Ultra High-Performance Liquid Chromatography−Tandem Mass

Apr 4, 2011 - Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States .... bufad...
0 downloads 9 Views 2MB Size
ARTICLE pubs.acs.org/ac

Ultra High-Performance Liquid ChromatographyTandem Mass Spectrometry for the Simultaneous Analysis of Asparagine, Sugars, and Acrylamide in Maillard Reactions Yu Zhang,†,§ Yiping Ren,‡ Jingjing Jiao,†,§ Dong Li,† and Ying Zhang*,† †

Department of Food Science and Nutrition, School of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, China ‡ Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China § Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114, United States

bS Supporting Information ABSTRACT: We developed an automated microwave digestion labstation (MDL) combined with ultra high-performance liquid chromatographytandem mass spectrometry (UHPLCMS/ MS) method under the control of positivenegative ion switching as a robust kinetic study tool for rapid and simultaneous quantification of asparagine, glucose, fructose, and acrylamide in Maillard reaction products. Maillard reactions were conducted in a potato model via MDL. The two-step simple pretreatment procedures included the addition of isotope internal standards 15N2-asparagine, 13C6-glucose, and D3-acrylamide, followed by appropriate dilution with the mobile phase and filtration. Analytes were separated on a Hypercarb column and monitored by MS/MS. Study of matrix effects indicated Maillard reaction products induce an ionization suppression of both positive and negative precursor ions, but quantitative results are corrected through the use of isotopically labeled internal standards. Using this method, the limit of detection (LOD) and limit of quantification (LOQ) ranges of all analytes were calculated as 0.040.6 and 0.11.1 μM, respectively. Excellent repeatability (RSD < 9.6%) and acceptable within-laboratory reproducibility (RSD < 9.2%) substantially supported the use of this method for sample analysis. The present kinetic tools, with 1050 min mimic of Maillard reactions and short instrumental run time (5.5 min per sample), were successfully validated and applied to simultaneous determination of acrylamide and its precursors and intermediates during Maillard reactions and kinetic elucidation. Furthermore, current tools of MDL combined with simple sample treatment procedures and UHPLCMS/MS analysis reduce sample analysis time and labor in the kinetic study.

A

crylamide has widely been found in various heat processing foods and evoked an international health alarm since 2002.1 It has been classified as probably carcinogen to humans by the International Agency for Research on Cancer.2 Exposure of acrylamide at high levels causes damage to the nervous system.3 Acrylamide is also considered as a reproductive toxic chemical4 with mutagenic and carcinogenic properties, as shown by in vitro and in vivo mammalian studies.5 Gas chromatography/mass spectrometry (GC/MS) and liquid chromatographymass spectrometry (LCMS) methods are generally acknowledged as the most appropriate methods for acrylamide analysis.68 Recently, rapid determination of acrylamide in food matrixes becomes increasingly urgent especially when large sample amounts need to be analyzed. Such rapid analysis technology relies on the improvement of both sample pretreatment procedures and instrumentation. Previous work reported fast combined extraction and cleanup pretreatment r 2011 American Chemical Society

procedures9 and coevaporation of acrylamide with water as a preparative method10 to avoid tedious and time-consuming pretreatment procedures. Compared to sample preparation, few studies focused on the improvement of instrumentation. Maillard reactions are indeed very complex, and even wellstudied initial reaction steps are difficult to describe on a kinetic basis, mainly because the Schiff base is difficult to determine quantitatively.11 To describe the kinetic behavior of acrylamide and further investigate its mechanism during Maillard reactions, changes of precursors, intermediates, acrylamide, and other final products should be simultaneously investigated. Proton transfer reaction mass spectrometry (PTR-MS),12 pyrolysis GC/MS, and Fourier transform infrared (FT-IR)13 spectrometry were Received: November 10, 2010 Accepted: March 3, 2011 Published: April 04, 2011 3297

dx.doi.org/10.1021/ac1029538 | Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry previously considered as useful tools to monitor real-time change of acrylamide contents in food matrixes or model systems. However, they are unavailable for simultaneous determination of other key compounds involved in Maillard reactions. Knol et al.14 established a kinetic model for elucidating the formation mechanism of acrylamide via LCMS/MS, high-performance liquid chromatography (HPLC) with refractive index detector, and an amino acid analysis kit to quantify acrylamide, reducing sugars, and asparagine contents, respectively. However, such an approach takes considerable time and needs large sample amounts. Meanwhile, the limit of quantification (LOQ) of the HPLC method is too restrictive and cannot be useful in trace quantification of reducing sugars. Besides, it is difficult to determine sugar levels in bread using LCMS/MS analysis due to restrictive LOQ.15 Thus, the development of a rapid method for simultaneous analysis of acrylamide, asparagine, and reducing sugars as an essential tool plays an important role in corresponding kinetic and mechanistic studies. The aim of this study was to develop a fast and sensitive microwave digestion labstation (MDL) plus ultra high-performance liquid chromatographytandem mass spectrometry (UHPLCMS/MS) method to simultaneously determine acrylamide, asparagine, glucose, and fructose levels in Maillard reaction products using an isotope dilution technique. The Maillard reaction products were prepared via the use of MDL with different heating times to mimic different stages of Maillard reactions.

’ EXPERIMENTAL SECTION Chemicals and Materials. Acrylamide, D-(þ)-glucose monohydrate, and D-()-fructose were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). L-Asparagine monohydrate was purchased from Biocity Science and Technology Inc. (Beijing, China). D3-Acrylamide, 13C6-glucose, and 15N2-asparagine were obtained from Cambridge Isotope Laboratories (Cambridge, MA). Working standards were prepared by making appropriate dilutions of stock solutions with 0.2% of formic acid in water and stored at 4 °C. The potato powder made of the Atlantis variety was provided by Sanjiang (Group) Potato Products Co., Ltd. (Lintao, Gansu, China). Maillard reactions were performed via MDL in a potato model. Microwave Digestion Labstation (MDL). The microwave heating reaction between asparagine and glucose was performed via an Ethos D MDL from Milestone Inc. (Shelton, CT). In this labstation, there are nine microwave sample vessels (including one reference vessel) in the carousel, which allows nine groups of reactions between substrates simultaneously under identical reaction conditions. The ATC-400CE automatic temperature and APC-55 automatic pressure control systems connected with the reference vessel allow real-time monitoring of internal temperature ((1 °C) and vapor pressure ((1 bar), respectively. In addition, a focused and high-sensitivity IR sensor is used for monitoring the surface temperature of all sample vessels inside the cavity. The reaction temperature and time and their limits can be modulated via a digital intelligent control panel connected with all of the above control systems. In our preliminary test, the maximum pressure of the asparagineglucose reaction was determined as 9.5 bar (950 kPa), which was safe (maximum safe pressure: 3500 kPa) for mimicking Maillard reactions.16 Preparation of Maillard Reaction Products. The substrate stock solutions, i.e., asparagine (0.2 M) and glucose (1 M), were

ARTICLE

prepared in phosphate buffer (0.1 M, pH 6.80). Maillard reactions were performed between equimolar levels of asparagine and glucose in a potato model system by MDL. The potato model system consisted of 20% potato matrix, in which the asparagine and glucose concentrations were adapted to a final equimolar level of 0.14 M after spiking with the above stock solutions. The volume of reaction solutions was made up to 10 mL by the phosphate buffer. Finally, all the microwave digestion vessels containing reactants were carefully sealed and then microwave-heated under a working power of 500 W at 180 °C after a prepared temperature programming as follows: room temperature f 120 °C (200 W, 5 min); 120 °C f 180 °C (500 W, 5 min). The microwave heating reaction was performed in triplicate repeats (n = 3) with different duration times (0.1, 1, 2, 3, 4, 5, 7.5, 10, 15, 25, and 40 min), which mimicked different stages of Maillard reactions. The asparagineglucose reaction with the absence of duration time was regarded as the control. At the end of heating, the digestion vessels filled with final reaction products were taken out and immediately cooled in prepared icewater to stop any further reaction. Sample Pretreatment Procedures. A mixed solution of internal standards including D3-acrylamide (10 μg/mL), 13C6glucose (1000 μg/mL), and 15N2-asparagine (100 μg/mL) was prepared in advance. An aliquot (10 μL) of Maillard reaction products with different heating duration times was sampled. Exceptionally, 40 μL of the reaction product with the heating duration time of 40 min was sampled. Then, 20 μL of mixed internal standard solution was added and the reaction solution was appropriately diluted with the mobile phase. Then, each sample solution was vortex-mixed and passed through 0.22 μm filtration membrane (Empire Science and Technology Co. Ltd., Hangzhou, China). Alternatively, a solid-phase extraction (SPE) cleanup step was conducted instead of membrane filtration. Two different kinds of SPE cartridges, i.e., Bond Elut-C18 cartridges (500 mg) (Varian, Palo Alto, CA) and Oasis HLB cartridges (200 mg) (Waters, Milford, MA), were used for the SPE cleanup. The aliquot of filtrate or SPE elution was finally transferred to an autosampler vial for UHPLCMS/MS analysis. To check whether there was a problem with acrylamide contamination from the filters or plastics, an aliquot (10 μL) of asparagine and glucose mixture without microwave heating was sampled and pretreated according to the above procedures. UHPLCMS/MS Analysis. The liquid chromatography was performed on an Acquity UHPLC system equipped with the micro vacuum degasser, autosampler, and column compartment (Waters, Milford, MA). Chromatographic separation was performed on a Hypercarb column (100 mm  2.1 mm i.d., 5 μm particle size, Thermo Electron, San Jose, CA) guarded with a precolumn of the same packing material (10 mm  2.1 mm i.d.). The mobile phase was formic acid (0.2%, v/v) at a flow rate of 0.2 mL/min. The chromatographic column was maintained at 40 °C with a run time of 5.5 min per sample. The sample injection volume was 5 μL. Tandem mass spectrometry was performed on a Quattro Ultima triple-quadrupole mass spectrometer (Micromass Company Inc., Manchester, U.K.) using the electrospray ionization (ESI) source. Nitrogen and argon were used as the nebulizer and collision gas, respectively. The analytes were detected by MS/MS in multiple reactions monitoring (MRM) mode. The detection of asparagine and 15N2-asparagine was operated in negative ion mode due to matrix peak interferences in positive ion mode. All of other analytes including isotope compounds were monitored in positive ion mode. 3298

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry

ARTICLE

Table 1. Retention Time (tR), MRM Transitions, Optimized Collision Energy, Dwell Time, and Channel Run Time of Analytes and Relevant Internal Standards tR (min)

traces (m/z)

collision energy (eV)

dwell time (s)

asparagine [M  H]

1.6

131.4 > 113.2

9

0.1

0 f 3.5

N2-asparagine [M  H] glucose [2M þ Na]þ

1.6 2.3

133.4 > 115.4 383.6 > 203.4

9 10

0.1 0.1

0 f 3.5 0 f 3.5

fructose [2M þ Na]þ

1.9

383.6 > 203.4

10

0.1

0 f 3.5

2.3

395.7 > 209.4

10

0.1

0 f 3.5

acrylamide [M þ H]þ

4.5

71.9 > 54.6

5

0.3

3.5 f 5.5

D3-acrylamide [M þ H]þ

4.5

74.7 > 58.4

5

0.3

3.5 f 5.5

compound 

15

C6-glucose [2M þ Na]þ

13

The ionization working parameters were optimized by the automatic optimization function of MassLynx v4.0 (Micromass, Manchester, U.K.) and finalized as follows: capillary voltage, 3.0 kV (ESI mode) and 3.5 kV (ESIþ mode); cone voltage, 45 V (ESI mode) and 50 V (ESIþ mode); source temperature, 120 °C; desolvation gas temperature, 350 °C; desolvation gas flow, 500 L/h; cone gas flow, 45 L/h; argon collision gas pressure to 3  103 mbar for MS/MS. The collision energy for each MRM transition was optimized. System control and data processing were performed by MassLynx v4.0. The retention time, MRM transitions, optimized collision energy, dwell time, and channel run time of each compound are shown in Table 1. Assessment of Matrix Effects in the Current Quantitative Method. Two sets of eight different levels of asparagine, glucose, fructose, and acrylamide were prepared to evaluate the absence or presence of matrix effects.17 The first set (set 1) was prepared to evaluate the MS/MS response for asparagine, glucose, fructose, and acrylamide in pure solvent solutions. The second set (set 2) was prepared in one of the Maillard reaction products and spiked with four standards after sample pretreatment. By comparing the absolute peak areas and slopes of the standard lines between these two different sets, the absence or presence of matrix effects was assessed. Set 1: five repeats of each level of analytes were conducted using analyte pure solvent solutions dissolved in the optimized mobile phase. Eight different levels (1, 5, 10, 20, 30, 50, 75, and 100 μM) of each standard were prepared. Aliquots (5 μL) of solutions were then directly injected into the UHPLCMS/MS system. Set 2: five repeats of each level of analytes were conducted in one of the Maillard reaction products. The sample matrixes were pretreated via the dilution of water and passed through 0.22 μm filtration membrane or followed by an SPE cleanup step (see details in the Sample Pretreatment Procedures section). Then, the sample filtration solutions were spiked with eight different levels (final concentration: 1, 5, 10, 20, 30, 50, 75, and 100 μM) of each standard and finally injected into the instrumental system. If one describes the peak areas obtained in pure standards in set 1 as A, and the corresponding peak areas for standards spiked after pretreatment into sample filtration solutions as B (set 2), the matrix effect (ME) can be calculated as the percentage ratio of B to A. To avoid negative values in the case of ion suppression, the ratio (B/A) is defined here generally as an “absolute” matrix effect.17

’ RESULTS AND DISCUSSION Kinetic Study Tools. A complete schematic diagram of current kinetic study tools is shown in Figure 1. Microwave heating provides a favorable medium for the occurrence of acrylamide and affects the formation and kinetics of acrylamide

channel run time (min)

distinguishingly due to its extraordinary heating style. This study uniquely used MDL to mimic the occurrence of Maillard reactions via microwave heating. Compared to the use of a microwave oven, this labstation allows parallel and simultaneous tests, more precise temperature and pressure control, faster reaction rate, and less matrix interference. The present kinetic tools cost 1050 min (different microwave heating duration times plus labstation temperature programming time) for the mimic of Maillard reactions. The separation using the Hypercarb column and UHPLCMS/MS quantification under positive negative ion switching mode allow simultaneous analysis of all analytes and rapid run time (5.5 min per run). Compared to previous kinetic study,14 current kinetic tools reduce considerable sample analysis time and labor effort. Besides, the use of simple potato matrix and MDL reduces matrix interference and simplifies sample treatment procedures. Optimization of LC Conditions. The simultaneous analysis performed on the present chromatographic system seems difficult because of the great polarity difference between acrylamide and reducing sugars. Besides, the separation between two sugar isomers (glucose and fructose) is a great challenge. Therefore, the selection of the chromatographic column should be wellconsidered. Four LC columns, i.e., (i) Shiseido Capcell Pak UG80 NH2 column (250 mm  4.6 mm i.d., 5 μm), (ii) Shiseido Capcell Pak C18 AQ column (150 mm  2.0 mm i.d., 3 μm), (iii) Waters Acquity UPLC BEH C18 column (50 mm  2.1 mm i.d., 1.7 μm), and (iv) Thermo Hypercarb column (100 mm  2.1 mm i.d., 5 μm), were investigated for the signal intensity and separation efficiency of analytes (Figure 2). Previous studies reported an LC column coated with amino-based proprietary bonding material is suitable for the separation of sugar compounds.1820 The chromatographic analysis using the UG80 NH2 column and the mobile phase of acetonitrile solution (75%, v/v) led to a fair separation between glucose and fructose in this study. However, the retention of asparagine and acrylamide was poor (Figure 2A). Conversely, appropriate retentions of acrylamide and asparagine were found when the C18 AQ column was available for use. However, glucose and fructose could not be separated (Figure 2B). This column allows the use of water as the mobile phase, which is suitable for simultaneous analysis of compounds with great polarity difference. Nevertheless, the failure of sugar separation limits its application. The UPLC BEH C18 column can bear the highest column pressure and obtain amazing analytical speed. But it is not suitable for the separation of sugar compounds, either (Figure 2C). Compared to all of the above columns, the use of the Hypercarb column not only ensured the requested separation between glucose and fructose, but also achieved favorable retention of acrylamide and asparagine (Figure 2D). Thus, the Thermo Hypercarb column was finally chosen. 3299

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry

ARTICLE

Figure 1. Schematic diagram of microwave digestion labstation (MDL) and UHPLC isotope dilution positivenegative ion switching MS/MS as kinetic study tools. AA, acrylamide; ASN, asparagine; FRU, fructose; GLU, glucose; I.S., internal standard.

Figure 2. Comparison of separation effects for asparagine, glucose, fructose, and acrylamide among four different candidate columns: (A) Shiseido Capcell Pak UG80 NH2 column (250 mm  4.6 mm i.d., 5 μm); (B) Shiseido Capcell Pak C18 AQ column (150 mm  2.0 mm i.d., 3 μm); (C) Waters Acquity UPLC BEH C18 column (50 mm  2.1 mm i.d., 1.7 μm); (D) Thermo Hypercarb column (100 mm  2.1 mm i.d., 5 μm). AA, acrylamide; ASN, asparagine; FRU, fructose; GLU, glucose. 3300

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry

ARTICLE

Figure 3. Representative UHPLCMS/MS chromatograms of simultaneous determination of asparagine, glucose, fructose, and acrylamide in Maillard reaction products. AA, acrylamide; ASN, asparagine; FRU, fructose; GLU, glucose. (A) The concentrations of analytes were at their LOQ levels: CAA = 0.1 μM, CGLU = 0.6 μM, CFRU = 1.1 μM, CASN = 0.8 μM. (B) The concentrations of analytes were at levels when samples were heated for 40 min: CAA = 0.8 mM, CGLU = 0.8 mM, CFRU = 0.5 mM, CASN = 1.3 mM. The monitoring channels presented from the top to the bottom in this figure indicate acrylamide (m/z 71.9 > 54.6), fructose (left peak) and glucose (right peak) (m/z 383.6 > 203.4), and asparagine (m/z 131.5 > 113.2), respectively.

The ionization efficiency of acrylamide can be significantly improved by adding formic acid (0.10.5%, v/v) into the mobile phase. Besides, formic acid is useful for the ionization of sugar compounds, which are not easily ionized. However, addition of formic acid reduces the pH of the mobile phase and affects chromatographic separation. Thus, the addition of ammonium acetate (NH4Ac, 20 mM) in formic acid (0.2%. v/v) could be considered. In the present study, formic acid (0.2%. v/v) without the addition of NH4Ac as the mobile phase was finally selected according to the separation and ionization efficiency of compounds. Optimization of MS/MS Conditions. Asparagine can be monitored under both ESIþ and ESI modes and eluted at the beginning of a chromatogram.15 In the present work, much less noise was observed under the ESI mode rather than ESIþ mode. The product ion scan of asparagine under the ESI mode is shown in Supporting Information Figure S1A. The fragment ion m/z 114 corresponds to the loss of ammonia, m/z 113 corresponds to the loss of water, whereas at m/z 70 the most likely fragmentation pattern would be the loss of ethylamide.15 The fragment ions m/z 115 and 71 of 15N2-asparagine were found, which are related to the loss of 15N-ammonia or water and 15N-ethylamide, respectively (Supporting Information Figure S2A). Previous studies suggested chlorine negative ion [M þ Cl]or cesium positive ion [M þ Cs]þ as the precursor ion of sugars.21,22 Saccharides can be ionized as ammonium or sodium ion adducts without the addition of sodium or ammonium ions into the mobile phase in advance. The sodium ion adducts are usually more abundant than the ammonium ion adducts.15,20 Supporting Information Figure S1, parts B and C, shows the fragmentation from the fragment scan of m/z 383, indicating the sodium ion adducts of glucose and fructose. They were monitored as monosodiated cluster ions [2M þ Na]þ fragmenting to monosodiated adduct ions [M þ Na]þ at m/z 203. Such fragment patterns indicated that successful chromatographic separation was the only way to the differentiation of glucose and fructose. Similarly, 13C6-glucose was detected at m/z 395 and fragmented at m/z 209 (Supporting Information Figure S2B). Besides, we have not found any other fragment ions from [2M þ Na]þ even when the collision energy exceeded 10 eV, indicating [M þ Na]þ was further fragmentized completely. Although

sodium adducts were not normally used for quantification as these types of adducts were difficult to be fragmentized by MS/MS, the difference between precursor ion and fragment ion was due to the absence of Mþ and not related to Naþ. The specificity of the fragment patterns of glucose and fructose was also coapproved by the similar fragment of 13C6-glucose. The fragment of acrylamide and D3-acrylamide were in agreement with previous work.10,16,23 The collision energy of all analytes was optimized according to the maximal abundance of their quantitative ions. Optimization of Sample Preparation and Pretreatments. To ensure the occurrence of equimolar-level Maillard reactions between asparagine and glucose, the original contents of these two substrates in potato matrix should be quantified in order to calculate their spiking levels. Using the current UHPLCMS/ MS method, the contents of asparagine, glucose, and fructose in potato matrix were quantified as 381, 162, and 145 μg/g, respectively (n = 3). To evaluate the precision of the above quantification, the concentrations of asparagine and sugars were comparatively analyzed by HPLC,14 which was performed on a Waters 2695 HPLC chromatograph with a Capcell Pak C18 AQ column (150 mm  2.0 mm i.d., 5 μm) protected by an RP18 guard column (4.0 mm  3.0 mm i.d., 5 μm), both purchased from Phenomenex Co. Other HPLC parameters included mobile phase (acetonitrile, 75%, v/v), flow rate (1.0 mL/min), injection volume (30 μL), column temperature (25 °C) ,and detectors (asparagine, diode array detector, 210 nm; glucose and fructose, differential refractive index detector). Results of HPLC analysis showed that the potato matrix contains 370 μg/g of asparagine, 153 μg/g of glucose, and 136 μg/g of fructose (n = 3). Statistical analysis demonstrated no significant difference of asparagine and sugars levels determined by the two methods (P > 0.05). Finally, an equimolar asparagineglucose model system was prepared via assembling two portions of asparagine and glucose from (i) potato matrix and (ii) complement of pure chemical solutions. The present study is devoted to rapid and easy-to-use sample pretreatment procedures. Current optimization work focused on whether an SPE process was a prerequisite. Results indicated that impurities were greatly eliminated using both Bond Elut-C18 cartridges (500 mg) and Oasis HLB cartridges (200 mg), and appropriate spiked recoveries (>80%) of acrylamide and sugars 3301

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry

ARTICLE

Table 2. Matrix Effect Data for Asparagine, Glucose, Fructose, and Acrylamide in Maillard Reaction Products

Table 3. Standard-Spiked Recovery, LOD, and LOQ Tests (n = 3)a asparagine

matrix effect (%)a nominal concn (μM)

asparagine

glucose

fructose

1

73.4 ( 6.6

64.8 ( 7.1

68.0 ( 4.3

81.7 ( 3.3

71.1 ( 8.7

61.9 ( 3.0

66.9 ( 3.8

83.4 ( 7.8

10 20

71.4 ( 4.6 75.2 ( 8.0

60.5 ( 5.6 69.4 ( 7.1

59.7 ( 3.4 63.0 ( 5.7

78.9 ( 8.1 89.3 ( 2.4

30

78.5 ( 4.2

67.3 ( 6.1

70.1 ( 2.9

80.2 ( 4.6

50

77.9 ( 5.9

73.5 ( 3.3

67.4 ( 5.7

81.9 ( 5.3

75

80.0 ( 6.3

66.6 ( 7.0

69.9 ( 6.0

82.7 ( 3.7

100

81.1 ( 9.1

74.2 ( 2.6

72.1 ( 4.4

90.9 ( 5.0

a

The test was performed in quintuplicate repeats (n = 5). Matrix effect expresses as the ratio of the mean peak area of an analyte spiked postextraction (set 2) to the mean peak area of the same analyte standards (set 1) multiplied by 100. A value of >100% indicates ionization enhancement, whereas a value of 0.05). Therefore, 13 C6-glucose was finally used for the internal standard of fructose. Besides, the quantification of glucose and fructose relied on their different retention and selectivity under optimal LC other than MS/MS conditions as described before. The possible cross-talk between two sugars may be checked via their separation and retention. On the basis of representative chromatogram of glucose and fructose (Figure 3B), the separation factor (R) and resolution factor between adjacent sugar peaks were calculated as 1.21 and 1.04, respectively. These results could ensure simultaneous quantification of glucose and fructose via the same MS/MS channel. LOD and LOQ are estimated as the concentration of analyte that produces a signal-to-noise (S/N) ratio of 3:1 and 10:1 in Maillard reaction products, respectively. In LOD and LOQ tests, samples were spiked with isotope internal standard mixture and gradually diluted until S/N = 3 and S/N = 10 of analyte peaks were reached, respectively, when samples were run in the UHPLCMS/MS system. The LOD and LOQ ranges of all analytes were determined as 0.0030.1 and 0.010.2 ng/g, respectively (Table 3). The quantification of asparagine, sugars, and acrylamide was achieved by means of eight-point calibration curves performed on a matrix of Maillard reaction products and application of internal standards. The minimum calibration concentrations of analytes were selected according to their respective LOQ values. The concentration ranges of analytes were 0.88 μM (asparagine), 0.66 μM (glucose), 1.16 μM (fructose), and 0.11 μM (acrylamide). Calibration results showed that asparagine and acrylamide were fitted linearly while glucose and fructose were calibrated quadratically (Supporting Information Table S2). All calibration curves of analytes were highly correlated (R2 > 0.998) within their concentration ranges. Standard-spiking recoveries of the current method were performed in Maillard reaction products with a 5 min heating time treatment. Samples were pretreated according to optimized procedures and spiked with the low (0.5 mM), intermediate (1 mM), and high levels (5 mM) of asparagine, glucose, and 3302

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry

Figure 4. Concentrations and kinetic profiles of (A) asparagine, (B) glucose, (C) fructose, and (D) acrylamide in Maillard reaction products with different heating time treatments. Each experiment was performed in triplicate repeats (n = 3). Conc., concentration.

fructose standards. Besides, the low, intermediate, and high spiking levels of acrylamide were 0.05, 0.1, and 0.5 mM, respectively. The internal standards 15N2-asparagine (100 μg/mL), 13 C6-glucose (1000 μg/mL), and D3-acrylamide (10 μg/mL) were also spiked for the quantification (see Sample Pretreatment Procedures Section). The recovery results are summarized in Table 3. The recovery ranges of analytes with low, intermediate, and high standard-spiking levels were 69.885.6%, 82.492.4%, and 84.295.3%, respectively. The repeatability and within-laboratory reproducibility were determined in accordance with the ISO 5725-2 criteria.26 The repeatability (n = 6) with an RSD range of 1.39.6% seems good via the determination of representative samples with 0.1, 5, 15, and 40 min heating time treatment (Supporting Information Table S3). In the within-laboratory reproducibility test, the above four representative samples were also selected and investigated. This experiment was repeated six times within a day for the intraday precision test and additionally performed once each day, continuously for 6 days, for the interday test. Results of both precision tests are shown in Supporting Information Table S4. The RSD ranges of analytes by the present method were 1.39.2% for the intraday test and 2.89.0% for the interday test, respectively. Excellent repeatability and reproducibility demonstrated the stability of the current quantitative method and long-time and large-amount analysis of samples. Considering all of the above method validation data, current kinetic tools and

ARTICLE

sample pretreatment procedures employed in the present work can be regarded as a rapid and robust quantification method, which was successfully applied to simultaneous analysis of asparagine, glucose, fructose, and acrylamide in Maillard reaction products and corresponding kinetic studies. Analysis of Maillard Reaction Product Samples and Kinetic Evaluation. To validate the application of current kinetic tools, this analytical method was applied to simultaneous quantification of all analytes in Maillard reaction products with different heating time treatments. Results appearing from Figure 4 showed great discrepancy of asparagine, glucose, and fructose levels in samples with different heating time treatments. The kinetics of asparagine, glucose, fructose, and acrylamide were clearly profiled. In the absence of heating (heating time, 0 min), the contents of two precursors was quantified as approximately 0.14 M, which is the addition level used in this study, while fructose and acrylamide could not be detected, indicating there is no contamination in the experiments from pretreatment procedures. The contents of two precursors were quickly reduced within the initial 2 min during microwave heating, indicating a strenuous reaction process. With the development of reaction, the content variation of asparagine and glucose were gradually gentle. Meanwhile, glucose was transferred into fructose via isomerization, which then reacted with asparagine. The kinetic profile of fructose indicated a quick increase at the beginning of the reaction followed by a steep decrease also within the initial 2 min. The elimination kinetics of glucose seems faster than asparagine because glucose is simultaneously involved in the isomerization and Maillard reactions. The quantitative analysis using the present UHPLCMS/MS method was successfully applied to simultaneous determination of four analytes during Maillard reactions and the kinetic elucidation. The kinetic profiles of acrylamide and its precursors and intermediates were in good correspondence with previous data.15 Compared to individual analysis of these analytes in previous work, the advantage of the current method was the simultaneous quantification of all relative compounds, which greatly reduced the time and cost. Besides, the present method also eliminated the error of results from different quantitative methods.

’ CONCLUSIONS This study developed kinetic tools for simultaneous determination of acrylamide, asparagine, glucose, and fructose in Maillard reaction products and corresponding kinetic elucidation. The analysis was performed by MDL combined with the isotope dilution UHPLCMS/MS method without significant interference from matrix effects. There was no need for the timeconsuming derivatization and SPE cleanup during sample pretreatments, which were prerequisites of well-known analytical methods. Isotope dilution using 15N2-asparagine, 13C6-glucose, and D3-acrylamide was appropriately used for accurate quantification of asparagine, glucose, fructose, and acrylamide, respectively. The present method was successfully applied to simultaneous determination of analytes during Maillard reactions and kinetic elucidation. Besides, current kinetic tools could be used for reduction studies on acrylamide, such as pH modification, improvement of heating processing parameters, and use of food additives, especially when asparagine, reducing sugars, and acrylamide in numerous samples need to be quantified. The advantages on rapid determination of current tools were obviously shown. Rapid pretreatment procedures and short 3303

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304

Analytical Chemistry run time (5.5 min per sample) ensure excellent efficiency of researches, suggesting a wide application for the mechanism and kinetic studies on the formation and reduction of acrylamide in Maillard reactions.

’ ASSOCIATED CONTENT

bS

Supporting Information. Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

ARTICLE

(21) Rogatsky, E.; Jayatillake, H.; Goswami, G.; Tomuta, V.; Stein, D. J. Am. Soc. Mass Spectrom. 2005, 16, 1805–1811. (22) Tannenbaum, H. P.; Roberts, J. D.; Dougherty, R. C. Anal. Chem. 1975, 47, 49–54. (23) Zhang, Y.; Ying, T. J.; Zhang, Y. J. Food Sci. 2008, 73, C60–C66. (24) Stadler, R. H.; Scholz, G. Nutr. Rev. 2004, 62, 449–467. (25) Zhang, Y.; Zhang, Y. Crit. Rev. Food Sci. Nutr. 2007, 47, 521–542. (26) ISO 5725-2:1994. Accuracy (trueness and precision) of measurements methods and results—Part 2, Geneva, Switzerland.

’ AUTHOR INFORMATION Corresponding Author

*Phone: 86 571 8898 2164. E-mail: [email protected].

’ ACKNOWLEDGMENT This study was financially supported by the National Natural Science Foundation of China (Grant No. 30972486) and the Eleventh Five-Year National Key Technology R&D Program of China (Grant No. 2009BADB9B07-2a). ’ REFERENCES (1) SNFA, Swedish National Food Administration. Acrylamide is formed during the preparation of food and occurs in many foodstuffs. Press release April 24, 2002. http://www.slv.se/templatesSLV/ SLV_Page____6182.asp. (2) Monographs on the Evaluation of Carcinogenic Risks to Humans; International Agency for Research on Cancer (IARC): Lyon, France, 1994; Vol. 60, pp 389433. (3) Tilson, H. A. Neurobehav. Toxicol. Teratol. 1981, 3, 445–461. (4) Dearfield, K. L.; Abernathy, C. O.; Ottley, M. S.; Brantner, J. H.; Hayes, P. F. Mutat. Res. 1988, 195, 45–77. (5) Dearfield, K. L.; Douglas, G. R.; Ehling, U. H.; Moore, M. M.; Sega, G. A.; Brusick, D. J. Mutat. Res. 1995, 330, 71–99. (6) Wenzl, T.; de la Calle, M. B.; Anklam, E. Food Addit. Contam. 2003, 20, 885–902. (7) Wenzl, T.; Lachenmeier, D. W.; G€okmen, V. Anal. Bioanal. Chem. 2007, 389, 119–137. (8) Zhang, Y.; Zhang, G. Y.; Zhang, Y. J. Chromatogr., A 2005, 1075, 1–21. (9) Mastovska, K.; Lehotay, S. J. J. Agric. Food Chem. 2006, 54, 7001–7008. (10) Chu, S. G.; Metcalfe, C. D. Anal. Chem. 2007, 79, 5093–5096. (11) Hedegaard, R. V.; Frandsen, H.; Skibsted, L. H. Food Chem. 2008, 108, 917–925. (12) Pollien, P.; Lindinger, C.; Yeretzian, C.; Blank, I. Anal. Chem. 2003, 75, 5488–5494. (13) Yaylayan, V. A.; Wnorowski, A.; Locas, C. P. J. Agric. Food Chem. 2003, 51, 1753–1757. (14) Knol, J. J.; van Loon, W. A. M.; Linssen, J. P. H.; Ruck, A.-L.; van Boekel, M. A. J. S.; Voragen, A. G. J. J. Agric. Food Chem. 2005, 53, 6133–6139. (15) Nielsen, N. J.; Granby, K.; Hedegaard, R. V.; Skibsted, L. H. Anal. Chim. Acta 2006, 557, 211–220. (16) Zhang, Y.; Fang, H. R.; Zhang, Y. Food Chem. 2008, 108, 542–550. (17) Matuszewski, B. K.; Constanzer, M. L.; Chavez-Eng, C. M. Anal. Chem. 2003, 75, 3019–3030. (18) Clarke, M. B.; Bezabeh, D. Z.; Howard, C. T. J. Agric. Food Chem. 2006, 54, 1975–1981. (19) Dye, C.; Yttri, K. E. Anal. Chem. 2005, 77, 1853–1858. (20) Wan, E. C. H.; Yu, J. Z. J. Chromatogr., A 2006, 1107, 175–181. 3304

dx.doi.org/10.1021/ac1029538 |Anal. Chem. 2011, 83, 3297–3304