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Analysis of acrylamide in food samples by capillary zone electrophoresis
Journal of Chromatography A, 1120 (2006) 199–204
Analysis of acrylamide in food samples by capillary zone electrophoresis E. Bermudo, O. N´un˜ ez, L. Puignou ∗ , M.T. Galceran Department of Analytical Chemistry, University of Barcelona, Mart´ı i Franqu`es, 1-11, E-08028 Barcelona, Spain Available online 23 November 2005
Abstract Conditions for the determination of acrylamide (AA) after derivatisation with 2-mercaptobenzoic acid by capillary zone electrophoresis were established. A derivatisation reagent-acrylamide ratio of 35:1 was selected as optimum and the reagent excess was not removed as it did not affect the determination of acrylamide by CZE. The best separation was achieved using a 40 mM phosphate buffer at pH 8.0, working at 25 kV in un-coated fused silica capillaries. Linear calibration curves over the range studied (0.3–100 g mL−1 ), the limit of detection (0.07 g mL−1 ), and both run-to-run (RSD values of 5.8 and 2.2% for concentration at low and medium concentration levels, respectively) and day-to-day precisions (up to 11.2 and 6.7% at low and medium concentration levels, respectively) were established. Finally, the applicability of the CZE proposed methodology was demonstrated by analyzing levels of acrylamide present in different foodstuff products such as home made french fries, breakfast cereals and biscuits. © 2005 Elsevier B.V. All rights reserved. Keywords: Acrylamide; Capillary electrophoresis; Food
1. Introduction The discovery [1–3] that cooking various foods at high temperatures leads to the formation of acrylamide at levels as high as milligrams per kilogram in the case of carbohydrate-rich foodstuffs such as potatoes, has caused considerable alarm [4,5]. These findings have attracted considerable attention world-wide because acrylamide has been classified as “probably carcinogenic to humans” by the International Agency for Research on Cancer (IARC) [6]. Following this discovery, a world-wide surveillance of this substance in various food products was immediately started. In addition, a joint effort was undertaken by the national food authorities and the food industry in order to gain a better understanding of the mechanisms of formation and of contamination levels of acrylamide in food. Numerous mechanisms have been discussed, predominantly the thermal degradation of the free amino acid asparagine via an Amadori product involving reducing sugars such as fructose and glucose [7–9]. Consequently, the Maillard reaction is proposed as most probable mechanism for acrylamide formation.
∗
Corresponding author. Tel.: +34 93 402 19 15; fax: +34 93 402 12 33. E-mail address: [email protected] (L. Puignou).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.10.074
Several methods have been developed in the past decades to determine acrylamide in water, biological fluids and noncooked foods, and the majority are classical methods based on high performance liquid chromatography (LC) or gas chromatography (GC) techniques [1,10,11]. However, these methods as such are not appropriate for the analysis of acrylamide in processed foods due to the complexity of these matrices. Recently, more selective analytical methods have been published that are mainly based on mass spectrometry (MS) as the detection technique, coupled with a chromatographic separation either by LC or GC, the latter in most cases after derivatisation of the analytes [12–15]. However, due to the relatively high cost of mass spectrometric instrumentation, the application of this approach might be beyond the means of some laboratories. Thus, methods such as LC-UV [12,15–17] and microemulsion electrokinetic chromatography (MEEKC) [18] could be an alternative. MEEKC is an electrophoresis technique which offers the possibility of separating a wide range of neutral, acidic and basic compounds so it is a good technique to determine acrylamide. However, MEEKC presents a higher complexity of application than capillary zone electrophoresis (CZE). Nevertheless, due to the chemical characteristics of acrylamide (high polarity and difficult ionisation), this last technique has not yet been applied to the analysis of this compound.
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The purpose of the work presented here was to develop a CZE method for the determination of acrylamide using a thioether derivative, 2-mercaptobenzoic derivative, proposed by Jezussek and Schieberle [19]. The present paper reports the optimisation of the experimental conditions. The influence of different parameters such as the concentration and pH of the aqueous buffer on the analysis were studied. The limit of detection (LOD) and run-to-run and day-to-day precision values at two levels of concentration comprise the figures of merit determined to establish the robustness of the method. Finally, the CZE developed method was applied to the determination of acrylamide in different foodstuff products such as french fries, breakfast cereals and biscuits. 2. Experimental 2.1. Chemicals and consumables Acrylamide standard (>99%) was purchased from Fluka (Buchs, SG, Switzerland). All the reagents used were of analytical grade. Methanol and sodium hydroxide were provided by Merck (Darmstadt, Germany). Sodium dihydrogen phosphate and sodium monohydrogen phosphate anhydrous were also obtained from Fluka. 2-Mercaptobenzoic acid was used as derivatising reagent and it was purchased from Sigma–Aldrich (Steinheim, Germany). Stock solution of acrylamide (1 mg mL−1 ) was prepared by dilution with Milli-Q water. The stock solution was stored at 4 ◦ C for a maximum of 4 weeks. Solid phase extraction (SPE) cartridges Strata-X-C (200 mg, 6 mL) and Isolute ENV+ (200 mg, 3 mL) were provided by Phenomenex (Torrance, USA) and IST (Hengoed, Mid-Glamorgan, UK), respectively. Syringe filters 0.45 m of nylon and nitrocellulose were purchased from Teknokroma (Barcelona, Spain). Water was purified with an Elix/Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Instrumentation The experiments were performed on a Beckman P/ACE 5500 capillary electrophoresis system (Fullerton, CA, USA) equipped with a diode array detection system. The electrophoretic separations were carried out using uncoated fused-silica capillaries (Beckman) with a total length of 57 cm (50 cm effective length) and 75 m I.D. A 40 mM phosphate buffer (pH 8.0) was used as carrier electrolyte. The buffer was filtered through a 0.45 m membrane filter, and degassed by sonication before use. A CE voltage of +25 kV was applied and the temperature was held at 25 ◦ C. Direct detection was carried out at 210 nm. Samples were loaded by hydrodynamic injection pressure assisted (3.5 kPa) during 5 s. The CE instrument was controlled using a Beckman P/ACE station software version 1.2. New capillaries were pre-treated by rinsing with 1 M sodium hydroxide for 30 min and water for 30 min, before being conditioned with the carrier electrolyte. At the beginning of each session, the capillary was rinsed with 1 M sodium hydroxide for 15 min, water for 15 min and finally with the carrier electrolyte
during 30 min. Between runs, the capillary was conditioned with carrier electrolyte during 5 min. 2.3. Sample preparation and clean-up procedure Samples were ground and homogenized using a supermixer blender system (Moulinex, Lyon, France) and an Ultraturrax T25 basic (IKA-Werke, Staufen, Germany), respectively. Subsamples of 2 g (1 g for potatoes samples) were weighed into 15 mL centrifuge tubes and 10 mL water was added. Each tube was shaken for 1 h on a rotating shaker (Rotatory Mixer 34526; Breda Scientific, Breda, The Netherlands). Then, the tubes were centrifuged at 4000 rpm for 30 min with a Selecta Centronic centrifuge (Selecta, Barcelona, Spain). In the case of french fries samples, centrifugation of 20,000 rpm was necessary and a Allegra 64R (Beckman, Fullerton, CA, USA) was used. A 5 mL aliquot of aqueous solution was filtered through a syringe filter of nitrocellulose (0.45 m). The defatting was carried out by adding 2 mL of hexane, and the organic phase was then removed. For clean-up, Strata-X-C SPE cartridges were conditioned with 4 mL of methanol followed by 4 mL of water. The cartridge was loaded with 3 mL of previous filtered extract. The extract was allowed to pass through the sorbent material. Then, the column was eluted with 3 mL of water and the eluent was collected for ENV+ SPE clean-up. These cartridges were previously conditioned with 3 mL of methanol followed by 3 mL of water. All of the eluant collected from the Strata-X-C was loaded and eluted with 1 mL of methanol:water (60:40). The eluent was filtered through a 0.45 m nylon filter and transferred into a glass vial for derivatisation. 2.4. Acrylamide derivatisation For derivatisation, the procedure described by Jezussek and Schieberle [19] was followed by including some modifications. Briefly, the extract mentioned above (1 mL) was adjusted to pH 8.0 by adding 5 L of 0.01 M sodium hydroxide. Then 100 L of a solution of 2-mercaptobenzoic acid in aqueous sodium hydroxide (154 mg in 10 mL of 1 M sodium hydroxide) was added for derivatisation, the solution was degassed with nitrogen gas to remove oxygen, and the reaction mixture was stirred in the dark for 3 h. Finally, the solution was filtered through a 0.2 m nylon filter and injected into the CE system. 3. Results and discussion 3.1. Acrylamide derivatisation and electrophoretic conditions As there is no previous work dealing with the determination of acrylamide (AA) by capillary zone electrophoresis, a preliminary study was performed using a 40 mM phosphate buffer (pH 7.0) as carrier electrolyte. Under these conditions, acrylamide derivatisation was studied following the procedure described by Jezussek and Schieberle [19]. In this procedure, the excess of the derivatising reagent (2-mercaptobenzoic acid) was removed by treatment with lead(II) acetate (a saturated solution
E. Bermudo et al. / J. Chromatogr. A 1120 (2006) 199–204
in water), and after centrifugation the supernatant was extracted with ethyl acetate. The organic phase was then dried over anhydrous Na2 SO4 and the solvent was removed. Finally, the residue was re-dissolved in an adequate media for injection. However, in order to reduce and simplify the derivatisation, we did not perform the extraction with ethyl acetate but instead directly injected the solution after treatment with lead(II) acetate. Nevertheless, following this procedure we observed that electric discharges occurred after performing three analyses by CZE, which prevented us from continuing with the analysis as the CE system caused the electrophoretic voltage to fail. When this occurred, we observed that a solid suspension in the inlet vial appeared that we attributed to the formation of less soluble lead(II) phosphate salts. At this point, we decided to simplify further the derivatisation procedure by suppressing the removal of 2-mercaptobenzoic acid step. So, after derivatisation with 2mercaptobenzoic acid the solution was directly injected in the CZE system. Fig. 1 shows the electropherogram obtained for an acrylamide standard of 20 g mL−1 . As can be seen, the excess of derivatisation reagent was not a problem as its signal appeared more than 3 min after the acrylamide peak. So, after this study we have simplified the AA derivatisation by removing the last steps of the procedure previously proposed [19], achieving a high recovery of the acrylamide derivative and a less time consuming procedure. The ratio derivatising reagent-acrylamide was also studied. For this purpose, 5 mL of a 20 g mL−1 AA standard solution was derivatised by adding different volumes (from 100 to 1000 L) of a derivatisation reagent solution (15.4 mg mL−1 ). Appropriate volumes of water were added in order to fix the total experimental volume to 6 mL and avoid a possible dilution effect. Fig. 2 shows the signal obtained for the AA derivative by CZE for derivatising reagent-acrylamide ratios ranging from 7:1 to 71:1. As can be seen, the AA derivatisation efficiency increased with the amount of 2-mercaptobenzoic acid. However, for derivatising reagent-acrylamide ratio higher than 35:1 a slight improvement was only achieved, so we selected 35:1 as optimal ratio for AA derivatisation.
201
Fig. 2. Effect of derivatisation reagent:acrylamide ratio on the acrylamide derivatisation process.
Once the acrylamide derivatisation was established, the electrophoretic conditions were optimised in order to improve the AA signal and reduce the analysis time. For this purpose, a phosphate buffer was used and its concentration was studied from 10 to 60 mM, and the electropherograms obtained are given in Fig. 3a. As can be seen, the AA derivative signal improved with the buffer concentration, although an important increase in the analysis time due to the reduction in the electroosmotic flow was observed. Moreover, at buffer concentrations higher than 40 mM the tail of acrylamide peak increased. As no variation in the AA relative signal was observed from 40 mM, this concentration buffer was chosen as the optimum value. The buffer pH was studied from 6.5 to 8.0 and the electropherograms obtained for each pH condition are given in Fig. 3b. As can be seen, no variation in the AA relative signal was observed although a slightly decrease in migration time was achieved when pH was raised, choosing pH 8.0 as optimum. In conclusion, a 40 mM phosphate buffer (pH 8.0) was selected as optimum carrier electrolyte for the CZE determination of AA. 3.2. Quality parameters Quality parameters using the optimized CZE method were calculated and the figures of merit are given in Table 1, where they are compared with those obtained by MEEKC [18]. The limit of detection (LOD), based on a signal-to-noise ratio of 3:1, was equivalent to a concentration of 0.07 g mL−1 , which correTable 1 Instrumental quality parameters Parameter
MEEKCa
LODs Concentration (g mL−1 ) Amount injected (pg)
0.7 10
0.07 1
– 3.4
5.8 2.2
– 11.6
11.2 6.7
Run-to-run precision (%RSD, n = 5) Low level (LOQ) Medium level (1 g mL−1 ) Fig. 1. Electropherogram of a 20 g mL−1 Acrylamide derivatised with 2mercaptobenzoic acid. Carrier electrolyte: 40 mM monohydrogen phosphatedihydrogen phosphate buffer (pH 7.0). Capillary voltage: +25 kV. Sample loaded by hydrodynamic injection (5 s, 3.5 kPa). Acquisition wavelength, 210 nm.
Day-to-day precision (%RSD, n = 5 × 3) Low level (LOQ) Medium level (1 g mL−1 ) a
Ref. [17].
CZE
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sponded to an injected mass of 1 pg (14 fmol). This LOD was 10 times lower than that previously obtained using MEEKC, with the advantage that the proposed CZE method is simpler and easier to apply. The calibration curve based on the peak area at concentrations between 0.3 and 100 g mL−1 was calculated and good linearity was obtained with a correlation coefficient higher than 0.999. For run-to-run precision, five replicate determinations of an acrylamide standard solution previously derivatised was carried out under optimum conditions, while day-to-day precision was calculated by performing 15 replicate determinations of the same solution in 3 days (five replicates each day). Precisions were calculated at two concentration levels (see Table 1). The relative standard deviation (RSD) obtained for run-to-run precision was 2.2% for medium concentration level and 5.8% for low concentration level (limit of quantitation, LOQ, four times LOD). The RSD value obtained for day-to-day precision were higher than those of the run-to-run precision but always lower than 11.2%. As can be seen when comparing the results using the medium concentration level (Table 1), the proposed CZE method had better precisions than those of the MEEKC method (6.7% compared with 11.6% for day-to-day precision), confirming the lower complexity of the methodology used in this work. From these results it can also be concluded that the derivatisation step added in this methodology did not negatively affect the precision of the present CZE method. 3.3. Application
Fig. 3. Effect of (a) buffer concentration and (b) buffer pH, on the determination of AA by CZE. Electrolyte: monohydrogen phosphate–dihydrogen phosphate buffer. Capillary voltage, +25 kV. Acquisition wavelength, 210 nm. Sample: acrylamide standard of 20 g mL−1 derivatised with 2-mercaptobenzoic. Sample loaded by hydrodynamic injection (5 s, 3.5 kPa).
In order to evaluate the applicability of the CZE method for the determination of acrylamide in food samples, the analysis of three foodstuffs was performed following the procedure described in Section 2. Several products such as brownishcoloured home made french fries, breakfast cereals, and biscuits were analysed, all of which are likely to contain higher levels of acrylamide than other industrial foods [20,21]. The extracted AA was quantified by the standard addition and external calibration methods. In the first case, three unspiked and four samples spiked at different concentration levels (100, 200, 300 and 500%)
Fig. 4. Electropherograms of (a) biscuit sample and spiked biscuit sample at a level of 300% and (b) home made french fries sample and the spiked sample at a level of 500%, obtained by CZE after acrylamide derivatisation with 2-mercaptobenzoic acid. Carrier electrolyte: 40 mM monohydrogen phosphate–dihydrogen phosphate buffer (pH 8.0). Other electrophoretic conditions as in Fig. 1.
E. Bermudo et al. / J. Chromatogr. A 1120 (2006) 199–204
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Table 2 Determination of acrylamide in different food products by CZE. Food product
External calibration AA concentration
Home made french fries potatoes Breakfast cereals Biscuits
(mg kg−1 )
4.75 1.33 3.28
were prepared by addition of accurately measured amounts of an acrylamide standard at the beginning of the clean-up process. Recoveries of this compound were estimated from the slope of the regression line of the added amount versus the measured amount. Fig. 4 shows, as an example, the electropherograms obtained by the proposed CZE method of a biscuit sample and a home made french fries sample, as well as the electropherograms of the same samples fortified at a 300% level (Fig. 4a) and at a 500% level (Fig. 4b), showing that no interferences appeared which allow to correctly quantify acrylamide peak. Samples were analyzed by triplicate using both quantification methods, and the results obtained are given in Table 2. A statistical paired-sample comparison analysis was performed with the results obtained using both quantification methods. For a 95% confidence level the results achieved were not significantly different so external calibration could be proposed as a fast and reliable method to quantify AA by CZE. From standard addition results, the recovery of the method was estimated and it was observed to be in the range 85–100% depending on sample. The AA concentration found in the food products analysed is in agreement with the values described for similar food samples. For instance, acrylamide levels ranging from 0.2 to 12 mg kg−1 have been reported in french fries [20], and the European Commission described levels in the range of 0.005–5.2 mg kg−1 , 0.005–3.3 mg kg−1 and 0.002–1.54 mg kg−1 for potato chips, biscuits and breakfast cereals, respectively [21]. So, the method developed in this work can be proposed for the analysis of acrylamide in foodstuff products with a high risk of causing acrylamide contamination in the industrial or homemade preparation of such products.
4. Conclusions A CZE method has been developed for the determination of acrylamide after derivatisation with 2-mercaptobenzoic acid in foodstuffs products. The previously established derivatisation procedure was improved by reducing several steps, allowing direct injection of AA derivative into the CZE system without the need to remove reagent excess. With this method, a limit of detection of 0.07 g mL−1 was obtained which involves a 10-fold enhancement when compared with that obtained by MEEKC. Good linearity (r2 > 0.999) and run-to-run and day-today precisions (RSD lower than 5.8 and 11.2%, respectively) were achieved. The addition of a derivatisation step did not negatively affect the precision of the established methodology. The results obtained show that the method can be used for quantita-
Standard addition Standard deviation
AA concentration (mg kg−1 )
Standard deviation
0.08 0.17 0.17
4.56 1.40 3.23
0.08 0.25 0.29
tive purposes in foodstuffs products. The application of CZE for the determination of acrylamide in french fries, breakfast cereals and biscuits, using external calibration and standard addition methods, has been demonstrated. As a consequence, external calibration can be successfully selected as a good strategy for the determination of AA in a large variety of samples. This less time-consuming method becomes especially appropriate for a routine screening of foodstuffs with a high risk of AA contamination due to their thermal processing. Acknowledgements This work has been carried out with support from the European Commission, Priority 5 on Food Quality and Safety (contract no. FOOD-CT-2003-506820 Specific Targeted Project), “Heat-generated food toxicants-identification, characterisation and risk minimisation”. This publication reflects the author’s views and not necessarily those of the EC. The information in this document is provided as is and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof use the information at its sole risk and liability. References [1] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. T¨ornqvist, Chem. Res. Toxicol. 13 (2000) 517. [2] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. T¨ornqvist, J. Agric. Food Chem. 50 (2002) 4998. [3] Swedish National Food Administration, www.slv.se/Download/ Document/approvedDocs/enginformationakryl.htm, 24 April 2002. [4] World Helath Organisation, Health Implications of Acrylamide in Food, Report of a Joint FAO/WHO Consultation, Department of Protection of the Human Environament, WHO, Geneva, Switzerland, 25–27 June 2002. [5] J. Ros´en, K.E. Hellen¨as, Analyst 127 (2002) 880. [6] IARC Monographs on the Evaluation of Carcinogen Risk to Humans, vol. 60, International Agency for Research on Cancer, Lyon, 1994, p. 389. [7] A. Becalski, B.P.Y. Lau, D. Lewis, S.W. Seaman, J. Agric. Food Chem. 51 (2003) 802. [8] T.M. Amrein, S. Bachmann, A. Noti, M. Biedermann, M. Ferraz Barbosa, S. Biedermann-Brem, K. Grob, A. Keiser, P. Realini, F. Escher, R. Amad´o, J. Agric. Food Chem. 51 (2003) 5556. [9] V.A. Yaylayan, R.H. Stadler, J. AOAC Int. 88 (2005) 262. [10] L. Castle, J. Agric. Food Chem. 41 (1993) 1261. [11] J. Tekel, P. Farkas, M. Kovac, Food Add. Contam. 6 (1989) 377. [12] T. Wenzl, M.B. de la Calle, E. Anklam, Food Add. Contam. 20 (2003) 885. [13] L. Castle, S. Eriksson, J. AOAC Int. 88 (2005) 274. [14] R. Weisshaar, Eur. J. Lipid Sci. Technol. 106 (2004) 786. [15] Y. Zhang, G. Zhang, Y. Zhang, J. Chromatogr. A 1075 (2005) 1.
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[16] S. Cavalli, R. Maurer, F. Hoefler, LC-GC Eur. 2 (2003) 9. [17] H. Terada, Y. Tamura, Shokuhin Eiseigaku Zasshi 44 (2003) 303. [18] E. Bermudo, V. Ruiz-Calero, L. Puignou, M.T. Galceran, Electrophoresis 25 (2004) 3257. [19] M. Jezussek, P. Schieberle, J. Agric. Food Chem. 51 (2003) 7866.
[20] M. Friedman, J. Agric. Food Chem. 51 (2003) 4504. [21] The EU monitoring database of acrylamide levels in food status December 2004, European Commission, Directorate-General, Joint Research Centre, Institute for Reference Materials and Measurements, Retieseweg, Geel, Belgium, http://www.irmm.jrc.be.
Analysis of acrylamide in food samples by capillary zone electrophoresis E. Bermudo, O. N´un˜ ez, L. Puignou ∗ , M.T. Galceran Department of Analytical Chemistry, University of Barcelona, Mart´ı i Franqu`es, 1-11, E-08028 Barcelona, Spain Available online 23 November 2005
Abstract Conditions for the determination of acrylamide (AA) after derivatisation with 2-mercaptobenzoic acid by capillary zone electrophoresis were established. A derivatisation reagent-acrylamide ratio of 35:1 was selected as optimum and the reagent excess was not removed as it did not affect the determination of acrylamide by CZE. The best separation was achieved using a 40 mM phosphate buffer at pH 8.0, working at 25 kV in un-coated fused silica capillaries. Linear calibration curves over the range studied (0.3–100 g mL−1 ), the limit of detection (0.07 g mL−1 ), and both run-to-run (RSD values of 5.8 and 2.2% for concentration at low and medium concentration levels, respectively) and day-to-day precisions (up to 11.2 and 6.7% at low and medium concentration levels, respectively) were established. Finally, the applicability of the CZE proposed methodology was demonstrated by analyzing levels of acrylamide present in different foodstuff products such as home made french fries, breakfast cereals and biscuits. © 2005 Elsevier B.V. All rights reserved. Keywords: Acrylamide; Capillary electrophoresis; Food
1. Introduction The discovery [1–3] that cooking various foods at high temperatures leads to the formation of acrylamide at levels as high as milligrams per kilogram in the case of carbohydrate-rich foodstuffs such as potatoes, has caused considerable alarm [4,5]. These findings have attracted considerable attention world-wide because acrylamide has been classified as “probably carcinogenic to humans” by the International Agency for Research on Cancer (IARC) [6]. Following this discovery, a world-wide surveillance of this substance in various food products was immediately started. In addition, a joint effort was undertaken by the national food authorities and the food industry in order to gain a better understanding of the mechanisms of formation and of contamination levels of acrylamide in food. Numerous mechanisms have been discussed, predominantly the thermal degradation of the free amino acid asparagine via an Amadori product involving reducing sugars such as fructose and glucose [7–9]. Consequently, the Maillard reaction is proposed as most probable mechanism for acrylamide formation.
∗
Corresponding author. Tel.: +34 93 402 19 15; fax: +34 93 402 12 33. E-mail address: [email protected] (L. Puignou).
0021-9673/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2005.10.074
Several methods have been developed in the past decades to determine acrylamide in water, biological fluids and noncooked foods, and the majority are classical methods based on high performance liquid chromatography (LC) or gas chromatography (GC) techniques [1,10,11]. However, these methods as such are not appropriate for the analysis of acrylamide in processed foods due to the complexity of these matrices. Recently, more selective analytical methods have been published that are mainly based on mass spectrometry (MS) as the detection technique, coupled with a chromatographic separation either by LC or GC, the latter in most cases after derivatisation of the analytes [12–15]. However, due to the relatively high cost of mass spectrometric instrumentation, the application of this approach might be beyond the means of some laboratories. Thus, methods such as LC-UV [12,15–17] and microemulsion electrokinetic chromatography (MEEKC) [18] could be an alternative. MEEKC is an electrophoresis technique which offers the possibility of separating a wide range of neutral, acidic and basic compounds so it is a good technique to determine acrylamide. However, MEEKC presents a higher complexity of application than capillary zone electrophoresis (CZE). Nevertheless, due to the chemical characteristics of acrylamide (high polarity and difficult ionisation), this last technique has not yet been applied to the analysis of this compound.
200
E. Bermudo et al. / J. Chromatogr. A 1120 (2006) 199–204
The purpose of the work presented here was to develop a CZE method for the determination of acrylamide using a thioether derivative, 2-mercaptobenzoic derivative, proposed by Jezussek and Schieberle [19]. The present paper reports the optimisation of the experimental conditions. The influence of different parameters such as the concentration and pH of the aqueous buffer on the analysis were studied. The limit of detection (LOD) and run-to-run and day-to-day precision values at two levels of concentration comprise the figures of merit determined to establish the robustness of the method. Finally, the CZE developed method was applied to the determination of acrylamide in different foodstuff products such as french fries, breakfast cereals and biscuits. 2. Experimental 2.1. Chemicals and consumables Acrylamide standard (>99%) was purchased from Fluka (Buchs, SG, Switzerland). All the reagents used were of analytical grade. Methanol and sodium hydroxide were provided by Merck (Darmstadt, Germany). Sodium dihydrogen phosphate and sodium monohydrogen phosphate anhydrous were also obtained from Fluka. 2-Mercaptobenzoic acid was used as derivatising reagent and it was purchased from Sigma–Aldrich (Steinheim, Germany). Stock solution of acrylamide (1 mg mL−1 ) was prepared by dilution with Milli-Q water. The stock solution was stored at 4 ◦ C for a maximum of 4 weeks. Solid phase extraction (SPE) cartridges Strata-X-C (200 mg, 6 mL) and Isolute ENV+ (200 mg, 3 mL) were provided by Phenomenex (Torrance, USA) and IST (Hengoed, Mid-Glamorgan, UK), respectively. Syringe filters 0.45 m of nylon and nitrocellulose were purchased from Teknokroma (Barcelona, Spain). Water was purified with an Elix/Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. Instrumentation The experiments were performed on a Beckman P/ACE 5500 capillary electrophoresis system (Fullerton, CA, USA) equipped with a diode array detection system. The electrophoretic separations were carried out using uncoated fused-silica capillaries (Beckman) with a total length of 57 cm (50 cm effective length) and 75 m I.D. A 40 mM phosphate buffer (pH 8.0) was used as carrier electrolyte. The buffer was filtered through a 0.45 m membrane filter, and degassed by sonication before use. A CE voltage of +25 kV was applied and the temperature was held at 25 ◦ C. Direct detection was carried out at 210 nm. Samples were loaded by hydrodynamic injection pressure assisted (3.5 kPa) during 5 s. The CE instrument was controlled using a Beckman P/ACE station software version 1.2. New capillaries were pre-treated by rinsing with 1 M sodium hydroxide for 30 min and water for 30 min, before being conditioned with the carrier electrolyte. At the beginning of each session, the capillary was rinsed with 1 M sodium hydroxide for 15 min, water for 15 min and finally with the carrier electrolyte
during 30 min. Between runs, the capillary was conditioned with carrier electrolyte during 5 min. 2.3. Sample preparation and clean-up procedure Samples were ground and homogenized using a supermixer blender system (Moulinex, Lyon, France) and an Ultraturrax T25 basic (IKA-Werke, Staufen, Germany), respectively. Subsamples of 2 g (1 g for potatoes samples) were weighed into 15 mL centrifuge tubes and 10 mL water was added. Each tube was shaken for 1 h on a rotating shaker (Rotatory Mixer 34526; Breda Scientific, Breda, The Netherlands). Then, the tubes were centrifuged at 4000 rpm for 30 min with a Selecta Centronic centrifuge (Selecta, Barcelona, Spain). In the case of french fries samples, centrifugation of 20,000 rpm was necessary and a Allegra 64R (Beckman, Fullerton, CA, USA) was used. A 5 mL aliquot of aqueous solution was filtered through a syringe filter of nitrocellulose (0.45 m). The defatting was carried out by adding 2 mL of hexane, and the organic phase was then removed. For clean-up, Strata-X-C SPE cartridges were conditioned with 4 mL of methanol followed by 4 mL of water. The cartridge was loaded with 3 mL of previous filtered extract. The extract was allowed to pass through the sorbent material. Then, the column was eluted with 3 mL of water and the eluent was collected for ENV+ SPE clean-up. These cartridges were previously conditioned with 3 mL of methanol followed by 3 mL of water. All of the eluant collected from the Strata-X-C was loaded and eluted with 1 mL of methanol:water (60:40). The eluent was filtered through a 0.45 m nylon filter and transferred into a glass vial for derivatisation. 2.4. Acrylamide derivatisation For derivatisation, the procedure described by Jezussek and Schieberle [19] was followed by including some modifications. Briefly, the extract mentioned above (1 mL) was adjusted to pH 8.0 by adding 5 L of 0.01 M sodium hydroxide. Then 100 L of a solution of 2-mercaptobenzoic acid in aqueous sodium hydroxide (154 mg in 10 mL of 1 M sodium hydroxide) was added for derivatisation, the solution was degassed with nitrogen gas to remove oxygen, and the reaction mixture was stirred in the dark for 3 h. Finally, the solution was filtered through a 0.2 m nylon filter and injected into the CE system. 3. Results and discussion 3.1. Acrylamide derivatisation and electrophoretic conditions As there is no previous work dealing with the determination of acrylamide (AA) by capillary zone electrophoresis, a preliminary study was performed using a 40 mM phosphate buffer (pH 7.0) as carrier electrolyte. Under these conditions, acrylamide derivatisation was studied following the procedure described by Jezussek and Schieberle [19]. In this procedure, the excess of the derivatising reagent (2-mercaptobenzoic acid) was removed by treatment with lead(II) acetate (a saturated solution
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in water), and after centrifugation the supernatant was extracted with ethyl acetate. The organic phase was then dried over anhydrous Na2 SO4 and the solvent was removed. Finally, the residue was re-dissolved in an adequate media for injection. However, in order to reduce and simplify the derivatisation, we did not perform the extraction with ethyl acetate but instead directly injected the solution after treatment with lead(II) acetate. Nevertheless, following this procedure we observed that electric discharges occurred after performing three analyses by CZE, which prevented us from continuing with the analysis as the CE system caused the electrophoretic voltage to fail. When this occurred, we observed that a solid suspension in the inlet vial appeared that we attributed to the formation of less soluble lead(II) phosphate salts. At this point, we decided to simplify further the derivatisation procedure by suppressing the removal of 2-mercaptobenzoic acid step. So, after derivatisation with 2mercaptobenzoic acid the solution was directly injected in the CZE system. Fig. 1 shows the electropherogram obtained for an acrylamide standard of 20 g mL−1 . As can be seen, the excess of derivatisation reagent was not a problem as its signal appeared more than 3 min after the acrylamide peak. So, after this study we have simplified the AA derivatisation by removing the last steps of the procedure previously proposed [19], achieving a high recovery of the acrylamide derivative and a less time consuming procedure. The ratio derivatising reagent-acrylamide was also studied. For this purpose, 5 mL of a 20 g mL−1 AA standard solution was derivatised by adding different volumes (from 100 to 1000 L) of a derivatisation reagent solution (15.4 mg mL−1 ). Appropriate volumes of water were added in order to fix the total experimental volume to 6 mL and avoid a possible dilution effect. Fig. 2 shows the signal obtained for the AA derivative by CZE for derivatising reagent-acrylamide ratios ranging from 7:1 to 71:1. As can be seen, the AA derivatisation efficiency increased with the amount of 2-mercaptobenzoic acid. However, for derivatising reagent-acrylamide ratio higher than 35:1 a slight improvement was only achieved, so we selected 35:1 as optimal ratio for AA derivatisation.
201
Fig. 2. Effect of derivatisation reagent:acrylamide ratio on the acrylamide derivatisation process.
Once the acrylamide derivatisation was established, the electrophoretic conditions were optimised in order to improve the AA signal and reduce the analysis time. For this purpose, a phosphate buffer was used and its concentration was studied from 10 to 60 mM, and the electropherograms obtained are given in Fig. 3a. As can be seen, the AA derivative signal improved with the buffer concentration, although an important increase in the analysis time due to the reduction in the electroosmotic flow was observed. Moreover, at buffer concentrations higher than 40 mM the tail of acrylamide peak increased. As no variation in the AA relative signal was observed from 40 mM, this concentration buffer was chosen as the optimum value. The buffer pH was studied from 6.5 to 8.0 and the electropherograms obtained for each pH condition are given in Fig. 3b. As can be seen, no variation in the AA relative signal was observed although a slightly decrease in migration time was achieved when pH was raised, choosing pH 8.0 as optimum. In conclusion, a 40 mM phosphate buffer (pH 8.0) was selected as optimum carrier electrolyte for the CZE determination of AA. 3.2. Quality parameters Quality parameters using the optimized CZE method were calculated and the figures of merit are given in Table 1, where they are compared with those obtained by MEEKC [18]. The limit of detection (LOD), based on a signal-to-noise ratio of 3:1, was equivalent to a concentration of 0.07 g mL−1 , which correTable 1 Instrumental quality parameters Parameter
MEEKCa
LODs Concentration (g mL−1 ) Amount injected (pg)
0.7 10
0.07 1
– 3.4
5.8 2.2
– 11.6
11.2 6.7
Run-to-run precision (%RSD, n = 5) Low level (LOQ) Medium level (1 g mL−1 ) Fig. 1. Electropherogram of a 20 g mL−1 Acrylamide derivatised with 2mercaptobenzoic acid. Carrier electrolyte: 40 mM monohydrogen phosphatedihydrogen phosphate buffer (pH 7.0). Capillary voltage: +25 kV. Sample loaded by hydrodynamic injection (5 s, 3.5 kPa). Acquisition wavelength, 210 nm.
Day-to-day precision (%RSD, n = 5 × 3) Low level (LOQ) Medium level (1 g mL−1 ) a
Ref. [17].
CZE
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sponded to an injected mass of 1 pg (14 fmol). This LOD was 10 times lower than that previously obtained using MEEKC, with the advantage that the proposed CZE method is simpler and easier to apply. The calibration curve based on the peak area at concentrations between 0.3 and 100 g mL−1 was calculated and good linearity was obtained with a correlation coefficient higher than 0.999. For run-to-run precision, five replicate determinations of an acrylamide standard solution previously derivatised was carried out under optimum conditions, while day-to-day precision was calculated by performing 15 replicate determinations of the same solution in 3 days (five replicates each day). Precisions were calculated at two concentration levels (see Table 1). The relative standard deviation (RSD) obtained for run-to-run precision was 2.2% for medium concentration level and 5.8% for low concentration level (limit of quantitation, LOQ, four times LOD). The RSD value obtained for day-to-day precision were higher than those of the run-to-run precision but always lower than 11.2%. As can be seen when comparing the results using the medium concentration level (Table 1), the proposed CZE method had better precisions than those of the MEEKC method (6.7% compared with 11.6% for day-to-day precision), confirming the lower complexity of the methodology used in this work. From these results it can also be concluded that the derivatisation step added in this methodology did not negatively affect the precision of the present CZE method. 3.3. Application
Fig. 3. Effect of (a) buffer concentration and (b) buffer pH, on the determination of AA by CZE. Electrolyte: monohydrogen phosphate–dihydrogen phosphate buffer. Capillary voltage, +25 kV. Acquisition wavelength, 210 nm. Sample: acrylamide standard of 20 g mL−1 derivatised with 2-mercaptobenzoic. Sample loaded by hydrodynamic injection (5 s, 3.5 kPa).
In order to evaluate the applicability of the CZE method for the determination of acrylamide in food samples, the analysis of three foodstuffs was performed following the procedure described in Section 2. Several products such as brownishcoloured home made french fries, breakfast cereals, and biscuits were analysed, all of which are likely to contain higher levels of acrylamide than other industrial foods [20,21]. The extracted AA was quantified by the standard addition and external calibration methods. In the first case, three unspiked and four samples spiked at different concentration levels (100, 200, 300 and 500%)
Fig. 4. Electropherograms of (a) biscuit sample and spiked biscuit sample at a level of 300% and (b) home made french fries sample and the spiked sample at a level of 500%, obtained by CZE after acrylamide derivatisation with 2-mercaptobenzoic acid. Carrier electrolyte: 40 mM monohydrogen phosphate–dihydrogen phosphate buffer (pH 8.0). Other electrophoretic conditions as in Fig. 1.
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Table 2 Determination of acrylamide in different food products by CZE. Food product
External calibration AA concentration
Home made french fries potatoes Breakfast cereals Biscuits
(mg kg−1 )
4.75 1.33 3.28
were prepared by addition of accurately measured amounts of an acrylamide standard at the beginning of the clean-up process. Recoveries of this compound were estimated from the slope of the regression line of the added amount versus the measured amount. Fig. 4 shows, as an example, the electropherograms obtained by the proposed CZE method of a biscuit sample and a home made french fries sample, as well as the electropherograms of the same samples fortified at a 300% level (Fig. 4a) and at a 500% level (Fig. 4b), showing that no interferences appeared which allow to correctly quantify acrylamide peak. Samples were analyzed by triplicate using both quantification methods, and the results obtained are given in Table 2. A statistical paired-sample comparison analysis was performed with the results obtained using both quantification methods. For a 95% confidence level the results achieved were not significantly different so external calibration could be proposed as a fast and reliable method to quantify AA by CZE. From standard addition results, the recovery of the method was estimated and it was observed to be in the range 85–100% depending on sample. The AA concentration found in the food products analysed is in agreement with the values described for similar food samples. For instance, acrylamide levels ranging from 0.2 to 12 mg kg−1 have been reported in french fries [20], and the European Commission described levels in the range of 0.005–5.2 mg kg−1 , 0.005–3.3 mg kg−1 and 0.002–1.54 mg kg−1 for potato chips, biscuits and breakfast cereals, respectively [21]. So, the method developed in this work can be proposed for the analysis of acrylamide in foodstuff products with a high risk of causing acrylamide contamination in the industrial or homemade preparation of such products.
4. Conclusions A CZE method has been developed for the determination of acrylamide after derivatisation with 2-mercaptobenzoic acid in foodstuffs products. The previously established derivatisation procedure was improved by reducing several steps, allowing direct injection of AA derivative into the CZE system without the need to remove reagent excess. With this method, a limit of detection of 0.07 g mL−1 was obtained which involves a 10-fold enhancement when compared with that obtained by MEEKC. Good linearity (r2 > 0.999) and run-to-run and day-today precisions (RSD lower than 5.8 and 11.2%, respectively) were achieved. The addition of a derivatisation step did not negatively affect the precision of the established methodology. The results obtained show that the method can be used for quantita-
Standard addition Standard deviation
AA concentration (mg kg−1 )
Standard deviation
0.08 0.17 0.17
4.56 1.40 3.23
0.08 0.25 0.29
tive purposes in foodstuffs products. The application of CZE for the determination of acrylamide in french fries, breakfast cereals and biscuits, using external calibration and standard addition methods, has been demonstrated. As a consequence, external calibration can be successfully selected as a good strategy for the determination of AA in a large variety of samples. This less time-consuming method becomes especially appropriate for a routine screening of foodstuffs with a high risk of AA contamination due to their thermal processing. Acknowledgements This work has been carried out with support from the European Commission, Priority 5 on Food Quality and Safety (contract no. FOOD-CT-2003-506820 Specific Targeted Project), “Heat-generated food toxicants-identification, characterisation and risk minimisation”. This publication reflects the author’s views and not necessarily those of the EC. The information in this document is provided as is and no guarantee or warranty is given that the information is fit for any particular purpose. The user thereof use the information at its sole risk and liability. References [1] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. T¨ornqvist, Chem. Res. Toxicol. 13 (2000) 517. [2] E. Tareke, P. Rydberg, P. Karlsson, S. Eriksson, M. T¨ornqvist, J. Agric. Food Chem. 50 (2002) 4998. [3] Swedish National Food Administration, www.slv.se/Download/ Document/approvedDocs/enginformationakryl.htm, 24 April 2002. [4] World Helath Organisation, Health Implications of Acrylamide in Food, Report of a Joint FAO/WHO Consultation, Department of Protection of the Human Environament, WHO, Geneva, Switzerland, 25–27 June 2002. [5] J. Ros´en, K.E. Hellen¨as, Analyst 127 (2002) 880. [6] IARC Monographs on the Evaluation of Carcinogen Risk to Humans, vol. 60, International Agency for Research on Cancer, Lyon, 1994, p. 389. [7] A. Becalski, B.P.Y. Lau, D. Lewis, S.W. Seaman, J. Agric. Food Chem. 51 (2003) 802. [8] T.M. Amrein, S. Bachmann, A. Noti, M. Biedermann, M. Ferraz Barbosa, S. Biedermann-Brem, K. Grob, A. Keiser, P. Realini, F. Escher, R. Amad´o, J. Agric. Food Chem. 51 (2003) 5556. [9] V.A. Yaylayan, R.H. Stadler, J. AOAC Int. 88 (2005) 262. [10] L. Castle, J. Agric. Food Chem. 41 (1993) 1261. [11] J. Tekel, P. Farkas, M. Kovac, Food Add. Contam. 6 (1989) 377. [12] T. Wenzl, M.B. de la Calle, E. Anklam, Food Add. Contam. 20 (2003) 885. [13] L. Castle, S. Eriksson, J. AOAC Int. 88 (2005) 274. [14] R. Weisshaar, Eur. J. Lipid Sci. Technol. 106 (2004) 786. [15] Y. Zhang, G. Zhang, Y. Zhang, J. Chromatogr. A 1075 (2005) 1.
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