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Analytica Chimica Acta 584 (2007) 322–332

Determination of acrylamide in Chinese traditional carbohydrate-rich foodsusing gas chromatography with micro-electron capture detector and isotope

dilution liquid chromatography combined with electrospray ionizationtandem mass spectrometry

Yu Zhang a, Yiping Ren b, Hangmei Zhao a, Ying Zhang a,∗a Department of Food Science and Nutrition, College of Biosystems Engineering and Food Science, Zhejiang University,

Hangzhou 310029, Zhejiang Province, PR Chinab Zhejiang Provincial Center for Disease Prevention and Control, Hangzhou 310009, Zhejiang Province, PR China

Received 14 May 2006; received in revised form 11 August 2006; accepted 13 October 2006Available online 15 November 2006

bstract

The present study developed two analytical methods for quantification of acrylamide in complex food matrixes, such as Chinese traditionalarbohydrate-rich foods. One is based on derivatization with potassium bromate and potassium bromide without clean-up prior to gas chromatog-aphy with micro-electron capture detector (GC-MECD). Alternatively, the underivatized acrylamide was detected by high-performance liquidhromatography coupled to quadrupole tandem mass spectrometry (HPLC-MS/MS) in the positive electrospray ionization mode. For both methods,he Chinese carbohydrate-rich samples were homogenized, defatted with petroleum ether and extracted with aqueous solution of sodium chloride.ecovery rates for acrylamide from spiked Chinese style foods with the spiking level of 50, 500 and 1000 �g kg−1 were in the range of 79–93%

or the GC-MECD including derivatization and 84–97% for the HPLC-MS/MS method. Typical quantification limits of the HPLC-MSMS methodere 4 �g kg−1 for acrylamide. The GC-MECD method achieved quantification limits of 10 �g kg−1 in Chinese style foods. Thirty-eight Chinese

raditional foods purchased from different manufacturers were analyzed and compared with four Western style foods. Acrylamide contaminant wasound in all of samples at the concentration up to 771.1 and 734.5 �g kg−1 detected by the GC and HPLC method, respectively. The concentrationsetermined with the two different quantitative methods corresponded well with each other. A convenient and fast pretreatment procedure will beptimized in order to satisfy further investigation of hundreds of samples.

2006 Elsevier B.V. All rights reserved.

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eywords: Acrylamide; Chinese traditional carbohydrate-rich foods; Micro-ele

. Introduction

Acrylamide (2-propenamide), a well-known neurotoxic com-ound, was detected in carbohydrate-rich fried or baked foodamples by the research groups from Swedish National Fooddministration (SNFA) and University of Stockholm in 2002

1]. The major mechanistic pathway for the formation of acry-amide in foods so far established is via the Maillard reaction

2–6]. It has been shown that the likely reactants which pro-uce significant levels of acrylamide in foods are asparaginend glucose [7]. Long-term exposure to acrylamide may cause

∗ Corresponding author. Tel.: +86 571 8697 1388; fax: +86 571 8604 9803.E-mail address: y zhang@zju.edu.cn (Y. Zhang).

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capture detection; Liquid chromatography tandem mass spectrometry

amage to the nervous system in both humans and animals to aertain extent [8,9], and acrylamide is also considered as a poten-ial genetic and reproductive toxin [10,11] with mutagenic andarcinogenic properties in experimental mammalians in both initro and in vivo study [12,13]. Meanwhile, the risk assessmentf acrylamide evaluated by the Scientific Committee on Toxic-ty Ecotoxicity and the Environment (CSTEE) of the Europeannion (EU) demonstrated that the exposure of humans to acry-

amide should be kept as low as possible with regard to thenherent toxic properties of acrylamide (neurotoxicity, geno-oxicity to both somatic and germ cells, carcinogenicity and

eproductive toxicity) [14].

These findings have attracted considerable interest and widettention all over the world. Recently in 2005, World Healthrganization (WHO) and Food and Agriculture Organization

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FAO) together announced that certain foods processed orooked at high temperature especially Western-style snacks con-ain considerable levels of acrylamide and may harm humanealth to a certain extent [15]. Under such situation, researchesn the analytical method and risk assessment of acrylamiden different food matrixes have once again become a hotspot.lthough considerable controversy exists regarding the expo-

ure levels relevant to carcinogenicity of acrylamide in humans12], the reports on the presence of acrylamide in European foodrompted the U.S. Food and Drug Administration (FDA) to ana-yze a variety of foods sold in the United States for the presencef acrylamide.

Chinese traditional carbohydrate-rich foods are an all-timeavorite in China, covering a wide range of products, such asried bread stick, clay oven rolls, hemp flowers, glutinous riceesame balls, steamed buns, etc. These foods were mainly pre-ared by high-temperature cooking such as grilling, roasting,aking, frying and deep-frying, which have similar process-ng style with lots of Western foods. Studies conducted soar indicated that moderate protein, high carbohydrate foodsuch as potatoes developed substantially higher levels of acry-amide under heating conditions [2,3]. Therefore, Chineseraditional carbohydrate-rich foods have probably high risk lev-ls of acrylamide under heating conditions. However, to our bestnowledge, few published papers in peer-review journals wereeported on the level of acrylamide under heating conditionsn Chinese traditional carbohydrate-rich foods. Furthermore,hina has a huge population, hundreds of millions of people tak-

ng various Chinese traditional carbohydrate-rich foods everyay, so it is very indispensable to investigate acrylamide thatrobably exists in these foods and provide warranty for analysisf health risk of Chinese people.

A great number of hitherto published methods have beeneveloped in the past years to quantitatively analyze the acry-amide monomer. Rosen and Hellenas (2002) reported a pioneertudy on the analysis of acrylamide in different heat-treatedoods using the isotope dilution liquid chromatography tan-em mass spectrometry (LC–MS/MS) technique [16]. Theyeveloped a mass spectrometry method for direct detection ofcrylamide, which would unequivocally verify the presence ofcrylamide in a range of heat-treated foods. Based on their val-dated methodology, Tareke et al. [17] thoroughly made thenvestigations of acrylamide in heated foodstuffs via comparisonetween GC–MS and LC–MS/MS. From then on, a sequence ofnalytical methods dealing with the analysis of acrylamide ineat-treated foods have been published in peer-review journals,eported by specific research groups or presented at internationalcientific conferences [18]. Hitherto, it can be summarized fromecent methodological studies that GC–MS and LC–MS/MSppeared to be acknowledged as the most useful and authori-ative method for acrylamide determination [19–22].

As for corresponding researches in our laboratory, a detailedeview article of different analytical methods employed to under-

tand the occurrence and determination of acrylamide in foodsas recently been published [23], which supplied detailed infor-ation on the sample pretreatment steps (extraction conditions,

erivatization, clean-up, etc.), chromatographic conditions and

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a Acta 584 (2007) 322–332 323

ass spectrometry parameters used by different laboratories.owever, based on conclusions of a recent inter-laboratory trial

24], updated papers on acrylamide research activities fromOAC [25–27] and EC/JRC expert workshop [28], many of

he methods do not perform well in difficult matrixes such asoffee. In addition, multiple responses are observed in eachf the mass transitions at different retention time but close tohat of acrylamide, which may cause interference. Some pre-ious studies [29,30] demonstrated the quantitative method ofcrylamide in coffee and corresponding foods. However, theonsiderable loss of the analyte was found in these matrixes andsignificant ion suppression effect led to a low response of acry-

amide under positive electrospray ionization conditions [31].s coffee problem was solved recently [32], laboratories are

ontinuously adapting their methods to achieve the required pre-ision and sensitivity for other complex matrix products. Mostf Chinese traditional carbohydrate-rich foods including someil-rich (e.g. Chinese fried bread stick) and protein-rich (e.g.gg omelet) products have relatively complex matrix systems.roblems relating to pretreatment steps, impurity interferencend ion responses become a challenge in the present study.

The objective of the present study was to develop and validateomplementary, highly sensitive and reliable analytical meth-ds for the determination of acrylamide in Chinese traditionalarbohydrate-rich foods. These methods should be applica-le to complex matrixes such as Chinese fried bread stick. AC method was developed based on derivatization of the tar-et analyte with bromination and detection by micro-electronapture detector (MECD). In addition, a confirmatory separa-ion and detection technique based on high-performance liquidhromatography (HPLC) coupled to tandem mass spectrometryMS/MS) of underivatized analyte was developed. To our bestnowledge, this is the first published GC-MECD method deal-ng with acrylamide levels in foods while HPLC method wasreviously reported [33]. The target of employing two differentnstrumental techniques was to compare the robustness of two

ethods especially with and without MS detection.

. Experimental

.1. Chemicals

Acrylamide (99%) and 13C3-labeled acrylamide (isotopicurity 99%) were purchased from Sigma–Aldrich (St. Louis,O, USA) and Cambridge Isotope Laboratory (Andover, MA,SA), respectively. Formic acid (96%) and methanol (HPLC-rade) were obtained from Tedia (Fairfield, OH, USA) anderck (Whitehouse Station, NJ, USA), respectively. All of other

olvents and chemicals such as potassium bromide, potassiumromate and sodium chloride used for the analysis of acry-amide were of analytical grade. Ethyl acetate and petroleumther should be redistilled before use. Water was purified withMilli-Q system (Millipore, Bedford, USA). All solutions pre-

ared for HPLC were passed through a 0.45 �m HNWP04700ylon membrane filter (Millipore, Bedford, USA) before use.

Acrylamide and 13C3-labeled acrylamide are potent cumula-ive neurotoxins in animals and men and may be carcinogenic.

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24 Y. Zhang et al. / Analytica C

hey are hazardous and should be handled carefully. Sampleretreatment procedures referring to organic reagent operationshould be carried out in a fume cupboard.

.2. Samples

Several representative samples of Chinese traditionalarbohydrate-rich foods were collected from two major super-arkets in Hangzhou, China. The analytical survey comprisedsequence of commercial products such as Chinese fried bread

tick, egg omelet, oil cake, etc. Considering protecting commer-ial benefits of Chinese traditional food manufacturers, brandsf tested samples were not shown and pointed out.

.3. Extraction

The Chinese traditional carbohydrate-rich samples wereulverized or homogenized in HL-2070 multi-function foodrocessor (Shanghai Herine Electric Appliance Co., Ltd., Shang-ai, China) or WH861 variable speed Waring blender (Taicangcience and Educational Instrument Factory, Taicang, Jiangsu,hina) prior to sampling while powdered food products were

ampled directly. As for sampling, 1.50 g of above-mentionedamples were weighted into 50 mL centrifuge tubes, 500 �L of3C3-labeled acrylamide internal standard solution (1 �g mL−1)as added and these tubes were placed for 10 min in order that

abeled acrylamide could adequately mix with sample matrixia osmotic effect. To make a defatting process, 20 mL of redis-illed petroleum ether was added and each tube was then cappednd shaken by hand or vortex briefly to mix the contents ofube. The tubes were then clamped and shaken in KQ3200Eltrasonic shaker (Kunshan Ultrasonic Instrument Co., Ltd.,unshan, Jiangsu, China) to mix the tube contents for 10 min.he supernatant petroleum ether was removed and the defat-

ing step was then performed again as described above. Sevenilliliters of sodium chloride (2 mol L−1) was added into the

esidue of each tube, which was capped and shaken in an ultra-onic shaker to extract the analyte for 20 min. The tubes wereentrifuged at 15,000 rpm for 15 min with a Microfuge 18 Beck-an Coulter centrifuge (Beckman Coulter Inc., Fullerton, CA,SA). The clarified aqueous layer was promptly removed byipette. The residues were extracted again by 8 mL of sodiumhloride and the extraction step was performed as describedbove. The supernatant fluids during two extraction steps wereemoved and merged by pipette for further use.

.4. Derivatization

Five milliliter of subsample aqueous solution from NaClxtraction and 0.6 mL sulfuric acid (10%, v/v) were sequen-ially added into a brown quantitative colorimetric cylinder. Theolume of solution was quantitatively fixed up to 10 mL with theddition of NaCl solution and the cylinder was then placed into

efrigerating cabinet for precooling (4 ◦C, 15 min). An aliquotf derivatization reactants, including 1 mL of 0.1 mol L−1 potas-ium bromate (KBrO3) and 1.50 g of potassium bromide (KBr)owder, were added to the precooled solution. The cylinder

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a Acta 584 (2007) 322–332

as shaken with a vortex blender and the reaction mixture wasllowed to stand for 30 min at 4 ◦C. The derivatization reactionas terminated by adding 0.1 mL of 0.1 mol L−1 sodium thio-

ulfate. A 4 mL aliquot of analyte solution was extracted twiceith 4 mL of redistilled ethyl acetate, and the combined extractsere dried over sodium sulfate. Aliquots of the dried extractsere transferred into 2 mL of amber glass autosampler vials forC-MECD analysis.

.5. GC-MECD analysis

GC-MECD was used for both method validation and quan-ification of acrylamide in Chinese traditional carbohydrate-richoods. Pretreated samples were analyzed on the chromato-raphic system including a 6890N network gas chromatographrom Agilent Technologies (Palo Alto, CA, USA) coupledo Agilent’s 6890N micro-electron capture detector connectedn-line. A 1 �L aliquot of pretreated solution (solvent: ethylcetate) was injected on-column with a 7683 automatic liquidampler and injector system (Agilent) onto a 19091N-113 HP-NNOWax capillary column (polyethylene glycol, 30 m length,.32 mm i.d., 0.25 �m film thickness, J&W Scientific, Agilent,A, USA). Separations were performed using nitrogen carrieras, applying the following temperature program: 110 ◦C (holdime 1 min), then at 10 ◦C min−1 to 140 ◦C (hold time 15 min)nd at 30 ◦C min−1 to the final temperature of 240 ◦C (hold timemin). The GC-MECD sample injector temperature and MS

nterface temperature were both held at 250 ◦C.

.6. Ethyl acetate extraction and clean-up

As for LC-ESI-MS/MS analysis, the analyte solution fromaCl extracts was then extracted by 15 mL of ethyl acetate for

hree times. The organic phase was removed from separatoryunnel, concentrated by rotatory evaporator and subsequently theoncentrated solution was evaporated to dryness under a gentletream of nitrogen. A 1.5 mL aliquot of ultrapurified water wasdded to the residue, and the solution was placed into an ultra-onic bath for 10 min. The re-dissolved liquid was ready for SPElean-up. Oasis HLB SPE cartridges (6 mL, 200 mg) purchasedrom Waters (Milford, MA, USA) were conditioned with 3.5 mLf methanol followed by 3.5 mL of water; the methanol andater portions were discarded prior to clean-up. Each cartridgeas loaded with 1.5 mL of re-dissolved extract. The extract was

llowed to pass through the sorbent material and discarded. Thenhe cartridge was eluted with 3 mL of water and the eluent wasollected. Aliquots of the eluent were transferred into 2 mL ofmber glass autosampler vials for HPLC-ESI-MS/MS analysis.

.7. Confirmatory GC–MS analysis

To confirm whether the analytes submitted for the quantitativenalysis of GC-MECD were brominated derivatives of acry-

amide, the analytes were qualified and confirmed by GC–MSnalysis. Brominated sample extracts prepared by aforemen-ioned pretreatment steps and terminated by adding sodiumhiosulfate were (see Section 2.4) submitted to an additional

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PE clean-up step performed according to Section 2.6. Sohat the derivatized extracts could be purified and co-extractiventerference could obviously be reduced prior to MS-basednalysis. The eluent was then collected and extracted thriceith 3 mL of redistilled ethyl acetate. The combined extractsere dried over sodium sulfate. Finally, 1 �L of the final test

olution was injected onto GC–MS, which was performed onHewlett-Packard (HP) 6890 gas chromatograph (GC) cou-

led to an HP 5973 benchtop mass selective detector (MSD)perated in selected ion monitoring (SIM) mode with positivelectron impact (EI) ionization. The analytical separation waserformed on a HP5-MS capillary column (polysiloxane poly-ers, 30 m × 0.25 mm, 0.25 �m, J&W Scientific, Agilent, CA,SA) and helium was chosen as the carrier gas at a flow ratef 1.0 mL min−1. Following injection, the column was held at0 ◦C for 2 min, then programmed at 10 ◦C min−1 to 200 ◦Cnd held for 5 min at 200 ◦C (total run time: 21 min). Injec-ions by the autosampler were made in splitless mode with aurge activation time of 1.0 min and an injection temperature of80 ◦C. The GC–MS interface transfer line was held at 280 ◦C.nder such conditions, the retention time of acrylamide and

3C3-acrylamide derivatives was 6.6 min. Ions monitored were/z 70, 149 and 151 for 2-bromopropenamide, and m/z 110 and54 for 2-bromo(13C3)-propenamide.

.8. HPLC-ESI-MS/MS analysis

Determination of acrylamide in Chinese traditional foods waserformed on a HPLC-MS/MS with the electrospray positiveonization (ESI+). In detail, a Waters 2695 HPLC quaternaryump system equipped with a 2695 micro vacuum degasser, a695 thermostated autosampler and a 2695 thermostated columnompartment was coupled with a Micromass Quattro Ultimariple-quadrupole mass spectrometer from Micromass Co. Com-any Inc. (Manchester, UK). Chromatographic separation wasarried out on an Atlantis dC18 column (210 mm length, 1.5 mm.d., 5 �m particle size; Waters, Milford, MA, USA) maintainedt 25 ◦C. The mobile phase was 10% methanol/0.1% formic acidn water with a flow speed of 0.2 mL min−1. A mobile phasequilibration time of ∼1.5 h was needed before analysis of stan-ards or samples began because too short an equilibration periodould result in shifting the retention time of acrylamide. The

onditions used for the electrospray source were as follows:apillary voltage, 3.5 kV; cone voltage, 50 V; source tempera-ure, 100 ◦C; desolvation gas temperature, 350 ◦C; desolvationas flow, 400 L h−1 nitrogen; cone gas flow, 45 L h−1 nitrogen;nd argon collision gas pressure to 3 × 10−3 mbar for MS/MS,hich gave a highest acrylamide response in this study. The

ollision energy (CE) was optimized for each multiple reac-ion monitored (MRM) transition. The HPLC-ESI-MS/MS runime was 10 min per sample. The collision energy for each

onitored transition was optimized in MRM mode. The tran-itions monitored according to previous studies [16,29] for

crylamide were 72 > 72 at 1 eV, 72 > 55 at 6 eV, 72 > 44 ateV and 72 > 27 at 15 eV. The transitions monitored for labeledcrylamide were 75 > 75 at 1 eV, 75 > 58 at 6 eV and 75 > 30 at5 eV.

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a Acta 584 (2007) 322–332 325

.9. Recovery experiments and quantification

Recovery rates for standard acrylamide were determinedrom spiked Chinese traditional food matrixes at spiking lev-ls of 50, 200 and 500 �g kg−1. For both methods, GC-MECDnd HPLC-ESI-MS/MS, recovery experiments were performedn three replicates. Quantification for GC-MECD analysis waserformed using external standard method. Calibration curvesere performed via injection of different levels of acrylamide

tandard (solvent ethyl acetate) after the derivatization of ana-yte (see Section 2.4), the concentration range of which was–250 ng mL−1. Quantifications of both method validation andcrylamide levels in real samples were evaluated by the statisticoftware of Single-instrument Cerity NDS for chemical QA/QCPalo Alto, CA USA). Quantification for LC-ESI-MS/MS wasperated using internal standard method. A calibration curveas made of the ratio Aaa/Ais against Caa/Cis (Aaa/Ais, the peak

rea ratio of acrylamide and 13C3-acrylamide; Caa/Cis, the con-entration ratio of acrylamide and 13C3-acrylamide). By addinghe same concentration of internal standard (50 ng mL−1) tohe sample and fixing the aliquot portion, concentration can besed as the abscissa of plot. Meanwhile, concentrations of acry-amide ranged from 1 to 200 ng mL−1 were studied. Quantitativealibration and calculation of acrylamide levels in samples forPLC-ESI-MS/MS were accomplished using the standard soft-are of MassLynx v4.0 (Micromass, Manchester, Lancashire,K), and the result data were transferred to an Access database

Microsoft, Bellevue, WA, USA).

.10. Data analysis

The integrated peak areas for the analyte and retention timeere saved in a report file via the above software. A custom-ritten Access macro converted the Analyst report file into a text

eport file, which was imported into Access database specificallyreated for this analysis. Statistical analysis was carried out in austom program developed using the Statistical Analysis SystemSAS) software (SAS Institute, Cary, NC, USA).

. Results and discussion

.1. Sample preparation and extraction

Spiking recovery tests might not adequately reflect the matrixnteractions of naturally embedded analytes. It is difficult tostimate extraction efficiencies from complex sample matrixesithout performing tracer studies with isotopically labeled

urrogate standards. Therefore, extraction of acrylamide frompiked Chinese traditional food samples was evaluated for sam-le homogenates with purified water and 2 mol L−1 of sodiumhloride (NaCl). Recoveries for individual acrylamide leveluantified by HPLC-ESI-MS/MS in various sample matrixessing water and NaCl were (25–43)% and (66–92)%, respec-

ively. The greatly improved extraction efficiency using NaClqueous solution is probably due to partial denaturation of pro-eins and inhibition of emulsification process [21,30,34,35]. Onhe other hand, similar to the experiences of private and offi-

326 Y. Zhang et al. / Analytica Chimica Acta 584 (2007) 322–332

Table 1Recovery rates for different kinds of further extracting approaches using ethylacetatea

Volume of ethylacetate (mL)

Recovery (%) Mean ± S.D. (%)b

1 × 15 35.4 38.3 30.4 34.7 ± 4.02 × 15 74.2 78.1 71.0 74.4 ± 3.63 × 15 93.4 90.7 96.4 93.5 ± 2.84 × 15 95.4 91.1 97.5 94.7 ± 3.3

a This method performance test was operated via considering recovery ratesad

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nd solvent costs in order to optimize the liquid–liquid extraction approachesuring the pretreatment of LC–MS/MS method.b Data were expressed as mean ± S.D. (n = 3).

ial food control laboratories, problems have been encounteredn the analysis of difficult matrixes due to interfering com-ounds in the characteristic acrylamide transitions (either forhe internal standard or the analyte) and concentrating diffi-ultly. A promising approach is to extract the analyte into aolar organic solvent, such as ethyl acetate. Sanders et al. [36]mployed ethyl acetate to extract acrylamide from the aqueoushase (removing interfering constituents such as salt, sugars,tarches, amino acids, etc.). The ethyl acetate extract couldhen be easily concentrated by rotatory evaporation. Therefore,ecovery rates for four different variations of further extractingolumes were tested, i.e. liquid–liquid extraction by (i) 15 mL;ii) 2 × 15 mL; (iii) 3 × 15 mL; (iv) 4 × 15 mL of ethyl acetate.orresponding results are shown in Table 1 and 3 × 15 mL ofthyl acetate was chosen as the optimal liquid–liquid extractingpproach according to the better recovery rate and solvent costecause no considerable recovery improvement presented forearly all of standards and sample matrixes using 4 × 15 mL ofthyl acetate compared to the extraction volume of 3 × 15 mL.fter condensation by rotatory evaporation, dryness by nitrogen,

edissolvation with purified water and SPE steps (see Section 2),he analyte extract was clean enough for direct injection into thePLC-ESI-MS/MS system.

.2. Modified derivatization of acrylamide

GC separation demands derivatization of acrylamide, whichs well done in most laboratories with hydrobromic acid (HBr)nd saturated Br2 solution [17,19,37–39]. The excess bromines then removed by addition of sodium thiosulfate until theolution becomes colorless so that the derivative reaction iserminated. As an alternative technique, derivatization withBrO3 and KBr was applied in the present study accord-

ng to Nemoto et al. (2002) [40] with some modifications.here are many advantages of this derivatization technique.se of the strong acid (HBr) and saturated Br2 solution cane avoided whereas these two solvents have been prepared dif-cultly and handled hazardously. Nevertheless, use of KBrO3nd KBr combination is relatively more convenient and safe,

nd the reaction is performed in about 30 min at cold stor-ge temperature with excellent reproducibility, i.e. less than0% GC-MECD area difference of derivatized standard inhree duplicates at three different days with the same con-

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ig. 1. The chromatograms of two bromination derivatives: (a) 2-romopropenamide (2-BPA) and (b) 2,3-dibromopropionamide (2,3-DBPA) byA) GC-MECD and (B) GC–MS.

entration. In addition, no further clean-up after derivatizations needed. Meanwhile, (KBrO3 + KBr) derivatives of acry-amide show excellent GC properties, i.e. sharp peak shapesnd good response in MECD. As described above, the twoerivatives 1 (2,3-dibromopropionamide, 2,3-DBPA, <5%) and(2-bromopropenamide, 2-BPA, >95%) are less polar compared

o the original compound and are therefore easily soluble inon-polar organic solvents like ethyl acetate. The GC-MECDhromatogram of two derivatives is shown in Fig. 1A and thetructures of 2-BPA and 2,3-DBPA were confirmed via GC–MSsee Fig. 1B). Disadvantages of (KBrO3 + KBr) derivatizationre the complex composition of the solvent that could influencehe reaction yield of the derivatives. Such derivatization shouldherefore be performed with an already cleaned extract as a finaltep before instrumental analysis.

.3. Optimization of clean-up

To further purify carbohydrate-rich sample extracts, solid-hase extraction (SPE) was performed following liquid–liquidxtraction. Most clean-up procedures consisted of the com-ination of several solid-phase extractions as for acrylamideetermination in previous studies. Becalski et al. [4] used a com-ination of three different cartridges: Oasis mixed-mode anionxchange (MAX) (Waters), Oasis mixed-mode cation exchangeMCX) and ENVI-Carb (graphitized carbon) (Supelco, Belle-onte, PA, USA). A similar combination of SPE cartridgesonsisting of Bond Elut C18, Bond Elut Jr-PSA (anion exchange)nd Bond Elut Accucat (all Varian) were chosen for clean-

p of samples, which were measured by LC–MS with columnwitching [41]. To simplify the clean-up procedures, a singlePE cartridge was chosen and the type and capacity of car-

ridge sorbent were optimized based on the effective clean-up.

Y. Zhang et al. / Analytica Chimica Acta 584 (2007) 322–332 327

Table 2SPE recovery test using some different commercially available cartridgesa

SPE cartridges Recovery (%)b

Acrylamide level (ng mL−1) 0.5 25 100

Varian Bond Elut-C18, 1 mL, 100 mg 30.3 ± 9.1 21.0 ± 2.2 11.3 ± 3.8Varian Bond Elut-C18, 3 mL, 500 mg 73.0 ± 9.0 88.1 ± 5.5 91.6 ± 5.2Oasis HLB, 3 mL, 60 mg 43.1 ± 6.5 39.2 ± 2.1 41.1 ± 0.7O

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mHtetAR.S.D. values lower than 1% for both standards (non-labeled andlabeled acrylamide) and different food matrixes. Fig. 3 showsthe chromatograms of acrylamide standard and isotope internalstandard by the two methods. For quantitative purposes, the two

asis HLB, 6 mL, 200 mg 90.3 ± 2.8

a This method performance test was operated via comparison of recovery in ob Data were expressed as mean ± S.D. (n = 5).

n the present study, some different commercially available SPEartridges such as non-polar stationary phase (Varian Bond Elut-18, 1 mL, 100 mg or 3 mL, 500 mg) or hydrophilic–lipophilicalanced copolymer (Oasis HLB, 3 mL, 60 mg or 6 mL, 200 mg)ere evaluated (Table 2). Results showed that acrylamide wasot completely adsorbed by cartridges with small size such asarian Bond Elut-C18 (1 mL, 100 mg) and Oasis HLB (3 mL,0 mg). Good adsorbability and recovery were found when usingasis HLB (6 mL, 200 mg) as SPE cartridge, i.e. (90.3 ± 2.8)%,

97.5 ± 0.4)% and (98.1 ± 5.2)% of SPE recovery (n = 5), in thecrylamide concentration of 0.5, 25 and 100 ng mL−1, respec-ively. Few proportions of the analyte were determined duringhe load process of SPE, i.e. 13.3%, 2.0% and 1.3%, under thehree selected concentrations. Oasis HLB cartridge belongs tohe main supporter of which is a hydrophilic–lipophilic balancend water-wettable reversed-phase sorbent for all compoundsnd all of general SPE needs produced by Waters (Milford,A, USA). Meanwhile, optimization of eluent with different

evels of methanol was studied and the maximum recovery ofcrylamide was obtained by the use of water as eluent (Fig. 2).eanwhile, an optimal volume (3 mL) of water was used as the

luent considering both recovery of clean-up and better sensi-

ivity. If the eluent volume was less than 3 mL, a bit amountf analyte was still adsorbed with the cartridges and not elutedhoroughly, which might lower the recovery of SPE clean-up.onversely, if the eluent volume was more than 3 mL, the final

ig. 2. The recovery of acrylamide by the use of different ratios of methanols eluent (n = 5). The value of methanol ratio 0 means the use of purified waters eluent. The tested levels of acrylamide were determined as (�) 0.5 ng mL−1;

) 25 ng mL−1; ( ) 50 ng mL−1. Data for the recovery of acrylamide by these of 50%, 75% and 100% of methanol as corresponding eluent were notetected when the tested level of acrylamide was designed as low concentration0.5 ng mL−1).

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o optimize the SPE cartridges during the clean-up of LC–MS/MS method.

luent was diluted and the analyte sensitivity was subsequentlyeduced.

.4. Method validation and comparison

Instrumental methods using standard solutions and completeethods using spiked sample surrogates were validated both forPLC-ESI-MS/MS of analyte and for GC-MECD of bromina-

ion derivates. For qualitative purposes, both of methods werevaluated by taking into account the precision of retention time,he interference of co-elution and peak purity of the analyte.

high repeatability of the retention time was obtained with

ig. 3. The chromatograms of (A) acrylamide standard (72 > 55), isotope inter-al standard (75 > 58) and TIC by HPLC-ESI-MS/MS and (B) 2-BPA byC-MECD.

328 Y. Zhang et al. / Analytica Chimica Acta 584 (2007) 322–332

Table 3Repeatability test of acrylamide in representative foods by the two methodsa

Heat-treated method Sample name GC-MECD HPLC-ESI-MS/MS

Level (�g kg−1) MU (%)b Level (�g kg−1) MU (%)b

Steaming Steamed buns 12.1 8.8 10.4 9.4Steamed dumplings 14.0 6.4 13.6 4.8

Frying Fried bread sticks 189.9 7.6 195.2 4.0Hemp flowers 79.6 9.4 78.7 8.1

Baking Clay oven rolls 31.6 10.2 30.2 6.0Chinese crisp cakes 33.2 10.4 29.0 5.3

Roasting Chinese battercakes 112.3 6.4 109.5 2.2

ducibty.

misrobCdrorrmf

t

heeaofrGmuibc

TP

S

I

I

Egg omelets 350.4

a This method performance test was performed in order to compare the reprob Data were expressed as mean and MU (n = 5). MU, measurement uncertain

ethods were validated by defining the linearity, LOD and LOQ,ntermediate precision and recovery. As for GC-MECD analy-is, the calibration curves (y = ax + b, a = 417089, b = −473)anged from 0.5 to 125 ng mL−1 (n = 3). Excellent linearity wasbtained with typical values for the correlation coefficient (R2)etween 0.999 and 1.000. Meanwhile, The calibration curve inhinese traditional carbohydrate-rich foods using internal stan-ard method (Aaa/Ais = 0.843Caa/Cis + 0.001) was linear over theange of 1–200 ng mL−1 with a coefficient of determination (R2)f 0.9992 (n = 3) as for the LC–MS/MS method. The calibrationange could be extended to higher amounts, but this was notoutinely done since corresponding linear ranges for both ofethods were sufficiently wide to measure acrylamide levels

rom current sample matrixes.As for the extract stability, acrylamide is not stable in con-

act with raw plant tissue or soil microorganisms [42]. Cooking

rmi

able 4recision and accuracy of the measurement of acrylamide in Chinese traditional food

tandard/sample Nom. conc.(�g kg−1)a

GC-MECD

Mean calc. conc.(�g kg−1)b

Accuracy (%)c

ntra-day assay (n = 6)Acrylamide std. 5 4.4 88.5

50 45.7 91.4500 471.3 94.3

Hemp balls – 36.7 –Crispy rice – 418.2 –Chinese potato crackers – 716.4 –

nter-day assay (n = 5)Acrylamide std. 5 4.5 90.5

50 46.1 92.1500 463.2 92.6

Hemp balls – 34.9 –Crispy rice – 412.9 –Chinese potato crackers – 726.9 –

a Nominal concentration of acrylamide.b Mean calculated concentration of acrylamide.c Accuracy was calculated as (mean calculated concentration)/(nominal concentratd Precision was expressed as R.S.D.

7.9 346.2 5.5

ility between the two methods.

alts the biodegradation of acrylamide so its stability in anxtract is dependent on pH, exposure to light, reactive co-xtractives and microorganisms. In our study, extracts werenalyzed within hours of their preparation. The repeatabilityf the method was estimated for determination of acrylamiderom some representative Chinese traditional carbohydrate-ich foods including steamed, fried, baked and roasted foods.ood repeatability was obtained for eight representative above-entioned samples by both of methods and the measurement

ncertainty (U) was estimated for the two techniques accord-ng to the formula, i.e. MU (%) = 2 × CVrepeatability (%), provedy previous study (Table 3, n = 5) [43]. The intermediate pre-ision of the two methods was determined by calculating the

elative standard deviations (R.S.D.) of the replicate measure-ents. Table 4 shows the results of a 5-day precision study

n which portions of three levels of acrylamide standard three

s

HPLC-ESI-MS/MS

Precision (%)d Mean calc. conc.(�g kg−1)b

Accuracy (%)c Precision (%)d

10.6 4.3 85.9 7.46.4 47.7 95.5 3.32.1 490.4 98.1 1.0

9.4 39.0 – 6.76.3 399.9 – 3.53.2 740.9 – 2.0

11.2 4.5 90.9 8.48.7 48.0 96.0 5.24.3 502.2 100.4 3.0

8.9 38.8 – 5.97.1 396.3 – 4.63.3 752.4 – 1.6

ion) × 100%.

Y. Zhang et al. / Analytica Chimic

Table 5Limit of detection (LOD, S/N = 3/1) and limit of quantification (LOQ,S/N = 10/1) for the instrumental and the complete methods as well as recover-ies and reproducibility (R.S.D., n = 3) for acrylamide-spiked Chinese traditionalfoods

Sample matrix Hemp balls Crispy rice Chinese potato crackers

GC-MECDa

InstrumentalLOD (�g kg−1) 0.15 0.10 0.15

Spiked Chinese foodsLOD (�g kg−1) 3 4 5LOQ (�g kg−1) 10 15 15Spiked recovery (%)b 79.1 85.4 92.7R.S.D. (%) 8.7 7.3 4.2

HPLC-ESI-MS/MSInstrumental

LOD (�g kg−1) 0.80 0.65 0.75

Spiked Chinese foodsLOD (�g kg−1) 1 2 1LOQ (�g kg−1) 4 5 5Spiked recovery (%)b 83.7 93.0 97.4R.S.D. (%) 6.4 4.9 1.4

a Acrylamide was analyzed as the corresponding bromination derivative (2-B

a

repicsto(

itmlsawwbttevfidbeazms

AHtttilw3vbtsplsfhM

tmLlodfTbaotr

3f

C2mtwFsmfceimcn

PA) in GC-MECD analysis.b Three representative samples were spiked as 50, 500 and 1000 �g kg−1 ofcrylamide standard.

epresentative products were repeatedly analyzed three timesach day for the inter-day precision study while the otherortion was repeatedly analyzed six times in day 1 for thentra-day precision study. The tested products were hemp balls,rispy rice and Chinese potato crackers. The intermediate preci-ion for acrylamide by both of techniques was excellent withhe values for the R.S.D. of each data set ranged 1.6–8.4%f GC-MECD and 3.2–11.2% of LC–MS/MS, respectivelyTable 4).

Recovery of methods was demonstrated in three tests employ-ng the method of standard addition. The matrixes includedhree kinds of special samples which represented the low, inter-

ediate and high levels of acrylamide containing foods fromocal supermarkets. Acrylamide standard was added to eachample at a certain level, which was close to the determinedcrylamide level of corresponding sample. Total acrylamideas then determined for each level. When total acrylamideas plotted against added acrylamide, the y-intercept calculatedy linear regression analysis was the incurred residue level inhe unspiked sample. Subtracting the incurred residue from theotal amount of acrylamide found indicated the spike recov-ry for each sample. Good recoveries were achieved for bothalidated methods (GC-MECD and HPLC-ESI-MS/MS), con-rming the reliability of the methods including the brominationerivatization prior to GC analysis. There was a discrepancyetween the recoveries obtained by the two methods consid-ring all of three matrixes. The bulky bromine substituents

djacent to the carbonyl group might hamper the derivati-ation reaction. The additional derivatization step in the GCethod can also be the reason for the slightly higher relative

tandard deviations compared to the HPLC method (Table 5).

blWd

a Acta 584 (2007) 322–332 329

lthough the GC-MECD method is more sensitive than thePLC-ESI-MS/MS method when employing standard solu-

ions, the opposite was found for quantification of Chineseraditional sample extracts. This finding can be explained byhe high-resolution potential of MS/MS efficiently suppress-ng matrix background in the mass chromatograms. Both theimit of detection (LOD) and limit of quantification (LOQ)ere evaluated by the statistical software of Chemstation forD (Agilent, Palo Alto, CA USA) for GC-MECD and MassLynx4.0 (Micromass, Manchester, Lancashire, UK) for LC–MS/MSased on the abundance of MRM transition (72 > 55), respec-ively. The matrix for LOD and LOQ calculations based ontep-by-step dilution of representative Chinese traditional sam-le preparation. Although the LOD of GC-based method isower than LC–MS/MS technique when employing standardolutions, the contrary results of LOD and LOQ were foundor quantitative analysis of acrylamide in sample extracts due toigh-resolution potential of MS/MS and relatively instability ofECD (Table 5).Recently, we participated in the acrylamide proficiency

esting organized by Food Analysis Performance Assess-ent Scheme (FAPAS) using the optimal pretreatment andC–MS/MS method in the present work. Compared to the

evel (1404 �g kg−1) reported by the organization, result fromur laboratory (No. 021) for acrylamide (1381 �g kg−1) inispatched test material with a z-score of −0.1 seemed satis-actory, which fulfill requirements from the organization [44].herefore, the suitability of GC-MECD could be confirmedy the certified LC–MS/MS method. Considering all of thebove data for method validation and comparison test, bothf methods and sample pretreatment procedures employed inhe present work can be regarded as selective, precise andobust.

.5. Acrylamide in Chinese traditional carbohydrate-richoods

Acrylamide contaminant was found in nearly all analyzedhinese traditional foods both by the GC-MECD (analyzed as-BPA by GC) and by the HPLC-ESI-MS/MS quantificationethod. Meanwhile, studies on comparison of acrylamide con-

ent between Chinese traditional carbohydrate-rich foods andestern style foods were also performed in the present work.ig. 4 shows chromatograms of acrylamide in representativeamples by the two methods. As for the LC–MS/MS chro-atogram, a non-identified peak at the retention time different

rom that of acrylamide is shown in Fig. 4A. This unknownompound, which could not be easily removed according to thextraction and clean-up methods in the present work, was presentn the acrylamide extract. As for the GC-MECD chromatogram,

any non-identified peaks presented because no additionallean-up steps were operated during the pretreatment. Fortu-ately, all of the above interferential peaks could be negligible

ecause the retention time of these impurity peaks did not over-ay with the response of both acrylamide and 2-BPA analyzed.

hen applied to the same samples the two methods despite theifferences in the methodology gave concordant results. How-

330 Y. Zhang et al. / Analytica Chimica Acta 584 (2007) 322–332

Table 6Quantitative analysis of acrylamide in steamed and fried Chinese traditional foods and comparison test of the GC-MECD method vs. the HPLC-ESI-MS/MS method

Acrylamide level (�g kg−1)a

HPLC-ESI-MS/MS GC-MECD Difference (%)b

SteamingSteamed buns 10.4 ± 0.5 11.6 ± 0.8 11.7Steamed dumplings 13.6 ± 0.3 14.0 ± 1.2 3.5Chinese noodles 6.1 ± 0.1 – –Steamed bread with meat paste 7.0 ± 0.4 – –Chinese porridge 3.2 ± 0.2 – –Western oatmeal 11.5 ± 0.8 12.4 ± 1.4 7.3

FryingRice crisps with five nuts 25.3 ± 1.4 26.9 ± 3.6 6.4Hemp balls 40.8 ± 5.4 38.7 ± 1.9 −5.1Fried bread sticks 1 196.6 ± 5.1 189.9 ± 5.9 −3.4Fried bread sticks 2 86.3 ± 1.4 80.7 ± 3.2 −6.5Fried bread sticks 3 136.9 ± 6.8 134.7 ± 3.3 −1.6Hemp sticks with fragrant oil 104.4 ± 4.2 108.2 ± 4.0 3.6Small hemp flowers 135.5 ± 5.5 135.7 ± 5.5 0.2Hemp flowers 1 78.7 ± 3.2 71.1 ± 3.3 −9.7Hemp flowers 2 577.2 ± 7.1 584.5 ± 6.1 1.3Glutinous rice sesame balls 45.3 ± 5.3 43.7 ± 3.1 −3.6Crispy rice 386.2 ± 9.2 382.2 ± 7.6 −1.0Western potato crisps 455.4 ± 3.6 433.9 ± 7.4 −4.7

a Data for the acrylamide level in food samples were expressed as mean ± S.D. (n = 3).b The difference of the two methods was calculated as (a1 − a2)/a2 × 100%. a1,

HPLC-ESI-MS/MS.

Fig. 4. The chromatograms of (A) acrylamide (72 > 55), spiked internal stan-dard (75 > 58) and TIC by HPLC-ESI-MS/MS and (B) 2-BPA by GC-MECD insteamed buns.

evcGfrafltphdfihhhtscLaTfttomp

acrylamide level measured by GC-MECD; a2, acrylamide level measured by

ver, acrylamide in some Chinese traditional foods processedia steaming could not be detected by GC because such foodsontain trace level of acrylamide, which is lower than LOQ ofC-MECD (Table 6). Compared to the trace levels of acrylamide

or most of Chinese steamed foods, there is a relatively wideange of acrylamide level (25.3–577.2 �g kg−1 by GC-MECDnd 26.9–584.5 �g kg−1 by LC–MS/MS) found in Chinese friedoods. In the present study, we especially focused on the acry-amide levels in hemp flowers because they are regarded ashe well-known and popular breakfast food for Chinese peo-le. A great difference of acrylamide level was found amongemp flowers purchased from different manufacturers becauseifferent processing conditions including oils, frying time andrying temperature were chosen. Compared to acrylamide leveln Western potato crisps fried with similar heat processing style,emp flowers contain a considerable level of acrylamide even aigher amount of acrylamide contaminant (Table 6). On the otherand, Fig. 5 reports data for acrylamide contaminant in Chineseraditional carbohydrate-rich baked and roasted foods. To oururprise, high level of acrylamide has been found in Chineseorn crisps (439.6 �g kg−1 by GC-MECD and 464.8 �g kg−1 byC–MS/MS) and potato crackers (771.1 �g kg−1 by GC-MECDnd 734.5 �g kg−1 by LC–MS/MS) during the investigation.herefore, this result should be attached importance to for both

ood safety departments and correlative infant food manufac-urers. Moreover, corresponding exposure assessment will need

o be conducted in order to gauge the utility of various controlptions. For instance, based on such contaminant risk assess-ent, it can be primarily realized whether modifications in

rocessing and cooking procedures are necessary.

Y. Zhang et al. / Analytica Chimic

Fig. 5. Different concentrations of acrylamide in Chinese traditionalcarbohydrate-rich foods processed under (A) baked conditions and (B) roastedconditions. (�) HPLC-ESI-MS/MS; (�) GC-MECD. The baked food samples:(1) Chinese northeast cakes; (2) Chinese northeast crispy cakes; (3) clay ovenrolls; (4) Chinese bread with milk and confiture; (5) salty toasts; (6) Beijingtraditional confiture; (7) papery cup cakes; (8) egg Shaqima cakes; (9) Chinesepuffed cakes; (10) Chinese baked cakes; (11) honey cakes; (12) fragrant ricecrisps. The roasted samples: (a) egg omelet; (b) milk battercakes; (c) Chinesetraditional “thousand-layer” cakes; (d) corn crisps; (e) Chinese flour crisps; (f)pwb

4

idbkcirslwtapam

ittf

ctnopvpcsic(pieii(r

A

(MSi

R

[[[

otato crackers; (g) Chinese southern battercakes; (h) fragrant fried samp; (i)heat cookies 1; (j) wheat cookies 2; (k) Western salty biscuits; (l) Frenchattercakes.

. Conclusion

The present study developed two analytical methods fordentification and quantification of acrylamide in Chinese tra-itional carbohydrate-rich complex food matrixes such as friedread sticks, hemp flowers and Chinese corn crisps. To our bestnowledge, these are the first GC-MECD method dealing withontaminant analysis of acrylamide and the first method employ-ng potassium bromate and potassium bromide as derivatizationeagents for acrylamide derivatization, i.e. 2-BPA, prior to GCeparation. Both methods are well suited for analysis of acry-amide at levels down to few micrograms per kilogram of sampleeight in Chinese carbohydrate-rich foods. The advantages of

he HPLC-ESI-MS/MS method are that no derivatization of

crylamide is necessary prior to clean-up. Furthermore, highrecision and low limit of quantification of the analyte werechieved by this method. On the other hand, the GC-MECDethod showed that no clean-up steps of acrylamide derivative

[[

a Acta 584 (2007) 322–332 331

s necessary prior to injection and was slightly more sensitivehan the HPLC method. Meanwhile, applying two complemen-ary methods is recommended as a quality assurance measureor analysis of acrylamide in various matrixes.

Extraction and derivatization are both proved to be the cru-ial steps in sample preparation, and further optimization ofhese processes should be performed. For instance, a conve-ient and fast pretreatment procedure should be optimized inrder to satisfy the investigation of hundreds of samples. Suchrocedure mainly depends on the optimization of extraction sol-ents and corresponding parameters. Furthermore, an additionalrotein precipitation step (e.g. treatment with potassium hexa-yanoferrate(II) trihydrate solution and zinc sulfate heptahydrateolution) may be performed when a protein-rich food matrixs submitted for acrylamide analysis. On the other hand, someorresponding studies referred to accelerated solvent extractionASE) as for the rapid extract method of acrylamide, whichrovides a fast and efficient extraction of acrylamide from var-ous food samples. Furthermore, fast analysis could then bemployed in a quality control environment closer to productionn a manufacturing facility or factory environment, for instance,n an on-line laboratory or enzyme linked immunosorbent assayELISA), allowing more efficient control and enabling moreapid response if needed.

cknowledgements

The authors thank Zhejiang Marine Fisheries InstituteHangzhou, Zhejiang, China) for providing the guidance of GC-

ECD technique. The financial support by National Naturalcience Foundation Council of China (Project No. 30540016)

s gratefully acknowledged.

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