fig. 3.14.2 TIC and mass chromatograms for n-methylcarbamate pesticides

ColumnMobile PhaseGradient ProgramFlow RateColumn TemperatureInjection VolumeProbe VoltageCDL TemperatureProbe TemperatureNebulizer Gas Flow RateCDL VoltageDeflector VoltageScan Range

: Shim-pack STR-ODS (150mmL. × 2.0mm I.D.): A: 0.2% Acetic Acid Solution B: Acetonitrile containing 0.2% Acetic Acid: 0% B (0min) → 100% B (20min): 0.2mL/min: 40˚C: 5µL: +4.5kV (APCI-Positive Mode): 230˚C: 200˚C: 2.5L/min: -30V: +30V: m/z 100-400 (1.2sec/scan)


18.50 19.00 19.50 20.00 20.50 min0

500

1000

1500

2000

2500

3000

Int.

19.4

00208.10

Fig. 3.14.3 SIM chromatogram of fenobucarb (40pg)

0.0 1.0 2.0 3.0 4.0 Conc0e3

100e3

200e3

300e3

400e3

500e3

600e3

700e3

800e3

Area

ID#:7 Mass:208.10 Name:Fenobucarb F(×)=175642.62*×+0.00 r2=1.000000

No.1234

Conc.0.0400.2001.0005.000

Area8951.84

36587.88176493.00877969.22

Fig. 3.14.4 Calibration curve for fenobucarb

87

Residual Pesticides

3.15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (1) - LC/MS

50 100 150 200 250 300 350 400 450 m/z0.0e6

2.5e6

5.0e6

7.5e6

10.0e6

12.5e6

Int.

215

218 237187172 395131 46928894 422107 48545136058 79 322305 340146

50 100 150 200 250 300 350 400 450 m/z0.0e6

2.5e6

5.0e6

7.5e6

10.0e6

Int.

200

399222 421244184 443260172 46730415467 49257 276143131 336 36795 320 353 38284 107

50 100 150 200 250 300 350 400 450 m/z0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

Int.

185

197207 227 391105 295167 257142 268 317 425280 369128 155 243 404341 49766 82 46945357 482

50 100 150 200 250 300 350 400 450 m/z0

2500

5000

7500

10000

12500

15000

Int.

274

170

184

393216

229

197408239 266128 313151 369 385 445299 43265 139117 355340104 47445991 500428

50 100 150 200 250 300 350 400 450 m/z0

5000

10000

15000

20000

25000

Int.

198

183

168

152

40728022855 141 24312292 213 430258 445314294 339 49669 368 464352 396 478

50 100 150 200 250 300 350 400 450 m/z0e3

100e3

200e3

300e3

400e3

500e3

Int.

183

243168153 265207113 228 28189 356297 368141 322193 461250 435 484416405 4997150

50 100 150 200 250 300 350 400 450 m/z0e3

50e3

100e3

150e3

200e3

250e3

300e3

350e3

Int.

168

228

250200183 266153 332 441131111 392348 48965 318 36397 424283 378 4748450 456

50 100 150 200 250 300 350 400 450 m/z0e3

100e3

200e3

300e3

400e3

500e3

600e3

700e3

Int.

198

419258 397280 435362296168112 232 31418655 378216 46033698 12877 489154

Metribuzin

APCI-positive mode APCI-negative mode

Metribuzin

DA DA

DK DK

DADK DADK

Fig. 3.15.2 Positive and negative APCI mass spectra of metribuzin and its metabolites

■ ExplanationMetribuzin, which is a triazine-group herbicide for annualweeds on potato, asparagus, and sugarcane fields, parksand roads, was included in an announcement made by theJapanese Environment Agency regarding the 67 types ofchemical substances that are suspected of disrupting theendocrine system. Metribuzin has not yet been subjectedto a thorough endocrinological investigation and soendocrine disruption has not been categoricallyestablished. It is, however, suspected of being anendocrine disruptor because of its reproductive toxicityand carcinogenicity.

In the 67 types of chemical compounds suspected ofbeing endocrine disruptors, 44 are herbicides,insecticides, or germicides. Of these 44, there are 22 that

are not registered or are invalid as pesticides in Japan.Metribuzin, however, is in active use and in order toinvestigate whether or not it is an endocrine disruptor, itis necessary to establish an easy analysis method andmonitor metribuzin in the environment.

Japan's Food Sanitation Law specifies residue thresholdlevels not only for metribuzin itself but also for thecombined total including its metabolites, desamino (DA),diketo (DK, methylthio-based desorption oxidant), anddesaminodiket (DADK). The analysis of metribuzin andits metabolites using atmospheric pressure chemicalionization (APCI) LC/MS is described here as anexample. Fig. 3.15.1 shows the structures of metribuzinand its metabolites.

NN

N

O

SCH3

NH2(H3C)3C

MetribuzinC8H14N4OS


NN

NH

O

SCH3

(H3C)3C

Desaminometribuzin (DA)C8H13N3OS

Exact Mass: 199.08Mol. Wt. : 199.27

NNH

N

O

O

NH2(H3C)3C

Diketometribuzin (DK)C7H12N4O2


NNH

NH

O

O

(H3C)3C

Desaminodiketmetoribuzin (DADK)C7H11N3O2


Fig. 3.15.1 Structures of metribuzin and its metabolites

88

3.15 Analysis of Metribuzin Using Positive and Negative Ion Atmospheric Pressure Chemical Ionization (2) - LC/MS

In positive APCI mass spectra for metribuzin, DA, andDK, protonated molecules can be observed as standardpeaks. The ion intensity is low for DADK protonatedmolecules (m/z 170) and the m/z 274 (M-H + 2Na +AcOH)+ ion is observed instead. In negative APCI massspectra, the (M-H)

_molecular ion type can be observed

as standard peaks for DA, DK, and DADK; metribuzinitself is observed as fragment ions m/z 199, 180, and 169,but the m/z 213 molecular ion type can hardly beobserved. These results are thought to reflect thedifferences in the proton affinity of the compounds. Inorder to analyze metribuzin and its metabolites, however,

the analysis of positive and negative ions is required. Fig.1.19.3 shows the results of analyzing metribuzin and itsmetabolites using positive and negative ions. DK andDADK were detected with negative ions m/z 183 and168, and metribuzin and DA were detected with positiveions m/z 215 and 200. The metribuzin, DA, DK, andDADK calibration curves (3.2 - 2,000 ppb, n = 5) showedgood linearity at Y = 38312 X + 48246 (r2 = 0.9998), Y =3146 X + 21011 (r2 = 0.9999), Y = 1527 X + 3320 (r2 =0.9999), and Y = 1596 X + 1764 (r2 = 0.9999)respectively, which means that highly accuratequantitative analysis is possible.

2.5 5.0 7.5 10.0 12.5 min

8000

9000

10000

11000

12000

Int.

2.5 5.0 7.5 10.0 12.5 min

8000

9000

10000

11000

12000Int.

2.5 5.0 7.5 10.0 12.5 min

7250

7500

7750

8000

8250

8500

Int.

2.5 5.0 7.5 10.0 12.5 min

7500

7750

8000

8250

8500

Int.

2.5 5.0 7.5 10.0 12.5 min0e3

50e3

100e3

150e3

200e3

250e3

Int.

2.5 5.0 7.5 10.0 12.5 min0e3

50e3

100e3

150e3

200e3

250e3

Int.

2.5 5.0 7.5 10.0 12.5 min0e3

250e3

500e3

Int.

2.5 5.0 7.5 10.0 12.5 min0e3

250e3

500e3

750e3Int.

2000 ppb

DK (m/z 183.2) DK (m/z 183.2)

DADK (m/z 168.2) DADK (m/z 168.2)

Metribuzin (m/z 215.2) Metribuzin (m/z 215.2)

DA (m/z 200.2) DA (m/z 200.2)

3.2 ppb

Fig. 3.15.3 Positive and negative SIM chromatograms of metribuzin and its metabolites (2,000ppb and 3.2ppb)

ColumnMobile PhaseGradient ProgramFlow RateColumn TemperatureInjection VolumeProbe VoltageCDL TemperatureProbe TemperatureNebulizer Gas Flow RateCDL VoltageDeflector VoltageScan Range

: Insertsil ODS-2 (150mmL. × 2.1mm I.D.): A: 0.2% Acetic Acid Solution B: Methanol containing 0.2% Acetic Acid: 30% B (0min) → 90% B (13-15min): 0.2mL/min: 40˚C: 50µL: +4.5kV (APCI-Positive Mode), -3.0kV (APCI-Negative Mode): 230˚C: 400˚C: 2.5L/min: -30V (Positive), +30V (Negative): +30V (Positive), -20V (Negative): m/z 215.2, 200.2 (Positive), m/z 183.2, 168.2 (Negative)


89

Residual Pesticides

3.16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (1) - LC/MS

■ ExplanationFluazifop, quizalofop-butyl, and other phenoxypropionic-acid herbicides are used widely throughout the worldbecause they have strong herbicidal effects at low doses.The active substance is carboxylic acid-based and inhibitsthe biosynthesis of fatty acids by acetyl-CoA carboxylaseinhibition. In Japan, the residue of these herbicides is anissue of concern and official testing methods forquizalofop-ethyl, cyhalofop-butyl, and fluazifop havebeen established. The total amount of quizalofop ismeasured using HPLC or LC/MS after hydrolysis ofquizalofop-ethyl, and the total amount of fluazifop ismeasured by performing GC or GC/MS on the estersformed from butyl esterification after hydrolysis.

The batch analysis of fluazifop, fluazifop-butyl,quizalofop, and quizalofop-ethyl using electrosprayionization (ESI) is described here. The ESI methodeffectively ionizes carboxylic acid-based herbicides(fluazifop and quizalofop) with negative ions and theester-based herbicides (fluazifop-butyl and quizalofop-ethyl) with positive ions. Fig. 3.16.1 shows the massspectra of these compounds. The deprotonated molecules(M-H)

_of the carboxylic acid-based herbicides in

negative ion mode and the protonated molecules (M+H)+

of the ester-based agricultural chemicals in positive ionmode can be confirmed.

N

O

F3C O COOH

CH3

50 100 150 200 250 300 350 400 450 500 550 m/z0

2500

5000

7500

10000

12500

15000

Int.

FluazifopC15H12F3NO4

Exact Mass : 327.07Mol. Wt. : 327.26

50 100 150 200 250 300 350 400 450 500 550 m/z0

25000

50000

75000

Int.

Cl O COOH

N

N

OCH3

QuizalofopC17H13ClN2O4

Exact Mass : 344.06Mol. Wt. : 344.75

100 150 200 250 300 350 400 450 500 550 m/z0

2500

5000

7500

Int.

Quizalofop-ethylC19H17ClN2O4

Exact Mass : 372.09Mol. Wt. : 372.80

Cl O COOC2H5

N

N

OCH3

50 100 150 200 250 300 350 400 450 500 550 m/z0e3

100e3

200e3

300e3

400e3

Int.

Fluazifop-butylC19H20F3NO4

Exact Mass : 383.13Mol. Wt. : 383.36

N

O

F3C O COOC4H9

CH3

ESI Positive

ESI Positive

ESI Negative

ESI Negative

326

254

588487438412198 272 344156 295 502 569415133 53556 102 241179 21778 468117 399 442375332 600

373

83

391

60414259 27911494 415238190163146 320262 306209 47034974 428 537508455 559 592226

343

345 381546271

467235 503525476

32059 403 49642836420089 536 584388220105 555254170 444123 29367 185151

384 425

402386106 224147 369126 279 42960

Fig. 3.16.1 ESI mass spectra of phenoxypropionic-acid herbicides

90

3.16 Analysis of Phenoxypropionic-acid Herbicides Using LC/MS (2) - LC/MS

When using a reversed-phase column, the retention timeof ester-based herbicides is longer than that of carboxylicacid-based herbicides. Therefore, it is possible to analyzeboth carboxylic acid-based and ester-based herbicides atthe same time by first selecting negative ion detection,and then, after the elution of carboxylic acid-based

herbicides is complete, switching to positive ion detection(Fig. 3.16.2).Good calibration curves were produced for eachsubstance at concentrations in the range 0.8ppb to500ppb. Fig. 3.16.3 shows the calibration curve and SIMchromatogram at 0.8ppb for fluazifop.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 min

50e3

100e3

150e3

200e3

250e3

300e3

350e3

400e3

450e3

Int.

each 500 ppb, 5 µL inj.

373.00(2.00)343.00(5.00)384.00(1.00)326.00(4.00)

Quizalofop

Fluazifop-butyl

ESI Positive (18.5-30min)

Quizalofop-ethyl

Fluazifop

ESI Negative (0-18.5min)

Fig. 3.16.2 ESI mass chromatograms of phenoxypropionic-acid herbicides

ColumnMobile PhaseGradient ProgramFlow RateColumn TemperatureInjection VolumeProbe VoltageCDL TemperatureBlock Heater TemperatureNebulizer Gas Flow RateQ-array DC VoltageQ-array RF VoltageScan RangeSIM

: Shim-pack VP-ODS (150mmL. × 2.0mm I.D.): A: 0.1% Formic Acid Solution B: Acetonitrile containing 0.1% Formic Acid: 20% B (0min) → 90% B (20-30min): 0.2mL/min: 40˚C: 5µL: -3.5kV (ESI- Negative Mode), +4.5kV (ESI-Positive Mode): 200˚C: 200˚C: 4.5L/min: -30V, 10V: 150: m/z 50-600 (1.0sec/scan): m/z 326, 343, 384, 373 (0.5sec/ch)


0 100 200 300 400 Conc0e3

250e3

500e3

750e3

Area

15.0 min

2500

2750

3000

Int.

326.00(1.00)

0.8ppb ~ 500ppb (n = 5)r2 = 0.99993

Y = (1786.70031)X + (16957.44868)

0.8 ppb

Fluazifop

Fig. 3.16.3 Calibration curve and SIM chromatogram of fluazifop

91

4.1 Aromatic Components of Alcohols - GC

■ ExplanationThe headspace method enables analysis of volatilecomponents in solids and liquids without complicatedpretreatment. The following are the advantages of theheadspace GC.

1) Components with low boiling points can be analyzedat high sensitivity.

2) Induction of components with high boiling pointsinto GC can be prevented, reducing the analysistime.

3) Contamination of GC injection port and column isminimized because non-volatile components are notinducted into the GC.

Here, several analysis examples for volatile componentsin sake and whisky will be introduced.

■ PretreatmentShop-sold sake and whisky were sealed in 5mL vials andkept at 100˚C for 60 min.


0

2

1

8

3

4

5

6

9(0.032ppm)

107

4 8 12 16(min)

Fig. 4.1.1 Analysis of brewage

0

1

8

3

4

5

6

9(0.587ppm)

10(0.637ppm)

7

4 8 12 16(min)

Fig. 4.1.2 Analysis of shochu

0

2

1

8

3

4

5

11

6

9(1.477ppm)

10(7.353ppm)

7

4 8 12 16(min)

■ Peaks 1 Acetaldehyde 2 Acetone 3 Ethyl Acetate 4 Ethanol 5 n-Propyl Alcohol 6 Isobutyl Alcohol 7 Isoamyl Acetate 8 Isoamyl Alcohol 9 Ethyl n-Caproate10 Ethyl n-Caprylate11 Ethyl n-Caprate

Fig. 4.1.3 Analysis of whisky

Instrument

Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPFsc + HSS-2B

: CBP20 (25m × 0.32mmI.D. df=0.5µm)

: 50˚C(5min)-10˚C/min-200˚C

: 230˚C

: 230˚C(FID)

: He(1.35mL/min)

: Split(1:16)

: 0.4mL

4. Aromas and Odors

92

4.2 Aromatic Components of Tea - GC

■ ExplanationVolatile components in solid samples like tea can beeasily analyzed using the headspace method. With thismethod, sample extraction by steam distillation is notrequired, as the sample is simply sealed to be analyzed.

■ Pretreatment3g of tea leaf in 10mL of distilled water was kept at100˚C for 60 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPFsc+HSS-2B

: CBP20 (25m × 0.32mmI.D. df=0.5µm)

: 50˚C(5min)-10˚C/min-200˚C

: 230˚C

: 230˚C(FID)

: He(1.4mL/min)

: Split(1:15)

: 0.4mL

0 4

4

8

8

5

9

12

14

13

16

16

(min)

Fig. 4.2.1 Analysis of refined green tea

0 4 8

95

1

13 14 15

12 16(min)

Fig. 4.2.2 Analysis of coarse green tea

0 4 8 12 16(min)

1

8

345

2

1411

139

■ Peaks 1 Acetaldehyde 2 Hexanal 3 Methyl n-Caproate 4 Isoamyl Alcohol 5 Ethyl n-Caproate 6 Hexyl Acetate 7 cis-3-Hexenyl Acetate or Hexanol 8 Methyl n-Caprylate 9 cis-3-Hexenol10 trans-2-Hexenol11 Ethyl n-Caprylate12 Ethyl Acetoacetate13 Linalool14 Benzaldehyde15 Ethyl n-Caprate16 Geraniol

Fig. 4.2.3 Analysis of black tea

93

Aromas and Odors

4.3 Essential Oil (Headspace Analysis) - GC

■ ExplanationThis is an analysis example for essential oil used asflavors for food products.

■ PretreatmentEssential oils were sealed in 5µL vials and kept at 40˚Cfor 30 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPF+HSS-2B

: CBP1 (25m × 0.53mmI.D. df=3.0µm)

: 50˚C(15min)-5˚C/min-200˚C

: 230˚C

: 230˚C(FID)

: He(10.5mL/min)

: Direct Injection

: 0.8mL

0 10 20 30 40(min)

Fig. 4.3.1 Analysis of orange oil

0 10 20 30 40(min)

Fig. 4.3.2 Analysis of lavender oil

0 10 20 30 40(min)

Fig. 4.3.3 Analysis of spearmint oil

94

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56

1. Limonene 2. 1,8-Cineole 3. Menthone 4. Terpinene-4-ol 5. β-Caryophyllene 6. Dihydrocarvone 7. Carvone

1 2 3 4 5 6 7

■ Peaks

Fig. 4.4.2 Analysis of spearmint oil

3 4 5 6 7 8 9 10 1. Limonene 2. 1,8-Cineole 3. trans-Sabinenehydrate 4. L-Menthone 5. Menthoturan 6. D-Isomenthone 7. Neo-Menthol 8. Terpinene-4-ol 9. β-Caryophyllene10. Menthol

1 2

0 4 8 12 16 20 24 28 32 36 40 44 48 52 56

■ Peaks

Fig. 4.4.1 Analysis of peppermint oil

4.4 Essential Oil (Direct Analysis) - GC

■ ExplanationHere, direct GC analysis examples of peppermint oil andspearmint oil used as flavorings are introduced.


Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: ULBON HR-20M

(50m × 0.25mmI.D. df=0.25µm)

: 60˚C-3˚C/min-220˚C

: 250˚C

: 250˚C(FID)

: He(1.4mL/min)

: Split(1:15)

: 0.2µL

1. Limonene 2. 1,8-Cineole 3. trans-Sabinenehydrate 4. L-Menthone 5. Menthoturan 6. D-Isomenthone 7. Neo-Menthol 8. Terpinene-4-ol 9. β-Caryophyllene10. Menthol

■ Peaks 1. Limonene 2. 1,8-Cineole 3. Menthone 4. Terpinene-4-ol 5. β-Caryophyllene 6. Dihydrocarvone 7. Carvone

■ Peaks

95

4.5 Diketones - GC

■ ExplanationThis introduces analysis examples using a headspacesystem with ECD for diketones contained in brewedproducts such as sake.

■ Pretreatment5mL of solution samples or 3g of solid samples weresealed in vials and kept at 60˚C for 40 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14APE+HSS-2B

: DB-WAX

(60m × 0.25mmI.D. df=0.25µm)

: 40˚C

: 200˚C

: 200˚C (ECD,Current 0.5nA)

: He(1.7mL/min)

: Split(1:15)

: 0.4mL

0 10 20 (min)

3

1 2

(519

ppb)

(333

ppb)

Fig. 4.5.1 Analysis of strong soy sauce

0 10 20 (min)

3

1

2

(217

ppb)

(55p

pb)

Fig. 4.5.2 Analysis of Japanese sake

0 10 20 (min)

3

2

(7pp

b)

1

(43p

pb)

■ Peaks 1 Diacetyl 2 2,3-Pentanedione 3 2,3-Hexanedione (internal standard)

Fig. 4.5.3 Analysis of shochu

Aromas and Odors

96

4.6 Fruit Fragrances - GC

■ ExplanationThis introduces several analysis examples using aheadspace system for various fruits fragrances. Theresults show how lower alcohol and esters formdistinctive fruit fragrances.

■ Pretreatment10g of fruit samples were sealed in vials and kept at 60˚Cfor 30 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPFsc+HSS-2B

: DB-WAX

(60m × 0.25mmI.D. df=0.25µm)

: 50˚C(5min)-10˚C/min-200˚C

: 230˚C

: 230˚C(FID)

: He(1.1mL/min)

: Split(1:18)

: 0.8mL

0 4 8 12 16 20 24(min)

5

2

3

8

641

12

7 111314

16

17

Fig. 4.6.1 Analysis of melon

0 4 8 12 16 20 24(min)

3 4 6

1517

19

1

2

5

911

12

■ Peaks 1 Acetaldehyde 2 Acetone 3 Methyl Acetate 4 Ethyl Acetate 5 Methanol 6 Ethanol 7 Methyl Butyrate 8 Ethyl Butyrate 9 2-Methyl-3-Butene-2-ol or n-propyl Alcohol10 Hexanal11 Isobutyl Alcohol12 Isoamy Acetate13 n-Amyl Acetate14 Methyl n-Caproate15 Isoamyl Alcohol or trans-2-Hexenal16 Ethyl n-Caproate17 Hexyl Acetate18 cis-3-Hexenyl Acetate19 Hexanol20 Methly n-Caprylate or cis-3-Hexenol

Fig. 4.6.2 Analysis of strawberry

0 4 8 12 16 20 24(min)

1

2 3 5

11

14

15

8

4 6 12

17

18

13

19 20

Fig. 4.6.3 Analysis of banana

97

Aromas and Odors

4.7 Vegetable Fragrances - GC

■ ExplanationThis introduces several analysis examples using aheadspace system for many vegetable fragrances. Theresults show how terpene compounds are a maincomponent in providing vegetables with earthy, freshfragrances.

■ PretreatmentSuitable amount of vegetable samples were sealed invials and kept at 40˚C for 30 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPF+HSS-2B

: CBP1 (25m × 0.53mmI.D. df=3.00µm)

: 50˚C(15min)-5˚C/min-200˚C

: 230˚C

: 230˚C(FID)

: He(10.5mL/min)

: Direct

: 0.8mL

0 10 20 30 40(min)

1 2 4

3

■ Peaks 1 α-Pinene 2 β-Pinene 3 Myrcene 4 Cineole/ Limonene

Fig. 4.7.1 Analysis of perilla (1.5g)

0 10 20 30 40(min)

2 341

Fig. 4.7.2 Analysis of parsley (2g)

0 10 20 30 40(min)

3 41

2

Fig. 4.7.3 Analysis of ginger (10g)

98

Hexanal (0.95ppm)

0 5 10 15min

0

2

4

6

8

mV

Fig. 4.8.2 Analysis of old rice

Hexanal (0.20ppm)

0 5 10 15min

0

2

4

6

8

mV

Fig. 4.8.1 Analysis of new rice

4.8 Flavor of Rice - GC

■ ExplanationIn order to analyze the flavor components of rice, thechromatograms of new rice and old rice (more than 3years old) were compared. 1g of polished rice and 1mL ofwater were put into a vial and, after heating at 100ºC for30min, the head space was analyzed. When quantitativeanalysis was performed for hexanal, which is said to be anoff-flavor constituent of rice, the results for new rice and oldrice differed significantly at 0.20ppm and 0.95ppmrespectively.

Note:Here, "new rice" refers to rice that was harvested inautumn and analyzed the following April. The brands ofnew rice and old rice used as samples here weredifferent.


Column

Column Temp.

Injection method

Detector

Carrier Gas

Split Ratio

Sample Heating Conditions

: GC-17AAFW ver.3 + HSS-4A

: DB-WAX (30m × 0.53mm I.D. df = 1µm)

: 50˚C(5min)-10˚C/min-200˚C

: Split 250˚C

: WFID 250˚C

: He, 30cm/sec

: 1:5

: 100˚C, 60min

99

Aromas and Odors

4.9 Flavoring Agent for Food Product - GC

■ ExplanationThis introduces several analysis examples using aheadspace system for flavoring agents that give sweetfragrances to cookies, etc. Examples of sweets are alsogiven.

■ PretreatmentSuitable amount of standard flavoring agent and sweetswere sealed in vials and kept at 130˚C for 40 min.


Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-14BPF+HSS-2B

: CBP1 (25m × 0.53mmI.D. df=3.0µm)

: 90˚C-6˚C/min-230˚C

: 300˚C

: 300˚C(FID)

: He(4.3mL/min)

: Direct

: 0.8mL

0 4 8 12 16 20(min)

13

12

Fig. 4.9.3 Analysis of chocolate (with nut cream) (3.9g)

0 4 8 12 16 20(min)

1211

31

25

6 8

7

9

1013

4

■ Peaks 1 Ethyl n-Butyrate 2 Isoamyl Acetate 3 Ethyl Isovalerate 4 Benzaldehyde 5 Isoamyl n-Butyrate 6 Allyl n-Caproate 7 Ethyl n-Heptanoate 8 Isoamyl Isovalerate 9 R-Menthol10 Ethyl n-Caprylate11 γ-Nonalactone12 Vanillin13 Ethyl n-Caprate

Fig. 4.9.1 Analysis of standard flavoring agent

0 4 8 12 16 20(min)

1

12

Fig. 4.9.2 Analysis of cookie (6.4g)

100

4.10 Analysis of Fishy Smell in Water (1) - GC/MS

■ ExplanationFishy smells are attributed to unsaturated aldehyde inuroglene Americane and has become a problem indrinking water supplies along with musty smell eversince vast outbreaks of it occurred in Lake Biwa in 1995. The 4 compounds of unsaturated aldehyde with carbonnumber 7 or 10 trans, cis-2,4-heptadienal and trans, cis-2,4-decadienal are the cause of this fishy smell. Thepurge & trap method is more effective than the headspacemethod to analyze these substances because of the lowvapor pressure. The threshold values of these substancesas odors are several 100ppb, and the lower detection limitof this method is several ppb.


1-1

1-2

2-1

2-2

TIC

*** CLASS-5000 *** report No. : 5 Data file : 970714.D11Sample : 10ppb 1Method file : AO.MET

9878391

1-1, 1-2

2,4-Heptadienal

2-1, 2-2

2,4-decadienal

0

0

MW : 110

MW : 15210 11 12 13 14 15 16 17 18 19

Fig. 4.10.1 TIC chromatogram of fishy smell components

Instrument

Column

Column Temp.

Inj.Temp.

I/F Temp.

Carrier Gas

-P&T-

Instrument

Sample Size

Trap Tube

Purge

Dry Purge

Thermal Desorption

: GCMS-QP5000

: DB-1701 (30m × 0.32mmI.D. df=1.0µm)

: 40˚C(8min)-20˚C/min-200˚C(5min)

: 230˚C

: 230˚C

: He(20kPa)

: Tekmar 3000J

: 5mL(35˚C)

: Tenax GR

: 11min

: 3min

: 225˚C, 8min

101

Aromas and Odors

4.10 Analysis of Fishy Smell in Water (2) - GC/MS

40

39

53 67

81

95110

60 80 100 120 140 160 180 200 40

41

55 67

81

95119

60 80 100 120 140 160 180 200

152

Fig. 4.10.2 Mass spectra

Ion set No.: 1466420

2-1

2-2

1-1

1-2

10 11 12 13 14 15 16 17 18 19

81.00110.00 ✽ 3.00152.00 ✽ 3.00

Fig. 4.10.3 SIM chromatogram of 100ppt

Ion set No.: 12900782

2-1

2-2

1-1

10 11 12 13 14 15 16 17 18 19

81.00110.00 ✽ 3.00152.00 ✽ 3.00

1-2

Fig. 4.10.4 SIM chromatogram of 1ppb

ID # 1 Mass No.: 110.00 Component: 2,4-heptadienalArea = 171214✽ Conc. + 3258.56 Contribution rate: 0.999998

10Area 6

101

2.0

1.0

0 1.00.5

Conc

123

Conc. (ppb) Area0.100 190071.000 175983

10.000 1715263

Fig. 4.10.5 Calibration curve for 2,4-heptadienal

ID # 2 Mass No.: 152.00 Component: 2,4-decadienalArea = 142136✽ Conc. + - 1729.78 Contribution rate: 0.999922

10Area 6

101

2.0

1.0

0 1.00.5

Conc

123

Conc. (ppb) Area0.100 190161.000 133221

10.000 1420285

Fig. 4.10.6 Calibration curve for 2,4-decadienal

Peak 1-1 mass spectrum Peak 2-1 mass spectrum

102

4.11 Analysis of Alcohols (1) - GC/MS

■ ExplanationThere are two headspace methods: static headspacemethod and dynamic headspace method. Generally, theterm headspace method refers to the static headspacemethod.The dynamic headspace method refers to a method wherepurge gas is continuously fed into the sample to purge outvolatile elements, and then the volatile elements areconcentrated onto the trapping agent. After concentration,target components are desorped and analyzed by GC/MS.This method enables microanalysis because it involvesthe concentration of the sample.Here, the difference between the static and dynamicheadspace methods will be shown using Japanese sakeand wine.A Chrompack CP4010 and a Tenax trapping set wereused in the dynamic headspace analysis.Sensitivity was clearly higher in dynamic headspaceanalysis.


Trap tube

Activated carbon tube

Water bath

Lab jack

Carrier in

Fig. 4.11.2 Schematic diagram of Tenax trapping set

Trap tube

Back flush vent

Split vent

Purge/desorption vent

Liquid nitrogen

Detector

He Gas

Fig. 4.11.1 CP4010 flow line diagram

Instrument

Column

Column Temp.

I/F Temp.

Carrier Gas

-HS-

Instrument

Sample Size

Sample Temp.

Thermostat

Injection

-TCT-

Instrument

Sample Size

Purge

Trap Tube

Precool

Thermal Desorption

: GCMS-QP5000

: DB-1701 (30m × 0.32mmI.D. df=1.0µm)

: 40˚C(5min)-5˚C/min-250˚C(5min)

: 250˚C

: He(35kPa)

: HSS-4A

: 10mL

: 60˚C

: 30min

: 0.8mL

: CP4010+Tenax Trap Set

: 20mL(Room Temp.)

: 20mL/min(5min)

: TenaxGR(0.1g)

: -150˚C(3min)

: 250˚C(5min)

103

Aromas and Odors

4.11 Analysis of Alcohols (2) - GC/MS

TIC 1 2

5 10

10

15

15

14

16

20 25 30

3

4 5 6

7

8

9 13

11 12

40000000

Fig. 4.11.6 TIC chromatogram of wine (TCT method)

TIC

5 10 15 20 25 30

7500000

1 2

10

15 16

37

9

13

9 1-Pentanol10 Ethyl Butanoate11 Ethyl 2-methylbutanoate12 Ethyl Isovalerate13 Isopentyl Acetate14 Limonene15 Ethyl Caproate16 Ethyl Caprylate

■ Peaks

Fig. 4.11.5 TIC chromatogram of wine (HS method)

TIC

5 10 15 20 25 30

400000001 2 3

4 5 6

7

8

9

10

13

14

14

15

15

17 18 19 20

TIC 68.00

138.0089.0099.00

3.003.00

Fig. 4.11.4 TIC chromatogram of Japanese sake (TCT method)

TIC

5 10 15 20 25 30

75000001

2

3 9 13

1 Ethanol2 Ethyl Acetate3 Isobutyl Alcohol4 1,1-Diethoxyethane5 Ethyl Propanoate6 Propyl Acetate7 Ethyl Isobutyrate8 Isobutyl Acetate

■ Peaks

Fig. 4.11.3 TIC chromatogram of Japanese sake (HS method)

104

4.12 Analysis of Strawberry Fragrances - GC/MS

■ ExplanationStrawberry fragrance components consist of fatty acidmethyl esters from C2 to C6. Old and new varieties ofmarketed strawberries were compared and the correlationbetween type and fragrance studied.Normally, steam distillation or the headspace method isused for pretreatment of fragrance components; however,sometimes problems occur with the heating process. Inthe case of strawberries, heat destroys cells and releaselarge amounts of special esters that are sometimesmistaken for fragrance components.Here, the Chrompack CP4010 + GCMS system (TCT +GCMS) was used to dry-air purge the sample withoutheating to enable optimum measurement of strawberryfragrances.


Fig. 4.12.1 TIC chromatogram of strawberry fragrance components (upper: old brand, lower: new brand)

TIC

4 6 8 10 12

3

34

5

21

5

67

67

8

8

9

9

60000000

10

10

11

11

14 16 18 20 22 24 26

TIC

4 6 8 10 12

60000000

14 16 18 20 22 24 26

1 Ethanol2 Acetone3 Acetic acid, Methyl ester4 Ethanoic acid, Methyl ester5 Butanoic acid, Methyl ester6 Butanoic acid, Ethyl ester

7 Hexanal 8 2-Hexenal 9 Hexanoic acid, Methyl ester10 Hexanoic acid, Ethyl ester11 C6H11OH

Old brand strawberry fragrances

New brand strawberry fragrances

■ Peaks

Instrument

Column

Column Temp.

I/F Temp.

Carrier Gas

-TCT-

Instrument

Sample Amount

Trap Tube

Pre Cool

Pre Flush

Thermal Desorption

: GCMS-QP5000

: DB-624 (60m × 0.25mmI.D. df=1.4µm)

: 40˚C(5min)-5˚C/min-230˚C(5min)

: 230˚C

: He(100kPa)

: CP4010(TCT Mode)

: 10g (Room Temperature)

: Tenax TA

: -150˚C, 5min

: 50˚C, 1min

: 250˚C, 10min, 20mL/min

105

Aromas and Odors

4.13 Analysis of Beverage Odors (1) - GC/MS

■ Explanation2,4,6-trichloroanisole (2,4,6-TCA), a cause of mustyodor, is contained in wood and paper-manufacturedpacking materials, and its transfer to food products anddrinking water may cause problem. The perceptualthreshold value of TCA in water is extremely low at theppt level. Conventionally, 2,4,6-TCA was analyzed bysolvent extraction or steam distillation method, but thesemethods require a lot of time and are extremelycomplicated; moreover, the poor collection rate wouldmake ppt-level measurement difficult.Here, measurement was conducted using a combinationof the Chrompack CP4010 and Tenax trapping set. In thismethod, the sample is purged to collect the targetcomponents in the trap tube. The trap tube is heated bythe TCT mode of the CP4010, and the desorpedcomponents are analyzed by GC/MS.This system setup is an offline one, so the Tenax unit iseasy to clean and there is no sample memory.


Trap tube

Activated carbon tube

Water bath

Lab jack

Carrier in

Fig. 4.13.1 Schematic diagram of Tenax trapping set

Trap tube

Back flush vent

Split vent

Purge/desorption vent

Liquid nitrogen

Detector

He Gas

Fig. 4.13.2 TCT main unit flow line diagram

Instrument

Column

Column Temp.

I/F Temp.

Carrier Gas

-TCT-

Instrument

Sample Size

Trap Tube

Purge

Thermal Desorption

: GCMS-QP5000

: DB-1701 (30m × 0.32mmI.D. df=1.0µm)

: 50˚C(2min)-30˚C/min-140˚C

-10˚C/min-220˚C

: 250˚C

: He(50kPa)

: GP4010(TCT Mode)

: 25mL(50˚C)

: Tenax GR

: 50˚C, 15min, 100mL/min

: 250˚C, 5min

106

4.13 Analysis of Beverage Odors (2) - GC/MS

Sample: 4.5ng/L Japanese sake

ID : 1Mass No. : 195.00Type : TargetComponent : 2,4,6-TCA

Retention time : 10.097Area : 11079Conc. : 4.550ng/L

Mass No.1 210.00

Area7624

Intensity ratio69

Fig. 4.13.5 Analysis of Japanese sake (4.5ng/L added)

Sample: 3ng/L black tea

Mass No.1 210.00

Area5544

Intensity ratio71

ID : 1Mass No. : 195.00Type : TargetComponent : 2,4,6-TCA

Retention time : 10.093Area : 7859Conc. : 3.020ng/L

Fig. 4.13.6 Analysis of black tea (3ng/L added)

Sample : 1ng/L SIM

Fig. 4.13.3 SIM chromatogram (1ng/L)

ID # 1 Mass No.: 195.00 Component: 2,4,6-TCAArea = 2424.61✽ Conc. contribution rate = 0.999816

Conc. (ng/L) Area1.0005.000

10.00020.00050.000

100.000

2637118562501051111

122666241154

Fig. 4.13.4 TCA calibration curve (1 to 100ng/L)

107

Aromas and Odors

4.14 Analysis of Fragrant Material (1) - GC/MS

■ ExplanationMany fragrant components are contained in foodproducts. These components are compounds of alcohols,esters, aldehydes, ketones, terpenes and others. Theamount and mixture ratio of these components determinethe aroma, and any aroma can be artificially synthesizedby mixing these components. Here, some 100-aromacomponents were mixed together and analyzed byGC/MS.


Column

Column Temp.

Inj.Temp.

I/F Temp.

Carrier Gas

Injection

: GCMS-QP5000

: DB-WAX (60m × 0.25mmI.D. df=0.25µm)

: 70˚C(5min)-3˚C/min-210˚C(30/min)

: 250˚C

: 230˚C

: He(180kPa)

: Split(100:1)

10 20 30 40 50 60 70

TIC 90000005

2

8

9

34

6

7

11

12 21

1823

25

26

29

35

27

53

36

28

30

31

3233

34

37

38

39

42

43

44

45

49

46

47

48

50

51

52

54

55

5657

58

59

63

62

60

6164

65

66

67

68

69

70

74

7576

80

81

82

84

85

91

878990

93

94

95

97

98

99

100

101

102

103

104105

106

107

96

92

88

86

77

7879

83

71

72

73

40

41

24

19

1317

2022

10

14

1615

10 20 30 40 50 60 70

TIC 90000005

2

8

9

34

6

7

11

12 21

1823

25

26

29

35

27

53

36

28

30

31

3233

34

37

38

39

42

43

44

45

49

46

47

48

50

51

52

54

55

5657

58

59

63

62

60

6164

65

66

67

68

69

70

74

7576

80

81

82

84

85

91

878990

93

94

95

97

98

99

100

101

102

103

104105

106

107

96

92

88

86

77

7879

83

71

72

73

40

41

24

19

1317

2022

10

14

1615

Fig. 4.14.1 TIC chromatogram of fragrant components

108


Compound Alcohol Ester Aldehyde Ketone Terepene Others1 Ethyl acetate

2 Diethyl acetal

3 Ethyl alcohol

4 Ethyl propionate

5 i-Butyl acetate

6 Chloroform

78 Ethyl n-butyrate

9 Ethyl 2-methyl butyrate

10 Ethyl i-valerate

11 n-Butyl acetate

12 n-Hexanal

13 i-Butyl alcohol

14 n-Amyl acetate

15 n-Butyl alcohol

16 Methyl i-amyl ketone

1718 n-Amyl propionate

19 Limonene

20 2-Methyl butyl alcohol

21 n-Amyl furmate

22 c-2-Hexenal

23 Ethyl caproate

24 n-Amyl alcohol

25 i-Amyl n-butyrate

26 n-Hexyl acetate

27 Methyl n-hexyl ketone

28 i-Amyl i-valerate

293031 Ethyl lactate

32 n-Hexanol

33 Ethyl n-hexyl ketone

34 Allyl caproate

3536 Methyl n-heptyl ketone

37 t-3-Hexenol

3839 Ethyl caprylate

40 Acetic acid

41 Furfural

42 Methyl n-octyl ketone

43 Tetrahydro furfuryl alcohol

44 Benzaldehyde

45 Ethyl nonanoate

46 Linalool

4748 Diethyl malonate

49 Methyl n-nonyl ketone

50 Ethytl levulinate

51 Methyl benzoate

52 Ehtyl caprate

53 l-Menthol

54

Table 4.14.1 Component names 1

109

Aromas and Odors


Compound Alcohol Ester Aldehyde Ketone Terepene Others55 Furfuryl alcohol

56 Ethyl benzoate

57 Phenyl diethyl acetate

5859 Methyl n-decyl ketone

60 Benzyl acetate

61 Methyl phenyl acetate

62 Dimethyl benzyl carbinyl acetate

63 Allyl caprate

64 Ethyl phenyl acetate

65 Allyl β-cyclohexyl propionate

66 Phenethyl acetate

67 Anethol

68 Caproic acid

69 Ethyl laurate

70 t-2-Decenal

71 Benzyl n-butyrate

72 Benzyl alcohol

73 Phenetyl propionate

74 i-Butyl phenyl acetate

75 Dimethyl benzyl carbinylbutyrate

76 Phenyl ethyl alcohol

777879 Phenyl ethyl propionate

80 Phenethyl i-valerate

81 Methyl n-tridecyl ketone

82 Anisaldehyde

83 γ-Nonalactone

84 Ethyl myristate

85 Triacetine

86 Methyl cinnamate

87 Benzylidene acetone

88 Ethyl cinnamate

89 γ-Decalactone

90 Eugenol

91 Phenethyl caproate

92 δ-Decalactone

93 Heliotropine

94 γ-Undecalactone

95 Anisalcohol

96 Cinnamy alcohol

97 Diethyl sebacate

9899 γ-Dodecalactone

100 Phenethyl octanoate

101 δ-Dodecalactone

102 TEC

103 Benzophenone

104 Ethyl vanillin

105106 vanilline

107 Benzyl benzoate

Table 4.14.2 Component names 2

110

Ca

MgNa

5. Inorganic Metals

5.1 Analysis of Inorganic Ions in Milk (1) - LC

■ ExplanationIon chromatography is the best method for analyzinginorganic ions in food products. In particular, use of adual flow line system allows simultaneous analysis ofanions and cations, which is useful in ion balancemeasurement.Here, an application example for analysis of inorganicions in milk will be introduced.

ReferencesShimadzu HPLC Food Analysis Applications Data Book(C190-E047)Yagi, Funato, Ito; Analytical Chemistry, 38 (11), 655(1989)

■ PretreatmentThe sample was injected into column afterdeproteinization with ultrafiltration membrane.

■ Analytical Conditions■ Anions

Column

Mobile Phase

Temperature

Flow Rate

Detection

■ Cations

Column

Mobile Phase

Temperature

Flow Rate

Detection

: Shim-pack IC-A3(150mmL. × 4.6mmI.D.)

: 8.0mM p-Hydroxy Benzoic Acid

3.2mM Tris Hydroxy Aminomethane

: 40˚C

: 1.5mL/min


: Shim-pack IC-C3(100mmL. × 4.6mmI.D.)

: 3.0mM Oxalic Acid

: 40˚C

: 1.2mL/min


1.H2PO4-

2.CI-

3.SO42-

2 ■ Peaks

0 5

3

1

10 15 (min)

Fig. 5.1.1 Analysis of inorganic anions

1.Na+

2.K+

3.Mg2+

4.Ca2+

■ Peaks

0 5 10 15 (min)

3

4

21

Fig. 5.1.2 Analysis of inorganic cations

111

5.1 Analysis of Inorganic Ions in Milk (2) - LC

DGU-12A LC-10AD (2)

CDD-6A (2)

CDD-6A (2)

CDD-6A (1)

CDD-6A (1)

FCV-12AH

SIL-10A

LC-10AD (1)

1st flow lineColumn (1)

Column (2)

Drain

2nd flow line

Fig. 5.1.3 Diagram of dual flow line system

Ca

MgNa Inorganic Metals

112

Air not added

Temperature(˚C)

Stage Time (sec)

Heat mode

Inner gas flow rate

1

2

3

4

5

6

7

8

0.20

0.50

0.50

1.00

1.00

0.0H

0.0H

1.00

70

120

400

500

700

700

2400

2600

3

30

20

10

10

3

3

2

lamp

lamp

lamp

lamp

step

step

step

step

Gas

Ar

Ar

Ar

Ar

Ar

Ar

Ar

Ar

Table 5.2.1 Heat program

Air added

Air not added

10.5ppb

10.4ppb

10.0ppb

10.0ppb

Added amountMeasurement results

Table 5.2.2 Measurement results for Pb in milk1

2

3

SDT.NO.

CONC.(ppb)

ABS.283.3nm

ABS. 0.14

0.000.0

[ABS]-X1✽ [C]+K0K0=0.0000, K1=0.0072

Furnace quantitative measurement/calibration curve methodCalibration curve

Sample No.

2

Sample No. Repeat No.

0.0721

0.0776

0.0238

0.0206

0.1048 5.12

0.0802

2 2

2 1

Peak height (Abs)

0.0749

Peak height (Abs) Area (Abs-sec) BKG height (Abs)

BKG height (Abs)

0.0921CV value (%)

5.12

CV value (%)

Conc. (ppb)

10.3523

20.0CONC.(ppb)

123

4.00008.0000

16.0000

0.02600.05200.1194

5.000.00

Sec

0.600

Abs

0.400

0.200

0.000

-0.200

Fig. 5.2.1 Peak profile and calibration curve of Pb in milk

5.2 Analysis of Pb in Milk Using Atomic Absorption Spectrophotometry - AA

■ ExplanationLead is harmful to human body and stricter regulationsare being applied to lead in food and pharmaceuticalproducts. Lead can be effectively detected byelectrothermal atomization with atomic absorption.Analysis of Pb in milk generally involves the flamemethod or electrothermal atomization where an acid isadded and the sample is thermally decomposed.However, these methods require time-consumingpretreatment.With direct analysis using electrothermal atomization,oxygen is often added during incineration to enhance thedecomposition of organic matter in milk. However, theoxygen causes the deterioration of the graphite tube.Here, the use of a platform tube, instead of the graphitetube, allowed accurate measurement without the additionof oxygen or air.


Wavelength

Lamp Current Low (mA)

Lamp Current High (mA)

Slit Width (nm)

Background Correction

: AA

: Pb 283.3nm

: 10

: 0

: 0.5

: BGC-D2

113

Ca


5.3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (1) - AA

■ ExplanationLead is harmful to human body and stricter regulationsare being applied to lead in food and pharmaceuticalproducts. Lead can be effectively detected by theelectrothermal atomization with atomic absorption. The13th revision of the Japanese Pharmacopoeia requires themeasurement of lead, instead of heavy metal, using theelectrothermal atomization method in purity tests forrefined white sugar.Here, analysis was performed in accordance with thePharmacopoeia, with pretreatment (see Table 5.4.1) andsample preparation using an autosampler for the standardaddition method.Table 5.3.1 shows the measurement parameters and Fig.5.3.2 shows the measurement results. Lead was notdetected in the analyzed white sugar, but 1ppb of leadwas clearly detected in the calibration curve. It can besaid that this analysis method is effective for the detectionof 0.5 ppm lead in white sugar (5ppb or less in processedsolution), which is specified in the standard.


Accurately load 0.050g of sample into polytetrafluoroethylene container

↓Add 0.5mL of nitric acid to dissolve sample

↓Seal container and heat for 5 hr at 150˚C

↓Cool, add water to accurately make 5mL of sample solution

↓Analyze using standard addition method for AA

(electrothermal)

Fig 5.3.1 Pretreatment for Pb in refined white sugar

Element :

Turret No. :

Lamp current Low (mA) :

Lamp current High (mA) :

Wavelength (nm) :

Slit width (nm) :

Lighting mode :

Final stage No. of concentration in oven : 5Concentration frequency : 1

Temperature Program

Autosampler Mixing Conditions

Pb

1

10

0

283.3

0.5

BGC-D2

Temperature (˚C)

Time (sec)

Heat mode

Sensitivity Gas Inner gas flow rate

Sampling Previous stage(sec)

1

2

3

4

5

6

7

Blank

0

1

2

3

0.20

0.20

1.00

1.00

0.00

0.00

1.00

0

0

0

0

0

2

0

Off

Off

Off

Off

Off

On

Off

Gas #1

Gas #1

Gas #1

Gas #1

Gas #1

Gas #1

Gas #1

110

250

600

600

600

2100

2600

30

10

20

20

5

3

2

0µL

100µL

100µL

100µL

100µL

0µL

0µL

20µL

40µL

60µL

200µL

100µL

80µL

60µL

40µL

200µL

200µL

200µL

200µL

200µL

Ramp

Ramp

Ramp

Step

Step

Step

Step

Regular

Regular

Regular

Regular

High

High

Regular

Adding Conc. (ppb)

Pb: 10ppb standard solution and pure water containing approximately 1.1mol/L of nitric acid

Inj. Vol. : 20µL

Sample amount TotalR1

(pure water)R2 (Pb: 10ppb

standard solution)

Lighting Conditions

Table 5.3.1 Measurement parameters

Instrument

Wavelength

Lamp Current Low (mA)

Lamp Current High (mA)

Slit Width (nm)

Background Correction

: AA

: Pb 283.3nm

: 10

: 0

: 0.5

: BGC-D2

114

5.3 Analysis of Pb in White Sugar Using Atomic Absorption Spectrophotometry (2) - AA

0.098

0.080

0.060

0.040

0.020

-0.001-0.018 1.000 2.000 3.302

Effective Calibration Curve-MSA (sugar)

Conc. (ppb)

Abs

orpt

ion

Correlation coefficient (r) =0.99997Abs=0.0293 Conc+0.000542

RES 3.6944 ppb0.0185

RSA

RSA

0.0883

-0.100

83.0000

88.00000.000 0.100 0.200 0.300 0.400

-0.0015 3.0000 600.0000 ppb

ppb

sugar

Absorption

sugar

sugar

sugar

0.0589

-0.100

83.0000

88.00000.000 0.100 0.200 0.300 0.400

-0.0006 2.0000 400.0000Absorption

sugar

sugar

CV = 0.4023%

REPAVG

0.08880.0885

-0.0027-0.0021

3.00003.0000

600.0000600.0000

ppbppb

CV = 1.1471%

REPAVG

0.05970.0593

-0.0004-0.0005

2.00002.0000

400.0000400.0003

ppbppb

sugar

RSA ppb0.0308

-0.100

83.0000

88.00000.000 0.100 0.200 0.300 0.400

-0.0314 1.0000 200.0000Absorption

Absorption

sugar

sugar

REP ppb0.0008 -0.0007 0.0000 0.0000

REP ppb0.0087 -0.0045 0.0000 0.0000

BLK ppb0.0070 -0.0043 0.0000 0.0000

sugar

NSA ppb0.0002 -0.0017 0.0000 0.0000sugar

sugar

AbsorptionType Actual Conc. Unit(ppb)BG




AbsorptionType Actual Conc. (ppb)BG

-0.100

83.0000

88.00000.000 0.100 0.200 0.300 0.400T

ime

(min

)T

ime

(min

)T

ime

(min

)T

ime

(min

)

Absorption-0.100

83.0000

88.00000.000 0.100 0.200 0.300 0.400

Tim

e

sugar

sugar

CV = 1.3869%

REPAVG

0.03000.0303

0.0001-0.0006

1.00001.0000

200.0000200.0000

ppbppb

CV = 24.9567%

REPAVG

0.00030.0003

-0.0014-0.0015

0,00000.0000

0.00000.0000

ppbppb

CV = 2.5254%

REPAVG

0.00670.0068

-0.0024-0.0034

0.00000.0000

0.00000.0000

ppbppb

Fig. 5.3.2 Measurement results for Pb in refined white sugar

115

Ca


5.4 Analysis of Cadmium in Rice (1) - AA■ ExplanationIn Japan, the concentration of cadmium in rice isregulated in accordance with the Food Sanitation Lawand directives issued by the Food Agency. Recently,however, as part of an international movement, aproposal to set a threshold level of 0.2ppm (mg/kg),which is lower than the level applied to circulation withinJapan, has been discussed at the Codex Committee (jointFAO/WHO food standards committee). The analysis ofcadmium in polished and unpolished rice withShimadzu's AA-6300 Atomic AbsorptionSpectrophotometer using the flame and furnace methodsis described here as an example. The AA-6300 uses anoptical double-beam (with the flame method) or anelectrical double-beam (with the furnace method) toensure both a stable baseline and high sensitivity. It isalso easy to switch between the flame method, whichoffers high speed, and the furnace method, which offershigh sensitivity, without the need for any special tools.

Table 5.4.1 Spectrophotometer parameters

■ PretreatmentA 5g sample was put into a beaker, sulfuric acid andnitric acid were added, thermal decomposition wascarried out, and a solution of 50mL was prepared usingthe sample. Although acid digestion was used here, othermethods that may be used include dry ashing, microwavedecomposition, and acid extraction. Alternatively, it ispossible to separate the alkaline elements and alkalineearth elements, which can cause interference, in thedecomposed liquid, and, in order to concentrate thecadmium, carry out chelate organic-solvent extraction.

■ Measurement Methods and ConditionsMeasurement was performed with the flame and furnacemethods. Measurement was also performed using anatom booster with the flame method in order to attain

■ Measurement ResultsFig. 5.4.1.1, 5.4.2.1 and 5.4.3.1 show the calibrationcurves for each analysis method. Table 5.4.3 provides acomparison of the 1% absorption values for each method.It can be seen that, compared to the standard flamemethod, using a booster with the flame method increasesthe sensitivity by a factor of 2.5 whereas using thefurnace method increases the sensitivity by a factor ofapprox. 300. Table 5.4.4 shows the measurement resultsobtained for polished and unpolished rice using each ofthe methods. The results obtained with each method areroughly the same. The lower quantitative limits forcadmium in rice that can be estimated from these resultsare approx. 0.10ppm with the flame method, approx.0.05ppm when using a booster with the flame method,and approx. 0.001ppm when using the furnace methodwith an injection volume of 10µL. This means thatquantitative measurement at the 0.2ppm level is possiblewith any of the methods.

Analysis wavelength

Slit width

Current

Lamp mode

228.8nm

2.0nm

8mA

BGC-D2

Table 5.4.4 Analysis results (in each type of rice)

Flame method without booster

0.073ppm

0.118ppm

Unpolished rice

Polished rice

Flame method

with booster

0.066ppm

0.127ppm

Furnace method

0.070ppm

0.118ppm

Table 5.4.2 Atomization parameters

Flame type

Burner height

Temperature program

Tube type

Injection volume

Total injection volume

Interference-suppression agent

Air-C2H2

7mm (without booster)11mm (with booster)

Drying: 120˚C (20s), 250˚C (10s)Ashing: 400˚C (20s)Atomization: 1,800˚C (3s)Cleaning: 2,600˚C (2s)

Pyro-covered tube

2 to 10µL (2µL here)

15µL

5µL of 100pm palladium nitrate

Flamemethod

Furnacemethod

Table 5.4.3 Comparison of 1% absorption values for each method (in solution)

Measurement method

Flame method without booster

Flame method with booster

Furnace method

1% absorption value

0.007 ppm

0.003 ppm

0.02 ppb

greater sensitivity.The main measurement conditions are given in Table5.4.1 and 5.4.2.

116

Fig. 5.4.1.1 Calibration curve for flame method without booster

5.4 Analysis of Cadmium in Rice (2) - AA

Fig. 5.4.2.1 Calibration curve for flame method with booster

Fig. 5.4.3.1 Calibration curve for furnace method

0.050ppm

Blank

Polished rice

Unpolished rice

Fig. 5.4.1.2 Signal profile for flame method without booster

0.050ppm

Blank

Polished rice

Unpolished rice

Fig. 5.4.2.2 Signal profile for flame method with booster

0.002ppm

Blank

Polished rice

Unpolished rice

Fig. 5.4.3.2 Signal profile for furnace method

117

Ca


5.5 Analysis of Inorganic Components in Powdered Milk (1) - ICP-AES

■ ExplanationThe microwave sample decomposition method is quickerthan the conventional wet decomposition method andtakes place in a sealed system to prevent externalcontamination and volatilization loss of components suchas As and Se. It is an extremely useful method todecompose the sample when the sample amount is small,or when a micro-amount element is to be analyzed.Here, powdered milk was liquidized using a microwavedecomposition unit and analyzed using ICP-AES. TheICP-AES, which causes little self-absorption and has awide dynamic range, enables analysis of majorcomponents like sodium and calcium, as well as minorcomponents such as cadmium and lead, in the samesolution. Arsenic, selenium and antimony can beanalyzed at higher sensitivity by using a hydridegenerator.

■ PretreatmentSee Fig. 5.5.1 for details of the operation flow formicrowave decomposition.


Fig. 5.5.1 Microwave decomposition flowchart

Instrument

High Frequency Output

Cooling Gas

Plasma Gas

Carrier Gas

Sample IntroductionSystem

Observation Method

: ICPS-7500

: HVG-1 (Hydride Generator)

: 1.2kW

: Ar 14.0L/min

: Ar 1.2L/min

: Ar 0.7L/min

: Coaxial Nebulizer/CycloneChamber, Hydride Generator

: Horizontal/Axial

0.5g sample (Teflon high-pressure container)

Preparative reaction (Approx. 4 hr)

Microwave decomposition (pressure mode) (Approx. 30 min)

Microwave decomposition (pressure mode) (Approx. 30 min)

Cooling (Approx. 1 hr)

Cooling (Approx. 2 hr)

Heating 190˚C (beaker) Heating 120˚C (evaporation to dryness)

Heating 120˚C (Approx. 10 min) Heating 70˚C (Approx. 4 min)Cooling

10mL measure up 10mL measure up25mL measure up

Coaxial nebulizer analysis

HVG analysis

10mL nitric acid, 3mL hydrogen peroxide

5mL hydrochloric acid (1:5)

5mL hydrochloric acid (1:4)

5mL hydrochloric acid (1:4)KI (20%wt/v) 0.5mL

Evaporate liquid down to few mL

Se As, SbNa, Mg, Al, Si, P, K, Ca, Cr, MnNi, Cu, Zn, Cd, Fe, Sn, Ba, Pb

Table 5.5.1 Powdered milk analysis results (µg/g)

Element

Na 1259

Mg 376

P 2238

K 4644

Ca 3960

Mn 0.34

Fe 75

Cu 2.8

Zn 23

Al 3.0

Measured value Element

Si 23

Ba 1.4

Ni 0.21

Sn 0.2

Cr 0.04

Cd 0.022

Pb <0.5

As 0.007*

Sb 0.002*

Se 0.03*

Measured value

* HVG hydride generator used

118

5.5 Analysis of Inorganic Components in Powdered Milk (2) - ICP-AES

■ ExplanationHere, a standard powdered milk was analyzed afterincineration. The results show that nearly all theinorganic components conformed to the guaranteedvalues.

SampleNon-fat Milk Powder (SRM 1549:NIST)Skim Milk Powder (CRM 063:BCR)

References- Standard Methods of Analysis for Hygienic Chemists

(Annotation), edited by the Pharmaceutical Society ofJapan, published by Kanehara & Co., Ltd

- Analysis Manual for the Standard Tables of FoodComposition in Japan 5th rev, edited by the ResourcesCouncil of the former Science and Technology Agency,published by the Japan Resources Association

■ Pretreatment1g of sample was placed on a platinum dish andincinerated to ash over 12 hours at 550˚C using anautoclave. 1mL of nitric acid was added to the incineratedsample to dissolve it. Finally, ultra pure water was addedto make 100mL of the sample solution.



Cooling Gas

Plasma Gas

Carrier Gas

Sample Introduction

Observation Method

: ICPS-8100

: 1.2kW

: Ar 14.0L/min

: Ar l.2L/min

: Ar 0.7L/min

: Coaxial Nebulizer

: Horizontal

Table 5.5.2 Analysis results for standard powdered milk (wt-%)

Element

Na 0.51

K 1.68

Ca 1.31

Mg 0.123

P 1.07

2.3Fe*

47.4Zn*

0.27Mn*

Quantitative value

NIST-SRM 1549 BCR-CRM 063

0.47±0.03

1.69±0.03

1.3 ±0.03

0.120±0.003

1.06 ±0.2

1.78±0.10

46.1±2.2

0.26±0.06

Guaranteed value

0.46

1.76

1.29

0.118

1.02

2.6

43

0.25

Quantitative value

0.457±0.016

1.78±0.07

1.26 ±0.03

0.112±0.003

1.04±0.03

2.06±0.25

(42)

(0.226)

Guaranteed value

*: µg/g ( ): Reference value

119

Ca


5.6 Analysis of Canned Beverage (Green Tea)- ICP-AES

■ ExplanationSamples like green tea, providing they contain nosediment, can be analyzed with high sensitivity by ICPemission analysis after they are directly injected withoutpretreatment. We conducted quantitative analysis of acommercially sold canned beverage (green tea) usingICPS-7510. Table 5.6.1 shows the quantitation results. Incomparing the directly injected sample with the sampleprocessed by the conventional wet decompositionmethod, we obtained almost the same quantitation results.

■ SampleCommercially sold canned beverage (green tea)

■ Sample Pretreatment1. Directly injected sample

After opening the can, immediately transfer 50 mL ofthe sample to a plastic container, and add 1 mL ofnitric acid and 0.1 ppm of the internal standard elementY. Adequately stir the mixture for use as the analysissample.

2. Wet decomposition methodAfter opening the can, immediately transfer 50 mL ofthe sample to a beaker, and bring it to a boil on a hotplate (190˚C). When the total volume is decreased toabout 10 mL, add 5 mL of nitric acid and 1 mL ofhydrochloric acid, and heat for about 2 hours. Afterallowing it to cool, add 0.1 ppm of the internalstandard element Y, bring the solution to 50 mL usingultrapure water, and use this as the analysis sample.


High Fequency Output

Coolant Gas Flow Rate

Plasma Gas Flow Rate

Carrier Gas Flow Rate

Sample Introduction

Chamber

Plasma Torch

Observation Method

: ICPS-7510

: 1.0 kW

: 14 L/min

: 1.2 L/min

: 0.7 L/min

: Ultrasonic Nebulizer

: Cyclone Chamber

: Standard Torch

: Axial

Table 5.6.1 Quantitation results for green tea (unit: mg/L)

Direct Injection Method(Quantitation Value)

Wet Decomposition Method(Quantitation Value)

PretreatmentElement

0.243

0.030

1.32

0.85

< 0.0005

< 0.0005

0.0055

0.082

0.0006

0.255

0.029

1.33

0.88

< 0.0005

0.002

0.0053

0.087

0.0007

Fe

Ni

Al

Mn

Pb

Sn

Cu

Zn

Cr

120

5.7 Analysis of Inorganic Components in Processed Food Products - ICP-AES

■ ExplanationThis is an analysis example for processed food. Variouselements included in food products are divided intoessential ones and harmful ones. The ICP emissionspectrometry, which allows simultaneous analysis ofthese elements, is quite useful for comprehending themutual relationships between elements.

SamplesTuna, bean curd dressed with liquid starch, vegetablesboiled in miso, rice and vegetable porridge, rice gruel

References- Standard Methods of Analysis for Hygienic Chemists

(Annotation), edited by the Pharmaceutical Society ofJapan, published by Kanehara & Co., Ltd

- Analysis Manual for the Standard Tables of FoodComposition in Japan 5th rev, edited by the ResourcesCouncil of the former Science and Technology Agency,published by the Japan Resources Association

■ PretreatmentHomogenize each sample in a homogenizer, take 10g foreach, add 10mL of nitric acid and 2mL of sulfuric acidand thermally decompose them until white smoke ofsulfuric acid appears. After cooling, measure up to100mL. Use these as samples.



Cooling Gas

Plasma Gas

Carrier Gas

Sample Introduction

Observation Method

: ICPS-8100

: 1.2kW

: Ar 14.0L/min

: Ar 1.2L/mln

: Ar 0.7L/min

: Coaxial Nebulizer/

Double Tube Chamber

: Horizontal

Table 5.7.1 Analysis results (µg/g: wet weight)

Tuna

560

77

460

747

23.3

0.13

2.10

1.16

< 0.005

< 0.1

Bean curd dressed with liquid starch

1150

874

303

603

145

1.01

3.25

2.39

< 0.005

< 0.1

Vegetables boiled in miso

1610

103

351

628

219

1.20

3.40

2.35

< 0.005

< 0.1

Rice and vegetable porridge

972

18.9

83

82

22.3

0.48

0.36

1.21

< 0.005

< 0.1

Rice gruel

10.3

10.9

47.8

38.9

10.5

0.43

0.21

1.21

< 0.005

< 0.1

Element

Na

Mg

P

K

Ca

Mn

Fe

Zn

Cd

Pb

Instrument





Sample Introduction

Chamber

Plasma Torch

Observation Method

: ICPS-7510

: 1.4 kW

: 16 L/min

: 1.4 L/min

: 0.7 L/min

: Coaxial Nebulizer

: Double Tube Chamber

: Standard Torch

: Horizontal

121

Ca


5.8 Analysis of Cooking Oil - ICP-AES

■ ExplanationUsing ICPS-7510, we conducted quantitative analysis ofcooking oil. The analysis can be performed easily by justdiluting the sample with a solvent such as xylene. Table5.8.1 shows the quantitation results. Fig. 5.8.1 shows thespectrum profile of Si, and Table 5.8.2 the calibrationcurve for Si.

■ SampleCooking oil

■ PretreatmentDilute the sample 3 times (w/w) with xylene for use asthe analysis sample. Prepare the calibration curve sampleby diluting a lubricating oil standard sample (Conostan)with xylene. Prepare Si by diluting silicone (KM-72F,containing 10.9% Si) with xylene.


Table 5.8.1 Quantitation results for cooking oil (unit : µg/g)

Na

<0.3

<0.3

Fe

0.07

0.56

Mg

0.36

18.9

P

1.23

120.5

Si

<0.03

<0.03

Element

Sample

Sample A

Sample B

Si 251.612nm

0

E0

.500

1.00

1.50

2.00 472

Vegetable oil

1ppm

Fig. 5.8.1 Si profile

1.500E 0

Si251.612 nm

1.000

0.500

0.0000.000

Coefficient :

0.002640.99999ppm

SDCorrelation

Unit

0.200

a= 0.0000e+000b= 0.0000e+000c= 0.0000e–001d= –4.8110e–003

0.400 0.600 0.800 1.000

Fig. 5.8.2 Si calibration curve

Instrument





Sample Introduction

Chamber

Plasma Torch

Observation Method

: ICPS-7510

: 1.2 kW

: 14 L/min

: 1.2 L/min

: 0.7 L/min

: Coaxial Nebulizer

: Cyclone Chamber

: Standard Torch

: Horizontal

122

5.9 Analysis of Pastry - ICP-AES

■ ExplanationAfter placing commercially sold pastry in solution, weconducted quantitative analysis of Na, Ca and Fe, usingthe ICPS-7510. The ICPS-7510 can perform multielement quantitation with simple sample pretreatment.Table 5.9.1 shows the quantitation results.

■ SampleCommercially sold pastry(pie, chocolate, dairy cream)

■ PretreatmentWeigh out pie at 0.4 g, or chocolate/dairy cream at 0.2 g,and heat the sample with 1% hydrochloric acid for about2 hours. After allowing the sample to cool, bring it to 100mL. Filter it through No. 5 type B filter paper, and use thefiltrate as the analysis sample.


Table 5.9.1 Quantitation results for pastries (unit : mg/100g)

Na

107.4

42.5

62.9

Mg

22.7

123.4

87.7

Fe

3.5

2.1

0.2

Element

Sample

Pie

Chocolate

Dairy Cream

5.10 Analysis of Nutrition Function Food Products (1) - ICP-AES

123

Ca


■ ExplanationFood with health claims, among the health food productsgenerally known as “supplements”, comprise two officialcategories. They are “food for specified health uses” and“food with nutrient function claims”, both of which mustsatisfy several standards including certain food-labelstandards or food-manufacturing standards. The food forspecified health uses have to be individually approved,and are permitted to display the special logo designed forthis category. The food with nutrient function claims, onthe other hand, have only to comply with certain food-manufacturing standards, and can be freely produced andsold as long as they are in accordance with the rulesspecifying appropriate nutritional constituents. The foodwith nutrient function claims are intended to supply the

Instrument





Sample Introduction

Chamber

Plasma Torch

Observation Method

: ICPS-7510

: 1.0 kW

: 14 L/min

: 1.0 L/min

: 0.7 L/min

: Coaxial Nebulizer

: Cyclone Chamber

: Standard Torch

: Horizontal/Vertical

Table 5.10.1 Standards for upper and lower limit of safe daily intake of food with nutrient function claims (minerals)

Table 5.10.2 Instrument and Analytical Conditions

Constituent

Calcium

Iron

Zinc

Copper

Magnesium

Upper Limit

600mg

10mg

15mg

5mg

300mg

Lower Limit

250mg

4mg

3mg

0.5mg

80mg

■ Sample- Commercially sold soft drink beverages (3 varieties)- Commercially sold fruit juice- Commercially sold processed food product (granules)

■ Sample PretreatmentAdd nitric acid to the appropriate amount of each sample,and then heat on a hot plate to perform decomposition.After decomposition, bring each to a fixed volume usingpure water for use as the analysis sample.

■ Calibration Curve SampleA standard solution (1000 ppm) for atomic absorptionanalysis was mixed and diluted with pure water toprepare the standard solution.

■ ResultsTables 5.10.3 and 5.10.4 show the quantitation results.

Reference Materials- Standards for the Food-labels of Food with Nutrient

Function Claims(March 27, 2001, Ministry of Health, Labour andWelfare Notification No. 97)

- Revision 5 Japan Food Standard Ingredients LabelAnalysis Manual(Public Corporation, Resources Association)

- Standard Hygiene Test Methods and Notations (ThePharmaceutical Society of Japan)

nutritional constituents that are necessary for healthygrowth and development of body, and the maintenance ofhealth. From April 1, 2004, in addition to calcium andiron, this type of food is allowed to display magnesium,zinc, and copper as nutrient function minerals. Table5.10.1 shows the food-manufacturing standards for thenutritional constituents of the food with nutrient functionclaims. We introduce here the results of analysis of thefood with nutrient function claims using ICP emissionspectrometer. Since the ICP emission analytical methodis highly sensitive and has a wide dynamic range,analysis of harmful trace elements can be conducted byanalyzing these nutritional constituents.

5.10 Analysis of Food with Nutrient Function Claims (2) - ICP-AES

124

Table 5.10.3 Quantitation results for soft drinks and fruit juice

�

Na

Mg

Ca

Fe

Cu

Zn

P

K

Element

Soft Drink A(Food with Nutrient Function Claims)

Soft Drink B(Food for specified health uses)

Soft Drink C(Food with Nutrient Function Claims)

Fruit Juice D(Food with Nutrient Function Claims)

Result

mg per 100 mL

µg per 100 mL µg per 100 mL µg per 140 mL µg per 180 mL

mg per 100 mL mg per 140 mL mg per 180 mL

Ingred. Label Value Result Ingred. Label Value Result Ingred. Label Value Result Ingred. Label Value

�

Cr

Se

Mn

Al

As

Cd

Pb

Ni

29

1.0

2.2

0.005

0.0008

0.003

1.2

15

�

0.1

4

1.8

2.8

< 1

0.04

< 0.5

< 0.1

�

30�

1�

2

�

3

0.40

111

0.9

0.0005

0.003

1.3

1.3

�

0.6

< 1

3.3

11.3

< 1

0.1

< 0.5

< 0.1

�

�

�

100�

1.0

�

12

1.8

271

2.0

0.004

0.056

15

28

�

2.3

< 1

20

37

< 1

< 0.4

< 0.7

2.6

�

12�

�

250�

2.0

�

4

7.5

7.2

0.085

0.01

0.038

11.8

171

�

< 0.1

< 2

58

71

< 2

< 0.5

< 0.9

1.4

�

1.8-10.8

Processed food E w/sugar containing vitamins

(Food with Nutrient Function Claims)

mg per granule (0.96 g)

µg per granule (0.96 g)

�

Na

Mg

Ca

Fe

Cu

Zn

P

K

Element Result Ingred. Label Value

Processed food F w/sugar containing iron citrate


mg per granule (0.4 g)

Result Ingred. Label Value

Processed food G w/yeast containing minerals


mg per 10 granules (2.5 g)

µg per granule (0.4 g) µg per 10 granules (2.5 g)

Result Ingred. Label Value

�

Cr

Se

Mn

Al

As

Cd

Pb

Ni

�

0.20

0.027

0.9

0.003

0.0001

0.0002

0.40

0.03

�

0.1

< 0.1

0.8

4.9

< 0.07

< 0.003

0.06

0.07

�

0.25

�

0.03

0.0003

0.001

2.8

0.0003

0.0002

0.0005

0.03

�

0.6

< 0.05

3.8

0.3

< 0.03

< 0.001

0.1

0.3

�

0.034�

�

�

3

�

2

160

260

5.4

0.004

8.5

2

3.7

�

53

23

72

365

< 0.2

0.03

1.6

1.2

�

2

150

250

5.0

7.5

�

50�

25

Table 5.10.4 Quantitation results for processed food products

5.11 Analysis of Plants - ICP-AES

125

Ca


■ ExplanationWe conducted quantitative analysis of plant standardsubstances (tea leaves, olives, citrus leaves) using theICP-7510. Table 5.11.1 shows the quantitation results.We obtained the results for each element, which were inaccordance with the guaranteed standard values.

■ SampleTea leaf powder standard substance NIES No. 7Olive powder standard substance BCR CRM-62Citrus leaf powder standard substance SRM1572

■ PretreatmentTo 0.5 g of sample, add nitric acid, hydrochloric acid anda small amount of hydrofluoric acid, and heat it fordecomposition. After allowing the sample to cool, bring itto a fixed volume of 100 mL for use as the analysissample.


Table 5.11.1 Quantitation results of plant standard substances (unit : µg/g)

Al

Ca

Cr

Cu

Fe

K

Mg

Mn

Ni

P

Pb

Zn

Element

767

3180

0.23

7.0

94.3

19400

1630

684

6.2

3710

1.0

32.0

Quant. Value

Tea Leaf(NIES No.7)

Olive(BCR CRM-62)

Citrus Leaf(NIST SRM1572)

775–20

3200–120

(0.15)

7–0.3

18600–700

1530–60

700–25

6.5–0.3

(3700)

0.8–0.03

33–3

Guar. Value

274

17700

1.1

46.0

298

3350

1140

55

1.1

1050

24.6

17.0

Quant. Value

(260)

(17500)

46.6–1.8

(280)

(3100)

(1200)

57–2.4

(1050)

25–1.5

16–0.7

Guar. Value

99

31300

0.78

17.0

81

18400

5790

21

0.50

1360

13.0

28

Quant. Value

92–15

31500–1000

0.8–0.2

16.5–1.0

90–10

18200–600

5800–300

23–2

0.6–0.3

1300–200

13.3–2.4

29–2

Guar. Value

Value in ( ) = reference value

Instrument





Sample Introduction

Chamber

Plasma Torch

Observation Method

: ICPS-7510

: 1.2 kW

: 14 L/min

: 1.2 L/min

: 0.7 L/min

: Coaxial Nebulizer

: Cyclone Chamber

: Standard Torch

: Axial

126

5.12 Analysis of Powdered Milk Using ICPM-8500 - ICP/MS

■ ExplanationTrace elements in powdered milk were analyzed usingthe ICPM-8500. The sample was decomposed using aclosed microwave decomposition system.

■ SampleCommercial powdered milk

■ Analyzed ElementsAl, Cr, Mn, Fe, Ni, Cd, and Pb

■ PretreatmentThe powdered milk was decomposed for analysis in thefollowing way.1 Approx. 0.5g of the sample was weighed into a

microwave decomposition container, 10mL of nitricacid and 3mL of hydrogen peroxide were added, andthis was left to stand for approx. 3 hours.

2 The lid of the container was closed, and a microwavedecomposition sequence lasting approx. 30 minuteswas run twice.

3 The container was allowed to cool, and the decomposedliquid was transferred to a Teflon beaker and heatedusing a hot plate.

4 When the volume of the liquid had reduced to approx. 2to 3mL, it was transferred to a plastic container anddistilled water was added to create a 50mL solution.50ppb of Y, In, and Tl were added as internal standardelements.

[References] (Quoted Sources)Analysis Manual for Standard Food Constituents Chart inJapan, 5th Edition (Shigen Kyokai)Methods of Analysis in Health Sciences (PharmaceuticalSociety of Japan)

■ Calibration-curve SampleStandard metal solutions for atomic absorption analysiswere mixed and diluted as appropriate with ultrapurewater. 50ppb of the internal standard elements wereadded and nitric acid was added to a concentration of0.5%.

■ Instrument and Conditions

Instrument

Plasma Unit:

Radio-Frequency Output

Coolant Gas Flow Rate (Ar)

Plasma Gas Flow Rate (Ar)

Carrier Gas Flow Rate (Ar)

Sample Injection Unit:

Nebulizer

Chamber

Plasma Torch

Sampling Depth

Sampling Interface

: ICPM-8500

: 1.2 kW

: 7 L/min

: 1.5 L/min

: 0.62 L/min

: Concentric Nebulizer

: Cooled Scott Chamber

: Triple-Tube Mini-Torch

: 5 mm

: Cu

Table 5.12.2 Quantitative results for powdered milk (µg/g)

Table 5.12.1 Analytical conditions for the ICP/MS method

■ AnalysisQuantitative analysis was performed with the calibration-curve method.

■ ResultsTable 5.12.2 shows the results of quantitative analysis.

Element

Al 27

Cr 52

55

57

60

111

208

Mn

Fe

Ni

Cd

Pb

M/Z INT

Y

Y

Y

Y

Y

2.3

0.11

0.28

7.8

0.33

0.01

0.04

In

TI

Quantitative value

Table 5.13.2 Results of plant analysis (µg/g)

M/Z

51

52

59

60

63

66

75

82

98

111

208

0.049

0.31

0.05

0.41

3.0

23.3

0.12

0.45

0.29

1.6

Element

Element

V

Cr

Quantitativevalue

Rice flour NIES No. 10

-

0.22*

Certified value

Co 0.02*

Ni 0.39±0.04

Cu 3.3±0.2

Zn 22.3±0.9

As 0.11*

Se 0.02*

Mo 0.42±0.05

Cd 0.32±0.02

Pb -

0.89

3.2

0.42

1.2

9.4

56

0.28

0.09

0.49

2.6

5.8

Quantitativevalue

Tomato . LeavesNIST . SRM1573

-

4.5±0.5

Certified value

0.6*

-

11±1

62±6

0.27±0.05

-

-

3*

6.3±0.3

0.19

0.81

0.06

0.77

14

29

3.1

0.04

0.12

0.11

12.0

Quantitativevalue

Citrus . LeavesNIST . SRM1572

-

0.8±0.2

Certified value

0.02*

0.6±0.3

16.5±1.0

29±2

3.1±0.3

-

0.17±0.09

0.03±0.01

13.3±2.4

* Reference values

127

Ca


5.13 Analysis of Plants Using ICPM-8500 - ICP/MS

■ ExplanationStandard plant samples were analyzed using the ICPM-8500. ICP/MS is an analysis method that offers a widedynamic range and a very high level of sensitivity andenables the batch analysis of trace inorganic constituents,such as Pb, Cd, As, and Se, in the same solution.

■ SampleRice flour (NIES No. 10)Tomato leaves (NIST SRM1573)Citrus leaves (NIST SRM1572)

■ Analyzed ElementsNi, Pb, As, Hg, Cd, Al, and Cr

■ Sample Preparation: Pressurized Decomposition0.1g of the samples were measured out in Teflonpressurized containers and 1mL of nitric acid was addedto each of them. After sealing the containers, they wereheated for 2 hours at 170°C. After being left to cool, Hoand Rh (10ppb each) were added as internal standardelements, and ultrapure water was added to make 50mLsample solutions.

■ Provision of Analysis SamplesTokyo University of Agriculture, Production andEnvironmental Chemistry Laboratory (Soil ScienceLaboratory)

[References] (Quoted Sources)46th Environment Agency Notice (environmental qualitystandards for soil)Partial amendment to enforcement regulations for theWater Pollution Control Law related to environmentalstandards promulgated on 8 March 1993

JIS K0102-1998 (Testing Methods for IndustrialWastewater)

■ Calibration-curve SampleA standard solution for atomic absorption analysis(1,000ppm) was mixed and diluted with ultrapure water.50ppb of internal standard elements were added and nitricacid was added to a concentration of 0.5%.

■ Instrument and Conditions

■ AnalysisQuantitative analysis was performed with the calibration-curve method.

■ ResultsFig. 5.13.2 shows the quantitative results. The measurementresults have been multiplied by the dilution factor toexpress them as concentrations in solids. Results for allthree samples correlated well with the certified values.

InstrumentPlasma Unit:Radio-Frequency OutputCoolant Gas Flow Rate (Ar)Plasma Gas Flow Rate (Ar)Carrier Gas Flow Rate (Ar)Sample Injection Unit:NebulizerChamberPlasma TorchSampling DepthSampling Interface

: ICPM-8500

: 1.2 kW: 7 L/min: 1.5 L/min: 0.62 L/min

: Concentric Nebulizer: Cooled Scott Chamber: Triple-Tube Mini-Torch: 5 mm: Cu

Table 5.13.1 Instrument and conditions

128

6. Others

6.1 Analysis of Organotin Compounds Using Capillary GC-FPD - GC

■ ExplanationTributyltin (TBT) and triphenyltin (TPT) are widely usedas antifouling coatings for ships and fishing nets.Although the production of coatings containing TBT wasstopped in Japan in 1997, it is still used in other countriesand is a cause of pollution in seawater and marine life.These compounds are also suspected of being endocrinedisrupters. The analysis of organotin compounds thathave undergone standard alkylation and deuterium-labeled organotin compound mixtures is described here asan example.


Fig. 6.1.2 Chromatogram of sea bass extract (0.2 to 0.4µg of standard product added to 2mL of final solution)

Instrument:

Column

Column Temp.

Inj. Temp.

Det. Temp.

Carrier Gas

Injection Method

Injection Volume

: GC-17AAFwver.3 (FPD-17c Sn Filter)

: DB-5 (30m × 0.25mm I.D. df = 0.25µm)

: 60˚C(1min)-20˚C/min-140˚C

-7˚C/min-280˚C(5min)

: 290˚C

: 300˚C

: He, 35kPa(1min)-150kPa(2.4mL/min)

: High-pressure splitless (1min)

: 3µL

0 5 10 15 20 25min

mV

0

2

1 23 45

6

7

8

910

1112

4

6 ■ Peaks1.Monobutyl Tin2.Dibutyl Tin(d18)3.Dibutyl Tin4.Tributyl Tin(d27)5.Tributyl Tin(TBT)6.Tetrabutyl Tin(TeBT)7.Monophenyl Tin8.Tripentyl Tin(TPeT)9.Diphenyl Tin(d10)10.Diphenyl Tin11.Triphenyl Tin(d15)12.Triphenyl Tin(TPT)

Fig. 6.1.1 Examples of extraction methods for organotin compounds

10g of fish↓

Extraction (1:1 mixture of 1N-methanol hydrochloride and ethyl acetate)↓

Extraction (3:2 mixture of ethyl acetate and hexane)↓

Column chromatography (anion-cation exchange resin)↓

Propylation (Grignard reagent: PrMgBr)↓

Column chromatography (Florisil)↓

Concentration: 0.2 to 2mL↓

GC or GC/MS

1L of water↓

Addition of 10mL of hydrochloric acid and 20g of NaCl↓

Extraction (hexane)

↓

Propylation (Grignard solution: PrMgBr)↓

Column chromatography (Florisil)↓

Concentration: 0.2 to 2mL↓

GC or GC/MS

129

Others

6.2 Analysis of Organotin in Fish (1) - GC/MS

■ ExplanationOrganotins such as tributyltin (TBT) and triphenyltin(TPT) are widely used as antifouling coatings for shipsand fishing nets, and the resulting pollution of seawaterand marine life has become an issue of concern. Althoughthese compounds are usually analyzed using GC-FPD,analysis using GCMS, which enables highly accuratequalitative determination, is described here as anexample.

Although tripentyltin (TPeT) is often used with GCmeasurement as the internal standard substance, this isnot an ideal selection because TBT, TPT, and TPeT havedifferent recovery rates. In this example, a deuterium-labeled compound, which makes full use of GC/MScharacteristics, is used as the internal standard substance.The advantage of the deuterium-labeled compound as astandard substance is that its properties are similar to thetarget compound but it does not exist in the sample.


Column

Column Temp.

Inj. Temp.

I/F Temp.

Carrier Gas

Injection Method

: GCMS-QP5000

: DB-1 (30m × 0.32mm I.D. df = 0.25µm)

: 50˚C(2min)-20˚C/min-140˚C-7˚C/min

-220˚C-15˚C/min-310˚C(6min)

: 280˚C

: 300˚C

: He (40kPa)

: Splitless (2min)

10g of fish

↓Extraction (1N ethanol hydrochloride)

↓Extraction (3:2 mixture of ethyl acetate and hexane)

↓Column chromatography (cation-anion exchange resin)

↓Propylation (Grignard reagent : PrMgBr)

↓Column chromatography (Florisil)

↓Sample: 2mL

↓GC/MS

1L of seawater

↓

Extraction (3:2 mixture of ethyl acetate and hexane)

↓

Propylation (Grignard reagent: PrMgBr)

↓

Sample: 2mL

↓GC/MS

Fig. 6.2.1 Extraction methods for organotin in fish and seawater

■ PretreatmentThe extraction methods for fish and seawater are shownbelow.

Constituent Selected ions (m/z)

295, 293, 316

277, 275, 291

291, 289

303, 305

366, 364

351, 349

d27-TBT

TBT

Tetra-BT

TPeT

d15-TPT

TPT

130

6.2 Analysis of Organotin in Fish (2) - GC/MS

41

50

57

69

121

110

83 97

100 150

135

165

179

221

197200

217235

250

270

277

291

300 350

Fig. 6.2.2 Mass spectrum of TBT

TIC1.MBT2.DBT-d183.DBT4.TBT-d27

5.TBT6.TetraBT3.MPT4.TPeT

9.DPT-d1010.DPT11.TPT-d1512.TPT

1 23

4 5

6

10

7

12

8

14

9 10

16 18

11 12

20

Fig. 6.2.6 SIM chromatogram of standard sample

4151

50

71

91

100

120

124145

150

171187

200

197

215 245

250

273

300 350299 345

351

Fig. 6.2.4 Mass spectrum of TPT

Mass number : 277.00 Constituent : TBTArea ratio=0.00070351 *Concentration ratio Contribution ratio=0.999907Area Ratio

8.010-1

4.0

0 0.5Conc Ratio

1.0103

1234

Concentration ratio Area ratio10.000

100.000500.000

1000.000

0.0060.0680.3460.706

Fig. 6.2.7 Calibration curve for TBT (10 to 1,000ppb)

289675

4

10.5 11.0

291.00277.00

IDTypeConstituent

Retention timeAreaConcentration

: 6 Mass number : 277.00: Target: T B T

: 10.788: 44583: 1.021ppm

Mass number AreaIntensity ratio1 291.00 18581 42

Fig. 6.2.9 SIM chromatogram for TBT in sea bass

189

46

50

57 66 85 97

100

111

122

136

150

154

167

200

187

212

220

231

254

250267

300 350332

318

314

295

Fig. 6.2.3 Mass spectrum of d27-TBT

41

50

57

7182 99

100

118120

148 160

150

202

200

204 226 239 252250

268 282 302 316300

342 276 355 380350

364

Fig. 6.2.5 Mass spectrum of d15-TPT

Mass number : 351.00 Constituent : TPTArea ratio=0.00370016 *Concentration ratio Contribution ratio=0.999963Area Ratio

4.0

2.0

0 0.5 1.0103Conc Ratio

1234

10.000100.000500.000

1000.000

0.0370.3811.8373.705

Concentration ratio Area ratio

Fig. 6.2.8 Calibration curve for TPT (10 to 1,000ppb)

Table 6.2.1 Quantitative results for tin in fish and seawater

0.068

Port W(µg/L)

0.078

0.173

Port K(µg/L)

0.019

Constituent

TBT

TPT

0.436 0.782

Sea bream(µg/g)

Sea bass(µg/g)

0.014 0.010

131

Others

6.3 Analysis of Shellfish Toxins (1) - LC

■ ExplanationIn recent years shellfish poisoned with paralytic shellfishtoxins are found in various regions, causing majordamage to the marine product industry and seriousproblems in food hygiene.Paralytic shellfish toxin is a neurotoxin produced by aphytoplankton dinoflagellate, and is known by thecomponent names such as saxitoxin or gonyautoxin.Post column derivatization fluorescent detection LCanalysis method was used by Nagashima and Oshima toanalyze this shellfish toxin.Here, this method is used in an analysis example forgonyautoxin (GTX) 1-4 standard sample.

ReferencesLC talk, No. 36 from Shimadzu Corporation (1995)Y.Nagashima, et.al.,Nippon Suisan Gakkai, 53 (5), 819 (1978).Y.Oshima, et.al., "Mycotoxins and Phycotoxins '88",Elsevier Science Publishers, New York, 1989, 319.


GTX-4(3.5)

0.0

3.0

6.0

9.0

12.0

15.0

18.0

GTX-3(2.5)

GTX-1(7)

GTX-2(5)

( ): Conc. in MU/mL

Fig. 6.3.1 Analysis example for gonyautoxin (GTX) 1-4 standard sample

Inj. Vol.: 10µL, concentration recorded at top of peak

- Separation conditions

Column

Mobile Phase

Temperature

Flow Rate

- Reaction Conditions

Primary

Reaction Reagent

Flow Rate

Temperature

Secondary

Reaction Reagent

Flow Rate

Temperature

- Detection

: STR ODS-2(150mmL. × 4.0mmI.D.)

: 10mM (Sodium) Phosphate Buffer

(pH 7.0) containing 4mM (Sodium)

Heptanesulfonate

: 40˚C

: 0.8mL/min

: 50mM (Sodium) Borate Buffer

(pH 9.5) containing 5mM Periodic Acid

: 0.4mL/min

: 60˚C

: 110mM of Phosphoric Acid Buffer

(pH 2.1)

: 0.4mL/min

: 40˚C


Ex : 330nm Em : 390nm

132

6.3 Analysis of Shellfish Toxins (2) - LC

M1 M2

1 2 4 5

6

789 10

13

14 15

11

12

10

R1

R2

3

1. 9. Degassing units2. Low-pressure gradient unit3. 10. Solvent delivery units4. Mixer5. Auto injector6. Column oven7. Analysis column

8. Chemical reaction tank11. Blender12. Fluorescent detector13. 14. 15. Reaction coilsM1, M2 Mobile phasesR1, R2 Reaction liquids

Fig. 6.3.2 Flow line

H

H

+

+HOH

OH

N

H2N

R1

R2 R3

N

NH3

N

O

OHN

HN

R1H

OHOHHH

OH

R2HHHH

OSO3-

OSO3-

R3HH

OSO3-

OSO3-

HH

STXneoSTXGTX1GTX2GTX3GTX4

Fig. 6.3.3 Structural formula of shell toxin components

133

Others

6.4 Analysis of Oxytetracycline - LC

■ ExplanationShop-sold pig liver was extracted using the officialgazette method and oxytetracycline was added to make asolution of 0.5ppm for analysis.

ReferenceOfficial Gazette extra No. 245 (December 26, 1995)

■ PretreatmentThe sample was pre-treated as shown in Fig 6.4.2 inaccordance with the official gazette.


Mobile Phase

Temperature

Flow Rate

Detection

: STR ODS-2(150mmL. × 4.6mmI.D.)

: 1M Imidazole Buffer/

Methanol = 77/23 (v/v)

: 40˚C

: 1.0mL/min


Ex : 380nm Em : 520nm

Oxytetracycline

mV

10

5

0

0 5 10 15 20min

Fig. 6.4.1 Analysis example of oxytetracycline

Sample 5g100mL McIlvaine buffer containing 0.01M EDTA2Na

Homogenize100mL hexane

Shake (5 min)

Centrifugal separation (3,500 rpm at room temperature for 10 min)

Lower layer splitting

Filtration

Filtrate 50mL

Sep-Pak Plus PS2Rinse with 30mL of distilled waterElution using 10mL methanol

Concentration to dryness (35 to 40˚C)2.5mL phosphoric acid buffer

HPLC

Fig. 6.4.2 Pretreatment flowchart for oxytetracycline

134

Sample 5g70mL acetonitrile70mL acetonitrile saturated hexaneHomogenize, centrifugal separation (2,600 rpm for 5 min)Transfer to separation funnel

Lower layer30mL acetonitrile saturated hexaneShake

Lower layer

10mL n-propanolConcentration to dryness under reduced pressure

ResidueDissolve in 5mL acetonitrileC18 cartridge

5mL acetonitrile poured down twiceCollect all of the acetonitrile

Concentration to dryness under reduced pressure

ResidueDissolve in 2mL Methanol/water (7:2) (6mL of liver or fat)

Test solution20µL

HPLC

Upper layer

Upper layer10mL acetonitrileShake

Lower layer

Fig. 6.5.2 Pretreatment flowchart for closantel

Closantel

mAbs20

15

10

10

5

5

0

0

0 10 20min

Ch2 24bnmCh2 24bnmCh2 24bnm

Ch2 36bnmCh2 36bnmCh3 36bnm

Fig. 6.5.1 Analysis example of closantel

6.5 Analysis of Closantel - LC

■ ExplanationShop-sold pig liver was extracted using the officialgazette method and closantel was added to make asolution of 1ppm for analysis.

ReferenceOfficial Gazette extra No. 245 (December 26, 1995)

■ PretreatmentThe sample was pre-treated as shown in Fig 6.5.2 inaccordance with the official gazette.


Mobile Phase

Temperature

Flow Rate

Detection

: STR-ODS-2(150mmL. × 4.6mmI.D.)

: Methanol/20mM Sodium

Dihydrogenphosphate Buffer (pH 3.3)

= 7/2 (v/v)

: 40˚C

: 1.0mL/min


135

Others

6.6 Simultaneous Analysis of Synthetic Antibacterial Agent (1) - LC

■ ExplanationHPLC is recognized as the best method for analysis offood-residual (especially fish and meat) antibacterialagents and antibiotics.

ReferencesHamada, Murakita; Shimadzu Review, 52 (2), 107 (1995)Murayama, Uchiyama, Saito, Food Hygiene Journal, 32,155 (1991)Milk Hygiene Volume 79, April 1993 (from the formerMinistry of Health and Welfare)

■ PretreatmentFig. 6.6.2 shows the method recommended by the formerMinistry of Health and Welfare, Japan (currently theMinistry of Health, Labour and Welfare).


Mobile Phase

Temperature

Flow Rate

Detection

: STR ODS-2(4.6mmφ×150mm)

: A: Water/Acetic Acid = 100/0.3 (v/v)

(NaClO4: included)

: B: Acetonitrile/Water/Acetic Acid

= 90/10/0.3 (v/v/v) (NaClO4 included)

Gradient Elution of 2 Liquids

: 40˚C

: 2.0mL/min

: Photodiode Array Detector

λ= 195nm to 600nm

0 10 20

1

3

2

45

6

7

8

9

10

11

1213 14

15

16 17

18

19

30 (min)

■ Peaks

1.ODX 2.CLP 3.SMR 4.TP 5.CDX 6.SDD 7.FZ 8.SMMX 9.TMP10.OMP

11.OA12.SDMX13.SQ14.MRT15.NA16.PYR17.PA18.DFZ19.NCZ

Fig. 6.6.1 Simultaneous analysis example for 19 synthetic antibacterial agent components

136

6.6 Simultaneous Analysis of Synthetic Antibacterial Agent (2) - LC

Acetonitrile layer Hexane layer

ResidueAcetonitrile layer

Acetonitrile layer

10µL acetonitrile layer - water layer

Residue

HPLC

Acetonitrile layer

Sample 5g

Hexane layer

Shake for 5 min

25mL acetonitrile

Ultrasound extraction for 30 sec

Centrifugal separation 3000 rpm for 5 min

10g anhydrous sodium sulfate

25mL acetonitrile

Homogenize, centrifugal separation 3000 rpm for 5 min

10mL 1-proponal

Concentration to dryness

1mL acetonitrile: water (4:6)

Ultrasound extraction for 30 sec

0.5mL acetonitrile saturated hexane

Centrifugal separation 3000 rpm for 5 min

25mL acetonitrile saturated hexane

Shake for 5 min

Residue

Fig. 6.6.2 Pretreatment flowchart for simultaneous analysis of 19 synthetic antibacterial agent components

6.7 Analysis of Enrofloxacin in Broiled Eel (1) - LC

137

Others

■ ExplanationEnrofloxacin is a type of the new quinolone syntheticantibiotics used to prevent and treat pneumonia and E.coli diarrhea in cows and pigs. However, in Japan, its usein farmed fish is not permitted. The analytical method forenrofloxacin in eel is prescribed in the notification“Enrofloxacin Analytical Method for Eel” (June 5, 2003,Ministry of Health, Labour and Welfare, Department ofFood Safety No. 0605002). The notification also specifiesboth HPLC using fluorescence detection and LC/MSusing electrospray ionization (ESI) as the analyticalmethods that can be used. We introduce here analysis ofbroiled eel (broiled without sauce), conducted by bothHPLC and LC/MS in compliance with these methods.


Pretreatment

Sample 10 g

Acetonitrile 30 mL

Homogenize

Centrifugal separation (3000 rpm, 15 min)

Acetonitrile 30 mL

Homogenize


Hexane 60 mL

Solvent distillation under low pressure (below 40˚C )

Water/Acetonitrile = 85/15 (v/v) 2 mL

HPLC LC/MS

Inject 5 µLInject 10 µL

Residue

Supernatant

Acetonitrile layer

Supernatant

Supernatant Residue

1 2

2

1

+

0 5 10 15min

0

0.5

1.0

(×100,000)

1

■ Peak1.Enrofloxacin

mV

Fig. 6.7.3 Chromatogram of broiled eel (spiked with 50 µg/L of enrofloxacin)

0 5 10 15min

0

0.5

1.0

(×100,000)

mV

Fig. 6.7.2 Chromatogram of broiled eel

Fig. 6.7.1 Structural formula of enrofloxacin

* McIlvain buffer (pH=3.0) preparation procedureA) Disodium hydrogen phosphate solution

Dissolve 71.63 g of disodium hydrogen phosphate (Na2HPO4 ⋅ 12H2O) in waterto make the volume 1L.

B) Citric acid solutionDissolve 21 g of citric acid (C6H8O7 ⋅ H2O) in water to make the volume 1L.Mix A and B at a ratio of 1:4 to obtain a pH of 3.0.

N

COOH

O

F

N

H3CH2CN

Column

Mobile Phase

Flow Rate

Column

Temperature

Detection

Injection Volume


: A: McIlvain Buffer Solution* (pH=3.0)

B: Acetonitrile

A/B = 85/15 (v/v)

: 1.0 mL/min

: 40˚C

: RF-10AXL Super (Cell Temperature 25˚C )

Ex: 285 nm Em: 460 nm

: 5 µL

6.7 Analysis of Enrofloxacin in Broiled Eel (2) - LC/MS

138

Fig. 6.7.4 shows an example of LC/MS analysis results ofbroiled eel using ESI positive mode. This is a SIMchromatogram (molecular weight 359) in which thedotted line indicates the pretreated sample, and the solid


Mobile Phase A

Mobile Phase B

Gradient Program

Flow Rate

Injection Volume

Column Temperature

Ionization Mode

Probe Voltage

Nebulizer Gas Flow Rate

Drying Gas

CDL Temperature

Block Heater Temperature

SIM


: 0.1% Formic Acid Aqueous Solution

: 90% Methanol - Water including 0.1% Formic Acid

: 0%B (0 min) → 100% B (20 – 25 min) → 0%B (25.01 – 35 min)

: 0.2 mL/min

: 10 µL

: 40˚C

: ESI Positive

: 4.5kV

: 1.5 L/min

: 0.15 MPa

: 200˚C

: 200˚C

: m/z 360

Fig. 6.7.4 SIM Chromatogram of broiled eel (spiked with 50 µg/L of enrofloxacin)

0 5 10 15 200.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0(×10,000)

360.00 (1.00)

■ Peak1.Enrofloxacin 1

min

line indicates the sample spiked with 50 mg/L ofenrofloxacin. The peak m/z360 is the enrofloxacinprotonated molecule.

6.8 Analysis of Malachite Green in Farmed Fish - LC

139

Others

■ ExplanationMalachite green is a type of organic pigment that is usedas a curative drug for the treatment of white spot diseaseor the tail rot in aquarium fish. However, in Japan, it isnot permitted to be used in farmed fish. The analyticalmethod for malachite green residue is prescribed in thenotification “Analytical Method for Malachite Green inFarmed Fish” (December 16, 2004, Ministry of Health,Labour and Welfare, Department of Food Safety No.1216002). The notification also specifies HPLC as theanalytical method and LC/MS as the confirmation testmethod to be used. We introduce here an example ofanalysis of commercially sold trout (malachite greenstandard addition sample) using the HPLC method incompliance with this notification.

■ Analysis of Commercial TroutFig. 6.8.2 shows the results of analysis of commerciallysold trout. Sample pretreatment was conducted accordingto the method described in the notification. The lowerlevel chromatogram is the result of the sample analysis,and the upper level chromatogram is the result of analysisof the sample (final acetonitrile solution) spiked with10µg/L of malachite green (equivalent to 2.0 µg/kg introut).


■ Pretreatment

Sample 10 g

Residue

Residue

Supernatant

Acetonitrile layer Hexane layer

20% NaCl 50 mLDichloromethane 50 mL

Hexane 80 mL

Acetonitrile 40 mL

Acetonitrile 40 mL

McIlvain buffer solution (pH3.0) 20 mL

Anhydrous sodium sulfate

Acetonitrile 2 mL

Dichloromethane layer Water layer

Homogenize 5min

Mix (5 min)

Mix (5 min)

Mix (5 min)

Filter

Solvent distillation 40˚C

HPLC (20 µL)



1

Supernatant 2

Supernatant +1 2

■ Peak1.Malachite green

1

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

-100

-50

0

50

100

150

200

250

300

350

400

450

Fig.6.8.2 Chromatograms of commercially sold trout(upper level) Commercially sold trout (spiked with 10 µg/L malachite green)(lower level) Commercially sold trout

Fig. 6.8.1 Malachite green structural formula (left) andabsorption spectrum (right)

300 400 500 600 700 nm

0.00

0.50

1.00

1.50

2.00

2.50

3.00

mAU

N

CH3

CH3

N

C H3

H3C +

N

CH3

CH3

N

C H3

H3C +

Column

Guard Column

Mobile Phase

Flow Rate

Column

Temperature

Detection

Injection Volume


: Shim-pack GVP-ODS (10 mmL. × 4.6 mm I.D.)

: A : 0.1M Citric Acid Buffer Solution (pH=3)

B : Acetonitrile

A/B = 1/1 (v/v)

: 0.6 mL/min

: 40˚C

: SPD-20AV 636 nm

: 20 µL

140

6.9 Analysis of New Type Quinolone Antibacterial Agents in Poultry - LC/MS

■ ExplanationIn Japan, some of the standards for food products andadditives were revised in accordance with NotificationNo. 369 issued by the Ministry of Health, Labour andWelfare on 26 November 2003. New standards and testmethods for the residual amounts of sarafloxacin anddanofloxacin in meat were established, and LC/MS isnow used for confirmation tests. In the example presentedhere, LC/MS is used in the analysis of new typequinolone antibacterial agents (sarafloxacin anddanofloxacin).

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

2.35

2.40

2.45

2.50

2.55

2.60

2.65

2.70

2.75

2.80

2.85(x1,000)

—386.00 (1.00)

---358.00 (1.00)

Danofloxacin

Sarafloxacin

Fig. 6.11.1 Analysis of standard samples (50 ppb for eachconstituent, 50 µL injected)

InstrumentsColumn

Mobile Phase

Flow RateTemperatureSample Store Temp

Ionization ModeApplied VoltageNebulizer Gas FlowDrying Gas PressureCDL Temp.Heat Block Temp.CDL VoltageQ-array VoltageSelected Ion Mass Number

: LCMS-2010A

: Shim-pack VP-ODS

(150mmL. × 4.6mm I.D.)

: A: 0.05%TFA-Water

B: Acetonitrile

A/B= 4/1 (v/v)

: 0.8mL/min

: 40˚C

: 5˚C

: ESI-Positive

: 4.5kV

: 1.5L/min.

: 0.2MPa

: 200˚C

: 200˚C

: S-Mode

: S-Mode

: m/z 358.00 (M+H)+ for Danofloxacin

m/z 386.00 (M+H)+ for Sarafloxacin


■ Confirming Addition of Regulated-Level ConcentrationAlthough unwanted peaks are obtained at retention timesdifferent to that of danofloxacin, the regulated-levelconcentration can be easily detected.

(×1,000)

---Poultry blank with regulated-level concentration added—Poultry blank

C19H20FN3O3

Exact Mass: 357.15Mol.Wt.:357.38

Danofloxacin

N

COOH

O

F

N

H3CN

3.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Danofloxacin

Fig. 6.9.2 Chromatogram for addition of regulated-levelconcentration (danofloxacin)

C20H17F2N3O3

Exact Mass: 385.12Mol.Wt.:385.36

Sarafloxacin

F

N

COOH

O

F

N

HN

(×1,000)

Sarafloxacin

2.34

2.35

2.36

2.37

2.38

2.39

2.40

2.41

2.42

2.43

2.44

2.45

2.46

2.47

2.48

2.49

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

---Poultry blank with regulated-level concentration added—Poultry blank

Fig. 6.9.3 Chromatogram for addition of regulated-levelconcentration (sarafloxacin)

141

Others

6.10 Analysis of Aminoglycoside Antibiotics (1) - LC/MS

■ ExplanationSome of the ministerial ordinances related to standards onthe constituents of milk and dairy products were revisedin accordance with Notification No. 170 issued by theMinistry of Health, Labour and Welfare on 26 November2003. New standards and test methods (LC/MS) for theresidual amounts of streptomycin and dihydrostreptomycin

Spectinomycin Streptomycin

Dihydrostreptomycin

Gentamycin C1a

Gentamycin C1

Gentamycin C2

Neomycin B

(min)6.0

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5(×100,000)

7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0

351.00(1.00)

292.70(2.00)

300.70(2.00)

225.70(5.00)

232.70(7.00)

239.70(3.00)

308.20(0.50)

Fig. 6.10.1 ESI mass chromatogram for aminoglycoside antibiotics

InstrumentsColumnMobile Phase AMobile Phase BFlow Rate Gradient ProgramColumn TemperatureInjection Volume

Ionization ModeApplied VoltageNeburizing Gas FlowDrying Gas PressureCDL TemperatureBlock TemperatureCDL VoltageSelected Ion Mass Number

: Shimadzu LCMS-2010A

: Shim-pack VP-ODS (150mmL. × 2.0mm I.D. )

: 5mM Perfluorobutyric Acid (PFBA)-Water

: Acetonitrile

: 0.4mL/min

: 10%B (0 min) → 40%B (15-20 min)

: 40˚C

: 10µL

: Positive ESI

: 4.5kV

: 1.5L/min

: 0.1MPa

: 200˚C

: 200˚C

: S-Mode

: m/z 351.0 for Spectinomycin m/z 225.7 for Gentamycin C1a

m/z 300.7 for Streptomycin m/z 232.7 for Gentamycin C2

m/z 292.7 for Dihydrostreptomycin m/z 239.7 for Gentamycin C1

m/z 308.2 for Neomycin B


in milk were established. In the example presented here,the analysis of these two constituents and of gentamycins,spectinomycin, and neomycin, constituents for whichLC/MS was already specified as the test method, isperformed.

6.10 Analysis of Aminoglycoside Antibiotics (2) - LC/MS

142

■ Samples with Standard of Regulated Concentration Added and Pork Blanks

6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8 Spectinomycin(m/z 351.0)

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0 Streptomycin (m/z 300.7)

(min)(min)

(×1,000) (×1,000)

Fig. 6.10.2 Samples with standard of regulated-level concentration added and pork blanks (1)

13.0 13.5 14.0 14.5 15.0 15.5 16.0 16.5

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.9

2.0

Neomycin B (m/z 308.2)

(min)

(×1,000)

9.0 9.5 10.0 10.5 11.0 11.5 12.0 12.5

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

(×1,000)

(min)

Dihydrostreptomycin(m/z 292.7)


(min)(min)

Gentamycin C1(m/z 239.7)

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.30

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

1.4

1.5

1.6

12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0

(×1,000) (×1,000)

Gentamycin C1a(m/z 225.7)


—Solid line : Regulated-level concentration added

---Dotted line: Pork blank





143

Others

6.11 Analysis of Fumonisin in Sweet Corn (1) - LC

■ ExplanationThe mycotoxin family member fumonisin is related tofusarium branch and is known to be the cause of equineleukoencephalomalacia and lung edema in pigs. Recentresearch also points to its involvement in humanesophageal cancer. Here, this component was analyzedusing pre-label fluorescent derivatization and detectionincorporating OPA agent.

ReferenceG.S.Shepland,et.al.,J.LiquidChromatogr.,13,2077 (1990)

■ Pretreatment200µL of thiol agent and 200µL of OPA agent was addedto 100µL of sample solution. After mixed and left to standfor 3 min, 10µL of the mixture was injected to HPLC.

Thiol Agent: 0.1M (Sodium) Borate Buffer (pH 9.2)containing 50mM 3-Mercaptopropionic Acid

OPA Agent: A/B = 1/4 mixtureA: 0.25M o-Phthalaldehyde Methanol SolutionB: 0.1M (Sodium) Borate Buffer (pH 9.2)


30

20

10

0

0 5 10 15 20min

Fum

onis

in B

2

Fum

onis

in B

1

Fig 6.11.1 Analysis example of fumonisin in sweet corn

ColumnMobile Phase

TemperatureFlow RateDetection

: STR ODS-II (150mmL. × 4.6mm I.D.): Methanol/50mM Citrate Buffer

(pH 4.3) (7/3, v/v): 40°C: 1.0mL/min: Fluorescence

Ex : 335nm Em : 440nm

144

6.11 Analysis of Fumonisin in Sweet Corn (2) - LC

Sample 20g

Filtration

Shake 10 min

Filtrate

Anion exchange SPE cartridge

Conditioning

Sample loading

Washing

Desorption

Eluent

10 mL

Methanol / water (3/1)50 mL

Residue

Evaporate to dryness

100µL to Derivatization

0.1M Borate buffer(pH 9.2) / Methanol(1/1) 500µL

Methanol 5mLMethanol / water (3/1) 5mL

Methanol / water (3/1) 8mLMethanol 3mL

1% Acetic acid methanol soln. 10mL

Fig. 6.11.2 Pretreatment flowchart for sweet corn

145

Others

6.12 Analysis of Aflatoxins (1) - LC

■ ExplanationAflatoxins are toxins produced by the Aspergillus familyof molds that grow in tropical and subtropical regions.Aflatoxins indicate a powerful acute toxicity (having adegenerative effect on the brain, liver and kidneys, etc.)and trace amounts of aflatoxins can cause liver cancer ifabsorbed over a long period of time. Aflatoxins includethe structurally similar B1, B2, G1 and G2 types and theirmetabolites M1 and M2, of which the most toxic withrespect to animals is B1. The Japanese Food SanitationLaw stipulates that the B1 aflatoxin exceeding 10 ppbmust not be detected in any food products. This sectionshows an example of analyzing an actual sample.


Mobile Phase


: Silica gel (5.5 µm particle diameter)(100 mm L. × 4.0 mm I.D.)

: Toluene/Ethylacetate/FormicAcid/Methanol = 90/5/2.5/2.5 (v/v)

: 40°C: 1.1mL/min: Fluorescence Ex : 365nm Em : 425nm

0 10 20 (min.)(b)(a)

0 10 20 (min.)

1

2

3

4

56

5

6

Fig. 6.12.1 Analysis of aflatoxins contained in cheese

■ Peaks

1. Aflatoxin B1

2. Aflatoxin B2

3. Aflatoxin G1

4. Aflatoxin G2

5. Aflatoxin M1

6. Aflatoxin M2

(a) Standard sample

(b) Content in cheese

146


0 10 20 30 (min.)

2

3

4

1

Fig. 6.12.2 Analysis of aflatoxins contained in peanuts

■ Peaks

1. Aflatoxin B1

2. Aflatoxin B2

3. Aflatoxin G1

4. Aflatoxin G2

0 10 20 30 (min.)

1

2

Fig. 6.12.3 Analysis of aflatoxins contained in corn

■ Peaks

1. Aflatoxin B1

2. Aflatoxin B2

■ Analytical ConditionsColumnMobile Phase


: Zorbax SIL (250mmL. × 2.1mmI.D.): Toluene / Ethyl Acetate / Formic Acid /

Methanol = 890 / 75 / 20 / 15 (v/v): 40°C: 0.37mL/min: Fluorescence Ex : 365nm Em : 425nm

■ Analytical ConditionsColumnMobile Phase


: Zorbax SIL (250mmL. × 2.1mmI.D.): Toluene / Ethyl Acetate / Formic Acid /

Methanol = 890 / 75 / 20 / 15 (v/v): 40°C: 0.37mL/min: Fluorescence Ex : 365nm Em : 425nm

147

Others


Aflatoxin analysis was performed with the help of Prof.Tetsuhisa Goto of Japan's National Food Research

Fig. 6.12.4 Extraction conditions for aflatoxins

Institute (supervised by the Ministry of Agriculture,Forestry and Fisheries).

Sample (75n)

← water

← Acetone (215mL)Blended for 30min

← diatomaceous earth (10n)StirredFiltered

Filtrate (200mL)

← 5% sodium chloride (100mL)

← hexane (100mL)Shaken for 5 min

hexane layer aqueous acetone layer.

← 5% sodium chloride (50mL)

← chloroform (100mL, 50mL)Shaken for 3 min

chloroform layer aqueous acetone layer.

← 5% sodium chloride (100mL)Shaken for 3 min

aqueous layer chloroform layer

Dehydrated through an anhydrous sodium sulfate columnconcentrated to about 10mL

A cidic alumina-silica gel-sodium sulfate column (5n, 15n, 15n layer)

Washed with benzene-acetic acid (9 : 1) (100mL)ether-hexane (3 : 1) (150mL)

Eluted with chloroform-methanol (97 : 3) (200mL)Evaporated to dryness in an N2 stream

Residue

Dissolved in chloroform (100µL)

Test solution for HPLC

148

6.13 Analysis of Aflatoxins Using LC/MS (1) - LC/MS

■ ExplanationOver 300 mold toxins, including aflatoxin, patulin andfumonisin, have been identified. HPLC and LC/MS areuseful for analyzing these toxins. LC/MS withoutstanding qualitative capabilities is especially useful for

analyzing multiple mold toxins, particularly aflatoxins.The Japanese MHLW notice on March 26, 2002stipulates LC and LC/MS as the analytical methods foraflatoxins in grains, legumes, seeds and nuts, and spices.

O O

O

O O

OCH3 O O

O

O O

OCH3 O O

O

O O

OCH3HO

O O

O O

O O

OCH3 O O

O O

O O

OCH3 O O

O O

O O

OCH3HO

O O

O

O O

OCH3

OH

O O

O

O O

OCH3

OH

B2aB2B1

G2aG2G1 M2

M1

Fig. 6.13.1 Structures of aflatoxins

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 min

5000

7500

10000

12500

15000

17500

20000

22500

25000

27500

In t.

331.00(1.00)329.00(1.00)315.00(1.00)313.00(1.00)

Fig. 6.13.2 SIM chromatogram of four varieties of aflatoxin(6 µL injected at 2.5 ng/mL. Analytical conditions conform to the MHLW notice.)

AFG2

AFG1

AFB2

AFB1

149

Others

6.13 Analysis of Aflatoxins Using LC/MS (2) - LC/MS

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z0e3

100e3

200e3

300e3

400e3

Int.331.2

Aflatoxin G2

Aflatoxin G1

Aflatoxin B2

Aflatoxin B1

287.1

269.5 313.4167.2 392.2241.4

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z0e3

100e3

200e3

300e3

400e3Int.

329.1

285.1

311.5259.3 371.3 415.3393.4239.8

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z0e3

250e3

500e3

Int.315.1

289.2133.0

100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 m/z0e3

100e3

200e3

300e3

400e3

500e3

Int.313.2

287.3

327.2279.3133.2 177.3

Fig. 6.13.3 Mass spectra for aflatoxin G2, G1, B2 and B1

InstrumentColumnMobile PhaseFlow RateColumn TemperatureSample Injection VolumeProbe VoltageNebulizer Gas Flow RateCDL VoltageCDL TemperatureBlock Heater TemperatureQ-Array VoltageDrying Gas Flow RateAnalysis Mode

LCMS-2010A

Shim-pack VP-ODS(150mmL. × 2.0mmI.D.)

Acetonitrile / Methanol / 10 mM Ammonium Acetate Aqueous Solution = 2/6/15 (v/v/v)

0.2 mL/min

40°C

6µL

+4.5kV(ESI-Positive Mode)

2.5L/min

0 V

300°C

250°C

Scan Mode

0.1MPa

SIM (m/z 313, 315, 329 and 331)

:

:

:

:

:

:

:

:

:

:

:

:

:

:

Table 6.13.1 Analytical Conditions

150

6.14 Analysis of Patulin Using LC/MS - LC/MS

■ ExplanationPatulin is a mycotoxin produced by Penicillium andAspergillus molds and is detected in fruit to which moldhas adhered. Some of the standards for food products andadditives were revised in accordance with NotificationNo. 369 issued by Japan’s Ministry of Health, Labour andWelfare (July 2003). New qualitative and quantitativetests using an LC-UV detector and confirmation testsusing LC/MS were established as test methods forpatulin. It is specified that the residual patulin content inproducts whose basic ingredient is apple juice or applejuice concentrate must not exceed 0.050 ppm.


Column

Mobile Phase

Flow Rate

Temperature

Detector

Sample Storage Temperature

Sample Injection Volume

Ionization Mode

Probe Voltage

Nebulizer-Gas Flow Rate

Drying-Gas Pressure

CDL Temperature


CDL Voltage

Q-Array Voltage

Selected Ion Mass Number

: Shimadzu LCMS-2010A

: Shim-pack VP-ODS (150 mmL. × 2.0 mmI.D.)


: 0.2 mL/min

: 40°C

: SPD-M10A (276 nm)

: 5°C

: 50 µL

: APCI-Positive

: 4.5 kV

: 2.5 L/min

: 0.05 MPa

: 250°C

: 200°C

: S-Mode

: S-Mode

: m/z 154.95 (M+H)+(Patulin)O

O

OH

O

C7H6O4


Fig. 6.14.1 Structure of patulin

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0

2.75

2.80

2.85

2.90

2.95

3.00

3.05

3.10

3.15

3.20

3.25

3.30

3.35

3.40µV (x1,000)

154.95 (1.00) Patulin

Fig. 6.14..2 Chromatogram for standard patulinsample (50 ppb, 50 µL)

UV: 276nm LC/MS (APCI)

6.0 7.0 8.0 9.06.0 7.05.0 8.0 9.0 minmin3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

(x1,000)(V Vx1,000)

12.5

10.0

7.5

2.5

5.0

0

-2.5

Regulated levelconcentration

Regulated levelconcentration

Blank

1/10 of Regulated levelconcentration 1/10 of Regulated level

concentration

Blank

Patulin

Patulin

Fig. 6.14.3 Chromatogram for patulin-added sample

6.15 Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (1) - LC/MS

151

Others

■ ExplanationDiarrhetic Shellfish Poison (DSP) toxins are present inthe dinoflagellates such as Dinophysis fortii andDinophysis acuminata. When bivalves take in these toxicplanktons, and humans ingest these shellfishcontaminated with these toxic planktons, these toxicsubstances, which are accumulated in the midgut gland ofthe shellfish, can cause acute gastroenteritis symptomsincluding vomiting, diarrhea and abdominal pain.Normally, the quantities ingested by humans do not causedeath. However, it is said that the toxic substances cannotbe decomposed by the level of heat processing done in

O

O O

O

O O

O

HOOC

OHOH

CH3

CH3

OH

OHH

CH2H3C

CH3

H3C

HOkadaic acid (OA)Exact MassMol. Wt.

: C44H68O13: 804.47: 805.00

O

O O

O

O O

O

HOOC

OHOH

CH3

CH3

OH

OHH

CH2H3C

CH3H3C

H3C

Dinophysistoxin -1 (DTX-1) Exact MassMol. Wt.

: C45H70O13: 818.48: 819.03

O

O

O

O

O

O CH3 CH3

CH3OH

OO

OH3C

OH

OH

CH3

H3CO

O

COOH

Pectenotoxin-6 (PTX-6)Exact MassMol. Wt.

: C47H68O16: 888.45: 889.03

Fig. 6.15.1 Structures of okadaic acid, dinophysistoxin-1 and pectenotoxin-6

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5Inten.(x100,000)

803. 4

804. 4

939. 3

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0Inten.(x100,000)

827. 4

769. 4

850. 5

828. 5787. 4

751. 3 787. 7

752. 4851. 7

841. 3733. 5

m/z 803 (M-H)-

m/z 827 (M+Na)+

m/z 849 (M+46)+

m/z 787 (M+H-H2O)+

m/z 769 (M+H-2H2O)+

m/z 751 (M+H-3H2O)+

m/z 733 (M+H-4H2O)+

O

O O

O

O O

O

HOOC

OHOH

CH3

CH3

OH

OHH

CH2H3C

CH3

H3C

Okadaic acid (OA) : C44H68O13Exact Mass: 804.47

Mol. Wt.: 805.00

H

Positive

Negative

Fig. 6.15.2 Positive ion and negative ion ESI mass spectra of okadaic acid

home cooking. Typical diarrhetic shellfish poisonsinclude Okadaic acid (OA), Dinophysistoxin (DTX),Pectenotoxin (PTX), and Yessontoxin (YTX). Theanalysis of these poisons is not easy, because, on top oftheir complicated structures, these toxins do not havechromophores. In Japan, the mouse bioassay is theofficial DSP testing method, but the analysis usingfluorescence derivatization followed by HPLC is also awell-known method. We introduce here examples ofanalysis of OA, DTX-1 and PTX-6 by LC/MS.

6.15 Analysis of Diarrhetic Shellfish Poison (DSP) by LC/MS (2) - LC/MS

152


Mobile Phase A

Mobile Phase B

Gradient Program

Flow Rate

Injection Volume

Column Temperature

Probe Voltage

Nebulizer Gas Flow Rate

Drying Gas

CDL Temperature


Monitored Ions

: Shim-pack VP-ODS (150 mmL × 2.0 mm I.D.)

: 5 mM Ammonium Acetate + 0.1% Formic Acid –Aqueous Solution

: Acetonitrile

: 10% B (0 min) → 90% B (15 - 20 min)

: 0.2 mL/min

: 2 µL

: 40˚C

: -3.5 kV (ESI-Negative Mode) + 4.5kV (ESI-Positive Mode)

: 1.5 L/min ←: 0.2MPa ←: 200˚C ←: 200˚C ←: m/z 803.25, 887.15, 817.25 (Negative Mode)

m/z 906.25, 911.15, 634.25, 850.30, 827.20, 864.30, 841.20 (Positive Mode)

OA DTX-1

PTX-6

OA DTX-1

PTX-6

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.00.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5

(x100,000)

817.25 (1.00)887.15 (1.00)803.25 (1.00)

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.00.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

1.1

1.2

1.3

(x1,000,000)

864.30 (1.00)841.20 (1.00)827.20 (1.00)850.30 (1.00)906.25 (1.00)911.15 (1.00)934.25 (1.00)

Positive ESI

Negative ESI

Fig. 6.15.3 DSP SIM chromatograms using positive ion and negative ion ESI

Printed in Japan 3295-10501-10A-IK

SHIMADZU CORPORATION. International Marketing Division3. Kanda-Nishikicho 1-chome, Chiyoda-ku, Tokyo 101-8448, Japan Phone: 81(3)3219-5641 Fax. 81(3)3219-5710Cable Add.:SHIMADZU TOKYO

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