Determination of Trace Acrylamide in Starchy Foodstuffs by HPLC

Article pubs.acs.org/JAFC

Determination of Trace Acrylamide in Starchy Foodstuffs by HPLC Using a Novel Mixed-Mode Functionalized Calixarene Sorbent for Solid-Phase Extraction Cleanup Wenfen Zhang,† Zhifen Deng,† Wenjie Zhao,*,§ Ling Guo,† Wei Tang,† Huifang Du,† Lin Lin,† Qiong Jiang,† Ajuan Yu,† Lijun He,§ and Shusheng Zhang*,† †

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou 450001, People’s Republic of China School of Chemistry and Chemical Engineering, Henan University of Technology, Zhengzhou 450001, People’s Republic of China

§

ABSTRACT: In this paper, a rapid and effective HPLC method, using tetraazacalix[2]arene[2]triazine-modified silica gel (NCSi) as solid-phase extraction (SPE) sorbent, was developed for the purification and determination of trace acrylamide in starchy foodstuffs. The main influence factors of SPE including amount of NCSi sorbent, sample flow rate, and volume and composition of washing solution were investigated and evaluated in the sample pretreatment step. The optimized purification effect was achieved at the sample flow rate of 3 mL/min with 100 mg of NCSi and 2 mL of washing solution (water, 100%). The HPLC separation was carried out on a C18 column (250 × 4.6 mm i.d., 5 μm) with a mobile phase of methanol/water (10:90, v/ v). The linear range of the calibration curve was 4−4000 ng/mL with s correlation coefficient of >0.9999. The intraday and interday RSDs (n = 5) of peak areas of acrylamide were 0.22 and 0.90% and the intraday and interday RSDs (n = 5) of retention times were 0.50 and 1.63%, respectively. In addition, overall recoveries through the extraction and NCSi-SPE purification ranged from 73.13 to 98%. Compared with the commercial SPE sorbents, NCSi featured excellent selectivity to retain polar and nonpolar interferences in the sample matrices. The improved method was simple, rapid, accurate, and promising for the determination of trace acrylamide in starchy foods with a complex matrix. KEYWORDS: tetraazacalix[2]arene[2]triazine-modified silica gel, SPE purification, HPLC, acrylamide, starchy foodstuffs

■

INTRODUCTION

For the cleanup of acrylamide from complex samples, accelerated solvent extraction (ASE), liquid−liquid extraction (LLE), and solid-phase extraction (SPE), alone or in combination with other purification steps, were conducted by many laboratories. ASE and LLE with conventional organic solvent are time-consuming and labor-intensive. Furthermore, they can easily lead to loss of acrylamide in the scavenging process and require large amounts of toxic solvents. To overcome these shortcomings, SPE has been developed and applied in the cleanup of acrylamide in coffee and coffee products,21,22 potato chips,23 and so on. As acrylamide is a small, polar, and hydrophilic molecule, a large amount of matrix interferences tends to be extracted simultaneously with acrylamide, which makes it difficult for analysis using a single conventional SPE method employing reversed-phase (RP) or ion-exchange sorbents. With the aim of solving this problem, multiple retention mechanism cleanup procedures were developed. However, most of them consist of the combination of several SPE cartridges. Bortolomeazzi et al.21 used a single SPE column consisting of 0.5 g of an in-house-prepared mixture of C18, strong cation (SCX), and anion exchange (SAX) sorbents in the ratio 2/1.5/1.5 (w/w/w) to extract and clean up roasted coffee samples.24 Becalski et al. used a combination of three different cartridges including Oasis MAX (mixed mode

Acrylamide, a colorless and odorless crystalline powder, is known to be formed by the Maillard reaction during heating of starchy foodstuffs. In April 2002, the Swedish National Food Administration (NFA) and researchers from Stockholm University found high acrylamide levels in carbohydrate-rich foodstuffs fried/baked at high temperatures.1 In recent years, research on acrylamide in different matrices has attracted extensive attention due to its demonstrated neurotoxicity, genotoxicity, and potential human carcinogenicity.2 In past years, a number of chromatographic methods have been developed to determine acrylamide such as gas chromatography (GC) with derivatization and high-performance liquid chromatography (HPLC) without derivatization.3,4 Nowadays, most analytical methods are based on GC-MS and LC-MS/MS,5−20 which appear to be acknowledged as the most useful and authoritative methods for the quantification of acrylamide in complex matrices. However, both GC-MS and LC-MS are high-cost and cannot be easily adopted by nonspecialized laboratories. On the contrary, HPLC with UV detection possesses the advantages of simplicity, lower cost, and strong maneuverability compared to MS techniques. Meanwhile, it is noteworthy that the matrix effect is a serious problem in the trace analysis of acrylamide in complex matrices such as chips and coffee,7,12,13 which make quantification and identification difficult. Therefore, removal of the matrix effect and sample pretreatment are of great importance in trace analysis. © 2014 American Chemical Society

Received: Revised: Accepted: Published: 6100

April 2, 2014 June 9, 2014 June 16, 2014 June 16, 2014 dx.doi.org/10.1021/jf501569q | J. Agric. Food Chem. 2014, 62, 6100−6107

Journal of Agricultural and Food Chemistry

Article

Figure 1. Chemical structure of the NCS stationary phase.36

Figure 2. Synthesis of NCSi sorbent.

orientation in the solid phase. Our previous work indicated that the NCS exhibited high selectivity toward various compounds under different conditions with different mechanisms including hydrophobicity, π−π stacking, hydrogen-bonding, inclusion, and anion-exchange interactions. A nNumber of compounds, including polycyclic aromatic hydrocarbons, nitrobenzene, organic bases, phenols, and inorganic anions, have been well separated on the NCS stationary phase.33 Recently, a NCSi (tetraazacalix[2]arene[2]triazine functionalized silica) SPE sorbent (Figure 2)34 was prepared utilizing preparation procedures similar to the NCS stationary phases in our laboratory. Because of the existence of multi-interaction, it is of great application value for purification or concentration trace analyte in a complex matrix. Furthermore, it has been successfully used for the extraction tobacco-specific N-nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, and a metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in rabbit plasma.35 In the present work, a novel multiple retention mechanism sorbent (NCSi) was used with the aim of developing a rapid and reliable sample preparation procedure for the determination of acrylamide in starchy foodstuffs. All of the main factors were optimized, and the results obtained by using the developed HPLC method based on NCSi SPE indicated that it is more suitable for the determination of acrylamide in the complex matrices.

anion exchange), Oasis MCX (mixed-mode cation exchange), and ENVI-Carb (graphitized carbon) to treat various samples (potato fries, potato chips, crispbread, instant coffee, coffee beans, cocoa, chocolate, and peanut butter). A similar combination of SPE cartridges consisting of Bond Elut C18, Bond Elut Jr-PSA (anion exchange), and Bond Elut Accucat was chosen for cleanup samples.24 However, these different SPE combination modes make the purification process cumbersome and expensive and even lead to loss of acrylamide in the cleanup process. Therefore, it is necessary to design a novel SPE sorbent with a multiple retention mechanism for the cleanup of acrylamide from complex sample matrices. Calixarenes, following cyclodextrins and crown ethers, are considered to be a typical representative of the third generation of host supramolecules. They consist of phenol units linked via methylene bridges and can also form inclusion complexes like other host supramolecules. There are a number of selective factors in the configuration of calixarenes such as cavity size, conformation, and substituents. Since Glennon and his coworkers reported the application of calixarenes as the stationary phase, it has attracted extensive attention.25−27 Our group has been comitted to the research of calixarene stationary phases for many years,28−31 and a novel multi-interaction and mixedmode stationary phase based on tetraazacalix[2]arene[2]triazine modified silica gel (NCS, Figure 1) has been synthesized in our laboratory in 2012.32 Being different from conventional calixarenes in which the aromatic rings are linked by methylene units, tetraazacalix[2]arene[2]triazine assembles aromatic rings by −NH− and adopts a 1,3-alternate conformation with two benzene rings nearly face-to-face parallel and two triazine rings tending to an edge-to-edge

■

MATERIALS AND METHODS

Chemicals, Materials, and Solutions. Tetraazacalix[2]arene[2]triazine was synthesized in accordance with the previously published procedures.32,36 3-Aminopropyltriethoxysilane was purchased from 6101

dx.doi.org/10.1021/jf501569q | J. Agric. Food Chem. 2014, 62, 6100−6107


Article

Jingchun Chemical Reagent Co., Ltd. (Shanghai, China). Acrylamide (>99%) was purchased from Research Institute of Fine Chemical Industry (Tianjin, China). HPLC grade methanol (MeOH), n-hexane, ethyl alcohol, and acetonitrile were provided by Fisher (Fair Lawn, NJ, USA). Deionized water was purified by a Milli-Q system from Millipore (Bedford, MA, USA). The polypropylene column tube and 20 μm PTFE sieve plates used for SPE were bought from DIKMA (Beijing, China). Commercially available MCX, MAX, HLB, and C18 cartridges (100 mg/3 mL) were obtained from Waters (Milford, MA, USA). Standard acrylamide was dissolved in water at a concentration of 40 μg/mL and stored at 4 °C as stock solutions. Five standard solutions at different concentration (4, 16, 40, 160, 400m and 4000 ng/mL) were prepared by diluting the standard stock solution with mobile phase. Instruments and Measurement. The comparison of retention behaviors of acrylamide on NCS and octadecyl silane (ODS) stationary phases and all determinations were carried out on an Agilent 1260 (Agilent, USA) consisting of a G1311C quaternary pump with a vacuum degasser, a G13116A temperature-controlled column oven, a G1329B autosampler, and a G1314F VWD UV detector. A Flash EA 1112 elemental analyzer and a Bruker Vector 22 IR spectrograph were used for characterization of the NCSi SPE sorbent. A centrifuge (Zhongda Instrument Plant, Jiangsu, China) was used for centrifugal separation. A Vortex mixer (Shanghai jingke industrial LTD, Shanghai, China) was used for mixing solutions. A rotary evaporator was used for enrichment (Yarong, Shanghai, China). The HPLC separations were performed on an Ultimate XB-C18 column (250 × 4.6 mm i.d., 5 μm) with a solution of MeOH/water (10:90, v/ v) at 0.5 mL/min. The wavelength was 200 nm, the injection volume was 20 μL, and the oven temperature was 30 °C. Under these chromatographic conditions, acrylamide and the food components in the tested samples were all baseline separated and eluted. Each separation and/or determination was performed in triplicate. Synthesis of NCSi SPE Sorbent. As illustrated in Figure 2, NCSi sorbent was prepared by a two-step modification process as previously described.32 First, aminopropyltriethoxyl-bonded silica gel (APS) was obtained. In the second step, APS was reacted with an excess of tetraazacalix[2]arene[2]triazine in anhydrous dimethylformamide (DMF) at 130 °C under nitrogen atmosphere. Different from the previous work, the spherical silica had a particle diameter of 40−60 μm instead of the previous 5 μm. The surface area is 500 m2/g. Retention Behaviors of Acrylamide on the NCS Stationary Phase. The retention behaviors were investigated on the previously studied NCS column (150 × 4.6 mm, 5 μm) and another four commercial columns (Agilent ZORBAX SB-C18, Waters XBridge Shield RP18, Ultimate XB-C18, and Hypersil ODS2) to evaluate the interactions between the NCS stationary phase and acrylamide. All tests were conducted at the same chromatographic conditions. Sample Preparation. Homogenization, Extraction, and Enrichment Process. All samples (fried potato chips, rice crust, instant noodles, biscuit) were purchased from the local supermarket. Dry samples were ground, mesh size 1 mm. One gram of homogenized samples was weighed and put into 15 mL centrifuge tubes in triplicate. To each centrifuge tube was added 10 mL of acetonitrile followed by 30 s of vortexing to extract the target acrylamide. All of the centrifuge tubes were centrifuged for 5 min at 4000 rpm. Then the clear supernatant was transferred into a glass tube, this process was repeated, and the two clear supernatants were combined. About 20 mL of extraction solution was rotary evaporated to near 3 mL after the addition of 2 mL of water under vacuum at 30 °C. SPE Cleanup. In this paper, a new purification procedure based on a novel homemade NCSi SPE sorbent is proposed for the analysis of acrylamide. The procedure consists of an acetonitrile extraction, centrifugation, and cleanup using homemade SPE cartridges. This method was compared with four different commercial SPE cartridges (C18, HLB, MAX, and MCX) with different interaction mechanisms to establish the best conditions for the determination of acrylamide in food products. To investigate the availability of the NCSi SPE for the cleanup of acrylamide in complex matrix, 100 mg of NCSi sorbent was packed

into a 3 mL SPE cartridge. The concentrated extracting solution (Sample Preparation) was passed through the five kinds of SPE cartridges, which had been preconditioned with 5 mL of MeOH and 5 mL of water, at the rate of 3 mL/min, and collected into a glass tube. The cartridges were then eluted with 2 mL of water and collected into the same glass tube, making the final volume approximately 5 mL. The 5 mL of collected aqueous solution was filtered through a 0.22 μm nylon filter (Agilent, USA) prior to HPLC analysis. All tests were performed in triplicate. Recovery Test. The extraction and purification were validated by recovery investigation. We spiked test samples at three concentration levels of 40, 400, and 4000 ng/g by adding acrylamide to blank samples or real samples. Then, the recoveries were determined by HPLC method after the spiked test samples were treated by extraction and SPE cleanup procedures.

■

RESULTS AND DISCUSSION Characterization of NCSi SPE Sorbent. In IR spectra of NCSi (Figure 3), the bands at 2937 and 2888 cm−1 are assigned

Figure 3. IR spectra of APS and NCSi sorbent.

to the stretching vibration of −CH2− groups. The characteristic absorption bands of the benzene rings appearing at 1449, 1518, and 1576 cm−1 confirmed that the macrocycle ligand was successfully immobilized on silica gel. Elemental analysis showed (Table 1) the bonding amount of tetraazacalix[2]arene[2]triazine on NCSi sorbent was about 200 μmol/g. SPE Optimization for Trace Acrylamide with the NCSi Sorbent. In this section, the main influence factors (amount of sorbent, flow rate of sample, composition and volume of washing solution) on the SPE recoveries (n = 3) of acrylamide Table 1. NCS Stationary Phase and NCSi SPE Sorbent parameters of the silica gel

NCS stationary phase NCSi SPE sorbent a

6102

element analysis

particle size (μm)

specific surface area (m2/g)

C (%)

H (%)

N (%)

bonded amounta (μmol/g)

5

300

11.25

1.56

5.69

250

40−60

500

8.83

1.28

4.72

200

Calculated from the carbon content. dx.doi.org/10.1021/jf501569q | J. Agric. Food Chem. 2014, 62, 6100−6107


Article

concentration of MeOH increasing from 0 to 20%; however, the interfering substances in the real samples increased along with the increasing proportion of MeOH. Meanwhile, a high concentration of MeOH may also result in loss of the target compounds. Therefore, a 100% water solution was selected as the optimal washing solution composition. For the selection of washing solution volume, various volumes (0.5−5 mL) of water solution were applied for the NCSi SPE process. The results display that the recoveries of acrylamide increased with the rise of the water solution volume from 0.5 to 2 mL. When the volume of water solution was >2 mL, the recoveries of acrylamide remained almost constant while the interference increased. As a result, 2 mL of water solution was chosen as the washing solution in this study. Comparison of SPE Cleanup by NCSi Sorbent and Commercial Sorbents (MCX, MAX, HLB, and C18). Comparison of SPE Cleanup by NCSi Sorbent and Commercial Sorbents. Concentrated extracting solution of 1 g of blank cookies (Sample Preparation) was passed through NCSi and four kinds of commercial SPE cartridges (MCX, MAX, HLB, and C18) possessing the same specifications as the homemade NCSi SPE cartridge according to the SPE cleanup procedures as previous described, respectively. About 5 mL of purged aqueous solution was collected in the five glass tubes (Figure 6), respectively. As shown in Figure 6, it was found that

are evaluated in detail to obtain the optimal purification conditions. The sorbent amount in the SPE cartridge is closely correlated with the purification effect. In this section, the influence of NCSi amount on the acrylamide’s extraction was studied with the amount ranging from 50 to 250 mg. As shown in Figure 4,

Figure 4. Recovery efficiency with different amounts of NCSi sorbent.

it is easily found that 100 mg of NCSi sorbent was enough to achieve satisfactory extraction and purification efficiency compared with 150 or 200 mg of NCSi sorbent. Thus, the optimal amount of sorbent was 100 mg. As is known, the flow rate of the sample is another critical factor that not only affects the purification effect of analytes but also controls the sample pretreatment time. In this section, flow rates ranging from 1 to 5 mL/min were investigated, and it was found that 3 mL/min was the optimal flow rate to provide higher extraction recovery (Figure 5).

Figure 6. Turbidity comparison of the purged liquors from NCSi and four commercial SPE cartridges.

the type of sorbent used played an important role on the purification efficiency: the purged liquor from NCSi is obviously clearer than those from the other four kinds of commercial SPE cartridges. The purged liquor from NCSi was clear, but the purged liquors from C18 and HLB showed a little turbidity and the liquors from MAX and MCX were a milky white. These results imply that the fat and weak polar matrices could coelute out together with acrylamide during the purification of real samples through MCX, MAX, HLB, and C18 SPE cartridges. Therefore, NCSi SPE was selected as optimal for the extraction and purification of acrylamide in the actual samples. To further evaluate the purification capacity of NCSi SPE sorbent, the above purged liquors from MCX and NCSi were

Figure 5. Recovery efficiency with different flow rates of sample.

A proper washing solvent is of great importance to reduce interfering substances and improve the recovery. Thus, the influence of the composition and volume of the washing solution on the purification of trace acrylamide (1 mL of working aqueous solution, 40 ng/mL) was studied. Different concentrations of MeOH solution (MeOH/water, v/v) at a constant volume (2 mL) were investigated. It was found that the recoveries showed no significant difference with the 6103



Article

defatted with hexane as described in the literature,37,38 respectively. The defatting procedures are described as follows: add 15 mL of n-hexane to the 5 mL of purged liquor, then vortex for 30 s, allow to stand, and remove the upper hexane to get rid of lipid. The remaining liquids were clear, and 5 μL of liquid was injected for HPLC separation. As expected, it was found that the chromatograms of the defatted and non-defatted liquors from NCSi showed no difference, and they were similar to the chromatogram of the defatted liquor from the MCX SPE cartridge (Figure 7). These results prove that NCSi SPE could omit the defatting step and reduce acrylamide loss during the purification process.

Figure 8. Chromatogram comparison of purged liquors from (A) NCSi without defatting step and from (B) C18, (C) HLB, (D) MAX, and (E) MCX with defatting steps. Conditions were as for Figure 7. Peak: 1, acrylamide.

different chromatographic columns. The retention properties of acrylamide are described in Table 2. From Table 2, NCS and Table 2. Retention Properties of Acrylamide on Different Chromatographic Columnsa chromatographic column NCS (150 × 4.6 mm, 5 μm) Agilent ZORBAX SB-C18 (150 × 4.6 mm, 5 μm) Waters XBridge Shield RP18 (250 × 4.6 mm, 5 μm) Hypersil ODS2 (250 × 4.6 mm, 5 μm) Ultimate XB-C18 (250 × 4.6 mm, 5 μm)

Figure 7. Chromatograms of purged liquors from MCX and NCSi with and without defatting steps in blank cookies. Conditions: column, Ultimate XB-C18 (250 × 4.6 mm i.d., 5 μm); mobile phase, MeOH/ water (10:90, v/v); flow rate, 0.5 mL/min; detection wavelength, 200 nm; injection volume, 20 μL; column temperature, 30 °C. Chromatograms: (A) NCSi defatted; (B) NCSi without defatting; (C) MCX defatted.

retention time (min)

symmetrical factor

half-peak width

6.183 4.253

0.826 0.563

0.2542 0.2442

7.054

0.745

0.1239

7.465

0.672

0.1653

8.487

0.929

0.1672

a

Chromatographic conditions are given under Instruments and Measurement.

Moreover, comparison of SPE purification efficiency between NCSi SPE and commercially available C18, HLB, MAX, and MCX SPE was carried out to further demonstrate the suitability of NCSi SPE for real samples. The sample cookies spiked at 40 ng/g acrylamide were performed under the extraction and SPE cleanup procedures. The 20 μL of purged liquor from NCSi and defatted liquor from the other four SPE sorbents was injected to perform HPLC separations. Their chromatograms are shown in Figure 8. The target peak of acrylamide by NCSi SPE cleanup showed more symmetry and higher UV response than those of the other commercially SPE cartridges, which makes the determination of trace acrylamide in complex matrix more accurate and sensitive. The NCSi sorbent exhibited high selectivity toward various interference and coprecipitation compounds with different mechanisms including hydrophobic, π−π stacking, hydrogen-bonding, inclusion, and anionexchange interactions. The proteins and carbohydrates were retained on NCSi depending on hydrogen-bonding and anionexchange interactions, whereas fat and nonpolar matrices were held on the basis of hydrophobic interactions. For acrylamide, there is a weak hydrogen-bonding interaction with NCSi, which leads to its being weakly retained on NCSi. Optimization of the Chromatographic Conditions. The chromatographic separation of acrylamide was performed by reverse-phase liquid chromatography using NCS and four

Ultimate XB-C18 provided better symmetry than the other columns, and Ultimate XB-C18 possessed higher theoretical plates than the others. Therefore, Ultimate XB-C18 (250 × 4.6 mm, 5 μm) was selected as the optimal column for the separation and determination of acrylamide in the real samples. However, it is noteworthy that the retention of acrylamide on the NCS column was a little bit stronger than those on the other ODS columns. Obviously, triazine rings and nitrogen bridges on the NCS stationary phase played an important role in acrylamide’s retention. This result again displays that weak hydrogen bonding interaction exists between acrylamide and NCS. We also carried out the HPLC separation of acrylamide with different mobile phases at different flow rates (detection wavelength at 200 nm). Chromatograms are shown in Figure 9. From Figure 9A,C,D, the retention time of acrylamide gradually reduced with the increase of MeOH content in the mobile phase from 5 to 15% at a flow rate of 0.5 mL/min. From Figure 9B,C,E, the retention time of acrylamide gradually decreases with the increase of flow rate from 0.4 to 0.6 mL/min with the mobile phase of 10% MeOH. Their retention times, peak areas, and theoretical plates were calculated and are presented in 6104



Article

quantification (LOQ) of this method were determined with signal/noise ratios of 3 and 10, respectively. The LOD and LOQ were about 5 and 20 ng/g at a detection wavelength of 200 nm, respectively. This LOD or LOQ was low compared to those of published works by GC-MS6 and LC-MS.39,40 The intraday and interday RSDs (n = 5) of peak areas (400 ng/mL) were 0.22 and 0.90% and the intraday and interday RSDs (n = 5) of retention time were 0.50 and 1.63%, respectively. Recovery and Application to Real Samples. The recovery investigations were carried out in accordance with the previous description. The standard acrylamide solutions at three levels (40, 400, and 4000 ng/mL) were added to real samples including potato chips, cookies, fried steamed buns, and rice crust. Then, the original samples and spiked samples were extracted, cleaned up by NCSi sorbent, and separated by HPLC. Typical chromatograms of potato chips are shown in Figure 10. The contents and recoveries of acrylamide were

Figure 9. Chromatograms of acrylamide with different mobile phases and flow rates. Conditions: (A) methanol/water (5:95, v/v), 0.5 mL/ min; (B) methanol/water (10:90, v/v), 0.4 mL/min; (C) methanol/ water (10:90, v/v), 0.5 mL/min; (D) methanol/water (15:85, v/v), 0.5 mL/min; (E) methanol/water (10:90, v/v), 0.6 mL/min; others as for Figure 7. Peak: 1, acrylamide.

Table 3. As can be seen from Table 3, along with the increase of flow rate, both the retention time and peak area decrease. When Table 3. Retention Time, Peak Areas, and Theoretical Plates of Acrylamide with Different Chromatographic Conditionsa chromatographic conditions (A) methanol−water (5:95, v/v), 0.5 mL/min (B) methanol−water (10:90, v/v), 0.4 mL/min (C) methanol−water (10:90, v/v), 0.5 mL/min (D) methanol−water (15:85, v/v), 0.5 mL/min (E) methanol−water (10:90, v/v), 0.6 mL/min

retention time (min)

peak area

theoretical plates (plates/m)

9.58

166.6

32004.57

10.492

207.2

31330.94

8.373

166.1

34315.10

7.659

178

34307.81

6.959

136.7

35955.18

Figure 10. Chromatograms of potato chips and spiked potato chips. Chromatograms: (A) potato chips; (B) potato chips spiked at 40 ng/ g; (C) potato chips spiked at 400 ng/g; (D) potato chips spiked at 4000 ng/g. Conditions were as for Figure 7. Peak: 1, acrylamide.

a

Other chromatographic conditions are given underInstruments and Measurement.

determined and are shown in Table 4. It can be seen from Table 4 that their recoveries ranged from 73.13 to 98%, indicating that the developed HPLC method based on NCSi SPE cleanup possessed high accuracy. The contents of acrylamide in these foods ranged from 13 to 28 ng/g; these values were much lower than the maximum residue limits (MRLs) recommended by the European Commission.41 These foods can be safely consumed. From the results above, it is concluded that the present SPE method based on NCSi sorbent is more suitable for the determination of acrylamide in samples with a complex matrix. In conclusion, an HPLC method based on NCSi-SPE cleanup has been successfully employed for the purification and determination of acrylamide in potato chip, cookie, fried steamed bun, and rice crust samples with complex matrices. The NCSi-SPE method enables a more efficient purification of acrylamide from a complex matrix due to its multi-interaction ability. The multi-interaction ability can selectively keep the main interferences (such as carbohydrates and proteins) from passing through NCSi while allowing acrylamide pass without any obstacle. The SPE procedure is simple and easy to operate; furthermore, NCSi-SPE offers higher accuracy for the determination of acrylamide in a complex matrix. The proposed

5.0% methanol was invoked as the mobile phase at a flow rate of 0.6 mL/min, the peak area reduced 23.2% compared with 0.5 mL/min. The flow rate of 0.4 mL/min shows the largest response, but it also holds the disadvantage of longer retention time and solvent cost. With the retention time, peak area, and theoretical plate taken into consideration, a 10% v/v methanol mobile phase and 0.5 mL/min flow rate were chosen as optimal conditions for our HPLC-UV analysis of acrylamide in the samples. Linear Regression Equation, LOD and LOQ of the Method. Method validation such as recovery, linearity, correlation coefficients, LODs, and LOQs were measured. The calibration curve was constructed by plotting peak area (y) versus the corresponding concentration of acrylamide (x, ng/ mL) and its linear regression equation was obtained as follows: y = 0.4357x + 0.2648 (n = 5, r 2 = 0.9999)

The results showed that good linearity was achieved in the range of 4−4000 ng/mL for acrylamide with a correlation coefficient of 0.9999. The limits of detection (LOD) and 6105



Article

Table 4. Analysis of Acrylamide in Various Foodstuffs spiking 40 ng/g

spiking 400 ng/g

spiking 4000 ng/g

sample

concentration (ng/g)

recovery (%)

RSD (%, n = 3)

recovery (%)

RSD (%, n = 3)

recovery (%)

RSD (%, n = 3)

cookies potato chips fried steamed buns rice crust

21 28 13 18

98 77.5 80.6 90.5

0.95 2.63 4.30 2.65

85.5 76.9 82.9 87.2

6.22 4.53 1.57 3.27

82.4 81.3 73.13 90.6

2.47 3.09 4.42 2.64

(11) Leung, K. S.; Lin, A.; Tsang, C. K.; Yeung, S. T. K. Acrylamide in Asian foods in Hong Kong. Food Addit. Contam. 2003, 20, 1105− 1113. (12) Fernandes, J. O.; Soares, C. Application of matrix solid-phase dispersion in the determination of acrylamide in potato chips. J. Chromatogr., A 2007, 1175, 1−6. (13) Arisseto, A. P.; Toledo, M. C. d. F.; Govaert, Y.; van Loco, J.; Fraselle, S.e; Degroodt, J.-M. A modified sample preparation for acrylamide determination in cocoa and coffee products. Food Anal. Methods 2008, 1, 49−55. (14) DeArmond, P. D.; DiGoregorio, A. L. Characterization of liquid chromatography-tandem mass spectrometry method for the determination of acrylamide in complex environmental samples. Anal. Bioanal. Chem. 2013, 405, 4159−4166. (15) Senyuya, H. Z.; Gökmen, V. Survey of acrylamide in Turkish foods by an in-house validated LC−MS method. Food Addit. Contam. 2005, 22, 204−209. (16) Bermudo, E.; Moyano, E.; Puignou, L.; Galcera, M. T. Determination of acrylamide in foodstuffs by liquid chromatography ion-trap tandem mass-spectrometry using an improved clean-up procedure. Anal. Chim. Acta 2006, 559, 207−214. (17) Karasek, L.; Wenzl, T.; Anklam, E. Determination of acrylamide in roasted chestnuts and chestnut-based foods by isotope dilution HPLC-MS/MS. Food Chem. 2009, 114, 1555−1558. (18) Bortolomeazzi, R.; Munari, M.; Anesea, M.; Verardo, G.; Goeyens, L. Rapid mixed mode solid phase extraction method for the determination of acrylamide in roasted coffee by HPLC−MS/MS. Food Chem. 2012, 135, 2687−2693. (19) Rosén, J.; Hellenäs, K. E. Analysis of acrylamide in cooked foods by liquid chromatography tandem mass spectrometry. Analyst 2002, 127, 880−882. (20) Aguas, P. C.; Fitzhenry, M. J.; Giannikopoulos, G.; Varelis, P. Analysis of acrylamide in coffee and cocoa by isotope dilution. Anal. Bioanal. Chem. 2006, 385, 1526−1531. (21) Bortolomeazzi, R.; Munari, M.; Anese, M.; Verardo, G. Rapid mixed mode solid phase extraction method for the determination of acrylamide in roasted coffee by HPLC−MS/MS. Food Chem. 2012, 135, 2687−2693. (22) Soares, C.; Cunha, S.; Fernandes, J. Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE clean-up. Food Addit. Contam. 2006, 23, 1276−1282. (23) Xu, L.; Zhang, L.; Qiao, X.; Xu, Z. J.; Song, J. Determination of trace acrylamide in potato chip and breadcrust based on SPE and HPLC. Chromatographia 2012, 75, 269−274. (24) Zhang, Y.; Ren, Y. P.; Zhang, Y. New research developments on acrylamide: analytical chemistry, formation mechanism, and mitigation recipes. Chem. Rev. 2009, 109, 4375−4397. (25) Glennon, J. D.; O’Connor, K.; Srijaranai, S.; Manley, K.; Harris, S. J.; McKervey, M. A. Enhanced chromatographic selectivity for Na+ ions on a calixarene-bonded silica phase. Anal. Lett. 1993, 26, 153− 158. (26) Brindle, R.; Albert, K.; Harris, S. J.; Troltzsch, C.; Horne, E.; Glennon, J. D. Silica-bonded calixarenes in chromatography: I. Synthesis and characterization by solid-state NMR spectroscopy. J. Chromatogr., A 1996, 731, 41−47. (27) Glennon, J. D.; Horne, E.; O’Connor, K.; Kearney, G.; Harris, S. J.; McKervey, M. A. Silica-bonded calixarenes in chromatography: II. Chromatographic retention of metal ions and amino acid ester hydrochlorides. J. Chromatogr., A 1996, 731, 47−56.

HPLC method based on NCSi-SPE is promising for use in the pretreatment and determination of acrylamide in starchy foodstuffs.

■

AUTHOR INFORMATION

Corresponding Authors

*(S. Zhang) Mail: Daxue Road 75, Zhengzhou 450052, P. R. China. Phone: 86 371 67763224. E-mail: [email protected]. *(W. Zhao) E-mail: [email protected]. Funding

We acknowledge the support of NSF of China (21275133 and 21305030). Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS

■

REFERENCES

We thank Dr. Julie Rimes and Dr. Simon Yang for assistance with editing the text.

(1) Swedish National Food Administration (http://www.slv.se). Information about Acrylamide in Food, April 24, 2002. (2) Scientific Committee on Toxicity Ecotoxicity and the Environment (CSTEE). Opinion on the results of the risk assessment of acrylamide. The 22nd CSTEE plenary meeting, Brussels, Belgium, March 6−7, 2001. (3) Poole, C. F.; Sye, W. F.; Zlatkis, A.; Spencer, P. S. Determination of acrylamide in nerve tissue homogenates by electron-capture gas chromatography. J. Chromatogr., A 1981, 217, 239−245. (4) Soares, C.; Cunha, S.; Fernandes, J. Determination of acrylamide in coffee and coffee products by GC-MS using an improved SPE cleanup. Food Addit. Contam. 2006, 23, 1276−1282. (5) Lee, M. R.; Chang, L. Y.; Dou, J. P. Determination of acrylamide in food by solid-phase microextraction coupled to gas chromatography−positive chemical ionization tandem mass spectrometry. Anal. Chim. Acta 2007, 582, 19−23. (6) Dunovská, L.; Č ajka, T.; Hajšlová, J.; Holadová, K. Direct determination of acrylamide in food by gas chromatography−high resolution time-of-flight mass spectrometry. Anal. Chim. Acta 2006, 578, 234−240. (7) Becalski, A.; Lau, B. P. Y.; Lewis, D.; Seaman, S. W. Acrylamide in foods: occurrence, sources and modeling. J. Agric. Food Chem. 2003, 51, 802−808. (8) Inoue, K.; Yoshimura, Y.; Nakazawa, H. Development of highperformance liquid chromatography−electrospray mass spectrometry with size-exclusion chromatography for determination of acrylamide in fried foods. J. Liq. Chromatogr. Relat. Technol. 2003, 26, 1877−1884. (9) Zhang, Y.; Ren, Y.; Zhao, H.; Zhang, Y. Determination of acrylamide in Chinese traditional carbohydrate-rich foods using gas chromatography with micro-electron capture detector and isotope dilution liquid chromatography combined with electrospray ionization tandem mass spectrometry. Anal. Chim. Acta 2007, 584, 322−332. (10) Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Törnqvist, M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem. 2002, 50, 4998−5006. 6106



Article

(28) Hu, K.; Qu, K.; Li, Y. B.; Ding, C. H.; Wang, X.; Zhang, J. X.; Ye, B. X.; Zhang, S. S. Investigation of the retention mechanism of naphthol and benzenediol on calix[4]arene stationary phase based on quantum chemistry calculations. J. Sep. Sci. 2008, 31, 2430−2433. (29) Hu, K.; Liu, J. W.; Tang, C.; Wang, C. J.; Yu, A. J.; Wen, F. Y.; Zhao, W. J.; Ye, B. X.; Wu, Y. J.; Zhang, S. S. Preparation, characterization and application of a new 25,27-bis-[2-(5methylthiadiazole)thioethoxyl]-26,28-dihydroxy-para-tert-butyl calix[4]arene stationary phase for HPLC. J. Sep. Sci. 2012, 35, 239− 247. (30) Zhao, W. J.; Hu, K.; Wang, C. J.; Liang, S.; Niu, B. L.; He, L. J.; Lu, K.; Ye, B. X.; Zhang, S. S. New oxo-bridged calix[2]arene[2]triazine stationary phase for high performance liquid chromatography. J. Chromatogr., A 2012, 1223, 72−78. (31) Hu, K.; Zhang, Y. H.; Liu, J. W.; Chen, K. K.; Zhao, W. J.; Zhu, W. X.; Song, Z. C.; Ye, B. X.; Zhang, S. S. Development of a new 25,27-bis(L-phenylalaninemethylester-N-carbonylmethoxy)-26,28-dihydroxy-para-tert-butylcalix[4]arene HPLC stationary phase and its application in food safety. J. Sep. Sci. 2013, 36, 445−453. (32) Zhao, W. J.; Wang, W. J.; Chang, H.; Cui, S. W.; Hu, K.; He, L. J.; Lu, K.; Liu, J. X.; Wu, Y. J.; Qian, J.; Zhang, S. S. Tetraazacalix[2]arene[2]triazine modified silica gel: a novel multiinteraction and mixed-mode stationary phase for high performance liquid chromatography. J. Chromatogr., A 2012, 1215, 74−81. (33) Zhang, S. S.; Zhao, W. J.; Liang, S.; Yu, A. J.; Hu, K.; Zhao, X. L.; Wu, Y. J. Preparation and application of tetraazacalix[2]arene[2]triazine modified silica stationary phase. Patent CN: 2011 1000 2890.6, 2011. (34) Zhang, S. S.; Zhao, W. J.; Chang, H.; Jiang, Q.; Deng, Z. F.; Lu, J. Preparation and application of tetraazacalix[2]arene[2]triazine modified silica sorbent for solid phase extraction. Patent CN: 201210458997, 2012. (35) Wang, L.; Yang, Ch. Q.; Zhang, Q. D.; Wang, S.; Lang, H. L.; Zhang, S. S.; Zhao, W. J.; Zhang, J. X. SPE-HPLC-MS/MS method for the trace analysis of tobacco-specific N-nitrosamines in plasma using tetraazacalix[2]arene[2]triazine modified silica sorbent. J. Sep. Sci. 2013, 36, 2664−2671. (36) Wang, M.; Yang, H. A general and high yielding fragment coupling synthesis of heteroatom-bridged calixarenes and the unprecedented examples of calixarene cavity fine-tuned by bridging heteroatoms. J. Am. Chem. Soc. 2004, 126, 15412−15422. (37) Gö k men, V.; Senyuva, H. Z.; Acar, J.; Sarıoğ l u, K. Determination of acrylamide in potato chips and crips by highperformance liquid chromatography. J. Chromatogr., A 2005, 108, 193−199. (38) Murkovic, M. Acrylamide in Austrian foods. J. Biochem. Biophys. Methods 2004, 61, 161−167. (39) Govaert, Y.; Arisseto, A.; van Loco, J.; Scheers, E.; Fraselle, S.; Weverbergh, E.; Degroodt, J. M.; Goeyens, L. Optimisation of a liquid chromatography−tandem mass spectrometric method for the determination of acrylamide in foods. Anal. Chim. Acta 2006, 556, 275−280. (40) Hoenicke, R.; Gatemann, W.; Harder, L. Analysis of acrylamide in different foodstuffs using liquid chromatography−tandem mass spectrometry and gas chromatography−tandem mass spectrometry. Anal. Chim. Acta 2004, 520, 207−215. (41) European commission recommendation. 2013/647/EU, Nov 8, 2013.

6107


Determination of Trace Acrylamide in Starchy Foodstuffs by HPLC

Recommend Documents