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Food and BioprocessTechnologyAn International Journal ISSN 1935-5130Volume 4Number 3 Food Bioprocess Technol(2010) 4:340-363DOI 10.1007/s11947-010-0470-x

Acrylamide in Foods: Chemistry andAnalysis. A Review

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REVIEW PAPER

Acrylamide in Foods: Chemistry and Analysis. A Review

Javad Keramat & Alain LeBail & Carole Prost &Nafiseh Soltanizadeh

Received: 13 February 2010 /Accepted: 3 November 2010 /Published online: 25 November 2010# Springer Science+Business Media, LLC 2010

Abstract Acrylamide is a potential cause of a widespectrum of toxic effects and is classified as probably“carcinogenic in humans”. The discovery of acrylamide inhuman foods has given rise to extensive studies exploringits formation mechanisms and levels of exposure and hasspurred search into suitable analytical procedures for itsdetermination in foodstuffs. However, the exact chemicalmechanisms governing acrylamide formation are not yetknown and cheap, convenient, and rapid screening methodsare still to be developed. Acrylamide in food is produced byheat-induced reactions between the amino group of aspar-agine and the carbonyl group of reducing sugars along withthermal treatment of early Maillard reaction products (N-glycosides). Similarly, the decarboxylated Schiff base anddecarboxylated Amadori compounds of asparagine as wellas the Strecker aldehyde have been proposed as directprecursors and intermediates of acrylamide. Correspondingchromatographic methods are used to determine variousstructural groups present in Maillard reaction modelsystems. Gas chromatography-mass spectrometry andliquid chromatography with tandem mass spectrometryanalysis are both acknowledged as the main, useful, andauthoritative methods for acrylamide determination. Thisreview is an attempt to summarize the state-of-the-artknowledge of acrylamide chemistry, formation mecha-nisms, and analytical methods. Special attention is given

to comparison of different chromatographic techniques,particularly the novel, simple, and low-cost methods of itsdetermination.

Keywords Acrylamide chemistry .Mechanism ofacrylamide formation . Acrylamide analysis

Introduction

In April 2002, the formation of acrylamide in starch-richfoods or high-temperature cooking, like with a variety ofbaked and fried foods cooked at high temperature, wasreported by researchers from the Swedish National FoodAdministration (SNFA) and the University of Stockholm.Since the Swedish report, similar findings have beenreported by numerous other countries, including, Norway,Switzerland, UK, and the USA (FAO/WHO 2004).Then,much interest was created about this compound, andmoderate levels of acrylamide (5–50 ppb) in heatedprotein-rich foods and higher contents (150–4,000 ppb) incarbohydrate-rich foods such as potato, beetroot, andselected commercial potato products were reported. Medianlevels of acrylamide were found at 1,200 ppb in potatochips, 450 ppb in French fries, and 410 ppb in biscuits andcrackers (Table 1; Tareke et al. 2002). Table 2 providessome information about contribution of food groups toacrylamide exposure.

Acrylamide is a potential cause of a wide spectrum oftoxic effects (Eriksson 2005; IARC 1994; European UnionRisk Assessment Report 2002; Manson et al. 2005),including neurotoxic effects as has been observed inhumans. Also, acrylamide has been found to be carcino-genic in animals, increasing incidences of a number ofbenign and malignant tumors identified in a variety of

J. Keramat :N. Soltanizadeh (*)Department of Food Science and Technology,Isfahan University of Technology,Isfahan 84156, Irane-mail: n_soltanizadeh@ag.iut.ac.ir

A. LeBail :C. ProstENITIAA,rue de la Geraudiere, BP 82225, 44322, Nantes, Cedex 3, France

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Table 1 Acrylamide content of various food products

Commodities Food item Number ofsamplesa

Meanconcentration,μg/kg

CV(%)

Reportedmaximum,μg/kg

Cereal and cereal-based products 3,304 (12,346) 343 156 7,834

Cereal and pasta, raw andboiled

113 (372) 15 71 47

Cereal and pasta processed(toasted, fried, grilled)

200 (634) 123 110 820

Cereal-based processedproducts, all

2,991 (11,327) 366 151 7,834

Bread and rolls 1,294 (5,145) 446 130 3,436

Pastry and biscuits 1,270 (4,980) 350 162 7,834

Breakfast cereals 369 (1,130) 96 131 1,346

Pizza 58 (85) 33 270 763

Fish and seafood (including, e.g.,breaded, fried, baked)*

52 (107) 25 180 233

Meat and offal (including,e.g., coated, cooked, fried)

138 (325) 19 174 313

Milk and milk products 62 (147) 5.8 119 36

Nuts and oilseeds 81 (203) 84 233 1,925

Pulses 44 (93) 51 137 320

Root and tubers 2,068 (10,077) 477 108 5,312

Potato puree/mashed/boiled 33 (66) 16 92 69

Potato baked 22 (99) 169 150 1,270

Potato crisps (US=chips) 874 (3,555) 752 73 4,080

Potato chips (US=French fries) 1,097 (6,309) 334 128 5,312

Potato chips, croquettes(frozen, not ready to serve)

42 (48) 110 145 750

Stimulated and analog 469 (1,455) 509 120 7,300

Coffee (brewed),ready-to-drink

93 (101) 13 100 116

Coffee (ground, instant,or roasted, not brewed)

205 (709) 288 51 1,291

Coffee extracts 20 (119) 1,100 93 4,948

Coffee decaffeinate 26 (34) 668 169 5,399

Coffee substitutes 73 (368) 845 90 7,300

Cocoa products 23 (23) 220 111 909

Green tea (roasted) 29 (101) 306 67 660

Sugar and honey (mainly chocolate) 58 (133) 24 87 112

Vegetables 84 (193) 17 206 202

Raw, boiled and canned 45 (146) 4.2 103 25

Processed (toasted, baked,fried, grilled)

39 (47) 59 109 202

Fruits, fresh 11 (57) <1 188 10

Fruits, dried, fried, processed 37 (49) 131 125 770

Alcoholic beverages (beer, gin, wine) 66 (99) 6.6 143 46

Condiments and sauces 19 (22) 71 345 1,168

Infant formula 82 (117) <5 82 15

Baby food (canned, jarred) 96 (226) 22 82 121

Baby food (dry powder) 24 (34) 16 125 73

Baby food (biscuits, rusks, etc.) 32 (58) 181 106 1,217

Dried food 13 (13) 121 266 1,184

Data were obtained from the Summary Report of the 64th Meeting of the Joint Food and Agriculture Organization of the United Nations/WorldHealth Organization Expert Committee on Food Additives (http://www.who.int/). Acrylamide occurrence data for different food items analyzedfrom 2002 to 2004 were provided from 24 countries. The total number of analytical results (single or composite samples) was 6,752 with 67.6%from Europe, 21.9% from North America, 8.9% from Asia, and 1.6% from Pacific. No data from Latin America and Africa were submittedaNumber of analytical results for individual plus composite samples. In parentheses, the total numbers of individual samples are shown

CV coefficient of variation

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organs (for example thyroid, adrenals; FAO/WHO 2004).Several observations led to the hypothesis that heating offood could be an important source of acrylamide exposureto humans, if the heating/frying is done with a frying pan,in an oven, or by microwave heating; but no acrylamide hasbeen detected in boiled food products (Törnqvist 2005).

There are two established legal limits for acrylamide. Oneconcerns drinking water (WHO 1996; EEC 1998), and theother involves the migration of acrylamide from packagingmaterials into food (EEC 1992). The latter is defined not tobe detectable within a limit of detection (LOD) of 10 μgacrylamide in 1 kg of food, while a daily intake of some tensof micrograms can be expected depending on dietary habits.This alarmed food producers as well as food controlauthorities. Valuable information about acrylamide and itstoxicological properties have been recently summarized inthe report released by the Scientific Committee on Food(SCF) (2002) and the Heatox Report (2007).

Most studies (Zhang et al. 2005; Vattem and Shetty2003; Schabacker et al. 2004) have focused on themechanisms of acrylamide formation in heat-treated foods.Since these mechanisms have not been completely under-stood, it is not yet possible to determine an effectivepathway for reducing its occurrence in different heatprocessing technologies by controlling critical steps of foodprocessing.

Acrylamide content in foods is defined as the net amountof acrylamide, i.e., what remains after formation anddegradation. It has been shown that prolonged storage andincrease of temperature and heating time enhance theacrylamide formation until it attains a maximum point ofacrylamide formation, then acrylamide degrades, and the

net amount of it decreases. The type of reaction responsiblefor the degradation is still unclear (Biedermann et al. 2002b;Biedermann et al. 2002c; Weibhaar 2004; Hoenicke andGatermann 2004; Hoenicke and Gatermann 2005; Delatouret al. 2004; Eriksson and Karlsson 2005). However, thestability of acrylamide at 190 °C has been evaluated bypyrolysis of 13C3-labeled acrylamide in a glucose/aspara-gine model reaction. At this temperature, acrylamidemainly occurs in the polymeric form with CH2CHCONH2

as the monomeric unit. Also, NMR experiments haveindicated that polymerization of acrylamide can easilyoccur. On the other hand, acrylamide may also react withsoft nucleophiles according to the hard and soft acid basetheory and can, therefore, be consumed in Michael typeaddition reactions (Stadler et al. 2004).

Rather than acrylamide, N-methylacrylamide and 3-buteneamide are the new compounds formed during bakingprocesses. These compounds are suspected toxicants infood products.

It is the main objective of most research efforts in thefield to determine how it is possibly formed or decomposedand how it can be accurately measured. Therefore, thisreview presents, in two separate sections, the recenthypotheses put forward and the factors involved inacrylamide formation mechanisms and the methods ofanalysis used for its determination.

Mechanisms of Acrylamide Formation

Becalski et al. (2003) developed two models. The first oneinvolved a mixture of the following six amino acids:

Table 2 Contribution of food groups to acrylamide exposure (%) in different countries for different age groups (Heatox Report 2007)

Commodities Country (age)

The Netherlands(1–97 years)

The Netherlands(1–6 years)

Sweden(18–74 years)

Germany(15–18 years)

Norway(16–79 years)

Belgium(13–18 years)

Potato products, fried 31 28 13 7 6 –

Potato crisps 15 18 11 30 17 –

Coffee 13 17 23 5 29 –

Biscuits 11 11 6 15 6 25

Bread 10 10 14 – 24 19

Ginger bread 6 8 – – – –

French fries – – 20 8 6 51

Crisp bread – – 8 3 – –

Toast – – – 11 – –

Special snack – – – 8 5 –

Cracker – – – 7 – –

Chocolate and chocolate-spread – – – – – 2

Others 14 8 5 6 7 –

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asparagine, aspartic acid, glutamine, glutamic acid, valine,and lysine along with glucose as a reducing sugar (Yang etal. 1999). The second model was a simplified versionthereof which consisted only of asparagine and glucose.These researchers found that acrylamide was not principallyformed from precursors (especially acrolein) present in theoil itself (Becalski et al. 2003). They also reported that, inmodel reactions consisting of a mixture of six amino acids,the yield of acrylamide at 175 °C was 3300 ng ofacrylamide per about 23 mg of each amino acid, but, itwas not formed in a mixture of aspartic acid, glutamine,and lysine under identical conditions. Nor was it detected insamples of amino acids and glucose heated at 120 °C or140 °C. They concluded that the presence of asparaginesand glucose at the right temperature should have importantroles in the formation of acrylamide and that its formationcould be expected at heating temperatures above 175 °C formore than 10 min.

In a similar study, Mottram et al. (2002) reported that asignificant quantity of acrylamide (221 mg per mol ofamino acid) formed when an equimolar mixture ofasparagine and glucose reacted at 185 °C in the phosphatebuffer. Their findings also confirmed that the presence ofasparagine and glucose was critical for the formation ofacrylamide.

The presence of asparagine with glucose or 2,3-butane-dione (one of several dicarbonyl compounds formed in theMaillard reaction) causes significant amounts of acrylamideto form in dry products, but only trace amounts would formwhen asparagine is replaced with other amino acids.Heating asparagine on its own at 185 °C does not produceacrylamide, confirming the requirement for the dicarbonylreactant to be present and the Strecker degradation to occur(Mottram et al. 2002). However, replacing glucose withother carbohydrates (D-fructose, D-galactose, lactose, andsucrose) concurrent with replacement of asparagine led to asignificant amount of acrylamide to be released (Stadler etal. 2002).

The above studies revealed that the type and quantity ofprecursors play important roles in acrylamide formation.Most amino acids other than asparagines do not take part inthe reactions leading to acrylamide formation. Also, thepresence of both the amino group (from asparagine) and thecarbonyl group (from reducing sugar) or the dicarbonylgroups (from Maillard reactions) is highly crucial foracrylamide formation. Temperature and solvent also haveimportant effects on acrylamide formation that must be dulyconsidered.

The above statement is applicable to such heat-treated,plant-based foods as cereals and potato which are rich inasparagine as a free amino acid (Magnin 1964; asparaginesaccounting for 40% and 14% of their total free amino acids,respectively; Borodin 1925). These foodstuffs are the most

likely ones to produce the highest amount of acrylamide inthe foods prepared from them.

The early Maillard intermediates such as N-glycosylas-paragines yield more acrylamide under milder reactionconditions than the binary mixtures of the precursors. Thedecarboxylated Schiff base and decarboxylated Amadoricompounds of asparagine have been proposed to be directMaillard precursors of acrylamide (Yaylayan et al. 2003;Zyzak et al. 2003). Also, the Strecker aldehyde has beendetected as another direct intermediate of acrylamide(Mottram et al. 2002). The pathway of acrylamideformation in a glucose/asparagine system occurs prior tothe Amadori rearrangement and, consequently, the amountof acrylamide released from N-glycosyl asparagine is about20 times higher than from the Amadori compound. Theprotection of the hydroxyl groups in the sugar ring and inthe carboxyl groups does not affect acrylamide formationfrom Amadori compound of asparagine. This is because theAmadori compound is not easily decarboxylated. Thesecompounds are the first stable intermediates generated as aresult of the early Maillard reaction leading to 1- and 3-deoxyosones, which further decompose to generate colorand flavor compounds (Ledl and Schleicher 1990). How-ever, in low-moisture systems limiting the reversibility ofthe initial step, the first stable intermediates are the N-glycosyl compounds, which mainly rearrange via thecorresponding Schiff base to the Amadori compound, the1-deoxyfructosyl derivative of the amino acid, which is nota favored Maillard intermediate to generate acrylamide. TheSchiff base may alternatively decarboxylate to the interme-diary azomethine yield, which after tautomerization leads tothe decarboxylated Amadori compound. The vinylogouscompounds are then released along with the correspondingaminoketone by a β-elimination reaction and cleavage ofthe carbon–nitrogen covalent bond (Yaylayan et al. 2003;Zyzak et al. 2003).

Decarboxylation of the Schiff base may proceed viathe zwitter ionic form which is more probable than theclassical Strecker degradation mechanism (Grigg et al.1988; Grigg and Thianpatanagul 1984; Schönberg andMoubacher 1952). Zyzak et al. (2003) reported evidencefor the decarboxylated Schiff base of asparagine, whichmay also represent the decarboxylated Amadori com-pound. They suggested acrylamide to be formed directlyfrom this compound. Yaylayan et al. (2003) suggested adecarboxylated Maillard intermediate as a direct precursorto acrylamide. The proposed pathway is based onIntramolecular cyclization of the Schiff base to theoxazolidine-5-one derivative. Such oxazolidine-5-oneshave been reported to easily decarboxylate, thus givingrise to stable azomethine yields, which after tautomeriza-tion reacts as a direct precursor to acrylamide (Manini etal. 2001).

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Investigations published in 2002 have revealed that theMaillard reaction is one major reaction pathway, particu-larly in the presence of asparagine, which directly providesthe backbone of the acrylamide molecule (Biedermann etal. 2002b; Becalski et al. 2003; Mottram et al. 2002; Stadleret al. 2002; Sanders et al. 2002; Weibhaar and Gutsche2002). However, other reaction pathways have also beensuggested, such as acrolein released by oxidative lipiddegradation leading to acrylic acid, which can react withammonia to give acrylamide. Acrylic acid can also begenerated from aspartic acid by the Maillard reaction(Zyzak et al. 2003; Gertz and Klostermann 2002; Stadleret al. 2003).

There are basically two major hypotheses put forward sofar regarding the formation of acrylamide from asparagine bythe Maillard reaction. Mottram et al. (2002) suggested that α-dicarbonyls were necessary as coreactants in the Streckerdegradation reaction affording the Strecker aldehyde as theprecursor to acrylamide. Stadler et al. (2004) proposedglycoconjugates, such as N-glycosides and related com-pounds formed in the early stage of Maillard reaction, as thekey intermediates leading to acrylamide. Yaylayan et al.(2003) and Zyzak et al. (2003) have supported thishypothesis by showing the importance of the Schiff base ofasparagine, which corresponds to the dehydrated N-glycosylcompound. The key mechanistic step is decarboxylation ofthe Schiff base which leads to the formation of Maillardintermediates that can directly release acrylamide (Fig. 3).

The conclusion to be drawn from the above observationsis that the main pathways for acrylamide formation can beclassified as follows.

Pathways of Acrylamide Formation

Asparagine Route of Acrylamide Formation via MaillardReaction

One of the major pathways of acrylamide formation is theasparagine route (Gökmen and Palazoğlu 2008). Also,different other routes have been suggested in conjunctionwith the Maillard reactions system in food products(Mottram et al. 2002; Stadler et al. 2002). Asparagine canprincipally be converted to acrylamide through thermaldecarboxylation and deamination reactions, but the pres-ence of reducing sugars is essential for these reactions tooccur. Many carbonyl-containing moieties can enhance asimilar transformation (Stadler et al. 2004; Becalski et al.2003; Yaylayan et al. 2003; Zyzak et al. 2003; Stadler et al.2003). In model studies, it has been shown that α-hydroxycarbonyls are much more efficient than α-dicarbonyls inconverting asparagine to acrylamide (Stadler et al. 2003).

Furthermore, according to the results obtained frommodel systems, fructose increases the acrylamide content

by about two times in comparison with other reducingsugars because its contains two α-hydroxylic groups ratherone as is the case with other sugars (Eriksson 2005).

The first step in the asparagine route is the formation ofSchiff bases from asparagine and reducing sugars (inter-mediate I). The main part of this compound, i.e., carbox-ylate ion, enhances the decarboxylation to intermediate II.The resulting negatively charged α-carbon is directlytransferred for removing the hydrogen from the α-hydroxygroup. Furthermore, the resulting α-hydroxy anion (astrong base within the molecule) will now be able toremove the acidic α-hydrogen in a six-member ringformation when the last step of the degradation toacrylamide is initiated (Fig. 1).

Zhang et al. (2005) reviewed the acrylamide formationmechanisms to announce that the asparagine pathway wasmainly responsible for acrylamide formation in cookedfoods after condensation with reducing sugars or a carbonylsource, the main intermediate product and molecularrearrangement products of which are shown in Fig. 2.

In addition, decarboxylated asparagine (3-aminopropio-namide) can generate acrylamide in the absence of reducingsugars. Structural considerations dictated that asparaginealone might be converted thermally into acrylamide throughdecarboxylation and deamination reactions. However, themain product of the thermal decomposition of asparagine ismaleimide, mainly due to the fast intramolecular cyclizationreaction that prevents the formation of acrylamide. On theother hand, asparagine (in the presence of reducing sugars)is able to generate acrylamide in addition to maleimide(Yaylayan et al. 2003; Zyzak et al. 2003).

Further evidence to support this pathway to acrylamideproduction was provided by Becalski et al. (2003). Theyreported that heating of asparagine-15N-amide hydrate andglucose at 175 °C produces a compound with similarcharacteristics to those of [15N] acrylamide and the yield ofreactants are similar to those obtained with unlabeledasparagine. This is in accordance with the results reportedby Stadler et al. (2002) who showed that 98.6% ofnitrogen-15 label was incorporated into acrylamide afterpyrolysis of 15N-amide-labeled asparagine with glucosewhile incorporation into acrylamide was observed when15N-α-amino-labeled asparagine was used in the samereaction. Therefore, it appears that only the amido nitrogenof asparagine is being incorporated into acrylamide in thisreaction and that acrylamide is formed through thedeamination and decarboxylation of asparagine and theformation of a C–C double bond. The exact nature of thereaction pathway is supposed to be the reaction ofasparagine with a carbonyl moiety followed by rearrange-ment(s) of an intermediate (Becalski et al. 2003).

Other possible pathways involve the Strecker reaction ofasparagine, with the Strecker aldehyde as the direct

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intermediate. Mottram et al. (2002) showed that a possiblepathway for acrylamide formation was from food compo-nents during heat treatment as a result of the Maillardreaction between amino acids and reducing sugars. It has

been found that asparagine, a major amino acid in potatoand cereals, has a crucial effect on the production ofacrylamide through Maillard reaction. Although most of theflavor and color generated during baking and roasting arethe products of the Maillard reaction, an importantassociated reaction is the Strecker degradation of aminoacids by those intermediates (Fig. 3), in which the amino

Fig. 1 A proposed mechanismwhich follows the Maillard re-action, for formation of acryl-amide from asparagine andreducing sugar (Eriksson 2005)

Fig. 3 Proposed pathways for the formation of acrylamide afterStrecker degradation of the amino acids asparagine and methionine inthe presence of dicarbonyl products from the Maillard reaction. Inasparagine, the side chain Z is –CH2CONH2; in methionine, it is –CH2CH2SCH3 (Mottram et al. 2002)

Fig. 2 Mechanism of acrylamide formation from a decarboxylatedAmadori product of asparagine, R=H or [CH(OH)]nCH2OH; n=0–3(Zhang et al. 2005)

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acid is decarboxylated and deaminated to form an aldehyde.Asparagine should be a particularly suitable reactant as italready has an amide group attached to a chain of twocarbon atoms compared with other amino acids that do nothave the correct carbon backbone for acrylamide.

Alternative Pathways of Acrylamide Formation

Even though the asparagine route (explained above) is thedominant route in the formation of acrylamide in foods,there are also alternative routes via the Maillard reactionsystem. The term Maillard reaction is a collective term tothe reaction between amines and carbonyl compounds. Theknowledge on the chemistry of color formation and toxicand mutagenic compounds is scant compared with flavorformation. The Strecker degradation is an importantpathway via Maillard reaction, in which amino acids reactwith dicarbonyl degradation products which are suggestedas a way of acrylamide formation. Another componentproduced from hexose degradation, as well as fructosylly-sine dehydration, is hydroxymethylfurfural (Kroh 1994;Ramírez-Jiménez et al. 2000). Figure 4 shows the routes ofacrylamide formation through the Maillard reaction.

Other pathways proposed as alternatives to the Maillardreactions include:

A: Acrolein (1-propenal) is a simple α- or β-unsaturatedaldehyde which is supposed to be a probable cytotoxiccompound (Casella and Contursi 2004). In food,particularly in oil and fat, an alternative pathway forthe formation of acrylamide through acrolein has beenproposed as the mechanism via acrylic acid (Gertz andKlostermann 2002; Stadler et al. 2003; Becalski et al.2002). In fact, recent reports on model reaction systemsdemonstrate that acrolein, together with asparagine, maygenerate appreciable levels of acrylamide under certainconditions, suggesting a critical role for acrolein in the

formation of acrylamide in lipid-rich foods (Zhang et al.2005; Yasuhara et al. 2003).

Acrylamide could be produced from oils and nitrogen-containing compounds present in foods. The most plausiblescheme includes the transformation of acrolein to acrylicacid and the final reaction of acrylic acid with ammonia,which could potentially be generated by pyrolysis ofnitrogen-containing compounds leading to the formationof acrylamide (Becalski et al. 2003).

In case methionine is used in place of asparagine in thereaction with butanedione, which is one of severaldicarbonyl compounds formed in the Maillard reaction(other amino acids do not produce acrylamide in reactionwith glucose or butanedione), Strecker aldehyde formedfrom methionine is methional, but acrolein could also beformed together with ammonia; subsequent oxidation ofacrolein to acrylic acid followed by amidation could thengenerate acrylamide (Fig. 3). However, since ammoniareacts readily with carbonyls and other Maillard intermedi-ates, this reaction might be limited (Mottram et al. 2002).

B: Aspartic acid, carnosine, and β-alanine can give rise toacrylamide formation through the formation of acrylicacid during their thermal decomposition in combina-tion with available ammonia to convert acrylic acid toacrylamide (Yaylayan et al. 2004; Stadler et al. 2003;Yaylayan et al. 2005; Sohn and Ho 1995).

C: Aminopropionamide has been identified as an inter-mediate during acrylamide formation from asparagine.This compound is also formed in reactions betweenasparagine and pyruvic acid and is a very effectiveprecursor to acrylamide formation (Stadler et al. 2004;Zyzak et al. 2003).

D: Pyruvic acid can be generated by dehydration anddesulfidation of serin and cysteine, respectively. It canthen be proposed as the reduction of pyruvic acid intolactic acid, with further dehydration into acrylic acid.Finally, acrylic acid is transformed to acrylamide(Yaylayan et al. 2005; Wnorowski and Yaylayan 2003).

E: Formation of acrylamide may occur by α-dicarbonyl-assisted Strecker dehydration. Compared with sugar/asparagine mixtures, co-pyrolysis of asparagine withvarious dicarbonyls such as α-diketones, α-ketoaldehydes, and glyoxal give relatively loweracrylamide concentrations, while acetol generates thehighest amount of acrylamide. However, the Streckeralcohol of asparagine (3-hydroxypropanamide) hasbeen found to generate acrylamide by a one-stepdehydration reaction, but only at concentrations lowerthan those reported for sugar/asparagine mixtures.These data suggest that the Strecker aldehyde ofasparagine via the alcohol has limited importance inthe formation of acrylamide (Stadler et al. 2004).

Fig. 4 Alternative formation routes of acrylamide via Maillardreaction system (Eriksson 2005)

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F: Benzaldehyde and styrene are formed as volatilecompounds during pyrolysis of Amadori compounds.As the latter compound represents the decarboxylatedAmadori compound of phenylalanine, acrylamide maybe formed from the decarboxylated Amadori com-pound of asparagine (Stadler et al. 2004).

G: The sugar-asparagine adducts, i.e., N-glycosylaspara-gine, generate high amounts of acrylamide, suggestingthe early Maillard reaction as a major source ofacrylamide (Stadler et al. 2002). Good evidence hasbeen provided that supports the early Maillard reactionas a main reaction pathway involving early decarbox-ylation of the Schiff base, rearrangement to theresulting Amadori product, and subsequent β-elimination to release acrylamide (Yaylayan et al.2004).

The early Maillard products such as N-glycosides havebeen found to be acrylamide precursors in thermaldecomposition reactions. On the basis of structural consid-erations, asparagine or the N-glycosides could be directprecursors to acrylamide under pyrolytic conditions. Theamino acid is the carbon source of acrylamide and theformation of the corresponding N-glycoside probablyfacilitates the decarboxylation step and heterolytic cleavageof the nitrogen–carbon bond to liberate acrylamide(CH2=CHCONH2) upon pyrolysis (Stadler et al. 2002).

In food processing systems that incorporate conditions ofhigh temperature and water loss, N-glycoside formationcould be favored. Maximum acrylamide formation bythermal decomposition of early Maillard intermediates hasbeen observed after an incubation period of 1 h at 180 °C(Stadler et al. 2004).

Corresponding Methods for Analysis of Model SystemProducts

Pyrolysis–Gas Chromatography/Mass Spectrometry

Pyrolysis techniques are based on the principle of increas-ing the amount of heat energy in the system, thus leading tothermal cracking of bands of non-volatile organic com-pounds. The pyrolysis–gas chromatography/mass spectrom-etry (Py–GC/MS) technique has a large potential forevaluation and analysis of complex samples, and usuallyno pre-treatment is needed. Polar compounds such asacrylamide can be derivatized directly on the filament inthe pyrolysis chamber (Zhang et al. 2005).

To testify the amide amino acid asparagine, 13Cisotope tracer technique is necessarily applied for themechanistic study of acrylamide. Py–GC/MS is also aconvenient method to perform experiments with labeledreactants. Position and label distribution in the common

products in the same (aqueous and pyrolytic) modelsystem are identical. Thus, the mechanisms of acrylamideformation under both conditions are similar (Yaylayan1999; Wnorowski and Yaylayan 2000). Yaylayan et al.(2003) identified niacinamide by Py–GC/MS assays with13C-labeled glucose in the reaction mixture of theasparagine/glucose model system. Niacinamide can formonly from the decarboxylated Amadori product ofasparagine with glyceraldehydes through cyclizationand dehydration reactions. During Py–GC/MS assays, itis easier for researchers to confirm the chemical structureof each intermediate or final product by using the massspectrometric data than the US National Institute ofScience and Technology (NIST) library in the Py–GC/MS software (Keyhani and Yaylayan 1996).

Fourier Transform Infrared Analysis

Fourier transform infrared (FT-IR) is a powerful tool foridentifying types of chemical bands in a molecule by producingan infrared absorption spectrum that is like a molecular “finger-print”. FT-IR is also the most useful method for identifyingchemicals present in organic samples.

When glucose/asparagine model reactions are per-formed, the facile formation of a decarboxylated asparagineAmadori product is detected by IR spectral analysis of amixture of asparagine and glyceraldehyde in methanol.Also, the carbonyl band of the Amadori product is indicatedby the appearance of the absorption band at 1,737 cm-1. Asthe reactions continue, bands at 1,493 and 1,580 cm-1

appear that indicate the decarboxylation reaction andformation of a decarboxylated Amadori product. Whenthe temperature of the mixture is raised to 180 °C, the datashow the disappearance of the amide I (1,674 cm-1) andamide II (1,615 cm-1) absorption bands, indicating cleavageof the asparagine moiety (Yaylayan et al. 1999a; Yaylayanet al. 1999b).

Other Methods of Analysis

Besides the chromatographic (Py–GC/MS) and spectro-scopic (FT-IR) techniques, other methods including high-performance liquid chromatography with ultraviolet diodearray detection, liquid chromatography with tandem massspectrometry (LC-MS/MS) (Robert et al. 2004; Terada andTamura 2003; Granvogl et al. 2004; Taubert et al. 2004),and time of flight mass spectrometry (Sanders et al. 2002)also play an important role in the elucidation of thegenerating mechanism of acrylamide during heat process-ing. The critical tools of these three techniques are UV scanspectrograms, multiple reaction-monitoring mode (MRM),quantitative analysis and precise mass assignment, respec-tively (Zhang et al. 2005; Hamdan et al. 2001).

Food Bioprocess Technol (2011) 4:340–363 347

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Methods of Acrylamide Analysis

Recent Analytical Methods

The discovery of acrylamide in human foods led to surveysexploring the levels of this potentially hazardous chemicaland spurred search into suitable analytical procedures for itsdetermination in foodstuffs (IARC 1994; Tareke et al. 2002;Becalski et al. 2003; Zhu et al. 2008; Ahn et al. 2002;Rosén and Hellenäs 2002; Tareke et al. 2000). The potentialpresence of acrylamide in foods was initially investigatedby employing derivatization of acrylamide (bromination ofa double bond) and subsequent gas chromatography-massspectrometry (GC-MS) detection (Andrawes et al. 1987;Castle 1993; Castle et al. 1991; United States Environmen-tal Protection Agency 1996). Later, several groups reportedusing the same techniques, but without derivatization(Biedermann et al. 2002b; Rothweiler and Prest 2003;Tateo and Bononi 2003). An LC-MS/MS-based methodwas then developed, and soon, a few more variants of thatprocedure appeared in the literature (Rosén and Hellenäs2002; Ono et al. 2003; Swiss Federal Office of PublicHealth 2002; Takatsuki et al. 2003; US Food and DrugAdministration 2003). Although MS is chosen as the maintechnique for GC- and High Performance Liquid Chroma-tography HPLC-based analysis, there is still a need todevelop a reliable, sensitive, rapid, and low-cost analyticalmethod for the determination of acrylamide without usingMS. Examples include the US EPA Method 8032A that usesliquid extraction and Gas Chromatography-Electron CapturedDetector (GC-ECD) for determinations in water and amethod by the German Health Agency (BGVV) (Results ofa BGVV Information Seminar 2002) that uses HPLC withUV detection for migration analysis of acrylamide from foodpacking materials (Dionex 2004). However, those methodsare not easily applied for determination of acrylamide in abroad range of foodstuffs.

Acrylamide does not show any specific wavelengthabsorption maxima (λmax). Therefore, by performing thesame type of bromination as for GC analysis, it is possible toachieve a detection limit down to 4μg/L by using LC, lowUV-detection (Brown and Rhead 1979; Brown et al. 1982).

Today, the detection limit is decreased to 0.5 μg/L bydirect injection on a LC-MS of the brominated derivate(Cavalli et al. 2004). Over the recent years, many morepapers and reviews have been published about theoccurrence and analytical methods of acrylamide in heatedfoods (Wenzl et al. 2003, Zhang et al. 2005; Castle andEriksson 2005; Eberhart et al. 2005; Kim et al. 2007; Pittetet al. 2004; Ren et al. 2006).

Becalski et al. (2003) determined the levels and sourcesof acrylamide in the Canadian food supply and developed aLC-MS/MS method. This method incorporated several

purification steps and might be useful for determination ofacrylamide by detection of its bromo derivative as arelatively clean extract is obtained (Castle 1993). Gökmenet al. (2005) developed a sensitive reversed-phase HPLC-DAD method for acrylamide analysis in potato-basedprocessed foods. Although a rapid and convenient mea-surement was successfully achieved, the method required arelatively complex sample pre-treatment including extrac-tion of acrylamide with methanol, purification with CarrezI, and Carrez II solutions, evaporation and solvent changeto water, and clean-up with an Oasis HLB solid phaseextraction (SPE) cartridge. It was also found that thesensitivity of the HPLC method was lower than those ofGC- and MS-based methods (Zhu et al. 2008).

Analysis for acrylamide by bromination and GC deter-mination is relatively advanced and is nowadays used foracrylamide determination in heated foods (Castle 1993;Castle et al. 1991; United States Environmental ProtectionAgency 1996; Bologna et al. 1999; Poole 1981). Recently,Zhang et al. (2006, 2007) developed a GC-ECD method foridentification and quantification of acrylamide in friedfoods such as potato crisps, potato chips, and fried chickenwings. The method showed a lower limit of detectioncompared with MS-based methods. Also, Zhu et al. (2008)developed a low-cost, convenient, sensitive, and accuratequantitative method for the determination of low-levelacrylamide in heat-processed starchy foods by GC-ECDusing the standard addition method. Hamlet et al. (2004)developed a rapid, sensitive, and selective analysis ofacrylamide in cereal products using bromination and GC-MS/MS. They also studied the kinetics of acrylamidebromination to develop and apply to MS/MS for selectivedetection with minimal sample clean-up. These authorsfound that using standard solutions of 2,3-dibromopropio-namide (2,3-DBPA) deteriorated the chromatographic per-formance due to the build-up of co-extracted materials.Therefore, they recommended deliberate conversion of 2,3-DBPA to 2-bromopropenamide (2-BPA) prior to injectionon the GC column. In most these methods, an aqueousextraction and clean-up procedure is based on using aderivatization step to form the brominated acrylamideadduct followed by GC/MS determination (Rosén andHellenäs 2002; Tareke et al. 2000; Castle 1993; UnitedStates Environmental Protection Agency 1996; US Foodand Drug Administration 2003).

Brandl et al. (2002) developed a rapid and convenientmethod for the sensitive determination of acrylamidesuitable for different kinds of starch-rich foodstuff, suchas potato chips, cereals, biscuits, cookies, crisp bread, etc.In contrast to the aqueous extraction of acrylamide fromfoods, they applied an organic solvent extraction method toavoid co-extraction of interfering food matrix components,especially starch; thus, they significantly simplified the

348 Food Bioprocess Technol (2011) 4:340–363

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clean-up procedure. An aqueous back-extraction of theprimary organic phase prior to the clean-up procedureenabled them to achieve a selective extraction of the targetanalyte. The method of analysis used for the resultingaqueous phase was liquid chromatography coupled withtandem mass spectrometry.

A method consisting of a fast, automated extractionusing accelerated solvent extraction (ASE) has beenpresented by Dionex (2004). According to this method,samples are extracted in 20 min using pure water with10 mM formic acid or acetonitrile. The extracts are directlyanalyzed by ion chromatography (IC) using a 4-mm ion-exclusion column and both UV and MS detection. Com-pared with conventional reversed phase columns, using thiscolumn allows for the separation of acrylamide from themany co-extractable compounds present in food samples.The advantages of the method include simplicity, speed ofanalysis, and a degree of automation that allows theanalysis of a large number of samples with minimal labor(Richter et al. 1996).

In principle, the analysis of acrylamide in different typesof food follows the same procedure, consisting of extrac-tion, derivatization (depending on the system of analysis),purification, concentration, and instrumental analysis (onthe basis of GC/MS of the brominated derivative and LC/MS of free acrylamide; Eriksson 2005; Tateo and Bononi2003). More detailed procedures on the extraction, clean-upand chromatographic techniques during the acrylamideanalysis are summarized in Table 3.

Extraction, Purification, and Derivatization

The high water-solubility of acrylamide means that extrac-tion from foods using plain water is very effective, with noneed for pH adjustment (Eriksson 2005).

Water at room temperature has been used as anextractant in most LC methods (Becalski et al. 2003; Rosénand Hellenäs 2002; Tareke et al. 2000). Heating orsonicating during the extraction should be avoided as thismay generate the SPE columns used in further clean-upsteps (US Food and Drug Administration 2003). Neverthe-less, Ahn et al. (2002) used heated water (at 80 °C) withoutany problem during clean-up.

Purification of water extracts for acrylamide analysis isbased the following principles: purification with SPEcolumns and chemical purification (deproteination) (Eriksson2005). However, the extraction solvent could be a mixture ofwater and organic solvents such as n-propanol or 2-butanone(Biedermann et al. 2002b). Recovery rates of 68–75.4% havebeen reported when pure methanol was applied for theextraction of baked food products (Tateo and Bononi 2003).Also, the extracted amount of acrylamide, using Soxhletextraction with methanol, far exceeded that by other

extraction techniques (>90%). However, one drawback isthe long extraction time of 10 days while no information isavailable on the potential acrylamide formation during theextraction (Wenzl et al. 2003). To overcome these problems,some researchers have extracted acrylamide selectively byusing ASE without co-extracting the starch, which leads tothe simultaneous simplification of the subsequent sampleclean-up (Brandl et al. 2002). Although solubility ofacrylamide in most organic solvents is lower than water,dichloromethane appears to be a promising extractionsolvent, particularly addition of 2% ethanol as a modifierimproves the results considerably. Re-extraction of theorganic phase with water delivers an aqueous solution whichis almost free of any interfering matrix components. Forinstance, fatty components remain in the organic phase; thus,defatting of the sample prior to extraction is not necessaryany more. Applying this method, nearly all of the targetfoodstuffs could be analyzed in a similar fashion withsatisfactory results (Brandl et al. 2002; Dionex 2004).

In using the ASE method, pure water, 10 mM formicacid solution, and acetonitrile were tested as the extractionsolvent in LC methods of acrylamide determination. Purewater extracts showed lower recoveries than the formicacid, but the formic acid extracts had a lower stability.Acetonitrile extracts were cleaner, as less material was co-extracted from the sample matrix. With three extractioncycles of 4-min durations, a yield of 95% in the first extractand an additional 8% in the second extraction of the samesample using 10 mM formic acid were achieved (Dionex2004). However, a mixture of water and acetone has alsobeen reportedly used as the extractant (Takatsuki et al.2003; Fauhl et al. 2002). Other researchers have also usedthe ASE device (Cavalli et al. 2002; Höfler et al. 2002).

Different mechanical methods can be used for the initialextraction steps that include shaking at high speeds on ahorizontal shaker (Becalski et al. 2003), using a rotatingshaker (US Food and Drug Administration 2003), occa-sional swirling (Ahn et al. 2002; Takatsuki et al. 2003), andmixing with a blender or mixing on a vortex (Fauhl et al.2002).

After extraction, the aqueous phase is centrifuged anddifferent laboratories have reported different centrifugationconditions, as described in Wenzl et al. (2003). Becalski etal. (2003) and FDA (2003) recommended combinedcentrifugation and filtration using a 5 kDa cut-off CentriconPlus-20 and 0.45 μm PVDF filters, respectively. Ono et al.(2003) used centrifugation filters with a 3 kDa cut-off afterclean-up of the extract by SPE.

To control the recoveries achieved and to keep track ofpossible losses during the extraction and purification steps,an internal standard is added to the food-extraction mixture.Similar to the GC-MS methods, isotopically labeled [13C3]-acrylamide (Tareke et al. 2002; Becalski et al. 2003), [D3]-

Food Bioprocess Technol (2011) 4:340–363 349

Author's personal copy

Tab

le3

Chrom

atog

raph

ic-based

metho

dsforthedeterm

inationof

acrylamidein

food

prod

ucts

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

Liquidchromatographymethods

Brandlet

al.

2002

Various

food

products

Accelerated

sampleextractio

nusing

dichloromethane

containing

2%ethanol,2gsample+IS+200μlwater

infive

cycles

(10min

each

at80

°C,

100bar),mixingof

combinedextract

with

5mlwater,usingaliquotof

aqueousextract

–Synergy

Polar-RP

Colum

n,150×

3mm

Mobile

phase:

water

containing

0.1%

acetic

acid;0.5ml/m

in;

columntemperature,40

°C

LC-M

S/M

S;

LOD,10

ng/

ml

Sheathgas,60

units;auxiliary

gas,10

units;corona

current,

5μA;vaporize

temperature,

350°C

;capillary

temperature,150°C

;m/z

transitio

ns(collisionenergy);

AA,75

>55;IS,75

>58

Rosén

and

Hellenäs

2002

Mashedpotato,

ryeflour,

crispbread,

potato

crisps

2–4gsample+IS+40

mlwater,

homogenization(2

min

at9,500min

-

1),centrifugatio

n(3600×

g,10

min),

extra-centrifugatio

nforpotato

chips

(10min

at16,800)afterprecipitatio

nby

freezing,pretreatmentof

SPEwith

1mlacetonitrile

andwashing

with

water,filtrationthrough0.22

μm

[2H3]acrylamide

–Hypercarb

HPLC

column,

50mm×2.1mm

Mobile

phase,

methanol/w

ater=20/

80(gradient);0.4mL/m

in;Inj,

10μL

LC–M

S/M

S;

LOD,

<10

μg/kg;

LOQ<30

μg/

kg

Capillaryvoltage,2kV

;cone

voltage,20

V;source

temperature,125°C

;desolvationtemperature,

400°C

;m/ztransitio

ns(collisionenergy);AA,72

>72

(0eV

),55

(9eV

),54

(16eV

),44

(20eV

),27

(14eV

);IS,75

>58

(9eV

)Syringe

filter

Ono

etal.

2003

Various

food

products

50gsample+IS+300–

400mlwater,

homogenization,

centrifugatio

n(20min

at48,000×g),freezing

and

meltin

gof

supernatant,centrifugatio

n(10min

at21,700×g),fractionatio

nof

0.5–

2mlsupernatanton

SPE

cartridge,

fractio

ncollected

and

centrifugatio

n(10min

at27,000×g),

filtrationof

supernatantthrough

0.22

μm

syringefilters,centrifugation

offiltrationwith

cut-offof

3kD

a(50min

at14000×

g)

[2H3]acrylamide

–AtlantisdC

18column,

150mm×

2.1mm

Mobile

phase:

methanol/w

ater=10/

90;0.1mL/m

in;runtim

e,10.2

min;Inj,2μL

LC–M

S/M

SLOD,0.2ng/

mLLOQ,

0.8ng/m

L

Ionsprayvoltage,5

.2kV

;turbo

gastemperature,450°C

;m/z

transitio

ns(collisionenergy);

AA,72

>55

(18eV

);IS,75

>58

(18eV

)

Roach

etal.

2003

Cereal,bread

crum

b,potato

chips,coffee

1gsample+IS+9mlwater,mixingfor

20min,centrifugatio

n(15min

at9,000rpm),centrifugatio

nof

5-ml

aliquotin

spin

filtrationtube

(2–

4min

at9,000rpm),pretreatmentof

OasisHLBSPEcartridges

(3.5

mLof

methanolfollo

wed

by3.5mLof

Water),extractloaded

(1.5

ml)and

collected

[13C3]acrylamide

–Synergi

Hydro-RP

80A

column,

250mm×2mm

Mobile

phase,0.5%

methanol/0

.1%

acetic

acid

inwater;0.2mL/m

in;

run-tim

e,10

min;Inj,20

μL

LC–E

SI-MS/

MS;LOD,

10μg/kg

Capillaryvoltage,4.1kV

;cone

voltage,20

V;source

temperature,120°C

;desolvationtemperature,

250°C

;m/ztransitio

ns(collisionenergy);AA,72

>72

(5eV

),55

(10eV

),27

(19eV

);IS,7

5>75

(5eV

),58

(10eV

),29

(19eV

)

Becalskietal.

2003

Potatochips,

potato

crisps,

cereals,

bread,

coffee

16gsample+IS+80

mlwater+10

ml

dichloromethane,mixing(15min),

centrifugatio

n(2

hat

24,000×g),

centrifugatio

nof

10mlsupernatant

(4hat

4,000×

g)in

5kD

acentrifuge

filter,passing5mlof

filtratethrough

OasisMAX

cartridgeconnectedwith

tandem

with

OasisMCX

cartridge,

loadingtheelutiononto

the

preconditio

nedENVl-carb

cartridge,

[13C3]-acrylam

ide

or[D

3]

acrylamide

–Hypercarb

column,

50×2.1mm

Mobile

phase,

15%

methanolin

1mM

ammonium

form

ate;

0.175ml/m

in;Inj,5–10

μl;

columntemperature,28

°C

MS/M

S;LOD,

~6μg/kg

Ionizatio

nmode,

positiv

e;desolvationtemperature,

250°C

;source

temperature,

120°C

;desolvationgasflow

,525L/h;cone

gasflow

,50

L/

h;collision

gaspressure,

2.6×10

-3mbar;ionenergy,

10V;MRM:dw

elltim

e;0.3s;cone

voltage,34

V;

massspan,0.1Da;

350 Food Bioprocess Technol (2011) 4:340–363

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Tab

le3

(con

tinued)

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

discarding

first1mlandcollecting

remaining

(f1),washing

cartridge

with

1mlwater

(f2)

and1.5ml10%

methanol(f3),analyzingf1,f2,f3

interchannel

energy,0.05

–0.1s;m/ztransitio

ns(collisionenergy);AA,72

>55

(11eV

),72

>54

(11eV

),72

>44

(14eV

),72

>27

(16eV

);IS,75

>58

(11ev)

Riedikerand

Stadler

2003

Breakfast

cereal,

crackers

Extractionwith

water,homogenization

usingadispersing

tool,

centrifugatio

n,mixed

with

acetonitrile

toprecipitate

co-

extractiv

es,acetonitrile

evaporation,

preconditio

ning

with

methanoland

water

(isolutemultim

ode,2,

2×2mL;

AccubondIISCX

1,1mL),residual

water

removal,extract(2

mL)loaded

andcollected,collected

(1mL)

extractchargedonto

thelatter

cartridge,

effluent

collected,filtration

throughasyringefilterunit

[13C3]acrylamide

–ShodexRSpak

DE-613

polymethacrylate

gelcolumn,

150×6mm

Mobile

phase,

0.01%

aqueous

form

icacid/m

ethanol=

6/4,

0.75

mL/m

insplit

to0.35

mL

aftertheLC

columnusinga

PEEKb;

T-piece,

runtim

e,12

min;Inj,50

μL

LC–E

SI-MS/

MS;LOQ,45

Capillaryvoltage,3.1kV

;cone

voltage,22

V;source

temperature,100°C

;desolvationtemperature,

350°C

;m/ztransitio

ns(collisionenergy);AA,72

>55

(11eV

),54

(20eV

),27

(20eV

)

Mestdaghet

al.2004

Frenchfries

1gsample+

IS+10

mlhexane,10

min

shaking,

centrifugatio

n(10min

at4000

rpm),hexane

removal,adding

10mlMilli-Q

water,20

min

shaking,

centrifugatio

n(20min

at4,000rpm),

ultrafiltratio

nthrough0.45-μm

mem

branefilter,preconditio

ning

ofcartridge(5

mlmethanoland5ml

water),loadingon

OasisHLBand

VarianBondElutAccuratecartridge

[D3]acrylam

ide

–AtlantisdC

18column,

150mm×

2.1mm

Mobile

phase,

92%

water

(containing0.1%

acetic

acid)and

8%water/m

ethanol(35/65,with

0.3%

fomaric

acid);0.15

ml/m

in

LC-M

S/M

Sm/ztransitio

ns(collision

energy);AA,72

>72

(5eV

),72

>55

(10eV

);IS,75

>58

(10eV

),75

>30

(20eV

)

Hoenickeet

al.2004

Various

food

products

Weighed

into

afilter,placed

ona

Witt'scher

pot,equipped

with

avacuum

pump,

defattedby

adding

iso-hexane

(80mL),spiked

with

IS,

incubatio

n(30min),extractio

nwith

water

(20mL)in

anultrasonic

bath

(60°C

,30

min)purificatio

nby

adding

acetonitrile

(20mL),CarrezI

andCarrezII(500

μLeach),

centrifugatio

n(4,500×g,

10min),

supernatantfiltrationthrougha

mem

branefilter

[2H3]acrylamide

–Merck

LiChrospher

100

CN

column,

250×4mm

Mobile

phase,

50%

acetonitrile

in1%

aceticacid

isocratic

for5min,

follo

wingrinsingwith

100%

acetonitrile

for5min,0

.7mL/m

in(spilt1:5),runtim

e,10

min;Inj,

10μLor

40μL

LC–M

S/M

S;

LOD,<10_g/

kg;LOQ,

<30_g/kg;

Electrosprayvoltage,5.5kV

;source

temperature,350°C

;m/ztransitio

ns(collision

energy);AA,72

>72,55,44

(18eV

);IS,75

>75,58,44

(18eV

)

Andrzejew

ski

etal.2004

Coffee

Spikedwith

IS,extractio

nwith

HPLC

gradewater,centrifuge

tubescapped

andshaken/vortexed(30s),

centrifugatio

n,aliquottransfer

toa

Maxi-SpinPVDFbfiltrationtube

(0.45μm)andcentrifugatio

nConditio

ning

(OasisHLB6cc

cartridge)

with

methanolandwater

(3.5

mLeach),filteredextract

(1.5

mL)loaded,water

(1.5

mL)

elutionandeffluent

transfer

onto

the

second

cartridge(BondElut-

Accucat),amarkplaced

onthe

[13C3]acrylamide

–Synergi

Hydro-RP

80A

column,

250mm×2mm

Mobile

phase,

0.5%

methanolin

water,0.2mL/m

in;runtim

e,10

min;Inj,20

μL

LC–M

S/M

S;

LOD,10

μg/

kg;

Capillaryvoltage,4.1kV

;cone

voltage,20

V;source

temperature,120°C

;desolvationtemperature,

250°C

;m/ztransitio

ns(collisionenergy);AA,72

>72

(5eV

),55

(10eV

),27

(19eV

);IS,7

5>75

(5eV

),58

(10eV

),29

(19eV

)

Food Bioprocess Technol (2011) 4:340–363 351

Author's personal copy

Tab

le3

(con

tinued)

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

outsideof

thetube

ataheight

equivalent

to1mLof

liquidabove

thesorbentbed,

conditioningwith

methanolandwater

(2.5

mLeach),

sampleloaded

andcollected

Gökmen

etal.

2005

Potatochips

2gsample+

IS+10

mlmethanol,

mixture

centrifugatio

n(10min

at11180×

g),supernatantclarification

with

CarrezIandIIaand

centrifugatio

n,drying

of2.5ml

supernatantundernitrogen,resolving

residuein

1mlwater,OasisHLB

cartridgepreconditio

ning

with

1mlo

fmethanoland1mlof

water,extract

loaded

(1ml)andcollected,filtration

througha0.45

μm

syringefilter

[13C3]acrylamide

–Atlantis

Mobile

phase,

1.0or

0.5ml/m

in;

columntemperature,25

°C;Inj,

20μl

DAD,LOD,

2.0μg/ml,

LOQ,4.0μg/

kg

226nm

with

peak

spectra190–

350nm

dC18,250×

4.6mm

Atlantis

HILIC,250×

4.6mm

ZorbaxSIL,250×

4.6mm

ZorbaxStable-

bond

C18,250×

4.6mm

HiChrom

5C18,

300×4.6mm

LunaC18,250×

4.6mm

Synergi

MAXRP,

250×4.6mm

Zhang,et

al.

2007

Various

food

products

1.5gsample+

IS,10

min

standing,

adding

20mlpetroleum

ether,10

min

shaking,

removal

ofpetroleum

ether

andrepeatingdefatting,adding

7ml

NaC

l(2

mol/L)and20

min

shaking,

centrifugatio

n(15min

at15,000

rpm),extractin

gtheresidue

with

8mlNaC

l,extractin

gtheNaC

lsolutio

nwith

15mlethylacetatefor

threetim

es,drying

organicphase

undernitrogen,adding

1.5mlwater

toresidue,

preconditio

ning

ofOasis

HLBcartridge(3.5

mlmethanoland

3.5mlwater),loading(1.5

ml)and

extractin

g

[13C3]acrylamide

–AtlantisdC

18

column;

210×

1.5mm

Mobile

phase,10%

methanol/0.1%

fomaricacid

inwater;0

.2ml/m

in;

columntemperature,25

°C

HPLC-ESI-MS/

MS

Capillaryvoltage,3.5kV

;cone

voltage,50

V;source

temperature,100°C

;desolvationgastemprature,

350°C

;desolvationgasflow

,400L/h

nitrogen;cone

gas

flow

,45

L/h

nitrogen;argon

collision

gaspressure,3×10

-

3mbar;m/ztransitio

ns(collisionenergy);AA,72

>72

(1eV

),72

>55

(6eV

),72

>44

(9eV

),72

>27

(15eV

);IS,75

>75

(1ev),75

>58

(6eV

),75

>30

(15eV

)

Kim

etal.

2007

Rice,bread,

corn

chips,

potato

chips,

biscuits,

candy,coffee

10gsample+

IS+98

mlwater,20

min

shaking,

centrifugatio

n(10min

at9,000rpm),conditioningC18

solid

-phaseextractio

ncartridge(5

ml

methanoland5mlwater),loading

andcollecting,

filtrationthrough

0.45

μm

mem

brane

[13C3]acrylamide

–AquaC18

HPLC

column,

2×

250mm

Mobile

phase,

aqueous0.2%

acetic

acid

and1%

methanol;0.2ml/

min;runtim

e,14

min;Inj,20

μl

LC-M

S/M

S;

LOQ,2μg/

kg

Capillaryvoltage,4.2kV

;source

temperature,120°C

;desolvationtemperature,

240°C

;desolvationgasflow

rate,650L/h

nitrogen;argon

gaspressure,2.5mbar;m/z

transitio

ns;AA,72>55;IS,

75>58

352 Food Bioprocess Technol (2011) 4:340–363

Author's personal copy

Tab

le3

(con

tinued)

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

Genga

etal.

2008

Fried

potato

chips,

biscuits,

Chinese

fried/

bakedfoods

2gsample+

10mlmethanol75%,

treatin

gwith

CarrezIandII,shaking

(45mint100rpm),centrifugatio

n(10min

at10000rpm),evaporating

5mlof

supernatantto

1mlunder

nitrogen,preconditio

ning

OasisHLB

cartridge(5

mlmethanoland5ml

water),loadingandelutingby

2ml

10%

methanol,filtrationof

extract

with

0.45

μm

syringefilter

acrylamide

–HC-75H+

column,

305mm×

7.75

mm

Mobile

phase:sulfuricacid

(5mM);

0.6ml/m

in;Inj,20

μl;column

temperature,50

°C

HPLC-D

AD;

LOD,30

μg/

kg

200nm

Liu

etal.

2008

Tea

1gsample+

IS+9mlwater,20

min

shaking,

adding

10mlacetonitrile

+4ganhydrousmagnesium

sulfate+

0.5gof

sodium

chloride,1min

shaking,

centrifugatio

n(5

min

at5,000rpm),separatin

gof

acetonitrile

layeranddrying

undernitrogen,

dissolving

theresiduein

05mlwater,

filtering

through0.45

μm

syringe

filter,preconditio

ning

ofOasisMCX

SPEcartridge(2

mlmethanoland

2mlwater),loadingandcollecting,

filtrationthrough0.22

μm

syringe

filter

[13C3]acrylamide

–ODS-C18

column,

250mm×

4.6mm

Mobile

phase,

10%

acetonitrile

and

90%

water

containing

0.1%

form

icacid;0.4ml/m

in;Inj,

LC-M

S/M

S;

LOD,1ng/

ml;LOQ,

5ng/m

l

Capillaryvoltage,1kV

;cone

voltage,20

V;1source

temperature,110°C

;desolvationtemperature,

400°C

;desolvationgasflow

,600L/h

nitrogen;cone

gas

flow

,50

L/h;argoncollision

gaspressure

to2×10

-3mbar;

m/ztransitio

ns(collision

energy)A

A,72

>55

(13eV

);IS,75

>58

(13eV

)

Gökmen

etal.

2009

Cookie,

potato

crisp,

bread

crisp

Aqueous

extractio

n,1gsample+

IS+

9mlof

10mM

fomaric

acid,treated

with

CarrezIandII,centrifugatio

n(10min

at5,000rpm),four

stage

extractio

nof

supernatantwith

out

Carrezclarification,

preconditio

ning

OasisMCX

cartridge,

extractloaded

andcollected,filtrationthrough

0.45

μm

nylonfilter.

[13C3]acrylamide

–AtlantisT3

column,

150mm×

4.6mm

Mobile

phase,10

mM

fomaricacid;

0.3ml/m

in;columntemperature,

25°C

LC-M

S,LOQ,

15μg/kg

Capillaryvoltage,2kV

;corona

current,5μA;drying

gas

temperature,350°C

;m/z

transitio

ns(collisionenergy);

IS,72

Methanolextractio

n,1gsample+IS

+9mlmethanol,centrifugatio

n(10min

at5,000×

g),supernatant

treatedwith

CarrezIandII,

centrifugatio

n(10min

at5,000rpm),

four

stageextractio

nof

supernatant,

drying

undernitrogen,reconstitution

ofresiduein

1mlwater,elution

throughpreconditio

nedOasisMCX

cartridge,

filtrationthrough0.45

μm

nylonfilter.

Gas

chromatographymethods

Tareke

etal.

2000

Fried

food

10gsample+

100mlwater,filtratio

nof

extractthroughglass-fiberfilter,

purificatio

non

carbograph

4column,

additio

nof

standard,brom

ination

N,N-D

imethyl

acrylamide

Bromination

HPPA

S1701

column,

25m×

0.32

mm

65°C

held

for1min,rampedat

15°C

/min

to250°C

,held

for

10min;Inj,2μL;splitless

GC–M

S;LOD,

5μg/kg

m/ztransitio

ns;AA,152,

150,

108,106;

IS,180,

178

Tareke

etal.

2002

Protein-rich

and

carbohydrate-

rich

foods

10gsample+

100mlwater,filtratio

nof

extractthroughglass-fiberfilter,

purificatio

non

carbograph

4column,

additio

nof

standard,brom

ination

N,N-dim

ethyl

acrylamideor

[13C1]acrylamide

Bromination

BPX-10column,

30m×0.25

mm

65°C

held

for1min,rampedat

15°C

/min

to250°C

,held

for

10min;Inj,2μL;splitless

GC–M

S;LOD,

5μg/kg

m/ztransitio

ns;AA,152,

150,

106;

IS,180,

155

Ono

etal.

2003

Various

food

products

50gsample+IS+300-400mlwater,

homogenization,

centrifugatio

n[2H3]acrylamide

Bromination

CP-Sil24

CB

Low

bleed/MS

85°C

held

for1min,rampedat

25°C

/min

to175°C

,held

for

GC–M

S;LOD,

0.2ng/m

Lm/ztransitio

ns;AA,52,150;

IS,155,

153

Food Bioprocess Technol (2011) 4:340–363 353

Author's personal copy

Tab

le3

(con

tinued)

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

(20min

at48,000×g),freezing

and

meltin

gof

supernatant,centrifugatio

n(10min

at21,700×g),fractionatio

nof

0.5-2mlsupernatanton

SPE

cartridge,

fractio

ncollected

and

centrifugatio

n(10min

at27,000×g),

filtrationof

supernatantthrough

0.22

μm

syring

filters,centrifugatio

nof

filtrationwith

cut-offof

3,000Da

(50min

at14,000×g)

column,

30m×

0.25

mm

6min,rampedat

40°C

/min

to250°C

,held

for7.52

min

Hoenickeet

al.2004

Various

food

products

Weighed

into

afilter,placed

ona

Witt’scher

pot,equipped

with

avacuum

pump,

defattedby

adding

iso-hexane

(80mL),spiked

with

IS,

incubatio

n(30min),extractio

nwith

water

(20mL)in

anultrasonic

bath

(60°C

,30

min)Purificationby

adding

acetonitrile

(20mL),CarrezI

andCarrezII(500

μLeach),

centrifugatio

n(4500×g,

10min),

supernatantfiltrationthrougha

mem

branefilter

[2H3]Acrylam

ide

–DB-W

AX

capillary

column,30

m×

0.25

mm

70°C

held

for1min,rampedat

20°C

/min

to230°C

,held

for

10min;Inj,1μL,splitless

GC–M

S/M

S;

LOQ,30

μg/

kg

m/ztransitio

ns;AA,89

>72,55;

IS,92

>75

Ham

letet

al.

2004

Bread

5gsample+IS+deionisedwater,1min

shaking,

adding

0.3mlglacialacetic

acid,treatin

gwith

CarrezIandII,

centrifugatio

n(20min

at1942

g)

[13C3]acrylamide

Bromination

Rtx®-50column,

30m×250μm

65°C

held

for2min,rampedat

15°C

/min

to250°C

,held

for

5min;Inj,1μl;splitless

GC-M

S/M

S;

LOD;

0.01

ng/m

l

Ionizatio

nmode,

negativ

e;argoncollision

gaspressure,

1.5mTorr;m/ztransitio

ns(collisionenergy);AA,149>

70(10eV

),151>70

(10eV

);IS,152>73

(10ev),154>73

(10eV

)

Pittet

etal.

2004

Cerealproducts

Weighted(15g)

into

a250mL

centrifuge

bottle,

spiked

with

IS,

samplesuspendedin

water

(150

mL)

andhomogenized

(30s),suspension

acidifiedto

pH4–

5by

additio

nof

glacialacetic

acid

(∼1mL),treated

successively

with

CarrezIandIIc

(2mLeach),centrifugatio

n(16,000g,

15min),brom

ination,

extract

transferredonto

aglass

chromatographycolumncontaining

calcinated

sodium

sulfateand

activ

ated

Florisil(5

geach),using

smallaliquotstakenfrom

hexane

(50mL),acrylamidederivativ

eeluted

with

acetone(150

mL),evaporated

to∼2

mLandthen

todryness(N

2),re-

dissolvedin

EtAcd

(400μL),

triethylam

ineadded(40μ

L)filtered

througha0.2μm

microfilter

[13C3]acrylamide

Bromination

ZB-W

AX

capillary

column,

30m×

0.25

mm

65°C

held

for1min,rampedat

15°C

/min

to170°C

,5°C

/min

to200°C

,40

°C/m

into

250°C

,held

for15

min;Inj,2μL,

splitless

GC–M

S;LOD,

2μg/kg;

m/ztransitio

ns;AA,149,

70;

IS,154,

110

354 Food Bioprocess Technol (2011) 4:340–363

Author's personal copy

Tab

le3

(con

tinued)

Study

Sam

ple

Extractionandclean-up

Internal

standard

Derivatization

Colum

nChrom

atographyparameters

DetectorLOD

andLOQ

Detectorparameters

Dunovskaet

al.2006

Potatocrisps,

breakfast

cereals,crisp

bread

3gsample+

IS+4.5mldeionized

water,30

min

ultrasonic

bath,adding

24mln-propanol,centrifugatio

n(5

min

at11000g),adding

5drop

oliveoil,drying,dissolving

the

residuein

2mlMeC

N,defatting

with

10and5mln-hexane,mixing1ml

MeC

Nwith

60mgPSA

sorbent,

centrifugatio

n(1

min

at11000rpm),

[D3]acrylamide

–IN

NOWxcapillary

column,

30m×

0.25

mm

70°C

for1.0min,20ºCmin

−1to

240°C

(heldfor10.5

min);

carriergas,heliu

m,1.0ml/m

in;

Inj,1μl;pulsed

splitless

1.0min,

4ml/min

−1

GC–H

RTOF

MSb;LOQ,

15and40

μg

kg−1

Acquisitio

nrate,2Hz;

pusher

interval,33

μs(30303

raw

spectras−

1);inhibitpush

value,

14;tim

e-to-digital

converter(TDC),3.6GHz;

massrange,

m/z45

–500;ion

source

temperature,220°C

;transfer

linetemperature,

240°C

;detector

voltage,

2,600V.

Serpenand

Gokmen

2007

Potatochips

2gsample+IS+20

mlm

ethanol,Carrez

clarification,

centrifugatio

n(10min

at10000rpm)using0.45

μm

microspin

PVDFcentrifuge

filter

[13C3]acrylamide

–HPIN

NOWAX

column,

30m×

250μm

Isotherm

alfor0min,rampedat

10°C

/min

from

80to

280°C

,Isotherm

alfor13

min;flow

ofcarriergas1.0ml/m

in;Inj,1μl;

splitless

GC-M

S;LOD,

15ng/g;

LOQ;5

0ng/g

Electronionizing,70

eV,m/z

transitio

ns;AA,71

>71,55,27;IS,74,58

Zhang

etal.

2007

Various

food

products

1.5gsample+

IS,10

min

standing,

adding

20mlpetroleum

ether,10

min

shaking,

removal

ofpetroleum

ether

andrepeatingdefatting,adding

7ml

NaC

l(2

mol/L)and20

min

shaking,

centrifugatio

n(15min

at15000rpm),

extractin

gtheresiduewith

8mlNaC

l

[13C3]acrylamide

Bromination

HPIN

NOWAx

capillary

column,

30m×

0.32

mm

110°C

for1min,10

°C/m

into

140°C

,140°C

held

for15

min,

rampedat

30°C

/min

to240°C

,andfinally

isotherm

alat

240°C

for7min;carriergas,nitrogen;

Inj,1μL,

GC-M

ECD;

LOD,10

μg/

kg

Ionizatio

nmode,

positiv

e;m/z

transitio

ns;AA,70,1

49,1

51;

IS,110,

154

Lee

etal.

2007

Frenchfries,

potato

chips

10gsample+100mlwater,

centrifugatio

n(10min

at5000

rpm),

dilutio

nof

1.5mlaliquotto

15ml

with

water,mixingwith

15mlbuffer

(pH

7),im

mersing

ofSPMEfiber

––

DB-W

AX

silica

capillary

column,

30m×

0.25

mm

Ram

pedat

15°C

/min

from

80°C

to220°C

,held

at220°C

for

2min;carriergas,heliu

m,1mL/

min;elutiontim

e,9.88

GC-PCI-MS/

MS;LOD,

0.1μg/L

Mass-to-chargeratio

(m/z)scan

rangefrom

40to

100u

The

microextractio

nfiberstested

herein

werecoated

with

75–>

μm

carbox

en/poly(dimethy

lsilo

xane)(CAR/PDMS);6

5μm

carbow

ax/divinylbenzene(CW/DVB);85

μm

polyacrylate(PA);10

0μm

poly(dim

ethy

lsilo

xane)(PDMS);65

μm

poly(dim

ethy

lsilo

xane)/diviny

lbenzene

(PDMS/DVB)

LOD

limitof

detection,

LOQ

limitof

quantification,

ISinternal

standard,Injinjectionvo

lume,

AAacrylamide,

SPEsolid

phaseextractio

n,SP

MEsolid

-phase

microextractio

na CarrezI,po

tassium

hexacyanoferrate

(II)trihyd

rate

solutio

n;CarrezII,zinc

sulfateheptahyd

rate

solutio

nbGC–H

RTOFMS:high

-resolutiontim

e-of-flig

htmassanalyzer

c SPME(Solidph

asemicroextractio

n).The

microextractio

nfibers

tested

herein

werecoated

with

75μm

carbox

en/poly(dimethy

lsilo

xane)(CAR/PDMS);65

μm

carbow

ax/divinylbenzene(CW/

DVB);85

μm

polyacrylate

(PA);10

0μm

poly(dim

ethy

lsilo

xane)(PDMS);65

μm

poly(dim

ethy

lsilo

xane)/diviny

lbenzene

(PDMS/DVB).

Food Bioprocess Technol (2011) 4:340–363 355

Author's personal copy

acrylamide (Becalski et al. 2003; Ahn et al. 2002; Rosénand Hellenäs 2002; Ono et al. 2003), and [13C1]-acrylamide(Takatsuki et al. 2003) have been used.

Most purification procedures consist in combiningseveral SPE. For instance, a combination of three differentcartridges: mixed-mode anion exchange, mixed-mode cat-ion exchange, and graphitized carbon have been used(Becalski et al. 2003). Takatsuki et al. (2003) also used asimilar combination of SPE cartridges for the clean-up ofsamples, which were measured by LC-MS with columnswitching. Also, a combination of SPE and filtration and/orultracentrifugation has been used to avoid blockage of thechromatographic system (Wenzl et al. 2003). However,Höfler et al. (2002) reported that both SPE and liquid–liquid extraction did not lead to any significant improve-ment in the analysis. Therefore, filtration through a 0.22-μm nylon filter is the only sample treatment used as clean-up procedure after extraction and before applying to HPLC.In contrast, other laboratories added acetonitrile to theaqueous extract and pipetted 0.5 ml Carrez I and Carrez IIonto the sample in order to obtain a clear sample (Wenzl etal. 2003).

One special aspect of the extraction procedure involvesthe swelling of the matrix in order to provide better accessfor the extraction solvents to potentially adsorbed orenclosed acrylamide. However, the side-effect associatedwith swelling is that it provides some time for thedevelopment of matrix/internal standard interactions. Forthis reason, the homogenized sample is mixed with waterand an internal standard solution and kept at a pre-specifiedtemperature for 10–20 min. Depending on the matrix,swelling yielded an increase in analyte recovery of up to100-fold (Biedermann et al. 2002b).

Although extraction at room temperature providessatisfactory results, hot water (60–80 °C) can be used toenhance the extraction. Increased recovery has also beenobserved by treating the sample in an ultrasonic bath(30 min at 60 °C; Schaller 2003). Problems with the highviscosity of the sample/water extraction mixture have beenreported to be solved by the addition of small amounts ofamylase to the mixture.

In derivatization methods, acrylamide is converted to2,3-dibromopropionamide which is volatile and can bedetected on a GC with an electron capture or an alkali flamedetector (Tekel et al. 1989; United States EnvironmentalProtection Agency 1996). This bromination is mostlyperformed by ionic reaction (Hashimoto 1976; Arikawaand Shiga 1980). The analysis has been suggested to beperformed on the more stable 2-bromopropenamideobtained after debromination of 2,3-dibromopropionamide(Andrawes et al. 1987; Martin et al. 1990). Applications ofSPE columns to obtain concentrated samples or utilizing amore sensitive derivatization technique may increase the

possibility for determination of residual acrylamide in manytypes of food (Kawata et al. 2001; Pérez and Osterman-Golkar 2003).

Bromination of acrylamide has the advantage that a morevolatile compound is produced and the selectivity ofdetermination is enhanced (Wenzl et al. 2003). However,some derivatization approaches are laborious and time-consuming. The procedure first reported by Hashimoto(1976) is carried out by adding a pre-prepared brominationsolution containing potassium bromide, hydrogen bromide,and bromine to either the pre-treated or raw aqueousextracts (Tareke et al. 2002; Ahn et al. 2002; Castle 1993;Castle et al. 1991; Ono et al. 2003). In this method, theyield of 2,3-DBPA is constant and >80% when the reactiontime is more than 1 h (United States EnvironmentalProtection Agency 1996).

Nemoto et al. (2002) improved the method by usingdifferent derivatization reagents including potassium bro-mide and sodium bromate in an acidic medium. Thenecessity of additional sample pre-treatment depends uponthe matrix. Matrices such as carbohydrate rich foods (e.g.,potato crisps or bread) require additional fractionation steps(Tareke et al. 2002; Tareke et al. 2000). Usually, the rawextract is subjected to fractionation on a graphitized carbonblack cartridge.

Bromination is frequently carried out overnight at 0 °C orslightly above the freezing point of water. It has also beenstated that application of isotopically labeled internal stand-ards allowed a reduction in the reaction time from overnight to1 h. This is in accordance with the methods proposed by otherscientists (Ono et al. 2003; Nemoto et al. 2002).

The excess of bromine is removed after the reaction bytitration with sodium thiosulfate solution (0.7–1 M) untilthe solution becomes colorless. The brominated acrylamideis less polar compared with the original compound and,therefore, non-polar organic solvents (usually ethyl acetateor a mixture of ethyl acetate and cyclohexane) are used forthe extraction of the analyte from the aqueous phase. Gertzand Klostermann (2002) reported that, on a DB-5 MScolumn, a transformation of 2,3-DBPA to 2-BPA does nottake place, so that it is not necessary to transform thedibrominated compound into the more stable 2-monobromopropenamide by adding triethylamine. Howev-er, more recent reports confirm that, in acrylamide analysiswith bromination and detection by GC-ECD in HP-INNOWAX capillary column, 2-BPA rather than 2,3-DBPAwas chosen as the quantitative analyte because the peakresponse of former was nearly 20 times higher than that ofthe latter (Zhang et al. 2006).

Defatting has to be included in some sample prepara-tions because of the influence of high fat content on theanalysis. In one method, the fatty compounds wereremoved by extracting with hexane or by using graphitized

356 Food Bioprocess Technol (2011) 4:340–363

Author's personal copy

carbon cartridges after swelling (Tareke et al. 2000; Tarekeet al. 2002). Other methods include a phase separation bycentrifugation followed by removal of the water fraction byazeotropic distillation (Biedermann et al. 2002b; Tateo andBononi 2003).

The effects of other factors, such as pH, on acrylamideextraction have been studied. Changes in pH have beenfound to have a significant effect on acrylamide extractionefficiency. A higher amount of acrylamide (three to fourtimes) was observed in food samples by changing pHtowards the alkaline pH (pH>12). One reason might be that,during normal water extraction, polyacrylamide stericallyhinders all of the acrylamide from getting into the solution.Alkaline pH can change the structure of the matrix andfacilitate the free acrylamide to get into the solution. Also,increasing pH releases the chemically bound acrylamide(bound with protein and carbohydrate) to become availablefor analysis with this extraction technique (Kim et al. 2001;Svensson et al. 2003).

Phase separation is usually carried out by centrifugationof the sample. Further clean-up is also performed byfractionation of the organic extraction on silica–gel car-tridges (Castle 1993; Castle et al. 1991). Since silica is ofhigh water adsorptivity, ethyl acetate has to be dried orreplaced by cyclohexane to avoid any change in silicaactivity. Florisil is also used as an adsorbent and a mixtureof acetone and hexane as the elution solvent (Nemoto et al.2002). Alternatively, gel permeation chromatography onBio-Beads S-X-3 gel is performed as the final sampleclean-up (Tareke et al. 2000). Recently, drying of theextract has been carried out by adding sodium sulfate(Tareke et al. 2002; Ahn et al. 2002). In addition, removalof residual water eliminates or decreases the effect ofinterferences from water-soluble co-extractants. The solventvolume has to be reduced to 30–200 μl to reach limits ofdetection in the range of 1–5 μgkg-1 before injection intothe GC column (Tareke et al. 2002; Tareke et al. 2000;Castle 1993; Castle et al. 1991).

Analyte Separation, Detection, and Quantification

Owing to the higher polarity of the non-derivatizedacrylamide, columns with polar phase, e.g., polyethylen-glycol, have to be used. A total of 1–2 μl samples is usuallyinjected in splitless mode, and analyte separation isperformed on standard GC capillary columns with a lengthof 30 m and an internal diameter of 0.25 mm (standard inGC-MS). The initial oven temperature is normally adjustedto 60 °C to 85 °C, and the heating rate is 15 °Cmin-1. Thefinal oven temperature is usually about 250 °C. This iswhile columns with middle to high polarity can be used inthe case of derivatized acrylamide. However, it is possibleto inject sample extracts into the GC in the split mode

(Biedermann et al. 2002b; Tateo and Bononi 2003; Wiertz-Eggert-Jörissen (WEJ) GmbH 2003).

The major drawback of GC analysis without derivatiza-tion is the lack of characteristic ions in the mass spectrumof underivatized acrylamide. In the electron ionizationmode, the major fragment ions are at m/z 71 and 55,respectively, which are also used for quantification. Co-extracted substances such as maltol or heptanoic acidproduce almost the same fragmentation pattern and may,therefore, interfere. Selectivity can be increased by chemicalionization using methane as the reagent gas (Biedermann etal. 2002b).

The original method using GC-MS with bromination isbased on adding methacrylamide as an internal standard tothe homogenized sample and then producing the derivative2,3-dibromo-2-methylpropionamide (Castle et al. 1991).Alternatively, methacrylamide can be derivatized separatelyand added to the sample directly before the final adjustmentof the solvent volume (Castle 1993). The latter is preferredbecause methacrylamide acts as a chromatographic internalstandard, which means that it can monitor potential changesin the performance of the instrument. However, Castle(2003) reported a large difference between the reactionkinetics of the bromination reactions of acrylamide andmethacrylamide.

Quantification is performed by adding different kinds ofinternal standards, ranging from propionamide to isotropi-cally labeled acrylamide. LOD is reported to be about 10–50 mgkg-1. Acrylamide may also be determined by positivechemical ionization with ammonia as the reagent gas andtandem mass spectrometric detection of the daughter ionsreleased from the single charged molecular ion adduct.Thus, LOD can be reduced by GC-MS/MS to 1–2 μgkg-1.

The methods usually used for acrylamide quantificationinclude external and internal standard methods. Theexternal quantitative analysis, however, revealed poorreproducibility. Zhang et al. (2006) suggested an improve-ment of the quantitative method by introducing the internalstandard, which is the most commonly used quantificationmethod for acrylamide. The commonly used internalstandards include isotope-labeled internal standard (e.g.,13C3-acrylamide or 2H3-acrylamide, etc.) and non-isotope-labeled internal standards (e.g., methacrylamide, etc.).Although the isotope-labeled internal standards are themost ideal ones of the type, they can only be used in MS-based analysis and satisfactory repeatability cannot beachieved until isotope-labeled acrylamide is used. Thiscould be due to the differing stability of the compounds andincomplete derivatization of structurally different internalstandards. Due to the large differences reported between thereaction kinetics of the bromination reactions of acrylamideand methacrylamide, a long bromination reaction time isrequired when methacrylamide is used as the internal

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standard (Wenzl et al. 2003; Castle and Eriksson 2005).The method of standard addition is also an accuratequantification method and especially useful when thematrix of the sample is very complex and extraction yieldsstrongly vary (Basilicata et al. 2005; Ito and Tsukada 2001).In cases where isotopically labeled acrylamide is used,repeatability of results is achieved by adding N,N-dimethy-lacrylamide. The properties of N,N-dimethylacrylamide areobviously far different from those of acrylamide. Conse-quently, the coefficient of variation (CV) of the acrylamiderecovery of spiked samples decreased from 26% to 7.5%when applying [13C3]-acrylamide (Tareke et al. 2002).Castle (1993) reported that 2,3-dibromopropionamidemight eliminate hydrogen bromide during injection orchromatographic separation. Others used dehydrobromina-tion instead of 2,3-dibromopropionamide (Andrawes et al.1987; Nemoto et al. 2002; Takata and Okamoto 1991).Another reason for the large CVs might be incompletederivatization of structurally different internal standards(methacrylamide and N,N-dimethylacrylamide). Mean-while, most laboratories use [D3]-acrylamide, [13C3]-acryl-amide or both together as internal standards.

The quantification is usually performed by the method ofstandard addition as follows: a set of GC peak areas of theanalyte obtained for each sample (one for unspiked and threefor spiked portions with different levels of standardsolutions) are plotted as along the y-axis, while the quantitiesof standard substances in the portions are plotted as the x-axis. A calibration curve is then prepared using the linearregression method to calculate the amount of the analyte inthe spiked portion of the sample (Zhu et al. 2008).

For the chromatographic separation of acrylamide, mostscientists have used reversed-phase chromatography (dif-ferent C18 columns; European Union Risk AssessmentReport 2002; Rosén and Hellenäs 2002; Takatsuki et al.2003). Different reversed-phase columns have been com-pared. Primisphere C18-HC is recommended because itprovides sufficient retention time for acrylamide to mini-mize matrix interference. An alternative to reversed-phasecolumns is the IC. An IonPac column is a combined ionexchange with size exclusion chromatography. The advan-tage is that there is a good separation of acrylamide frommatrix compounds of even untreated sample extracts (Ahnet al. 2002; Cavalli et al. 2002; Höfler et al. 2002).

For detection of acrylamide after LC separation, tandemmass spectrometry is most often the method of choice.There are just a few exceptions in which UV at 202 nm(incurring a lack of selectivity) and single quadruple MS (inthe single ion monitoring mode) are used (Höfler et al.2002). This lack of selectivity would hamper the determi-nation of acrylamide in complex matrices. A solution couldbe the use of two-dimensional (Fauhl 2003) and/ormultidimensional LC (Takatsuki et al. 2003) that use four

different columns for separation of the analyte from theinterference.

LC-MS/MS, working in MRMs, in which the transitionfrom a precursor ion to a product ion is monitored, has ahigh selectivity. MRM means that the transition from aprecursor ion, which is separated in the first quadruple, to aproduct ion, generated by collision with argon in the secondquadruple, is monitored in the third quadruple. Thetransition 72→55 has been usually selected for quantifyingacrylamide because it shows a relatively high intensity(Becalski et al. 2003; Ahn et al. 2002; Rosén and Hellenäs2002; Tareke et al. 2000). Other transitions, such as 72 →54, 72→44, and 72→27, have been used in some cases forconfiguration. For the detection of the isotopically labeledacrylamide used as internal standard, the monitoredtransitions are 75→58 for [D3]- and [13C3]-acrylamideand 37→56 for [13C1]-acrylamide. Despite the selectivityoffered by MS/MS, interference can still occur. Peaksshowing identical retention times to acrylamide anddeuterated acrylamide have been observed. This problemcould be solved by increasing the pH of the solution fromwhich acrylamide was extracted into an organic solvent(e.g., the ASE device used during extraction; Swiss FederalOffice of Public Health 2002). Becalski et al. (2003) alsoreported the existence of an early eluting compound thatinterferes when transition 72→55 is used for the detectionof acrylamide. Increasing the column length from 100 to150 mm and applying Isolute Multi-Mode cartridges duringsample preparation eliminated the problem. Further detailson the chromatographic conditions and the optimumparameters used for the MS/MS and UV detectors arereported in Wenzl et al. (2003).

Strategies for Reduction of Acrylamide Levels in Food

Different strategies for reducing acrylamide levels in foodhave been suggested. Removing or reducing of reactants isone of them. When one of the reactants (asparagine orglucose) is at lower concentration, formation of acrylamidewill be reduced. Decreases in asparagine content may beachieved by: (a) selecting cultivars (e.g., potatoes, cerealgrain) that contain lower levels of asparagine; (b) elimina-tion of enzymes which control biosynthesis of asparagineby suppressing genes that encode them; (c) hydrolysis ofasparagine to aspartic acid and ammonia by acid- and/orasparaginase/amidase-catalysis; (d) modification of aspara-gine to N-acetylasparagine via acetylation, so formation ofN-glycoside intermediates, which form acrylamide, isprevented (Friedman 1978, 2001).

Meanwhile, researchers have determined two possibleways to reduce the level of sugar in potatoes. Duringstorage of potatoes, the amount of sugar increases, thus

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using fresh potatoes could result in less acrylamide beingformed. Also, storing potatoes below 8–10 °C can increasethe formation of reducing sugars, and the presence of thesereducing sugars together with asparagine may lead toacrylamide formation. The variety of potato with relativelylower amounts of reducing sugars and asparagine alsoaffects the amount of acrylamide formation (FAO/WHO2004).

In another procedure, the formation of acrylamide willbe reduced with disruption of reaction. There is a time–temperature relationship to the formation of acrylamide infood, thus changing the temperature or duration of cookingwill affect the level of acrylamide. It has been suggestedthat, when the temperature of food rises above 120 °C, therate of acrylamide formation increases rapidly with tem-perature over a limited range (Claus et al. 2008). Acrylam-ide formation in food is also pH-dependent and optimumpH for acrylamide formation in food is about 7. In acidicpH, acrylamide formation is inhibited. Lowering the pH ofthe food system to reduce acrylamide generation mayattribute to protonating the α-amino group of asparagine,which subsequently cannot engage in nucleophilic additionreactions with carbonyl source (Zhang and Zhang 2007).Other inhibitors of acrylamide formation are the asparagi-nase enzymes that disrupt the formation of acrylamide.Another factor is water activity that seems to be a criticalfactor (Taeyman et al. 2004).

Destroying and/or trapping of acrylamide after itsformation is another strategy which can be done via (a)hydrolysis of the amide group of acrylamide to acrylic acidand ammonia by acid- or enzyme-catalysis; (b) polymeri-zation of monomers of acrylamide to polyacrylamide inprocessed foods by means of using UV light or radiation(Friedman 1997); (c) reaction of acrylamide with SH-containing amino acids, esters, peptides, and proteins(Friedman 1996). Also, some compounds like NaCl andCaCl2 could decrease the amount of acrylamide formed(Açar et al. 2010; Pedreschi et al. 2010).

Conclusion

Only a limited number of methods have been so farproposed in the literature on the determination of acrylam-ide in food products. As for the recognition of methods,mainly two general methods of analysis (LC-MS/MS orGC-MS) are used, and it is still difficult to determine whichone is more reliable.

By comparing methodologies, large differences arefound among the extraction procedures and clean-upstrategies, e.g., variation in extraction, in swelling con-ditions, in temperature and time of extraction, in mechan-ical treatment, and in centrifugation or the use of SPE for

both GC- and LC-based methods, especially in LC samplepreparation. GC-MS after bromination is the best approachso far, because this method is a relatively mature coupledtechnique with adequate sensitivity and multiple ionconformation.

Application of GC-MS/MS or coupling to a high-resolution MS would even further lower the detection limitof certain foods to 1–2 μg/kg. Determination of acrylamideusing LC-MS/MS may avoid derivatization and has theadvantages of rather high sensitivity and stability.

Also, research should be focused on cheap, convenient,and rapid screening methods that are reliable and robustwhich could be employed in most laboratories. In thisrespect, GC-ECD method has been developed for identifi-cation and quantification of derivatized acrylamide in heat-processed starchy foods, while it requires a relatively low-cost instrumentation to perform compared with MSdetection-based methods. The ASE method provides a fastand efficient extraction of acrylamide from various foodsamples along with simple and rapid sample preparationwithout SPE clean-up and concentration prior to GC-ECDanalysis. In addition, the standard addition method has beenreported as a suitable quantification method for thedetermination of acrylamide in heat-processed foods.

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