Application of matrix solid phase dispersion in the determination of acrylamide

Application of matrix solid phase dispersion in the determination of acrylamide, Application of matrix solid phase dispersion in the determination of acrylamide, Application of matrix solid phase dispersion in the determination of acrylamide

Available online at www.sciencedirect.com Journal of Chromatography A, 1175 (2007) 1–6 Application of matrix solid-phase dispersion in the determination of acrylamide in potato chips Jos´e O Fernandes ∗ , Cristina Soares REQUIMTE, Servi¸co de Bromatologia, Faculdade de Farm´acia, Universidade Porto, Rua An´ıbal Cunha, 164, 4099-030 Porto, Portugal Received 24 July 2007; received in revised form October 2007; accepted October 2007 Available online 18 October 2007 Abstract A sample preparation technique based on matrix solid-phase dispersion (MSPD) was applied to the determination of acrylamide in potato chips Samples (0.5 g) were ground and dispersed in C18 (2 g), transferred to an empty column, and after a previous clean-up with n-hexane to eliminate fat, acrylamide was eluted with water (4 ml + ml) After bromination of the extract the samples were analysed by GC–MS in selected ion monitoring mode The method presents good recoveries and the limits of detection and of quantification were 12.8 and 38.8 ␮g kg−1 , respectively In order to evaluate the performance of the MSPD-GC/MS developed method, 17 potato chips samples were simultaneously analysed by an alternative method based on the extraction of acrylamide with hot water The results obtained by using both methods were very similar, with a correlation factor (r) of 0.9985 The reported MSPD extraction method revealed to be simpler and faster then previous GC–MS methods used to quantify acrylamide in potato chips © 2007 Elsevier B.V All rights reserved Keywords: Matrix solid-phase dispersion (MSPD); Acrylamide; Food analysis; GC–MS Introduction Acrylamide is a toxic compound that is formed in certain foodstuffs, particularly those rich in carbohydrates, during processing or cooking at high temperatures [1,2] Since the announcement by the Swedish National Food Administration that acrylamide was found in heat-treated foods [3], it became a priority human health issue and several studies were reported concerning the compound toxicology, formation and detection [for reviews see [4–7]] Regarding the quantification of acrylamide, the already available methods mostly employ GC–MS or LC-MS/MS, after application of multi-step sample preparation processes However, these methods are usually subject to noteworthy difficulties that are mainly related with the chemical properties of the compound, especially with its strong polar character and the low molecular weight This makes acrylamide quantification in complex matrices, such as foodstuffs, a difficult task with respect to both the extraction procedure and the final determination ∗ Corresponding author Tel.: +351 222078910; fax: +351 222003977 E-mail address: josefer@ff.up.pt (J.O Fernandes) 0021-9673/$ – see front matter © 2007 Elsevier B.V All rights reserved doi:10.1016/j.chroma.2007.10.030 Extraction procedures adopted are generally labour-intensive and time-consuming In order to obtain satisfactory analyte recoveries, efforts to isolate the compound include repeated extractions of the analyte from the matrix, centrifugation, and pooling of the supernatants This stage of the analytical protocol requires the use of relatively large volumes of solvents, which represents a significant hindrance considering the subsequent solvent evaporation and disposal In many cases, the mixing of sample and solvent produces emulsions that may decrease the extraction efficiency and lengthen the time the analyst needs to complete the procedure Due to the high polarity of acrylamide water is usually the extracting solvent of choice [3,8–11] However, the use of water brings out some inconveniences such as the co-extraction of the great majority of other food components like sugars, proteins, amino acids and organic acids According to some authors organic solvents could adequately replace water in order to restrict the co-extraction of these compounds [12–15] As water does not selectively extract the targeted substance, tedious and time-consuming clean-up procedures are needed to partially isolate the analyte from other matrix components The most frequently used purification techniques include the addition of Carrez solution to eliminate proteins [10,14,15], extraction with organic solvents to eliminate fats [9,16–19] and J.O Fernandes, C Soares / J Chromatogr A 1175 (2007) 1–6 the utilisation of solid-phase extraction (SPE), alone or in combination with other purification steps specially with difficult matrices like coffee [6,9,20], as a clean-up technique of the extracts The described sample preparation techniques need a large amount of sample, sorbent(s) and high-quality organic solvents, and require intensive manipulation of the extracts These methods are therefore expensive, both in terms of time and material consumption, and their sample throughput is too low to meet the challenges of modern food analysis The development of faster, more cost effective and environment friendly procedures is therefore a pressing demand Many studies have demonstrated the feasibility of modern extraction techniques, such as matrix solid-phase dispersion (MSPD) which meets the above-listed requirements, for the isolation of different types of drugs, microcontaminants and naturally occurring compounds from a wide variety of samples MSPD is fast, relatively analyte- and matrixindependent and, even with a subsequent less effective clean-up, it enables the isolation of cleaner extracts than those obtained with classical methods [21] The principles and basic procedures of MSPD have been described in several reviews, which also list a number of applications [22–26] MSPD can be described as a process for the extraction of target compounds from solid, semi-solid and liquid matrices that was introduced by Barker et al [27] MSPD combines aspects of several analytical techniques, performing sample disruption while dispersing the components of the sample on a solid support, thereby generating a chromatographic material suitable for the extraction of compounds from the dispersed sample According to Kristenson et al [22], C18 -MSPD performed with a g solid support blended with 0.5 g of sample and finally analysed with GC – electron-capture detection (ECD) (or GC–MS) presents practical limits of detection (LODs) of 5–50 ␮g kg−1 and good extraction efficiency (recovery 70–105%) for several contaminants in food The main objective of this work was the development of a MSPD-GC/MS procedure for the analysis of acrylamide in potato chips, one of the foodstuffs more susceptible to exhibit high levels of this contaminant The results obtained by using the developed method were compared with those furnished by a previously developed analytical procedure that resorted to a liquid extraction approach Labs (Andover, MA, USA) as a mg ml−1 methanol solu˚ 55–105 ␮m was from tion The preparative C18 sorbent 125 A, Waters (Milford, MA, USA), Florisil 60–100 mesh (pesticide residue analysis grade) was purchased from Sigma (Steinheim, Germany), ethylenediamine-N-propylsilane (PSA) bonded silica bulk was from Supelco (Bellefonte, PA, USA), aminopropyl silica gel and silica gel, both for preparative chromatography were acquired from Fluka (Buchs, Switzerland) and Merck (Darmstadt, Germany), respectively The MSPD empty columns were made of polyethylene with a reservoir of 25 ml, and a single frit with a pore size of 10 ␮m were acquired from Symta (Madrid, Spain) n-Hexane and ethyl acetate (pesticide residue analysis grade) and acetonitrile (ultrapure grade), were from Fluka Potassium bromide (IR spectroscopy grade) and bromine (analytical grade) were from Merck Sodium chloride (analytical grade) was from J.T Baker (Deventer, The Netherlands) Potassium hexacyanoferrate(II) trihydrate, zinc sulphate heptahydrate, sodium sulphate anhydrous and acetic acid (glacial) were obtained from Panreac (Barcelona, Spain) Hydrobromic acid 48% and sodium tiosulphate volumetric solution mol l1 were from Riedel-de Hăaen (Seelze, Germany) 2.3 Standards and reagents A stock solution of AA (2 g l−1 ) was prepared by dissolving the compound in acetonitrile, and appropriately diluted to prepare working standard solutions at and mg l−1 Working standard solutions of 13 C3 -AA (I.S.), were prepared with acetonitrile at 10 mg l−1 All stock and working solutions were stored at ◦ C The saturated bromine–water solution was prepared by adding bromine (∼3 ml) to 200 ml of water until precipitation became visible Carrez I solution and Carrez II solution were prepared by dissolving 15 g of potassium hexacyanoferrate(II) trihydrate in 100 ml of water, and 30 g of zinc sulphate heptahydrate in 100 ml of water, respectively 2.4 Equipment Seventeen samples of potato chips were collected from several local supermarkets A portion representative of each sample (≈20 g) was taken, ground in an electric grinder (Moulinex, Ecully, France) and stored at ◦ C GC–MS analyses were performed in a gas chromatograph (Agilent GC-6890N), equipped with a split–splitless injector, and coupled to a mass-selective detector (Agilent MSD-5975N) (Agilent, Palo Alto, CA, USA) The analytical separation was performed in a capillary column MDN-12 (30 m × 0.25 mm I.D., 0.25 ␮m) from Supelco Sample introduction were performed with an automatic injector (model 7683B) from Agilent All the filtration steps and MSPD extractions were made in a vacuum manifold (Visiprep Solid Phase Extraction Manifold) from Supelco with capacity for 12 columns Evaporation of the solvents was performed in a Băuchi Rotavapor (model RE 111) (Flawil, Switzerland) The evaporation, under a stream of nitrogen, was carried out on a Pierce (model Reacti-therm 18790) (Rockford, IL, USA) 2.2 Chemicals 2.5 GC–MS operating conditions Acrylamide (AA) (99% purity) was acquired from Aldrich (Steinheim, Germany) The internal standard [13 C3 ]acrylamide (13 C3 -AA) (99% purity) was purchased from Cambridge Isotope 2.5.1 Gas chromatography Carrier gas: helium (constant flow at ml min−1 ) Sample injection volume: ␮l (splitless, pulsed pressure 32 psi, 60 s) Experimental 2.1 Sampling J.O Fernandes, C Soares / J Chromatogr A 1175 (2007) 1–6 Injector temperature: 280 ◦ C Oven temperature: 85 ◦ C (1 min), 15 ◦ C min−1 to 280 ◦ C, hold 10 (total of 24 min) Transfer line: 240 ◦ C 2.5.2 Mass spectrometry Electron energy, 70 eV (EI mode) Mode of acquisition: selected ion monitoring (SIM), m/z 106, 150 and 152 for 2,3-dibromopropionamide (2,3-DBPA) and m/z 110, 153, 155 for [2,3-13 C3 ]dibromopropionamide (2,3-13 C3 -DBPA) The ions m/z 150 for 2,3-DBPA, m/z 155 for 2,3-13 C3 DBPA were used for quantification and the others for confirmation The identity of the peak was confirmed by retention time and by comparing the relative abundance ratios of the confirmatory ions with those of a standard solution 2.7 Preparation of standards for calibration 2.7.1 MSPD-GC/MS Aliquots of the mg l−1 working standard AA solution (equivalents to to 0.75 ␮g of AA corresponding to to 1500 ␮g kg−1 in the samples) were placed in a glass mortar with g of C18 and 0.25 ␮g of I.S After blending using a glass pestle, the mixtures were treated parallel to the samples, according to the described procedure 2.7.2 LE-GC/MS Aliquots of the mg l−1 working standard AA solution (equivalent to to ␮g of AA corresponding to to 1500 ␮g kg−1 in the samples) were placed in 50 ml centrifugal tubes with ␮g of I.S and the volume was made up to 20 ml with water These solutions were treated parallel to the samples, according to the described procedure 2.6 Sample preparation 2.8 Bromination of the samples and standards 2.6.1 Matrix solid-phase dispersion (MSPD-GC/MS) An aliquot of 0.5 g of the previously ground sample, g of C18 sorbent and 0.25 ␮g of I.S (25 ␮l of the 10 mg l−1 I.S solution) were placed in a glass mortar and blended together using a glass pestle to obtain the complete disruption and dispersion of the sample on the solid support When blending was complete (after 2–3 min), the sample was packed into an empty column containing a polyethylene frit at the bottom A second frit was placed on the top of the sample before careful compression with a syringe plunger The packed column was placed in a vacuum manifold, and the mixture was defatted with 20 ml of n-hexane The flow was controlled so that the n-hexane was in contact with the sample during approximately Cartridge drying was performed with vacuum aspiration during After drying, acrylamide was extracted with ml water holding the flow to permit a soaking step of This water was completely collected and a second aliquot of ml of water was added observing again a soaking step The second elution volume was collected to the same vial and the column was kept under vacuum aspiration for to collect all the water The flow control was important to ensure reproducibility In Fig a diagram of the method is presented All analysed samples were prepared in duplicate To ml of the aqueous extracts g of KBr was added The solutions were then acidified with HBr until pH 1–3 (100–150 ␮l) and ml of saturated bromine solution were added The derivatization reaction took place in an ice bath, protected from light, for at least h The excess bromine was then decomposed by the addition of mol l−1 Na2 S2 O3 solution until the yellow colour of the extracts disappeared (50–150 ␮l) The solutions obtained were saturated with g of NaCl and the acrylamide derivative was extracted twice with 10 and ml portions of ethyl acetate/n-hexane 4:1 (v/v) The volume of the organic phase was reduced to ml under a stream of nitrogen at 40 ◦ C, and a small quantity of anhydrous Na2 SO4 was added Finally, it was centrifuged at 3000 rpm during min, the upper layer was transferred to another vial and evaporated to 0.5 ml under a gentle stream of nitrogen The extracts were then injected twice in the gas chromatograph 2.6.2 Liquid extraction (LE-GC/MS) To evaluate the performance of the MSPD method, all the samples were also analyzed after extraction by a previously optimized liquid extraction procedure with hot water Briefly, acrylamide was extracted with water at 65 ◦ C (2 g of sample + ␮g of I.S to 20 ml of water) after a swelling time of 15 min, proteins were eliminated by addition of Carrez solutions followed by the centrifugation of the solutions and vacuum filtration and, finally, most of the water was eliminated (until ml) in a rotary evaporator before derivatization All samples were also prepared in duplicate Results and discussion 3.1 Method development Aiming at screening the presence of AA in largely consumed (in Portugal) food products, we have previously developed a GC–MS methodology that was based on relevant work published in this field [3,10] Briefly, AA was extracted from the samples with water at 65 ◦ C, Carrez solutions were added in order to precipitate proteins, the solvent partially evaporated and finally the compound was brominated, extracted twice by ethyl acetate:n-hexane 4:1 (v/v) and then analysed by GC–MS in SIM mode This methodology was applied to potato chips, cereal based products and chocolate, but revealed to be inadequate to analyse coffee samples, for which some additional clean-up steps were required [20,28] Although the extraction of acrylamide from food matrices by using these methods could be considered efficient, it was also time-consuming and laborious With an increased demand for acrylamide analysis in our laboratory, a faster sample J.O Fernandes, C Soares / J Chromatogr A 1175 (2007) 1–6 Fig Schematic diagram of the MSPD extraction method applied and described in this work preparation method became mandatory The good results reported in several studies that used MSPD as a sample treatment procedure for extracting/purifying contaminants from a variety of solid, semi-solid, viscous and liquid foodstuff, and the recent successful experiences of our group with the referred technique [29,30], encouraged us to develop an MSPD method to extract acrylamide from food samples 3.2 Preliminary extraction assays Preliminary experiments were carried out to assess the effect of several sorbents on the yield and selectivity of the MSPD process Taking into account the strong hydrophilicity of acrylamide, besides the most commonly used ODS (C18 ), several polar sorbents were assayed namely florisil, silica gel, and the bonded phases PSA and aminopropylsilane (NH2 ) For all the experiments, 0.5 g of the samples (with added internal standard 13 C -AA) were dispersed with g of the sorbent, and after trans3 fer to an empty cartridge the elution was carried out with water under controlled vacuum It was found that the type of sorbent used had a big influence on the extraction Florisil was firstly eliminated because it did not permit the flow of water through the mixtures preventing the obtaining of results In opposition to our initial expectations, the C18 sorbent exhibited the best performance, not only in terms of extraction yield but also concerning the time required for this operation: when using PSA, NH2 or silica gel the elution of a ml volume needed 20 whereas with C18 this operation was carried out in less than Water was initially selected as the extraction solvent because of the acrylamide solubility in it at any temperature We decided not to use hot water to avoid the co-extraction of undesired components from the matrix The extraction volume was defined as recommended by Barker [24], ml of water divided in two aliquots of ml each with a soaking step between them We should note a lack was observed in the reproducibility not only when using only or ml (3 + 3) of water but also when the soaking step of was not performed The results obtained with the C18 sorbent were enhanced when a previous clean-up with 20 ml of n-hexane was performed before the extraction with water (Fig 2) We concluded that the removal of the fats (in potato chips for instance the quantity of fats correspond to approximately 40% in total weight) with nhexane, insures a more adequate contact of the water with the food matrices, improving the extraction yield of acrylamide Under the conditions established, greater sample/sorbent ratios were tested in order to increase the sensibility of the method However, the use of more than 0.5 g of sample for g of C18 provoked the clogging of the frits and the obstruction of the flow J.O Fernandes, C Soares / J Chromatogr A 1175 (2007) 1–6 Fig Chromatogram of a potato chip sample (0.5 g) mixed with g of C18 and: (A) previously deffated with 20 ml of n-hexane followed by extraction with ml + ml of water, (B) extracted with ml + ml of water Table AA concentration of the potato chips samples analysed using the two methods described Samples 10 11 12 13 14 15 16 17 Acrylamide concentration in chips (␮g kg−1 ) MSPD-GC/MS LE-GC/MS 412.70 241.80 186.42 480.35 440.55 471.18 928.74 284.03 459.01 507.73 478.90 1828.75 1052.96 785.22 1452.29 943.79 336.62 387.46 223.08 191.41 484.74 449.02 495.26 908.97 290.03 497.25 555.06 511.42 1890.86 1140.39 833.37 1513.61 993.96 364.62 All samples were prepared in duplicate and injected twice using both methods The developed MSPD-GC/MS method was evaluated in terms of linearity, precision, recovery, and limits of detection and quantification The obtained results are presented in the next sections The performance of the method was also evaluated by comparing the results obtained in the analysis of different samples of potato chips with the results obtained for the same samples when these were analysed by the liquid extraction method routinely used in our laboratory The obtained results are presented in Table 3.3 Method assessment 3.3.1 Linearity The linearity of the method was tested several times using standard (calibrating) solutions treated with the same method developed for the samples Usually, seven standards were simultaneously prepared and treated in parallel with a set of samples The range of concentrations corresponded to 0–1500 ␮g l−1 AA in the sample The quantity of added I.S was 0.25 ␮g, corresponding to 500 ␮g kg−1 in the sample for all samples and standards Calibration curves were constructed by plotting the AA/I.S area ratio against the concentration of AA in the standard The correlation coefficients were usually higher than 0.999 3.3.2 Precision To study the intraday precision three aliquots (0.5 g) of the same sample were prepared simultaneously and submitted to the overall method and injected in triplicate in the same day The RSD obtained was 2.6% To study the interday precision of the method, three aliquots of the same sample were submitted to the overall developed method in three different weeks and injected in triplicate in different days The RSD obtained was 5.5% 3.3.3 Recovery Six aliquots (0.5 g) of the same sample that presented 240 ␮g kg−1 of AA were prepared simultaneously To the samples 0, 0.025, 0.050, 0.125, 0.500 and 0.750 ␮g of acrylamide (0, 25, 50, 125, 500 and 750 ␮l of the mg l−1 AA solution) and 0.25 ␮g of I.S were added The samples were treated as described for the overall method and injected twice The AA recovery from the spiked solutions varied between 98% to 111% These samples were also used to test the linearity of the method in real samples The correlation coefficient obtained was higher than 0.998 and the concentration of the sample achieved by extrapolation of the curve was 233 ␮g kg−1 (Fig 3) 3.3.4 Limit of detection and limit of quantification The limit of detection (LOD) and the limit of the quantification (LOQ) of the method were calculated using the calibration curve parameters The LOD was established using LOD = 3.3 × (s/S) and the LOQ was established using LOQ = 10 × (s/S), where s is the standard deviation of the intercept and S is the slope of the curve [31,32] The LOD and the LOQ obtained for this method were 12.8 ␮g kg−1 and 38.8 ␮g kg−1 , respectively 6 J.O Fernandes, C Soares / J Chromatogr A 1175 (2007) 1–6 and more expeditious manner, which makes it an advantageous alternative for the routine analysis of acrylamide The validation of this methodology in other matrices, namely cereal products, chocolate and baby foodstuffs is currently being evaluated Acknowledgement The authors are thankful to FCT for financial support in the framework of the project POCI/AGR/61543/2004 References Fig Linearity of the MSPD-GC/MS method applied to a real sample Fig Graphical comparison of the two methods used to extract AA from the samples 3.3.5 Comparison between MSPD-GC/MS and LE-GC/MS methods All the samples analysed in this study were submitted to the described MSPD-GC/MS method and to the LE-GC/MS method routinely used in our laboratory The results obtained are presented in Table The comparison of the two methods was made using a regression line (Fig 4) In the y axis are the results obtained with the conventional method and in the x axis are the results for the MSPD method Analysing Table and Fig we can conclude that a close similarity of the results obtained by both methods Taking into account all the advantages described when using MSPD, this method can be used with confidence in the determination of acrylamide in potato chips Conclusions MSPD extraction technique was for the first time applied to the extraction of acrylamide from foodstuff (potato chips samples) The developed method employed C18 as dispersive agent and the extraction of acrylamide were accomplished by water The obtained extracts were further brominated and analysed by GC–MS The method exhibit noteworthy analytical features, namely the carrying out of the analysis in an easier, simpler [1] D.S Mottram, B.L Wedzicha, A.T Dodson, Nature 419 (2002) 448 [2] R.S Stadler, I Blank, N Varga, F Robert, J Hau, P.A Guy, M.-C Robert, S Riediker, Nature 419 (2002) 449 [3] E Tareke, P Rydberg, P Karlsson, S Eriksson, M Tornqvist, J Agric Food Chem 50 (2002) 4998 [4] K Skog, J Alexander (Eds.), Acrylamide and Other Hazardous Compounds in Heat-Treated Foods, CRC Press, 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Bromination of the samples and standards 2.6.1 Matrix solid- phase dispersion (MSPD-GC/MS) An aliquot of 0.5 g of the previously ground sample, g of C18 sorbent and 0.25 ␮g of I.S (25 ␮l of the. .. obtained results are presented in the next sections The performance of the method was also evaluated by comparing the results obtained in the analysis of different samples of potato chips with the