Alternative extraction techniques for analysis of acrylamide in food

Alternative extraction techniques for analysis of acrylamide in food, Alternative extraction techniques for analysis of acrylamide in food, Alternative extraction techniques for analysis of acrylamide in food

ARTICLE IN PRESS LWT 39 (2006) 392–398 www.elsevier.com/locate/lwt Alternative extraction techniques for analysis of acrylamide in food: Influence of pH and digestive enzymes Sune Erikssona,b,Ã, Patrik Karlssonb a Department of Environmental Chemistry, Stockholm University, S-10691 Stockholm, Sweden b AnalyCen Nordic AB, P.O Box 905, S-53119 Lidkoăping, Sweden Received 29 July 2004; received in revised form December 2004; accepted March 2005 Abstract Acrylamide in food is normally measured as ‘‘water-soluble free acrylamide’’ However, it is shown that the technique of extraction, according to the method for extraction of dietary fibres or at high pH can affect the results This has to be accounted for, particularly in the assessment of exposure and in studies of bioavailability and, in the long term, health risk assessment r 2005 Swiss Society of Food Science and Technology Published by Elsevier Ltd All rights reserved Keywords: Acrylamide; Extraction methodology Introduction Acrylamide is a reactive difunctional monomer containing a reactive electron deficient double bond and an amide group, which undergoes reactions typical of those functionalities It exhibits both weak acidic and basic properties Many of the reactions of acrylamide are reversible, such as the addition of ammonia, amines, phosphines and bisulphites Alkaline conditions permit the addition of mercaptans, sulphides, ketones, nitroalkanes, and alcohols to acrylamide Alcohol reactions involve, for instance, polymeric alcohols such as poly (vinyl alcohol), cellulose and starch (American Cyanamid, 1969; Habermann, 1991) Since the discovery of acrylamide formation during heating of foods (Tareke, Rydberg, Karlsson, Eriksson, & Toărnqvist, 2000, 2002; Rosen & Hellenaăs, 2002) was rst published, many different analytical methods, using both LC-MS/MS and GC-MS, have been applied for the analysis of acrylamide in foods Different methods ÃCorresponding author AnalyCen Nordic AB, P.O Box 905, S-53119 Lidkoăping, Sweden Tel.: +46 51088700; fax: +46 51066438 E-mail address: sune.eriksson@analycen.se (S Eriksson) have shown to give similar results at different laboratories, and they also have been demonstrated through different proficiency programs (Tareke et al., 2002; Rosen & Hellenaăs, 2002; Ahn et al., 2002; Clarke, Kelly, & Wilson, 2002; Ono et al., 2003; Wenzl, de la Calle, & Anklam, 2003; Wenzl et al., 2004) Most applied methods for analysis of acrylamide in food products comprise extraction with water, which means that what is analysed is ‘‘water-soluble free acrylamide’’ The importance of pH for formation of acrylamide in foods was shown by Rydberg et al (2003), Jung Choi and Ju (2003), Gama-Baumgartner, Grob, and Biedermann, (2004), and Pedreschi, Kaack, and Granby (2004) However, an effect of pH during extraction of acrylamide when performing the analysis of acrylamide has, to our knowledge, not been discussed Different studies have exhibited that added acrylamide in foods is bound or adsorbed to food components in unknown ways (Biedermann, Biedermann-Brem, Noti, & Grob, 2002a; Biedermann, Noti, BiedermannBrem, Mozzetti, & Grob, 2002b; Rydberg et al., 2003) It was not studied to the extent that it is possible to release this acrylamide for analysis, except in some trials with enzymatic hydrolysis (Jezussek & Schieberle, 0023-6438/$30.00 r 2005 Swiss Society of Food Science and Technology Published by Elsevier Ltd All rights reserved doi:10.1016/j.lwt.2005.03.002 ARTICLE IN PRESS S Eriksson, P Karlsson / LWT 39 (2006) 392–398 2003) showing no effect of using enzymatic treatment (protease and/or a-amylase) Bologna, Andrawes, Barvenik, Lentz, and Sojka (1999) even showed recoveries deviating from their standard recoveries on beans, sugar beet, corn and potato, and found that acrylamide is more stable in potatoes than in the rest of the tested crops So far it has not been possible to use this knowledge when performing calculations of intake of acrylamide in food (Svensson et al., 2003; Dybing & Sanner, 2003; Konings et al., 2003), since most analyses of food products concerned ‘‘water-soluble free acrylamide’’ The present report studies whether there is an influence of pH and different enzymes in extracting solvents on the amount of free acrylamide measured in foods Experiments with pH extractions of free acrylamide in polyacrylamide are also discussed, as a comparison with food products Materials and methods In these experiments, commercial food product samples were used, which were bought in the regular food stores in Sweden They were extracted with water, at different pHs and, in the same way as for analysis of dietary fibre (Nordic Committee on Food Analysis, 1989), both with enzymes (heat stable a-amylase, protease and amyloglucosidase) and without enzymes In the same way, extraction with pepsin (Everitt et al., 1980) had been applied in order to investigate whether the fraction of extracted acrylamide was affected (This method was commonly used for analysing pepsindigestible protein in animal feed in Sweden.) Both systems are in vitro methods imitating the digestion 393 system, the dietary fibre method for measurement of dietary fibre in carbohydrates, and the pepsin method to measure the part of the protein, which is solubilized by pepsin (Table 1) Equipment (except glassware and normal laboratory equipment): Water-baths (1) boiling and 3771 1C and (2) shaking, capable of maintaining 6070.5 1C; Fibertec system E1023 Filtration Module with incubation asks, Foss Tecator, (Hoăganaăs, Sweden); SPE column OASIS HLB 0.2 g, Waters Corp, (Milford, MA, US); SPE column Isolute Multimode, Argonaut Technologies, Inc., (Redwood City, CA, USA); Syringe filter 0.2 mm, Sartorius Minisart hydrophilic; HPLC-MSMS: Agilent 1100 with Micromass Quattro Ultima MSMS, equipped as earlier described (Tareke et al., 2002) Reagents: Sodium hydroxide solution 0.275 and mol/l; Phosphate buffer, 0.08 mol/l, pH 6.070.1; Hydrochloric acid solution 0.325, and 7.5 mol/l; Enzymes: Amyloglucosidase solution EC 3.2.1.3, from Aspergillus niger, Sigma-Aldrich, (St Louis, MO, US); Protease, (Subtilisin from Bacillus lichiformis) EC 3.4.21.62, Sigma-Aldrich, (St Louis, MO, US), 150 mg dissolved in ml 0.08 mol/l phosphate buffer; Heat stable amylase, Thermamyls 120 L Type L, EC 3.2.1.1, Novozymes A/S, (Bagsvaerd, Denmark); Pepsin, from porcine gastric mucosa, 2000 FIP-U/g, EC 3.4.23.1 Merck, (Darmstadt, Germany), diluted to 12.5 FIP-U/ ml in water (new solution every day) Standards: Internal standard solution (13C3)acrylamide, 99%, Cambridge Isotope Laboratories, (Andover, MA, US) diluted to mg/ml with water; Standard solutions: Acrylamide, Aldrich 99+, Sigma-Aldrich, (St Louis, MO, US), diluted in steps between 0.001 and 0.5 mg/ml with water Polyacrylamide 50 wt% in water solution, Mw 1500, Aldrich (Milwaukee, WI, US) Table Summary of enzymes used Enzyme EC-code* Incubation temperature (1C) Incubation pH Working path* Method Heat-stable a-amylase EC 3.2.1.1 100 6.0 Dietary fibre Protease EC 3.4.21.62 60 7.5 Amyloglucosidase EC 3.2.13 60 4.4 Pepsin EC 3.4.23.1 37 0.15–0.3 mol/l HCl Endohydrolysis of 1,4-a-glucosidic linkages in polysaccharides containing three or more 1,4-a-linked D-glucose units Hydrolysis of proteins with a broad specificity for peptide bonds, and a preference for a large uncharged residue in P1 Hydrolyses peptide amides Hydrolysis of terminal 1,4-linked a-Dglucose residues successively from nonreducing ends of the chains with release of b-D-glucose Preferential cleavage: hydrophobic, preferably aromatic residues in P1 and P10 positions * NC-IUBMB, 1992–1999 Dietary fibre Dietary fibre Pepsin ARTICLE IN PRESS 394 S Eriksson, P Karlsson / LWT 39 (2006) 392–398 2.1 Extraction methods 2.1.1 Dietary fibre extraction for acrylamide determination Accurately weigh ground/milled 1–3 g test portions to the nearest 0.1 mg into Fibertec incubation flasks Add 0.08 mol/l phosphate buffer (50 ml), a magnetic rod, and mix by magnetic stirring Add heat stable amylase (0.1 ml), mix, and incubate in a boiling water-bath for 30 Adjust pH to 7.570.1 at room temperature Incubate in 60 1C water-bath until the temperature has risen to 60 1C Add protease solution (0.1 ml), mix, and incubate 30 in the 60 1C water-bath Adjust pH to 4.470.1 at room temperature Incubate in 60 1C waterbath until temperature has risen to 60 1C Add amyloglucosidase solution (0.1 ml), mix, and incubated for 30 in the 60 1C water-bath Cool down to room temperature Internal standard 1.0 ml mg/ml (13C3)acrylamide was added The extract was centrifuged and filtered through a 0.2 mm syringe filter to get ml of filtrate, which was further purified on SPE and analysed as described below [(13C1)acrylamide and (13C3)acrylamide standard had 80–90% recovery during this enzymatic treatment.] 2.1.2 Extraction with pepsin In 215 ml, 38–39 1C temperated water, sample (5.0 g) was added in a way, that it was completely soaked Add 7.5 M HCl (5 ml) and pepsin-solution (25 ml) Incubate for 48 h at 37 1C, and add a further 7.5 mol/l HCl (5 ml) after 24 h This solution was then analysed for acrylamide, after addition of the internal standard, (13C3)acrylamide 2.1.3 pH adjustments The homogenized samples (normally 10 g) were extracted with water (100 ml), and the internal standard, (13C3)acrylamide was added Adjustment and measuring of pH were done at ambient temperature, with HCl or NaOH solutions on a magnetic stirrer No further control of the pH was done after the adjustment of pH The exposure for the buffered solution was 15–30 before the sample was centrifuged, and the extract was further treated as described below 2.2 Acrylamide analysis with LC-MSMS For comparison, samples were also prepared and analysed on contents of free acrylamide as described earlier (Tareke et al., 2002) with some modifications The homogenized samples (normally 10 g) were extracted with water (100 ml), and the internal standard (13C3)acrylamide was added The samples were centrifuged, the supernatant filtrated through a 0.2 mm syringe filter, to get ml of filtrate Samples of ml of filtrate from the different work-up procedures were added on an SPE column, Oasis HLB 0.2 g column, Waters (Milford, MA, US) as described by others (Roach, Andrzejewski, Gay, Nortrup, & Musser, 2003; Andrzejewski, Roach, Gay, & Musser, 2004) The recovery was ca 100% (SD ¼ 7:5%) compared to internal standards for most food matrices Detection levels decreased from 10 to mg/kg and reproducibility was high (CVE5%) as shown earlier (Tareke et al., 2002) for water extraction Others used more complex extraction methods, which increased the CV to 20% at 3–50 mg/kg and to 10% above 50 mg/kg The column was washed with one column volume of methanol followed by five volumes of water before each use The extract was further cleaned with Isolute SPE column, as described previously (Tareke et al., 2002) The extract was then used directly for HPLC-MS/MS analysis On every analytical occasion a blank sample of water, which had passed through the analytical work-up procedure, was included (all blank samples, included water extraction, enzyme treatment, pH adjustment to give maximum mg/kg) The use of isotope-substituted internal standard was essential for the reproducibility considering that the absolute recovery varies between food matrices The suppression in the LC-MS analysis, which had been earlier observed (Rydberg et al., 2003), had decreased by adding the OASIS column It should be remembered that the internal standard does not compensate for a different behaviour of the analyte, for instance, in extraction efficiency, formation from other compounds or reaction with matrix before work up Laboratory plastic materials (like SPE columns) may be contaminated by monomeric acrylamide This stresses the need for blank samples To minimise the effect of this contamination the sample concentration should be as high as possible, and dilutions should be done after the use of SPE columns Results and discussion  Extraction at different pHs: pH in the range of 2–7.5, gives the same result as normal water extraction At higher extraction pHs, the recovery of acrylamide increases gives a maximum of around 12 and above (n ¼ 25) How much the increase is depends on the type of product Examples from studied food samples shows that pH 12 gives between 1.1 and 1.6 (potato products, n ¼ 5; x ¼ 1:3), 1.1–4.1 (bread products, n ¼ 10; x ¼ 2:0), 3.1–3.6 (hamburger, n ¼ 3; x ¼ 3:3) and 1.7–3.6 (coffee, cocoa, dried fruits, n ¼ 6; x ¼ 2:5) times higher measured amounts of acrylamide compared to normal water extraction and extraction at pH Table summarizes the above results Figs and show typical curves for two kinds of bread and the relationship between yield of ARTICLE IN PRESS S Eriksson, P Karlsson / LWT 39 (2006) 392–398 395 Table Result summary with a high pH extraction Product group Number of products Yield ratio pH 12/pH Mean ratio x Potato products Bread products Hamburgers, meat Mixed products, coffee, cocoa, dried fruits 10 1.1–1.6 1.1–4.1 3.1–3.6 1.7–3.6 1.3 2.0 3.3 2.5 Dark christmas soft bread 1400 Table Extra controls performed at pH 12 Type of control Number of tests Results Blank Mixture of asparagine and fructose Mixture of asparagine and glucose 0.5% asparagine and 0.5% fructose to bread 0.5% asparagine and 0.5% glucose to bread Standard pH adjusted Mixture of acrylic acid and ammonia 10 at different conc p1 mg/kg o3 mg/kg at different conc o3 mg/kg No increase No increase different conc Same result o3 mg/kg 1200 Serie1 AA (µg/kg) 1000 800 600 400 200 0 10 11 12 13 14 15 pH Fig Analysed contents of acrylamide in Swedish dark Christmas soft bread, after extraction at different pH Table Comparison of acrylamide content after dietary fibre extraction, with and without enzymes Product Number of samples Dietary fibre, without enzymes Yield ratio with water extraction Dietary fibre, with enzymes Yield ratio with water extraction Potato products Bread products Mixed products, coffee, cocoa, dried fruits 1.0–1.3, x ¼ 1:2 1.1–2.1, x ¼ 1:6 1.3–2.4, x ¼ 1:8 1.0–1.4, x ¼ 1:2 1.2–2.1, x ¼ 1:5 1.2–2.8, x ¼ 2:0 Whole kernal bread 1200 1000 Serie1 AA (µg/kg) 800 600 400  Fig Analysed contents of acrylamide in Swedish whole kernel bread after extraction at different pH Procedure according to extraction for dietary fibre gives the same acrylamide content, irrespective of whether enzymes are present or not (n ¼ 18) (Table 4) Pepsin extraction with different kinds of bread, coffee, cocoa and dried fruits showed no difference in results compared to normal water extraction (n ¼ 10) Liberation of residual acrylamide monomer from polyacrylamide, Mw 1500, increases drastically with pH, starting at pH or 10 (Fig 3) acrylamide and pH Table summarizes the controls, which have been carried out at pH 12 Procedure according to methods for dietary fibre gives the same or higher acrylamide content as compared to extraction with water, (1.0–2.8, n ¼ 18; x ¼ 1:5) The results regarding the yield at low pH is in agreement with Calbiani, Careri, Elviri, Mangia, and Zagnoni (2004) who extracted acrylamide at acid pH, with the same result as that of normal water extraction 200  0  pH 10 11 12 13 14 15 ARTICLE IN PRESS S Eriksson, P Karlsson / LWT 39 (2006) 392–398 396 Regarding the results of polyacrylamide, Ahn and Castle (2003) earlier looked at the thermal stability of polyacrylamide In this study, the stability during hydrolytic conditions was noted, and their results cannot be directly compared with the present findings The normal analytical method for acrylamide monomer in food is not appropriate for analysing residual acrylamide in polyacrylamide Many authors have published methods for that purpose, cf Table These methods included the use of organic solvents, which shrank the polyacrylamide (polyacrylamide is soluble in polar liquids such as ethylene glycol, formamide and 250000 Serie1 AA (µg/kg) 200000 150000 glycerol, and insoluble in alcohols, acetone and aliphatic or aromatic hydrocarbons; Hunkeler & Herna´ndez Barajas, 1997) so that the residual acrylamide will be released into the solution In our experiments (not shown), polyacrylamide had to be extracted 1–2 days with only water, in order to have an effective extraction of the residual acrylamide in polyacrylamide When increasing pH to 10–12 in a solution of polyacrylamide, it starts to deamidate (Muller, 1981; Morawetz, Sawant, & Suen, 1981; Sawant & Morawetz, 1984) and swell (Mallo, Candau, & Cohen, 1985) This swelling can open up the polyacrylamide structure, so that residual acrylamide can be released into the solution Polyacrylamide chemistry, reviewed by Caulfield, Qiao, and Solomon (2002), described the hydrolysis of polyacrylamide under different pH conditions and other ways of hydrolyzing it, without finding any proof of formation of free acrylamide from polyacrylamide Our findings not interfere with their review conclusions Conclusion 100000 50000 12 pH 10 pH pH pH pH pH pH pH Fig Release of acrylamide from polyacrylamide at different pH Digestion of what we eat is a complex process It includes production and the use of many enzymes from our mouth, stomach and the intestine system (Brody, 1999), and there is a difference of around pH units during food digestion, with the lowest pH in the stomach of about pH 1–2 (Fallingborg, 1999) The methodology for analysis of acrylamide, used by most laboratories (Wenzl et al., 2003), implies measurement of free water-soluble acrylamide in most cases It has so far been assumed that the water-soluble acrylamide is equal to the acrylamide taken into the body The enzymes used here have given amounts of extractable acrylamide similar to the yields obtained at normal water extraction A higher amount of acryla- Table Extraction for acrylamide in polyacrylamide/copolymers of acrylamide and different gels Extraction Reference 0.1 g swollen in 25 ml water overnight at ambient temperature Methanol (250 ml) was added, and left to stand for one day at ambient temperature 0.1–0.2 g was added to acetonitrile (10 ml) Agitated until supernatant was clear 80% methanol in water (100 ml) was added to 10 g of dry polyacrylamide (100–120 mesh),and shaken for h Extracted with DMSO (1 mg/ml) and diluted (1:10) with water Sample (5 g) was added to 80% methanol in water (50 ml), and shaken for 3–4 h Diluted 100–2000 times with water Sample (1.0 g) was added to, depending on the type of polymer Castle (1993) Herna´ndez-Barajas and Hunkeler (1996) MacWilliams, Kaufman, and Waling (1965) Saroja et al (2000) Skelly and Husser (1978) Smith and Oehme (1993) Tseng (1990) a) 1% acetonitrile in water (100 ml), b) 50:50 (v/v) acetonitrile-methanol (10 ml), stirred for 30 ml diluted to 100 ml with water, c) acetonitrile-tetrahydrofuran (10 ml), stirred for 30 ml diluted to 100 ml with water Polymer inversion by using different mixing techniques, with an excess of water Ver Vers (1999) ARTICLE IN PRESS S Eriksson, P Karlsson / LWT 39 (2006) 392–398 mide in food samples can be made chemically available by changing pH during the extraction One of the reasons can be that, as in polyacrylamide, it is a sterical hinderance for all of the acrylamide to get into solution, during normal water extraction By changing the pH, the structure of the matrix can be changed and made to facilitate free acrylamide to get into solutions The seemingly higher content of acrylamide in food measured by the commonly used method may have an impact on estimation of the bioavailability of acrylamide in food, and, finally, health risk assessment of intake of acrylamide from food Studies of the bioavailability have to be performed so that the uptake from food can be correlated to acrylamide contents, which are relevant and reliable This means, that the analytical methods have to be evaluated and standardized by comparing different ways of performing the extraction of acrylamide in food products In the meantime, the extraction technique should be given when measured acrylamide contents in food are reported Acknowledgments This project is financed through AnalyCen R&D, and is executed in cooperation with Stockholm University (M Toărnqvist with support from The Swedish Research Council FORMAS) Thanks also to Martin Zachrisson, Annika Larsson, and Helena Larsson, AnalyCen Nordic, Lidkoăping for performing all the extractions with different methods References Ahn, J S., & Castle, L (2003) Tests for the depolymerization of polyacrylamides as a potential source of acrylamide in heated foods Journal of Agricultural and Food Chemistry, 51, 6715–6718 Ahn, J S., Castle, L., Clarke, D B., Lloyd, A S., Philo, M R., & Speck, D R (2002) Verification of the findings of acrylamide in heated foods Food Additives and Contaminants, 19(12), 1116–1124 American Cyanamid (1969) Chemistry of acrylamide, Bulletin PRC 109 Wayne, N.J., US: Process Chemicals Department, American Cyanamid Co Andrzejewski, D., Roach, J A G., Gay, M L., & Musser, S M (2004) 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