RESEARCH PAPER

Quick supramolecular solvent-based microextractionfor quantification of low curcuminoid content in food

Noelia Caballero-Casero & Miraç Ocak &

Ümmüham Ocak & Soledad Rubio

Received: 31 July 2013 /Revised: 26 September 2013 /Accepted: 30 September 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract There is a need to monitor the consumption ofcurcuminoids, an EU-permitted natural colour in food, toensure that acceptable daily intakes are not exceeded,especially by young children. This paper describes a sensitivemethod able to quantify low contents of curcumin (CUR),demethoxycurcumin (DMC) and bis-demethoxycurcumin(BDMC) in foodstuffs. The method was based on a single-step extraction by use of a supramolecular solvent (SUPRAS)made up of reverse aggregates of decanoic acid, and directanalysis of the extract by use of liquid chromatography–photodiode array (PDA) detection. The extraction involvedthe stirring of 200 mg foodstuff with 600 μL SUPRAS for15 min. No cleanup or concentration of the extracts wasrequired. Curcuminoid solubilisation occurred via dispersionand hydrogen bonding. The method was used for thedetermination of curcuminoids in different types of foodstuff(snack, gelatine, yoghurt, mayonnaise, butter, candy and fishproducts) that encompassed a wide range of protein, fat,carbohydrate, sugar and water contents (0.85–11.04, 0–81.11, 0.06–75, 0.06–79.48, and 10.08–85.10 g, respectively,in each 100 g of food). Method quantification limits for thefoodstuffs analysed were in the ranges 2.9–7.7, 2.8–11.2 and3.3–9.0 μg kg−1 for CUR, DMC and BDMC, respectively.The concentrations of curcuminoids detected in the foodstuffs

and the recoveries obtained from fortified samples were in theranges ND–284, ND–201 and ND–61.3μg kg−1, and 82–106,89–106 and 90–102 %, for CUR, DMC and BDMC,respectively. The relative standard deviations were in therange 2–7 %. This method enabled quick and simplemicroextraction of curcuminoids with minimal solventconsumption, while delivering accurate and precise data.

Keywords Curcuminoids . food . supramolecular solvent .

microextraction . food additives

Introduction

Modern analysis of food samples involves analytical andoperational challenges that require innovative strategies.Accurate results must be delivered at set regulatory limits,and quality control laboratories are continuously demandingfaster, simpler, safer and cheaper analytical methods.

Sample handling still has a basic function in thedevelopment of comprehensive methods for food analysis.Much effort has been devoted in the last two decades toreducing solvent organic consumption and simplifying sampleextraction and cleanup. In solid food sample treatment, the useof auxiliary energy to enhance solvent extraction (e.g.pressurised liquid extraction (PLE) [1], microwave-assistedextraction (MAE) [2], ultrasound-assisted extraction, (UAE)[3]) and the use of alternative solvents (e.g. supercritical fluids[4]) have become familiar in many analytical labs. Thesetechniques are faster and more environmentally friendly thantraditional solvent extraction, but problems related to the needfor dedicated equipment, cost, and matrix-dependent resultsstill remain. In the last few years, some attention hasbeen focused on other alternative solvents, includingroom temperature ionic liquids [5] and supramolecularsolvents (SUPRAS) [6].

Published in the topical collectionMicroextraction Techniques with guesteditors Miguel Valcárcel Cases, Soledad Cárdenas Aranzana and RafaelLucena Rodríguez.

N. Caballero-Casero : S. Rubio (*)Department of Analytical Chemistry, Institute of Fine Chemistry andNanochemistry, University of Córdoba, Edificio AnexoMarie Curie,Campus de Rabanales, 14071 Córdoba, Spaine-mail: qa1rubrs@uco.es

M. Ocak :Ü. OcakFaculty of Art and Science, Department of Chemistry,Karadeniz Technical University, 61080 Trabzon, Turkey

Anal Bioanal ChemDOI 10.1007/s00216-013-7409-9

Supramolecular solvents are nano-structured liquidsgenerated spontaneously from aqueous or hydro-organicsolutions of amphiphiles via a self-assembly process. Thisprocess is induced by changes in the temperature [7] or thepH of the solution [8], or by the addition of salts [9] or a non-solvent for the amphiphile [10]. Both the special arrangementand high concentration of amphiphiles in the orderedaggregates render SUPRAS very attractive for the developmentof generalised sample treatments able to deal with a range offood types and analytes. The ordered aggregates have regionsof different polarity that provide a variety of interactions foranalytes; the type of interaction can be tuned by varying thehydrophobic or the polar group of the amphiphile; and intheory, because amphiphiles are ubiquitous in nature andsynthetic chemistry, we could design the most appropriateSUPRAS for a specific application. This last property is veryattractive for multiresidue analysis, as shown in a recent reportdealing with the determination of sulfonamides in meat [11].Another benefit is that the high concentration of amphiphiles inthe SUPRAS, and therefore the large number of binding sitesthey offer, means these solvents have the potential to extractanalytes from a variety of food types with an efficiency thatshould be matrix-independent. Reported uses of SUPRAS forextracting contaminants [12–14] from solid food samples,although scarce so far, have proved that SUPRAS enabledevelopment of efficient, fast, simple, cheap and safe sampletreatments. There is no need for dedicated equipment, and onlyminute volumes of solvent, obtained by means of easilyaccessible synthetic procedures, are required.

This paper deals with the development of a generalisedsample treatment for extracting curcuminoids from differentfoodstuffs before LC–PDA. Curcuminoids are the majorbioactive constituents of Curcuma species. The groundrhizomes of turmeric (Curcuma longa L .) contain 3–15 %curcuminoids, namely curcumin (CUR), demethoxycurcumin

(DMC) and bis-demethoxycurcumin (BDMC) [15].Commercial curcumin, obtained by means of solventextraction and purification of turmeric rhizomes, is widelyused as a natural food colour additive in Europe and NorthAmerica [16]. It consists of a mixture of CUR, DMC andBDMC (e.g. 71.5 %, 19.4 % and 9.1 %, respectively [17]).Table 1 shows the chemical structures of curcuminoids, theirionisation constants, and their ability to form hydrogen bonds.

The acceptable daily intakes (ADI) for curcumin set by theEuropean Food Safety Authority (EFSA) and the Joint FAO–WHO Expert Committee on Food Additives (JECFA) are 3and 0–3 mg, respectively, per kg body weight [18]. EuropeanDirective 94/36/EC [19] prescribes maximum limits forcurcumin of between 20 and 500 mg kg−1. Suitable analyticalmethods are required to ensure ADIs are not exceeded,especially by young children, and to comply with EUlegislation. Analysis of foodstuffs permitted to containcurcumin quantum satis may be also required to ensurelabelling compliance.

Analytical methods for curcuminoids have been mainlydeveloped for turmeric roots and biomatrices other than food,to study their pharmacological properties and biologicaleffects [20, 21]. Scotter has written an excellent review ofthe methods available for the determination of permittedadded natural colours in food, including those for curcumin[22]. As pointed out by this author, methods for determiningindividual curcuminoids in food are limited. As far as we areaware, only three methods have dealt with the determinationof individual curcuminoids in food [23–25], and because therewas until very recently a lack of commercially availablepure standards, much effort has been devoted to thepreparation of standards from turmeric extracts, whichis very time consuming.

LC–electrospray–mass spectrometry (LC–ESI–MS) hasbeen proposed for the determination of curcuminoids in food

Table 1 Chemical structure, ionisation constants, and number of proton acceptors and donors for curcuminoids

Compound name Stucture pKaa H+ acceptors/donors a

Curcumin 8.11 ± 0.46 6 / 2

Demethoxycurcumin 8.31 ± 0.46 5 / 2

Bis-Demethoxycurcumin 8.50 ± 0.46 4 / 2

a Source: https://scifinder.cas.org

N. Caballero-Casero et al.

and validated for tea and candies [23]. Sample treatmentinvolved extraction with methanol assisted by ultrasonication.For samples with low expected curcuminoid content, themethanolic extract was dispersed in water and then subjectedto SPE cleanup, methanol elution, evaporation to dryness, andmethanol reconstitution in a small volume. Two moremethods for curcuminoid determination have been reported,both based on LC–PAD detection [24, 25]. In the first [24], acomprehensive chromatographic separation of curcuminoidsand annatto was reported, and this was used in a single spikingexperiment involving mince fish (average recovery 85 %).Sample treatment included grinding with Celite and HCl inthe presence of ascorbyl palmitate, followed by a hexane washto remove oils and extraction into acetonitrile in the presenceof antioxidants. The water content of the samples was knownto significantly affect the separation of curcuminoids. Thesecond method [25] was applied to a range of foodstuffs.Sample treatment involved the extraction of 0.5 g of driedfood with 10 mL methanol for 6 h, and, although globalrecoveries for curcuminoids were in the range 84.7–98.9 %for solid samples, no recoveries for individual curcuminoidswere given. No quantitation limits were reported, but ourestimation from spiking experiments gives values ofapproximately 2 mg kg−1, which is not low enough to analysefoodstuffs with low curcuminoid content.

The research here presented evaluated the ability of aSUPRAS consisting of reverse aggregates of decanoic acidto efficiently extract curcuminoids from food, using lowsolvent volumes. Foodstuffs were selected to cover thedifferent food categories for which the addition of thesenatural yellowish colorants is authorised [19] and toencompass a wide range of protein, fat, carbohydrate, waterand sugar contents. The objective was to develop a simple andfast method able to deal with both high and low curcuminoidcontent. The variables affecting the efficiency of theSUPRAS-based microextraction were studied, the analyticalfeatures of the LC–PDA method were evaluated, and themethod developed was used for the determination ofcurcuminoids in non-spiked and spiked foodstuffs.

Experimental

Chemicals

All chemicals were of analytical reagent grade and were used assupplied. Decanoic acid and HPLC grade acetonitrile wereobtained from Sigma-Aldrich (Barcelona, Spain).Tetrahydrofuran (THF), hydrochloric acid and acetic acidglacial were supplied by Panreac (Sevilla, Spain). Ultra-high-quality water was obtained from a Milli-Q water purificationsystem (Millipore, Madrid, Spain). Standards for curcumin,demethoxycurcumin and bis-demethoxycurcumin were

purchased from Sigma (St. Louis, MO, USA). Stock individualsolutions of curcuminoids at a concentration of 1 g L−1 wereprepared in methanol and stored under dark conditions at−20 °C in an amber flask. Working solutions were preparedby proper dilution of the stock solutions with methanol.

Apparatus

The liquid chromatographic system used consisted of aThermoQuest spectra system (San Jose, CA, USA) equippedwith a SCM 1000 vacuum membrane degasser, a P2000binary pump, an AS3000 autosampler and a Spectra SystemUV6000 LP detector. In all experiments, a PEEK Rheodyne7125NS injection valve with a 20 μL sample loop was used(ThermoQuest, San Jose, CA, USA). The analytical columnwas an Ultrabase C18 (5 μm, 250 mm×4.6 mm) fromAnalisis Vínicos (Tomelloso, Spain). A homogeniser-disperser Ultra-Turrax T25 Basic from Ika (Werke, Germany),a vortex-shaker REAX Top equipped with an attachment (ref.549-01000-00) for 10microtubes fromHeidolph (Schwabach,Germany), and a high speed brushless centrifuge MPW-350Requipped with an angle rotor 36×2.2/1.5 mL (ref. 11462)from MPW Med-Instruments (Warschaw, Poland) were usedfor sample preparation. A digitally regulated centrifugeMixtasel equipped with an angle rotor 4×100 mL (ref.7001326) from JP-Selecta (Abrera, Spain) was used forsupramolecular solvent production.

Supramolecular solvent production

The following procedure was used to obtain a SUPRASvolume (~10 mL) able to treat 16 food samples. Decanoicacid (0.9 g), tetrahydrofuran (15 mL) and hydrochloricsolution 0.01 mol L−1 (15 mL) were mixed in a 50 mL glasscentrifuge tube. The mixture was centrifuged at 3500 rpm(2400 g) for 10 min. The SUPRAS formed was less densethan water, and was separated as a new liquid phase at the topof the solution. It was extracted by use of a syringe, and storedin a closed glass vial until use. Under these conditions, it wasstable at room temperature for at least one month. Figure 1shows a schematic of the synthetic procedure.

Determination of curcuminoids in food samples

Sample preparation

Samples belonging to different food categories (snack,gelatine, yoghurt, mayonnaise, processed fish, butter andcandy) were purchased in local supermarkets in Córdoba(South Spain) and were stored under dark conditions at 4 °Cuntil analysis. The whole content of consumer-size packageswas used for sample treatment. Samples, except candies, werehomogenised by use of a high-speed Ultra-Turrax for 5–

Quick supramolecular solvent-based microextraction

10 min. Because of the high sugar content of candiesthe addition of ultra-high quality water at a sample/water ratio (w/w ) of two was required, beforehomogenisation by use of the Ultra-Turrax for 10 min.After homogenisation, 200 mg subsamples were takenfor analysis and recovery experiments, which wereperformed in triplicate. The subsamples were fortifiedat different concentration levels (0.1–2 mg kg−1) byadding minute volumes (20–40 μL) of curcuminoidstandard solutions in methanol. Fortified samples wereleft to stand at room temperature and in dark conditionsfor 90 min before analysis. Curcuminoids in sampleswere stable during this period of time. For subsamplerepresentativity studies, the whole content of the samplewas fortified.

Supramolecular solvent-based microextraction

Subsamples (200 mg) were mixed with 600 μL SUPRAS in asafe-lock 2 mL microtube from Eppendorf Ibérica (Madrid,Spain). Four or five 3 mm diameter glass balls wereintroduced in the microtube to improve sample dispersionduring extraction, which was performed by means of vortex-shaking at 2500 rpm for 15 min. The mixture was thencentrifuged at 15000 rpm for 5–10 min to separate the solventfrom the solid residue. Finally, the SUPRAS containingthe target analytes was transferred to a glass vial, fromwhich 20 μL was injected into the chromatographicsystem. A schematic of the sample treatment procedureis shown in Fig. 2.

Liquid chromatography–photodiode array detection

The three curcuminoids were separated and quantifiedby use of LC with PDA detection. The mobile phaseconsisted of an aqueous solution of acetic acid 2 % (A)

and acetonitrile (B), and was pumped at a constant flowof 1 mL min−1. A linear gradient from 45 % A to 65 %A in 15 min was used for separation. Reconditioning ofthe column took approximately 5 min. The columntemperature was set at 40 °C. Calibration curves were runfrom standards dissolved in methanol, in the concentrationranges 0.8 μg L−1–80 mg L−1, 0.7 μg L−1–80 mg L−1 and0.5 μg L−1–80 mg L−1 for CUR, DMC and BDMC,respectively. Quantitation was performed by means ofmeasuring peak areas at 425 nm.

Light microscopy photograph of SUPRAS

Stored at 4 ºC until use

SUPRAS phaseSeparation

Decanoic acid (0.9 g)Tetrahydrofuran (15 mL)

0.01 M HCl (15 mL)

SUPRAS droplets

Centrifugation

Fig. 1 Schematic of the procedure for synthesising decanoic acid-based SUPRAS

Sample homogenization

Homogenization with ultraturrax (5-10 min)

SUPRAS based-microextraction

200 mg subsamples + 0.6 mL SUPRAS

Vortex-shaking (15 min)

SUPRAS separation from the sample residue

Centrifugation at 15000 rpm (5-10 min)

HPLC-PDA-Detection (λ = 425nm)

Retention time

BDMC DMC C8.5 min 10.3 min 13 min

Fig. 2 Schematic of the procedure for determination of curcuminoids infood

N. Caballero-Casero et al.

Results and discussion

Supramolecular solvent production and binding capabilities

Decanoic acid, like other alkyl carboxylic acids, isslightly soluble in water (0.15 g L−1 at 20 °C) and verysoluble in THF, where it forms reverse aggregates via asequential-type self association model [26]. Addition ofdecanoic acid (typically at concentrations below 8 %, w/v ) to a hydroorganic solution of water and THF (water/THF (v/v ) ratios between 1.5 and 50) causes theformation of oily droplets that flocculate and produceconglomerates of individual droplets. These conglomeratesseparate from the bulk solution as a new liquid phase,named SUPRAS. The process does not occur fordecanoate, so the pH of the solution should be belowfour to ensure the presence of decanoic acid (pKa=4.8±02). Figure 1 shows a microphotograph of theindividual droplets that make up the SUPRAS. Theseconsist of decanoic acid, THF and water, in proportionsthat depend on the THF/water ratio (v/v ) of thesynthetic solution.

The volume of solvent produced was a linear and anexponential function of the amount of decanoic acid andTHF, respectively, in the synthetic solution. The followingequation was derived: y = (1.04±0.02)ae (0.0473±0.0009)b,where y is the volume of SUPRAS in μL, a the amount ofdecanoic acid in mg, and b the percentage of THF (v/v ).

Decanoic acid-based SUPRAS are expected to efficientlyextract curcuminoids by use of low solvent volumes, on thebasis of:

1. the types of interaction they can establish, namelydispersion forces between the hydrocarbon chain of theacid and the aromatic structure of curcuminoids, andhydrogen bonds between the polar groups of thesurfactant and analytes; and

2. the large concentration of surfactant in the SUPRAS (e.g.0.2–0.8 mg μL−1).

Curcuminoids are only stable at acidic pH, rapidlydecomposing at pHs above neutral, so the SUPRAS is aconvenient medium for analyte stabilisation.

Optimisation of the supramolecular solvent-basedmicroextraction

Optimisation was performed by extracting 200 mg ofsnack fortified with 2 mg kg−1 of each curcuminoid.Unfortified samples were analysed for curcuminoids,and the concentration found subtracted from those offortified samples. Experiments were performed intriplicate. Selection of the optimum conditions was on

the basis of recoveries and method quantitation limits(MQLs) obtained. MQLs were calculated from theinstrumental quantitation limits (0.8, 0.7 and 0.5 μg L−1 forCUR, DMC and BDMC, respectively), the volume ofSUPRAS used for extraction, the recoveries obtained andthe sample weight used for analysis. The variablesinvestigated were SUPRAS composition and volume,and time required to reach equilibrium conditions. Aftersample centrifugation, two phases were alwaysobserved: the insoluble sample matrix components atthe bottom, and the SUPRAS extract containing thethree curcuminoids and other solutes from the sample.Table 2 shows the results obtained.

As commented above, the decanoic acid, THF andwater content of the SUPRAS depends on the THF/water ratio in the synthetic solution. SUPRAScomposition remains invariable when the amount ofdecanoic acid in the synthetic solution is increased atconstant THF/water ratios. Both the water and the THFcontent of the SUPRAS increase as the THF/water ratiodoes, so the decanoic acid in the SUPRAS becomesmore diluted. Table 2 shows the recoveries obtained,and the MQL estimated for curcuminoids, by use of600 μL SUPRAS synthesised in solutions containingTHF in the range 10–70 %. Maximum recoveries andminimum MQL were obtained by use of SUPRASsynthesised in 50 % THF and 50 % water, so thisSUPRAS was selected for further experiments. Thewater solution used for synthesis was adjusted topH 2, so the pH of the water group of the reverseaggregates in the SUPRAS was acidic and was thereforea suitable medium to stabilise the curcuminoids.

The effect on recovery of the volume of SUPRASused for the microextraction of curcuminoids wasassessed in the range 200–800 μL. Volumes below600 μL were not able to completely wet the subsamplesof snacks, candies, fish products and some otherfoodstuffs. The minimum volume enabling quantitativerecoveries (i.e. 600 μL) was selected, to keep MQLvalues as low as possible (Table 2).

Only 15 min vortex shaking (vibration motion=2500 rpm)was necessary to reach equilibrium conditions (Table 2).This is a consequence of the high extraction efficiencyof the SUPRAS, probably caused by the different typesof interaction it offers and the high number of bindingsites.

Representativity of the food subsamples used foranalyses was investigated by spiking 50 g aliquots ofsnack with 2 mg kg−1 of each curcuminoid, and thenanalysing six 200 mg subsamples. The standarddeviation for the different curcuminoids was in therange 2–3 %, so the subsamples used were believedrepresentative of the whole sample.

Quick supramolecular solvent-based microextraction

Analytical performance

Sensitivity and linearity

Calibration curves were run using standard solutions inmethanol. No differences in peak areas or retentiontimes were observed for the curcuminoids injected intothe SUPRAS or methanol. The linear range wasconfirmed by visual inspection of the plot residualsversus analyte amount; the residuals were randomlyscattered within a horizontal band, and a randomsequence of positive and negative residuals wasobtained. Linearity was achieved in the ranges0.8 μg L−1–80 mg L−1, 0.7 μg L−1–80 mg L−1 and0.5 μg L−1–80 mg L−1 for CUR, DMC and BDMC,respectively. Correlation coefficients were in the range0.996–0.999, indicating good fits.

Practical quantification limits [27] were estimated from sixindependent complete determinations of curcuminoids intypical low-level foodstuffs. When these foodstuffs couldnot be obtained, an estimate of the background signal wasmade at a representative part of the readout adjacent to thecurcuminoid signal in the curcuminoid-containing sample.

Quantification limits were calculated by use of a signal-to-noise ratio of 10. The values obtained for the different types offoodstuff analysed are shown in Table 3, and were in theranges 2.9–7.7, 2.8–11.2, and 3.3–9.0 μg kg−1 for CUR,DMC, and BDMC, respectively, enabling the determinationof low curcuminoid content in food.

Selectivity

Possible interference from matrix components that couldco-elute with the three curcuminoids was assessed bycomparison of the slopes of the calibration curves (n =6), obtained from standard solutions in methanol, withthose obtained from 200 mg of different foodstuffsfortified with known amounts of curcuminoids (0.1–2 mg kg−1 to 200 mg subsamples) and run using thewhole procedure. No statistically significant differencebetween these slopes was revealed by use of a student’st-test [28]. The experimental t-values were in theinterval 1.8–2.9, and were below the critical t-value(3.36, significance level =0.01). Therefore, matrixcomponents were not expected to interfere with thedetermination of curcuminoids.

Table 2 Mean recoveries and method quantification limits obtained for curcuminoids as a function of the composition and volume of SUPRAS and theextraction time

Variable CUR DMC BDMC

Recoverya±Sb (%) MQL (μg kg−1) Recoverya±Sb (%) MQL (μg kg−1) Recoverya±Sb (%) MQL (μg kg−1)

THF (%)

10 42±2 5.6 47±1 4.4 40±3 3.7

20 55±3 3.6 50±2 4.1 53±2 2.8

30 67±2 3.5 70±2 2.9 65±2 2.3

40 80±1 2.9 77±1 2.7 90±1 1.6

50 99±1 2.4 100±1 2.1 98±1 1.5

60 84±1 2.8 92±1 2.3 88±1 1.7

70 76±2 3.1 80±1 2.6 80±1 1.8

SUPRAS volume (mL)

200 – – –

400 – – –

600 101±2 2.3 98±2 2.1 102±1 1.4

800 98±1 3.2 99±1 2.8 100±2 2.0

Extraction time (min)

5 88±2 2.7 84±3 2.5 87±1 1.7

10 95±2 2.5 88±2 2.4 88±2 1.7

15 100±1 2.3 98±1 2.1 98±1 1.5

20 99±1 2.4 103±1 2.0 102±1 1.4

30 102±1 2.3 99±1 2.1 101±1 1.4

a 200 mg of snack spiked with 2 mg kg−1 of each curcuminoidb Standard deviation, n =3

N. Caballero-Casero et al.

Precision

The precision of the method was evaluated by extractingeleven independent samples of snack fortified with2 mg kg−1 curcumin. The precision, expressed as relativestandard deviation (RSD), was between 3 and 7 for the threecurcuminoids.

Analysis of foodstuffs

The method here proposed was used to determine CUR,DMC and BDMC in fortified and unfortified foodstuffsbought in different local supermarkets in Córdoba(Spain). Spiking of samples at 0.1 mg kg−1 wasperformed for each curcuminoid. The protein, fat,carbohydrate, sugar, and water contents of the selectedfoodstuffs, expressed as g per 100 g of food, were inthe ranges 0.85–11.04, 0–81.11, 0.06–75, 0.06–79.48and 10.08–85.10, respectively, and thus encompassed awide range of compositions (see Table 3 for detailedcomposition). It is reasonable to expect that the resultshere obtained can be extrapolated to other foodstuffswithin these composition ranges.

Table 3 shows the concentrations found and therecoveries obtained, with the corresponding standarddeviations, for the three curcuminoids in the differentfoodstuffs analysed. The concentrations of CUR, DMC

and BDMC were in the ranges ND–284 μg kg−1, ND–201 μg kg−1 and ND–61.3 μg kg−1, respectively. Thecontent of individual curcuminoids in most samplesanalysed was below 100 μg kg−1, thus revealing theneed for sensitive methods to evaluate ADI values forcurcuminoids, especially the values prescribed for youngchildren, and to ensure labelling compliance. Recoveriesranged between 82–106, 89–106 and 90–102 % forCUR, DMC and BDMC, respectively, with relativestandard deviations in the range 2–7 %.

Figure 3 shows the chromatograms obtained for someof the unfortified foodstuffs analysed. No coelution ofother matrix components was observed for curcuminoidsin any of the samples analysed, except for BDMC infoodstuffs including gelatine and mayonnaise (peak a inFig. 3). The shoulder observed in the chromatographicpeak for these samples corresponded to a clear peak at370 nm. Therefore, for exact quantification, the rightside of the chromatographic peak should be extrapolatedto the baseline.

Differences between the retention times measured forthe analytes from standards and fortified samples werelower than 0.7 % in all cases, and the repeatabilityobtained for consecutive measurements of this variablefrom samples, expressed as relative standard deviations(n =6), was 0.35, 0.43 and 0.5 % for CUR, DMC andBDMC, respectively.

Table 3 Mean concentration and recovery, with their respective standard deviations, obtained for the determination of curcuminoids in non-fortified andfortified samples, and practical method quantitation limits obtained for each type of foodstuff analysed

Foodstuff CUR DMC BDMC

Concentrationfoundh±S i

(μg kg−1)

Recoveryk±S i

(%)MQLl

(μg kg−1)Concentrationfoundh±S i

(μg kg−1)

Recoveryk±S i

(%)MQLl

(μg kg−1)Concentrationfoundh±S i

(μg kg−1)

Recoveryk±S i

(%)MQLl

(μg kg−1)

Snacka 7.5±0.1 103±4 2.9 3.41±0.07 99±5 2.8 ND 102±3 3.3

Gelatineb 284±9 101±2 3.5 201±5 102±4 5.6 61.3±0.2 98±5 6.0

Yoghurtc 46±1 106±7 4.0 34.1±0.6 99±4 4.5 7.69±0.02 101±3 4.2

Mayonnaised 83±2 91±6 4.3 70±1 80±2 7.0 21.62±0.06 90±7 8.7

Buttere 7.0±0.4 102±5 4.2 ND 106±6 3.5 ND 97±7 2.5

Candyf ND 91±4 4.0 ND 87±3 8.5 ND 90±4 3.9

Fishg 28±1 82±5 7.7 12±1 89±7 11.2 12±1 90±5 9.0

ND, not detected

Nutritional composition: (g per 100 g food): a Water: 5.44; fat: 2.97; protein: 8.03; carbohydrate: 75.00; sugar: 9.38; bWater: 84.39; fat:0; protein: 1.22; carbohydrate: 14.19; sugar: 13.49; c Water: 85.10; fat: 0.39; protein: 10.19; carbohydrate: 3.60; sugar: 3.24; dWater:54.30; fat: 19; protein: 0.9; carbohydrate: 23.9; sugar: 4.34; eWater: 15.87; fat: 81.11; protein: 0.85; carbohydrate: 0.06; sugar: 0.06;f Water: 10.08; fat: 0.08; protein: 5.97; carbohydrate: 12.7; sugar: 79.48; gWater: 53.08; fat: 13.25; protein: 11.04; carbohydrate: 21.18;sugar: 2.50hMean for three independent determinations from the analyses from unfortified samplesi Standard deviationk Sample spiked with 0.1 mg kg−1 of each curcuminoidl Calculated from six independent complete determinations

Quick supramolecular solvent-based microextraction

Conclusion

A SUPRAS made up of reverse aggregates of decanoicacid was here proposed for the quick and inexpensivemicroextraction of curcuminoids from foodstuffsbelonging to different categories (snack, gelatine,yoghurt, mayonnaise, butter, candy and fish products)and featuring a wide range of protein (0.85–11 %), fat(0–81.11 %), carbohydrate (0.06–75 %), sugar (0.06–79.48 %) and water (10.08–85.10 %) contents. Thetwo structure-derived extraction properties of SUPRAS,namely the different polarity regions able to provideseveral types of interaction, and the large number ofbinding sites, make SUPRAS excellent candidates for useas general extractants in food analysis. Valuable assets of thematrix-independent sample treatment here developed are: ittakes approximately 15 min and several samples can besimultaneously treated; it requires only a small amount ofsample (0.2 g), made representative of the bulk byhomogenisation, and a small, eco-friendly SUPRAS

volume (600 μL); it is low in cost; and conventionallab equipment is used.

As commented in the Introduction, only three methodshave been previously reported for simultaneous determinationof CUR, DMC and BDMC in food [23–25]. The maindisadvantages of these methods are the complex sampletreatments required (extraction, SPE cleanup or hexane wash,evaporation to dryness, etc [23, 24]), the time-consumingprocedures involved (e.g. extractions take 6 h [25]), theinability to provide validation for a wide range of foodstuffs[23, 24] or the individual determination of curcuminoids infood [25], and/or the high LODs obtained, making themunsuitable for the determination of low curcuminoid contentsin food [25].

The analytical method here proposed is sensitiveenough to determine low curcuminoid contents in foodand is suitable for estimating acceptable daily intakesand for monitoring labelling compliance, using sampletreatment and quantification equipment available to mostenforcement laboratories.

0 2 4 6 8 10 12 14 16 18 20 0 2 4 6 8 10 12 14 16 18 20

0 2 4 6 8 10 12 14 16 18 200 2 4 6 8 10 12 14 16 18 20

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Yogurt

a

a a

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Fig. 3 Chromatograms obtained for the determination of curcuminoids in gelatine, candy, yoghurt and mayonnaise. The concentrations obtained for (a)BDMC, (b) DMC and (c) CUR are given in Table 3

N. Caballero-Casero et al.

Acknowledgments The authors gratefully acknowledge financialsupport from Spanish MICINN (Project CTQ2011-23849) and from theAndalusian Government (Junta de Andalucía, Spain, Project P09-FQM-5151). FEDER also provided additional funding. N. Caballero-Caseroacknowledges Spain’s MINECO for award of a doctoral fellowship(BES-2012-052170).

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