Article pubs.acs.org/JAFC
Simultaneous Analysis of PhIP, 4′-OH-PhIP, and Their Precursors Using UHPLC−MS/MS Yan Yan,† Mao-Mao Zeng,† Zong-Ping Zheng,† Zhi-Yong He,† Guan-Jun Tao,† Shuang Zhang,† Ya-Hui Gao,‡ and Jie Chen*,†,§ †
Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China School of Food Science and Technology, Jiangnan University, Wuxi 214122, People’s Republic of China § Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, People’s Republic of China ‡
S Supporting Information *
ABSTRACT: A novel method allowing simultaneous analysis of PhIP, 4′-OH-PhIP, and their precursors (phenylalanine, tyrosine, creatine, creatinine, glucose) has been developed as a robust kinetic study tool by using ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC−MS/MS). A direct hydrochloric acid (HCl) extraction was applied to achieve the simultaneous extraction of all seven analytes, with the mean recoveries ranging from 60% to 120% at two concentration levels. Then, an Atlantis dC18 column selected from four different chromatographic columns was ultimately used to separate these compounds within 15 min. The limits of detection range of allseven analytes were calculated as 0.14−325.00 μg L−1. The intra- and interday precision of the proposed method were less than 15.4 and 19.9%, respectively. The proposed method was successfully applied to depict the kinetic profiles of PhIP, 4′-OH-PhIP, and their precursors in pork model, reducing the analysis time and cost in the kinetic study. KEYWORDS: heterocyclic aromatic amines, precursors, simultaneous analysis, meat model, UHPLC−MS/MS
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INTRODUCTION 2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) and 2-amino-1- methyl-6- (4-hydroxyphenyl) imidazo [4,5-b] pyridine (4′-OH-PhIP) are heterocyclic aromatic amines (HAAs) often identified in a variety of meat products such as pork, beef, and chicken.1−4 They are reported to be mainly produced from creati(ni)ne, sugars, amino acids, or some nitrogenous bases and nucleotides.5−7 Over the past few years, a number of studies have detected much higher concentrations of PhIP than any other HAAs in meat, and it has attracted attention as a potential carcinogenin animals and a mutagen being able to bind the DNA in human.8−12 Moreover, the International Agency for Research on Cancer had classified the PhIP as a possible human carcinogen.13 4′-OH-PhIP, the hydroxyl derivative of PhIP, has been reported to be present in a variety kinds of meat products and could induce low mutagenicity in S. typhimuricm TA 98 WITH S9 mix.1,6,14,15 To the best of our knowledge, few studies have addressed this new mutagen. In evaluating HAA formation mechanisms, the use of kinetic studies to clarify the different reaction stages of HAAs has become increasingly notable. Several studies have investigated the HAA generation process from a kinetic perspective.16,17 For example, Arvidsson et al.18 conducted a detailed kinetic evaluation of five HAAs (IQx, MeIQx, 7,8-DiMeIQx, 4,8DiMeIQx, and PhIP) at four temperature levels (150, 175, 200, and 225 °C) and fit the kinetic data for IQx and PhIP to a firstorder model. In another study, the formation kinetics of PhIP were evaluated in four kinds of meat heated to 180 and 220 °C.8 These studies all used the formation kinetics of HAAs as the only research target. Nevertheless, to completely describe © 2014 American Chemical Society
the kinetic behavior of HAAs and further evaluate their formation mechanisms, the changes in their precursors, such as amino acids, sugars and creati(ni)ne, should also be considered. Several recent studies of HAA formation have noted the effects of the HAA precursor levels and proven that the HAA levels generated in meat products were closely related to their precursors.19,20 In these studies, precursor contents were generally determined to establish a relationship with the formation of HAAs. However, different analytical methods were often separately used to detect the HAAs and their precursors. There is lack of a published method for simultaneously detecting HAAs and their precursors. For example, Borgen et al.21 published an investigation on the effects of precursor composition on HAA formation. In this study, the author used HPLC with a photodiode array UV and a fluorescence detector to quantify HAAs, ion-exchange chromatography to analyze the free amino acids and enzymatic method to determine the glucose and creati(ni)ne. This separate analytical strategy requires a number of analytical runs, which takes considerable time and involves large amounts of reagents and equipment. Therefore, a rapid method for the simultaneous analysis of HAAs and their precursors is in high demand for research regarding to kinetic and mechanistic studies of HAA formationboth in model systems and in food. The simultaneous determination of HAAs and their precursors is challenging due to the difference in their Received: Revised: Accepted: Published: 11628
August 6, 2014 October 19, 2014 October 19, 2014 October 19, 2014 dx.doi.org/10.1021/jf503776e | J. Agric. Food Chem. 2014, 62, 11628−11636
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Model System. The dried fresh meat model system was established according to an earlier study, with some modification.8 Pork was purchased at a local market and freeze-dried for 48 h. The lyophilized samples of raw pork (0.7 g) were mixed with diethylene glycol (3 mL) in the glass reaction vials and heated at 195 °C for 0− 120 min using a thermostat-controlled oil-bath with electronically controlled temperature and temperature probe from RKC Instrument Inc. (Tokyo, Japan). The heating block was preheated for 2 h before sealing and inserting the vials. The temperature was checked every 30 s, and temperature fluctuations were within 2 °C. After heating, the vials were immediately cooled in an ice-bath to stop any further reaction. The model system experiments were performed in triplicate. Sample Preparation Procedures. The content of the reaction vials was diluted and homogenized with 12 mL of 0.1 M hydrochloricacid (HCl) (four times for 15 s). Then the homogenate was extracted for 15 min using ultrasound. After the centrifugation of the mixture (11363 g for 20 min at 4 °C), the supernatant was collected. The extraction procedures were repeated twice, and then the extracting solutions were combined and the volume was fixed to 25 mL with the HCl solution. Ethanol was added to 10 mL of acidic solution with an end concentration of 80% to remove the protein.29 The protein-free extract obtained was concentrated until it was dry in a rotary evaporator at 39 °C and then dissolved in 2 mL by solution of 0.1% formic acids and 50% methanol in water before being passed through a 0.22 μm filtration membrane. Alternatively, the SPE purification procedure with three cartridges (Supelclean LC-18, Oasis MCX, and STYRE-SCREEN SSH2P) was implemented instead of the membrane filtration. For MCX cartridge, columns were first activated continuously with 6 mL of methanol, 6 mL of distilled water, and 6 mL of 0.1 M HCl solution, and were then rinsed sequentially with 6 mL of 0.1 M HCl solution and 6 mL of methanol after loading the sample. The retained analytes were finally eluted by a 6 mL mixture of methanol and ammonium hydroxide (25%) at a ratio of 95:5. For the LC-18 column, after conditioned with 6 mL of methanol and water, the columns were loaded the sample. First 1 mL of the sample solvents were discarded. And the subsequent sample solvents and the eluates from the 6 mL of the methanol were collected. With regard to the SSH2 P cartridge, similar activate procedures as MCX were implemented. After loading the sample, the cartridges were then washed with water and 50% methanol in water. The target compounds were ultimately eluted with 6 mL of the methanol. All the elution obtained from the SPE procedure were then evaporated to dryness under a stream of nitrogen and then resuspended in 300 μL of the mixed solution (0.1% formic acids and 50% methanol in water). Finally, the filtrate or SPE elution was diluted to proper concentrations and transferred to an autosampler vial for UHPLC−MS/MS analysis. UHPLC−MS/MS Analysis. The chromatographic analysis was performed on a Waters Acquity UPLC system equipped with a quaternary pump system, micro vacuum degasser, autosampler and column compartment setting the temperature at 35 °C. The separation was carried out on a Waters Atlantics dC18 column (250 × 4.6 mm2 i.d., 3.0 μm) (Milford, MA), which was selected from three other columns: Waters Acquity UPLC BEH C18 (50 × 2.1 mm2 i.d., 1.7 μm), Waters Acquity UPLC HSS T3 (100 × 2.1 mm2 i.d., 1.8 μm) and Waters XSELECT HSS T3 (150 × 4.6 mm2 i.d., 3.5 μm). The gradient elution was achieved with a binary mobile phase of methanol (A) and formic acid (0.1%, v/v) (B) at a flow rate of 0.5 mL min−1. The gradient elution0−10 min 5−100% A phasewas finally returned to its initial composition and equilibrated for 5 min before the next injection. Separation and stabilization were achieved in 15 min. The single injection volume was set at 1 μL. The MS/MS detection was performed on a Waters Quattro Premier XE triple quadrupole mass spectrometer (TQD) (Milford, MA) using an electrospray ionization (ESI) source in positive mode. The data acquisition was carried out by MassLynxv 4.1 SCN 805 software from Waters. The capillary and extractor voltages were 3.5 kV and 3 V, respectively. Nitrogen (99.9% of purity) was used as the desolvation gas with the gas flow set to 800 L h−1 at a temperature of 350 °C. The flow rate of the cone gas was set to 50 L h−1 at a source temperature of 130 °C. Argon (99.9% of purity) was used as the collision gas at a flow
polarities. The present extraction method, as well as the column and mobile phase used for the retention and isolation of HAAs or sugars, may not suitable for their simultaneous analysis in a single run. Meanwhile, due to the absence of a chromophore, the amino acids, sugar and creati(ni)ne often need to be derivatized with different derivatization reagents, which complicates detection.22−24 To solve the extraction and retention problems, a suitable extraction solvent and a special column that promotes superior polar compound retention should be used. LC−MS/MSa very sensitive technique for HAA determination at trace levels25,26could eliminate the need for derivatization. Recently, this technique has been used as a tool to determine both amino acids and sugars in some published studies.27,28 In the present study, a robust method for the simultaneous quantification of two HAAs (PhIP and 4′-OH-PhIP) and their precursors (phenylalanine, tyrosine, creatine, creatinine, and glucose) in pork samples was established using ultra highperformance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC−TQD−MS/MS). The appropriate extraction solvents and chromatographic columns for the simultaneous extraction and isolation of compounds with different polarities were compared and selected. The method was validated with regard to linearity, detection limitations, matrix effects, and recoveries. Finally, the new kinetic tools were applied to a kinetic study of HAAs and their precursors in fresh pork model systems with different heating time treatments.
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MATERIALS AND METHODS
Reagents and Standard Solutions. Optima LC/MS methanol, acetonitrile and HPLC-grade formic acids (99.9%) were obtained from Thermo Fisher Scientific (Waltham, MA). Distilled water was purified using a Milli-Q filtration system from Millipore Corp. (Bedford, MA). All of the reagents were filtered through a 0.22 μm nylon or cellulose filter before injecting them into the UHPLC−MS/MS system. The SPE used Supelclean LC-18 columns were ordered from Supelco Corp. (St. Louis, MO). The Oasis MCX cartridges (60 mg, 3 mL) were obtained from Waters Corp. (Milford, MA) and the STYRE-SCREEN SSH2P columns were acquired from Sepax Technologies (Newark, NJ). The screw cap Tuf-Bond Teflon-fitted glass reaction vials (50 mL of capacity) were purchased from Pierce Company (Rockford, IL). All of the other chemicals used in this study were of analytical grade and purchased from Sinopharm Chemical Reagent Beijing Co. Ltd. (Beijing, China). Creatine, creatinine, and two HAAs standards, PhIP and 4′−OHPhIP (Figure 1), were purchased from Santa Cruz Biotechnology, Inc.
Figure 1. Structures of PhIP and 4′-OH-PhIP. (Santa Cruz, CA). L-phenylalanine (Phe), L-tyrosine (Tyr) and Dglucose (Glc) were obtained from Sigma-Aldrich Company (St. Louis, MO). The standard stocking solutions of each compound (about 125 mg L−1 for HAAs, about 1000 mg L−1 for amino acids, creatine, creatinine, and glucose) were prepared and stored at −20 °C in the dark. The working standards of all of the above-mentioned chemicals were prepared by making the appropriate dilutions of stock solutions with a mixed solution of 0.1% formic acids and 50% methanol in water, and stored at 4 °C. 11629
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Table 1. Retention Time and MS/MS Parameters of the HAAs and Their Precursors compound PhIP 4′-OH-PhIP Phe Tyr creatine creatinine Glc a
dwell time (secs) 0.037 0.037 0.037 0.037 0.037 0.037 0.037
cone voltage (V) 54 54 22 24 28 34 14
quantitation transitiona 225.03 241.03 165.98 181.98 131.97 114.03 382.90
> > > > > > >
210.01 (42) 225.76 (32) 119.68 (40) 122.73 (24) 89.83 (16) 44.02 (20) 202.73 (20)
proposed assignation [M + H − ̀ CH3] [M + H − ̀ CH3]+ [M + H − COOH − H]+ [M + H − COOH − NH2 + 2H]+ [M + H − NH2 − CN]+ [M + H − NH2 − CO − HCN]+ [2M + Na − M]+
conformation tranitiona
+
165.98 181.98 131.97 114.03
> > > >
102.80 (28) 90.81 (34) 43.77 (22) 85.84 (16)
Collision energy (V) is given in parentheses.
rate of 0.13 mL min−1. The specific MS/MS parameters (compound transitions, collision energy, cone voltage, and dwell times) were optimized with 1 ng μL−1 target compounds (Table 1). Validation. The proposed method was validated in terms of linearity, recovery, limit of detection (LOD), limit of quantification (LOQ), and matrix effects. The calibration curve was established by plotting the peak area ratios of standards to the internal standard against seven analyte concentrations. The recoveries of the sample preparation were determined by analyzing blank samples spiked with two concentration mixed standards (low and high) and calculated as [(levels in spiked samples − levels in blank samples)/amount added] × 100.The precision of the overall method was studied by implementing intraday (repeatability) and interday (reproducibility) precision experiments. Intraday precision was investigated at a low spiked level in a recovery study with three replicates, whereas interday precision was evaluated at a high spiked level in a recovery study that was accomplished over three consecutive days. The matrix effects were evaluated using the slope comparison method.30 The food sample extracts, which were spiked with diminishing concentrations of the standards, were used to establish standard addition calibration curves. Then, the slopes of these curves (A1, A2,···An) were compared with the slopes obtained from the pure standards (B1, B2,···Bn) at the same concentration levels, and the slope ratios Rn were equal to An/Bn. The matrix effects could then be decided with Rn as the ionization suppression effect (1) or without matrix effects (= 1).
separation between creatinine and Glc limit its application (Figure 2C). Using the same CSH technique, the Atlantics dC18 column is improved by the designer to pursue a simultaneous retaining and isolating of both the polar and nonpolar compounds with symmetrical peaks and proper retaining efficient. Compared with all of the above-mentioned columns, the use of the dC18 column not only achieved favorable retention for all analytes, but also offered acceptable separation between creatinine and Glc. Therefore, this column was ultimately selected (Figure 2D). In order to improve the separation between Glc and create(ni)ne, different mobile phase and gradient conditions were tested. Due to the surface charge used in the packing to enhance the polar compound retention, no significant improvements were observed. However, without the baseline separation for all analytes, the quantifications are acceptable, since these compounds could be separated by their molecular ions in MRM mode, according to a statement from a previous study.26 Therefore, a simple gradient condition was finally used to simultaneously detect the two HAAs and their precursors. The ESI−MS/MS parameters were optimized by injecting 1 ng μL−1 of the standard solution into the ESI source. Both the positive and negative ion modes were evaluated, and the results showed that all of the analytes were detectable in the positive mode. Amino acids could also be detected in the negative mode with relatively low sensitivity. Thus, the positive ion mode was ultimately preferred. As for the ionization, saccharides were suggested to possess multiple ionization options. Previous studies have reported cesium positive ions [M + Cs]+ or monosodiated cluster ions [2M + Na]+ as the precursor ions of sugars.27,31 Thus, the fragmentations of Glc were determined in this study. Supporting Information (SI) Figure S1 shows the fragmentation of Glc from monosodiated cluster ions [2M + Na]+(m/z 383) to monosodiated adduct ions [M + Na]+ (m/z 203)a transition that was ultimately chosen for the quantification of Glc in multiple reaction monitoring (MRM) mode. The ionization conditions for all seven targets are summarized in Table 1. Sample Extraction and Clean-Up. To obtain the best extraction efficiency for PhIP, 4′-OH-PhIP, and their precursors, including Phe, Tyr, creatine, creatinine, and Glc, four solvents were tested with the same samples: water, 0.1% formic acids in water, 0.1 M HCl, and the solution of 0.1% formic acids and 50% methanol in water. The recoveries of all seven compounds after extraction are summarized in Table 2, which shows that all four solvents could successfully extract all of the target compounds with recoveries over 56.7%. Compared with the other three solvents, 0.1 M HCl could extract more PhIP and 4′-OH-PhIP, and was thus chosen.
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RESULTS AND DISCUSSION UHPLC−MS/MS Determination. Given that the polarities of HAAs, sugars and creati(ni)ne are considerably different, the simultaneous detection of the HAAs and their precursors in a single run appears difficult. Therefore, the selection of a suitable chromatographic column is of great importance. Four columns, including Waters Acquity UPLC BEH C18 column, Waters Acquity UPLC HSS T3 column, Waters XSELECT HSS T3 column, and Waters Atlantis dC18 column were tested for obtaining well isolation and the suitable sensitivity of analytes (Figure 2). The BEH C18 column, while widely used for the isolation of HAAs in previous studies,26 was not suitable for the retention of Glc, creatine, creatinine, and Tyr, which are eluted before 1 min and could not have been isolated from their matrixes (Figure 2A). Both the UPLC HSS T3 and Select HSS T3 columns were developed by manufacturers to promote polar compound retention, and a charged surface hybrid (CSH) technique that controlled a small amount of charges on the surface of the fixed phase was applied to the latter column to further enhance the retention efficiency, according to the product description. The appropriate retentions of HAAs and amino acids were found when a UPLC HSS T3 column was used, but for Glc, creatine, and creatinine, the retention remained poor (Figure 2B). Conversely, the Select HSS T3 column provided proper retentions for all of the target compounds, but the poor symmetry for Glc and the bad 11630
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Figure 2. Comparison of separation effects for PhIP, 4′-OH-PhIP, and their precursors (Phe, Tyr, creatine, creatinine, and Glc) in same standard concentrations among four different candidate columns: (A) Waters Acquity UPLC BEH C18 (50 × 2.1 mm2 i.d., 1.7 μm), (B) Waters Acquity UPLC HSS T3 (100 × 2.1 mm2 i.d., 1.8 μm), (C) Waters XSELECT HSS T3 (150 × 4.6 mm2 i.d., 3.5 μm) and (D) Waters Atlantis dC18 (150 × 4.6 mm2 i.d., 3 μm). The m/z of fragment and signal sensitivity values for each compounds channels were enclosed in these figures. 11631
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Table 2. Recoveries (Mean ± SD) of HAAs and Their Precursors by Extraction with Different Solvents compound PhIP 4′-OH-PhIP Phe Tyr creatine creatinine Glc
water 79.8 56.7 72.0 98.8 98.6 82.4 65.8
± ± ± ± ± ± ±
16.2 6.9 7.8 10.3 14.7 8.7 9.6
0.1% formic acid in water 73.4 57.9 71.6 96.8 99.2 80.5 61.4
± ± ± ± ± ± ±
15.3 6.1 9.5 14.5 8.8 7.7 6.2
0.1 M HCl 100.7 70.9 72.6 105.7 99.7 82.7 64.1
± ± ± ± ± ± ±
0.1% formic acid and 50% methanol in water
7.3 12.3 9.8 10.0 12.4 16.8 11.4
72.0 57.5 71.0 97.8 97.3 79.7 63.7
± ± ± ± ± ± ±
12.2 9.4 6.1 12.3 9.8 7.6 11.7
The SPE procedure used for purification was tested using three different SPE cartridges. The spiked solution obtained from the 0.1 M HCl extraction was used to evaluate the performance of every cartridge. The results are expressed by recovery rates and are summarized in Figure 3. As the figure
Figure 3. Recoveries of PhIP, 4′-OH-PhIP, and their precursors (Phe, Tyr, creatine, creatinine, and Glc) for SPE cleanup procedure using three different cartridges. The error bars were calculate from the three repeated tests (n = 3).
Figure 4. Representative UHPLC−MS/MS chromatograms of simultaneous determination of PhIP, 4′-OH-PhIP, and their precursors (Phe, Tyr, creatine, creatinine, and Glc) in a fresh pork model after heating and spiking with standards.
shows, the HAAs were retained strongly on the MCX column (recoveries higher than 95%), agreeing with the previous study of MCX cartridge selected to purify the meat sample in HAAs analysis.32 However, this column was found to be unsuitable for retaining the Glc. Appropriate spiked recoveries (>70%) for Glc were observed using both LC-18 and SSH2P columns, similar to the previous study which obtained appropriate recoveries of acrylamide and sugars using these two columns,28 whereas the poor retaining efficiency for creatine and creatinine limited the application of these two types of columns. Therefore, the necessity of the SPE procedure should be considered for optimization purposes. The samples were then alternatively pretreated without SPE. After being spiked with the standards, the extraction solution was diluted and filtrated through a 0.22 μm membrane and then injected into the UHPLC−MS/MS. The chromatograms demonstrated that no impurities were found in any of the channels, except for Glc (Figure 4). Because the small interferential peak appearing in the Glc channel could be isolated to baseline from the Glc peak, this interference would not affect the quantification. Therefore, without the interference (including macro-interference, e.g., protein and fat), the
pretreatment procedures could be simplified by omitting SPE cleanup. The simplified sample pretreatment used in the present study could contribute to the establishment of a rapid and robust quantification method for the simultaneous analysis of HAAs and their precursors, and thus may save analytical time in kinetic studies. Validation of the Proposed Method. The characteristics of the proposed method were demonstrated by a validation procedure studying linearity, LOD, LOQ, recovery, precision, and matrix effects. The linearity was evaluated by injecting different concentrations of standards and plotting the peak area versus the concentrations. As Table 3 shows, the detector response for individual compounds was linear over a broad concentration, with coefficients higher than 0.991. Glc was a fitted quadratic with a coefficient of 0.996 (SI Figure S2), which confirms an earlier study that reported the quadratic fitting of both Glc and sucrose.27 11632
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Table 3. Validation Parameters of the Proposed Method compound
linear range (μg L−1)
coefficients (r2)
LOD (μg L−1)
LOQ (μg L−1)
PhIP
1.15−147.50
0.995
0.14
0.58
4′-OH-PhIP
3.95−253.00
0.995
1.98
7.91
Phe
8.44−1080.00
0.999
0.52
2.11
Tyr
8.44−1080.00
0.999
4.22
16.88
creatine
41.41−42400.00
0.998
5.18
20.70
creatinine
115.23−7375.00
0.991
3.60
14.40
Glc
----c
----c
325
650
spiked level (μg g−1) 0.2 0.8 0.8 3.0 100.0 400.0 150.0 600.0 3000.0 6000.0 3000.0 6000.0 20.0 80.0
mean recovery %a (RSD%) 106.8 115.6 68.4 72.7 72.1 89.7 102.0 122.2 101.5 119.0 80.1 100.8 64.3 59.2
(15.1) (15.4) (14.3) (19.9) (9.9) (6.8) (5.5) (19.4) (13.5) (3.6) (9.4) (0.8) (15.4) (13.2)
matrix effectsb (Rn ± SD) 0.82 ± 0.07 0.78 ± 0.06 0.89 ± 0.07 0.84 ± 0.11 0.73 ± 0.08 0.67 ± 0.05 0.72 ± 0.07
a Intraday and interday precisions are given in parentheses (n = 3). bMatrix effects was expressed as the slope ratios (Rn) of standard spiked calibration curve to pure standard calibration curves at same analyte concentration. A value of >1.00 shows ionization enhancement, while
