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Liquid Chromatography-Tandem Mass Spectrometry for Determination of Aflatoxin B1, Deoxynivalenol and Zearalenone in Artificial Porcine Gastrointestinal Digestive Juice
CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 43, Issue 1, January 2015 Online English edition of the Chinese language journal
Cite this article as: Chin J Anal Chem, 2015, 43(1), 1–6.
RESEARCH PAPER
Liquid Chromatography-Tandem Mass Spectrometry for Determination of Aflatoxin B1, Deoxynivalenol and Zearalenone in Artificial Porcine Gastrointestinal Digestive Juice WANG Rui-Guo, SU Xiao-Ou*, FAN Xia, WANG Pei-Long, GAO Zhong-Wu, ZHANG Yu Institute of Quality Standards and Testing Technology for Agricultural Products, Chinese Academy of Agricultural Science, Key laboratory of Agrifood Safety and Quality, Ministry of Agriculture, P. R. China, Beijing 10081, China
Abstract: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of aflatoxin B1 (AFB1), deoxynivalenol (DON) and zearalenone (ZEA) in artificial porcine gastrointestinal digestive juices, and successfully applied to the in vitro evaluation of adsorption efficiency of mycotoxin adsorbent. The formula feed was digested by artificial gastric and small intestinal juices in vitro, and then the mycotoxins and adsorbent were added in specific ratios. After incubation and centrifugation, the supernatant was diluted 10-fold and then analyzed by LC-MS/MS. The three mycotoxins were separated on a reversed-phase C18 column using a gradient elution program with 0.2 mM ammonium acetate aqueous solution and 0.1% formic acid in methanol as mobile phases. Qualitative analyses were performed under multiple-reaction monitoring mode, and quantitative analyses were carried out by using isotope internal standard method. Under the optimum conditions, the limit of quantification for AFB1, DON and ZEA was 1, 50 and 40 μg L–1 in gastric digested juice and 0.3, 50 and 20 μg L–1 in intestinal digested juice respectively, and the relative standard deviations (RSDs) were less than 5.0%. Then the thermal stability was investigated by incubating the analytes at 39.0 °C ± 0.5 °C for 1, 2, 5 and 10 h, and the experiment results showed that the three mycotoxins were stable under these conditions. Furthermore, the method was used to evaluate the binding efficacies of eight mineral adsorbents and five organic adsorbents. The mineral binders demonstrated binding efficacies of 85.13%–96.50%, 8.11%–14.71% and 13.67%–29.97% for AFB1, DON and ZEA in gastric digestive juice, and 76.15%–92.96%, 12.29%–31.31%, 0%–23.16% in intestinal digestive juice, respectively. The organic adsorbents exhibited binding efficacies of 7.42%–16.65%, 6.68%–16.24% and 18.56%–38.96% for AFB1, DON and ZEA in gastric digestive juice, and 8.65%–13.42%, 3.83%–23.49%, 24.88%–4.76% in intestinal digestive juice, respectively. Key Words: Liquid chromatography–tandem mass spectrometry; Artificial porcine gastrointestinal digestive juice; Mycotoxin; Adsorbent
1
Introduction
Mycotoxins are secondary metabolites produced by different fungal species, which can cause series of toxic response and reduce animal’s production performance when ingested by higher animals[1]. Animal feeds are very susceptible to contamination of mycotoxins[2,3]. Mycotoxin adsorbent is an effective agent for detoxifying moderate or low level of mycotoxin in animal feeds[4]. The most important
index for evaluating mycotoxin adsorbent is its adsorption rate for specific toxins. However, a universal method for evaluating the adsorption rate of mycotoxin adsorbents is currently unavailable. At present, the evaluation of mycotoxin adsorbent adsorption rate is limited by the detection method of toxin and adsorption targets. The adsorption rates of adsorption agents can be determined in vitro or in vivo. The in vitro determination methods generally use a buffer solution as medium to determine the adsorption capacity of a mycotoxin
________________________ Received 7 August 2014; accepted 4 October 2014 *Corresponding author. Email: [email protected] This work is sponsored by the National Science & Technology Program in Rural Areas during the 12th Five-year Plan Period of China (No. 2011BAD26B0405). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60794-0
WANG Rui-Guo et al. / Chinese Journal of Analytical Chemistry, 2015, 43(1): 1–6
adsorbent. This method is widely used in preliminary evaluation of adsorbents and screening for potential adsorption agents, but has low confidence level due to inconsistency with in vivo tests[5]. The in vivo determination methods utilize indirect indexes, for instance, animal production performance or biomarkers, to evaluate adsorbent effect. However, evaluation results are generally unstable because the analysis is affected by many factors and test conditions[6]. Meanwhile, high performance liquid chromatography (HPLC) is generally used to evaluate the adsorption rate of mycotoxin adsorbents[7,8], but exhibits relatively lower sensitivity compared with LC-MS/MS. Moreover, HPLC analysis also involves multiple steps and a complex process with poor ability to simultaneously determine multiple toxins. Enzyme-linked immunosorbent assay has also been used for evaluation[9–11], but shows low reliability. Aflatoxin B1 (AFB1) is usually used as the target in evaluating the adsorption rate of mycotoxin[12] because it is more easily adsorbed than other toxins[13]. Seventy-five percent of the feeds are contaminated simultaneously by various toxins, mainly including deoxynivalenol (DON) and zearalenone (ZEA)[14,15]. However, the adsorption of most mycotoxin adsorbents for DON and ZEA is not good, and even invalid[16]. In vivo experiments also showed that adsorbents with good adsorption for AFB1 usually had poor binding effect in feeds contaminated simultaneously with various mycotoxins[17]. Moreover, other ingredients in feeds are likely to interfere in the adsorption of mycotoxins because of the nonspecificity of adsorbents for mycotoxins[18]. Thus, the key for evaluating the adsorption rates of mycotoxin adsorbents is the evaluation system and detection method of mycotoxins. Currently, liquid chromatographytandem mass spectrometry (LC-MS/MS) has been widely used in simultaneous detection of various mycotoxins in foods and feeds[19]. However, the reports on LC-MS/MS application for in vitro determination of absorption rate of mycotoxin adsorbents are not available. In this work, we prepared artificial porcine gastric and small intestinal juices by simulating the pig gastrointestinal environment and the characteristics of formula feed matrix. The effects of other feed ingredients were taken into account when evaluating the adsorption rate of mycotoxin adsorbents. LC-MS/MS was used to detect the mycotoxins AFB1, DON and ZEA. The experimental results proved that the proposed strategy could comprehensively and accurately determine the adsorption rates of mycotoxin adsorbents.
2 2.1
3K15 high speed refrigerated centrifuge (Sigma, USA), ZWY-200D constant temperature shaking incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., China) and D37520 high-speed centrifuge (Kendro Laboratory Products, Inc., USA) were used in the experiment. AFB1, DON and ZEA standards were from FERMENTEK Ltd. (purity ≥ 99%, Israel). Isotope internal standards 13 C17-AFB1, 13C15-DON, and 13C18-ZEA were obtained from ROMER. Ultrapure water was obtained from a MilliQ Gradient System (Merck Millipore, China). Acetonitrile, methanol, ammonium acetate, and formic acid were of high performance liquid chromatography grade and purchased from Fisher Scientific (USA). The commonly used agents such as pepsin, trypsin, porcine bile salt, sodium chloride of analytical pure grade were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). The eight montmorillonite adsorbents were purchased from Zhejiang Sanding Technology Co., Ltd. (China), Panzhihua Xingjiahuanbao Technology Co., Ltd. (China), Chifeng Hezhengmei Chemical Co., Ltd. (China), Chifeng Wuhuatianbao Mineral Material Co., Ltd. (China), Zhejiang Fenghong New Material Co., Ltd. (China), Shouguangzhonglian Fine Montmorillonite Co., Ltd. (China), Inner Mongolia Tianyuan Montmorillonite Development Co., Ltd. (China), and R&l Chemical Inner Mongolia Co., Ltd. (China), respectively. The five yeast cell wall adsorbents were obtained from Guangdong Jiangmen Center for Biotech Development Co., Ltd. (China), Beijing Youlibao Biotechnology Co., Ltd. (China), ICC Brazil (Brazil), Shanghai of Dinghu Biological Technology Co., Ltd. (China), and Angel Yeast Co., Ltd. (China), respectively. The formula feed for growing pigs was kindly provided by China National Feed Quality Control Center (Beijing). 2.2 2.2.1
Preparation of solutions Preparation of mixed standard solution of mycotoxins
Mixed standard stock solutions (5, 50 and 20 mg L–1) of AFB1, DON and ZEA were prepared by pipetting 50, 500, and 200 μL aliquots from 1000 mg L–1 stock solutions of AFB1, DON, and ZEA respectively, and then diluting to 10 mL with acetonitrile. The mixed stock solution was kept at −20 °C for further use. 2.2.2
Preparation of mixed isotope internal standard solution
Experimental Apparatus and reagents
TQD UPLC-tandem mass spectrometer (Waters, USA), RVC 2-18 desktop concentrator (CHRIST Inc., Germany),
Aliquots of 500 μg L–1 13C17-AFB1 (400 μL), 25 mg L–1 C15-DON (400 μL), and 25 mg L–1 13C18-ZEA (200 μL) were placed into a 2-mL centrifuge tube, dried in a concentrator at 1500 rpm under 40 °C, and then redissolved with the injection solution (0.2 mM aqueous ammonium acetate-acetonitrile-
13
WANG Rui-Guo et al. / Chinese Journal of Analytical Chemistry, 2015, 43(1): 1–6
formic acid (95:4.9:0.1, V/V)) and subjected to ultrasonic treatment for 2 min. The resulting solutions were diluted to 100 mL with the injection solution, and stored at 4 °C. 2.2.3
Preparation of artificial porcine gastric juice
Sodium chloride (2 g) and pepsin (3.2 g) were initially dissolved with sufficient amount of water, then 7 mL of concentrated hydrochloric acid (36.5%) was added, and the obtained mixture was diluted to 1000 mL with water to prepare the artificial gastric juice. Certain amount of formula feed for growing pigs was weighed into a conical flask, and the artificial gastric juice was added in a ratio of 1:3 (w/V). The pH of the resulting solution was adjusted to 2.0 ± 0.5 with diluted hydrochloric acid, and then the solution was incubated in a constant temperature shaking incubator at 39.0 °C ± 0.5 °C and 220 rpm for 1 h, and left to stand for 1 min. The supernatant was transferred and centrifuged at 10000 rpm for 10 min. The supernatant was collected into a 1000 mL reagent bottle and kept at 4 °C for further use. 2.2.4 Preparation of artificial porcine small intestinal juice Monopotassium phosphate (6.8 g) was dissolved in 500 mL water, and the pH of the resulting solution was adjusted to 6.8 with 0.1 M sodium hydroxide. Trypsin (10 g) was dissolved in water, mixed with the monopotassium phosphate solution, and then 3 g of porcine bile salt was added and diluted with water to 1000 mL to obtain an artificial porcine small intestinal juice. Three volumes of artificial small intestinal juice were added to the residual feed precipitate when preparing artificial porcine gastric digested juice. The resulting artificial gastric juice was incubated in a constant temperature shaking incubator at 220 rpm under 39.0 °C ± 0.5 °C for 1 h and then left to stand for 1 min. The supernatant was centrifuged at 10000 rpm for 10 min. Then the supernatant was collected into a 1000 mL reagent bottle and kept at 4 °C.
swirling. The concentrations of free toxins in the balanced system were determined by LC-MS/MS. The adsorption rate of each adsorbent was determined in triplicate by using the samples without the mycotoxin adsorbent as matrix calibration group. The concentrations of the free toxins in the artificial small intestinal juice were determined by the same methods as mentioned above. The adsorption rates of the mycotoxin adsorbents (%) were calculated as follows: Y = (1 − Ceq/C0) × 100% (1) where, Y is the adsorption rate, C0 is the initial concentration of toxin (matrix calibration group), and Ceq is the concentration of free toxin in the balanced system. 2.4 Chromatographic and mass spectrometric conditions An Acquity UPLC BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 µm; Waters, USA) was used to analyze the mycotoxins. A column temperature of 40 °C, a flow rate of 0.40 mL min–1, and an injection volume of 5 μL were used for the analysis. The mycotoxins were eluted with 0.2 mM ammonium acetate aqueous solution (mobile phase A) and 0.1% formic acid–methanol (mobile phase B) following the gradient elution program as follows: 0–0.5 min, 90% A; 0.5–1.5 min, 70% A; 1.5–2.5 min, 40% A; 2.5–3.5 min, 30% A; 3.5–4.0 min, 20% A; 4.0–4.2 min, 90% A; 4.2–5.0 min, 90% A. The mycotoxins were analyzed using an electrospray ionization (ESI) mass spectrometry after separation by UPLC. The ion source temperature was set at 150 °C and desolvation agent temperature at 450 °C with N2 as desolvation and cone gas at flow rate of 900 and 20 L h–1, respectively. Positive ion mode was used to detect AFB1 and DON and negative ion mode was used to detect ZEA. A 0.75 kV capillary voltage was applied. Multiple-reaction monitoring (MRM) mode was adopted for the detection, and the parameters for detection ion, collision energy, cone voltage are presented in Table 1.
3 2.3
Results and discussion
Sample treatment 3.1
An aliquot of 40 μL mixed standard stock solution of the mycotoxins was placed into a 50-mL screw-capped plastic centrifuge tubes, dried under nitrogen flow at 40 °C, and redissolved with 5 mL artificial porcine gastric juice. The resulting solution was added with an accurately measured mycotoxin adsorbent (4.0 mg ± 0.1 mg), vortexed for 1 min, and then immediately heated to 39.0 °C ± 0.5 °C in a water bath. The mixtures were incubated in a constant temperature shaking incubator at 39.0 °C ± 0.5 °C and 220 rpm for 1 h and then immediately cooled down. Aliquots (1 mL) of the mixtures were placed into 2-mL centrifuge tubes and centrifuged at 13000 rpm for 5 min. The supernatant (50 µL) and isotope internal standard solution (450 μL) were mixed by
Optimization of conditions for mass spectrometry
The mass spectrometric conditions for the analysis of AFB1, DON, and ZEA and their respective isotopic internal standards were optimized using methanol-water (50:50, V/V) as mobile phase with different sampling mode combinations. Full scan was performed under positive- and negative-ion modes to select the appropriate quasi-molecular ion peaks and ionization modes. The quasi-molecular ions of AFB1 and DON ([M + H]+) were obtained under positive ionization mode, whereas the quasi-molecular ion of ZEA ([M ‒ H]‒) was obtained under negative ionization mode because it contains a phenolic hydroxyl group. The ionization mode for DON with the addition of acetate ions[20] or removal of
WANG Rui-Guo et al. / Chinese Journal of Analytical Chemistry, 2015, 43(1): 1–6
hydrogen[21] had been reported. However, our study showed that a good response for DON could be obtained with the addition of hydrogen. The MS parameters including the characteristic ions of the three toxins (Table 1) were optimized based on the ion scan diagrams of the blank and standard solutions. 3.2
standard methods were calculated (Table 3). In quantitative determination of the three mycotoxins, a stronger matrix effect and poor precision were observed using the external standard method, whereas less matrix effect and good precision were obtained using the internal standard method. Although the quantification of the target analytes could be achieved with calibration curve using matrix spiked with standards, considering that the matrix effect might have negative influences on the linearity, accuracy and precision of the method[22], and the differences in matrix effects of various adsorbents might exist, in the present study, the internal standard method was adopted to eliminate the interference of matrix effect.
Optimization of liquid chromatography conditions
The composition and proportion of the mobile phase not only affect the chromatographic behavior of the target compounds, but also affect the ionization efficiency and sensitivity. The effects of four mobile phases, including 0.2 mM ammonium acetate/0.1% formic acid-methanol (A), 0.2 mM ammonium acetate/acetonitrile (B), 0.1% formic acid/ 0.1% formic acid-methanol (C), and 0.1% formic acid/ acetonitrile (D), on the separation and peak signal intensities of the three mycotoxins were investigated. The highest signal peak response value for the three mycotoxins with good peak separation was achieved using mobile phase A (Table 2). By further optimizing mobile phase conditions, sample detections were completed within 5 min using gradient elution. Figure 1 shows the separation of three mycotoxins and their isotopic internal standards. 3.3
3.4
Linear equation, correlation coefficient, limit of quantification and precision
Aliquots (100, 40, 20, 10, 5 and 2.5 μL) of the mixed standard stock solutions of the mycotoxins were accurately pipetted, dried at room temperature under nitrogen flow, and dissolved with 5 mL of the artificial gastric juice and small intestinal juice to prepare a series of matrix calibration standard solutions. The solutions were diluted 10 folds, added with the isotopic internal standard solutions, and analyzed by LC-MS/MS. The calibration curve was constructed using the ratios of target compound peak area and internal standard peak area at different concentration levels. The limit of detection (LOD) was calculated as a signal to noise ratio of 3:1 using blank sample. The linear equations and LODs for each analyte in the matrixes are shown in Table 4. The mixed standard stock solution of the mycotoxins (40 µL) was analyzed in parallel for six times. RSDs of 0.11%, 0.23% and 0.24% were obtained for AFB1, DON, and ZEA in artificial gastric juice matrix, and 0.24%, 0.16%, and 0.07% for AFB1, DON and
Matrix effect
Given that 10-fold diluted samples were injected into the system and no purification was conducted, a strong matrix effect was observed in the LC-MS/MS analysis. The peak areas of the porcine artificial gastric and small intestinal juices spiked with high, moderate, and low levels of mycotoxin were compared with those of the standard solutions with the same concentrations, and the matrix effects in external and internal
Table 1 Optimized MS/MS parameters of AFB1, DON and ZEA and their isotopic internal standards Scan mode
Analyte
Parent ion (m/z)
AFB1
2.67
313.10
13
2.67
330.09
DON
2.02
297.22
13
2.02
312.43
ZEA
3.28
317.22
13
3.28
335.07
Positive ion
Negative ion
Retention time (min)
C17-AFB1
C15-DON
C18-ZEA
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
Dwell time (s)
241.04* 284.86 255.10 249.06* 231.07 262.80
46 46 46 20 20 25
36 22 36 12 10 12
0.106 0.106 0.106 0.106 0.106 0.106
175.06* 187.12 140.04
38 38 40
26 22 30
0.106 0.106 0.106
*Quantification ions.
Table 2 Effect of mobile phase systems on the signal responses of AFB1, DON and ZEA Mobile phase system Analyte
0.2 mM ammonium acetate/0.1% formic acid-methanol (%)
0.2 mM ammonium acetate/acetonitrile (%)
0.1% formic acid/0.1% formic acid-methanol (%)
0.1% formic acid/acetonitrile (%)
AFB1 DON ZEA
100 100 100
78.5 24.7 57.3
22.5 36.4 18.2
47.3 30.7 15.3
WANG Rui-Guo et al. / Chinese Journal of Analytical Chemistry, 2015, 43(1): 1–6
standard peak area after incubation for 1 h was set as 100%, and the ratios at other time points were found to be 95.9%–111.3%, indicating that no degradation was detected after incubation at 39 °C ± 0.5 °C for 10 h and the mycotoxins had high stability under the experimental conditions. 3.6
Fig.1
The practical samples were treated as described in section 3.2 in a ratio of pig gastrointestinal fluid to feed of 3:1 (w/w), corresponding to the addition of 0.24% of the mycotoxin adsorbent in the formula feed contaminated with mycotoxins at the level of AFB1 120 μg kg–1, DON 1200 μg kg–1 and ZEA 480 μg kg–1, which was close to the conditions in practical productions. The adsorption efficacies of eight montmorillonite adsorbents and five yeast cell wall adsorbents were evaluated by the proposed method. The mineral binders demonstrated binding efficacies of 85.13%–96.50%, 8.11%–14.71%, and 13.67%–29.97% for AFB1, DON, and ZEA in gastric digestive juice, as well as 76.15%–92.96%, 12.29%–31.31%, and 0%–23.16% in intestinal digestive juice, respectively. The organic adsorbents exhibited binding efficacies of 7.42%–16.65%, 6.68%–16.24%, and 18.56%–38.96% for AFB1, DON, and ZEA in gastric digestive juice, as well as 8.65%–13.42%, 3.83%–23.49%, and 24.88%–4.76% in intestinal digestive juice, respectively. The measured results were in accordance with the adsorption properties of the two types of adsorbents for different toxins, and were consistent with the results in literatures[4–6]. The experiment results indicated that the proposed method can be used to determine the adsorption rates of mycotoxin adsorbents.
Chromatograms of quantification ions of AFB1, DON and ZEA and their isotopic internal standards in artificial gastric juices (40 μg L–1 for AFB1, 400 μg L–1 for DON, 160 μg L–1 for ZEA)
ZEA in small intestinal juice, respectively, which satisfied the precision requirement of the instrument. 3.5
Analysis of practical samples
Stability
The stability of the mycotoxins in the detection system is an important factor in evaluating the adsorption efficiency of the adsorbents. In the present study, the stability of the three mycotoxins incubated in artificial porcine gastric and small intestinal juices at 39.0 °C ± 0.5 °C for 10 h were investigated. The samples were treated following the procedure in section 2.3 without the addition of mycotoxin adsorbents, and then incubated in the shaking incubator for 10 h. The samples after incubation for 1, 2, 5 and 10 h were analyzed by the proposed method. The ratio of mycotoxin peak area and internal
Table 3 Matrix effects of external calibration (EC) and isotope dilution (ID) methods (n = 5) Analyte
Addition concentration (μg L–1)
Injection concentration (μg L–1)
100 50 20 1000 500 200 400 200 80
10 5 2 100 50 20 40 20 8
AFB1
DON
ZEA
Artificial gastric juice
Artificial intestinal juice
ECa MEc/RSDd
IDb ME/RSD
EC ME/RSD
ID ME/RSD
45.0%/7.2% 49.2%/5.1% 48.4%/12.7% 58.4%/5.4% 59.5%10.1% 64.3%13.0% 13.6%/7.3% 10.4%/9.1% 8.7%/16.5%
106.9%/1.6% 116.4%/1.3% 110.3%/1.9% 107.0%/1.7% 105.3%/1.9% 111.2%/2.8% 110.2%/2.2% 107.6%/0.9% 93.9%/2.5%
55.0%/9.7% 58.7%11.2% 60.2%/13.1% 48.1%/5.5% 42.0%/9.6% 37.8%/10.1% 20.2%/6.3% 23.9%/14.8% 25.1%/21.3%
102.2%/1.6% 114.7%/1.8% 114.0%/2.5% 102.1%/0.8% 104.8%/1.6% 108.0%/1.2% 103.5%/1.0% 115.4%/2.4% 110.5%/2.6%
a: External calibration (EC); b: isotope dilution (ID); c: Matrix effect (ME); d: Relative standard deviation (RSD).
Table 4 Calibration equations and LODs of AFB1, DON and ZEA Solution system Artificial gastric juice
Artificial small intestinal juice
Analyte
Linear range (μg L–1)
Calibration equation
Correlation coefficient (R2)
LOD (μg L–1)
AFB1 DON ZEA AFB1 DON ZEA
2.5–100 50–1000 40–400 2.5–100 50–1000 20–400
y = 0.072x + 0.039 y = 0.003x + 0.253 y = 0.007x − 0.113 y = 0.067x + 0.085 y = 0.007x − 0.368 y = 0.006x − 0.014
0.9991 0.9996 0.9994 0.9989 0.9983 0.9992
1.0 50.0 40.0 0.3 50.0 20.0
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Cite this article as: Chin J Anal Chem, 2015, 43(1), 1–6.
RESEARCH PAPER
Liquid Chromatography-Tandem Mass Spectrometry for Determination of Aflatoxin B1, Deoxynivalenol and Zearalenone in Artificial Porcine Gastrointestinal Digestive Juice WANG Rui-Guo, SU Xiao-Ou*, FAN Xia, WANG Pei-Long, GAO Zhong-Wu, ZHANG Yu Institute of Quality Standards and Testing Technology for Agricultural Products, Chinese Academy of Agricultural Science, Key laboratory of Agrifood Safety and Quality, Ministry of Agriculture, P. R. China, Beijing 10081, China
Abstract: A rapid liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of aflatoxin B1 (AFB1), deoxynivalenol (DON) and zearalenone (ZEA) in artificial porcine gastrointestinal digestive juices, and successfully applied to the in vitro evaluation of adsorption efficiency of mycotoxin adsorbent. The formula feed was digested by artificial gastric and small intestinal juices in vitro, and then the mycotoxins and adsorbent were added in specific ratios. After incubation and centrifugation, the supernatant was diluted 10-fold and then analyzed by LC-MS/MS. The three mycotoxins were separated on a reversed-phase C18 column using a gradient elution program with 0.2 mM ammonium acetate aqueous solution and 0.1% formic acid in methanol as mobile phases. Qualitative analyses were performed under multiple-reaction monitoring mode, and quantitative analyses were carried out by using isotope internal standard method. Under the optimum conditions, the limit of quantification for AFB1, DON and ZEA was 1, 50 and 40 μg L–1 in gastric digested juice and 0.3, 50 and 20 μg L–1 in intestinal digested juice respectively, and the relative standard deviations (RSDs) were less than 5.0%. Then the thermal stability was investigated by incubating the analytes at 39.0 °C ± 0.5 °C for 1, 2, 5 and 10 h, and the experiment results showed that the three mycotoxins were stable under these conditions. Furthermore, the method was used to evaluate the binding efficacies of eight mineral adsorbents and five organic adsorbents. The mineral binders demonstrated binding efficacies of 85.13%–96.50%, 8.11%–14.71% and 13.67%–29.97% for AFB1, DON and ZEA in gastric digestive juice, and 76.15%–92.96%, 12.29%–31.31%, 0%–23.16% in intestinal digestive juice, respectively. The organic adsorbents exhibited binding efficacies of 7.42%–16.65%, 6.68%–16.24% and 18.56%–38.96% for AFB1, DON and ZEA in gastric digestive juice, and 8.65%–13.42%, 3.83%–23.49%, 24.88%–4.76% in intestinal digestive juice, respectively. Key Words: Liquid chromatography–tandem mass spectrometry; Artificial porcine gastrointestinal digestive juice; Mycotoxin; Adsorbent
1
Introduction
Mycotoxins are secondary metabolites produced by different fungal species, which can cause series of toxic response and reduce animal’s production performance when ingested by higher animals[1]. Animal feeds are very susceptible to contamination of mycotoxins[2,3]. Mycotoxin adsorbent is an effective agent for detoxifying moderate or low level of mycotoxin in animal feeds[4]. The most important
index for evaluating mycotoxin adsorbent is its adsorption rate for specific toxins. However, a universal method for evaluating the adsorption rate of mycotoxin adsorbents is currently unavailable. At present, the evaluation of mycotoxin adsorbent adsorption rate is limited by the detection method of toxin and adsorption targets. The adsorption rates of adsorption agents can be determined in vitro or in vivo. The in vitro determination methods generally use a buffer solution as medium to determine the adsorption capacity of a mycotoxin
________________________ Received 7 August 2014; accepted 4 October 2014 *Corresponding author. Email: [email protected] This work is sponsored by the National Science & Technology Program in Rural Areas during the 12th Five-year Plan Period of China (No. 2011BAD26B0405). Copyright © 2015, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(15)60794-0
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adsorbent. This method is widely used in preliminary evaluation of adsorbents and screening for potential adsorption agents, but has low confidence level due to inconsistency with in vivo tests[5]. The in vivo determination methods utilize indirect indexes, for instance, animal production performance or biomarkers, to evaluate adsorbent effect. However, evaluation results are generally unstable because the analysis is affected by many factors and test conditions[6]. Meanwhile, high performance liquid chromatography (HPLC) is generally used to evaluate the adsorption rate of mycotoxin adsorbents[7,8], but exhibits relatively lower sensitivity compared with LC-MS/MS. Moreover, HPLC analysis also involves multiple steps and a complex process with poor ability to simultaneously determine multiple toxins. Enzyme-linked immunosorbent assay has also been used for evaluation[9–11], but shows low reliability. Aflatoxin B1 (AFB1) is usually used as the target in evaluating the adsorption rate of mycotoxin[12] because it is more easily adsorbed than other toxins[13]. Seventy-five percent of the feeds are contaminated simultaneously by various toxins, mainly including deoxynivalenol (DON) and zearalenone (ZEA)[14,15]. However, the adsorption of most mycotoxin adsorbents for DON and ZEA is not good, and even invalid[16]. In vivo experiments also showed that adsorbents with good adsorption for AFB1 usually had poor binding effect in feeds contaminated simultaneously with various mycotoxins[17]. Moreover, other ingredients in feeds are likely to interfere in the adsorption of mycotoxins because of the nonspecificity of adsorbents for mycotoxins[18]. Thus, the key for evaluating the adsorption rates of mycotoxin adsorbents is the evaluation system and detection method of mycotoxins. Currently, liquid chromatographytandem mass spectrometry (LC-MS/MS) has been widely used in simultaneous detection of various mycotoxins in foods and feeds[19]. However, the reports on LC-MS/MS application for in vitro determination of absorption rate of mycotoxin adsorbents are not available. In this work, we prepared artificial porcine gastric and small intestinal juices by simulating the pig gastrointestinal environment and the characteristics of formula feed matrix. The effects of other feed ingredients were taken into account when evaluating the adsorption rate of mycotoxin adsorbents. LC-MS/MS was used to detect the mycotoxins AFB1, DON and ZEA. The experimental results proved that the proposed strategy could comprehensively and accurately determine the adsorption rates of mycotoxin adsorbents.
2 2.1
3K15 high speed refrigerated centrifuge (Sigma, USA), ZWY-200D constant temperature shaking incubator (Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd., China) and D37520 high-speed centrifuge (Kendro Laboratory Products, Inc., USA) were used in the experiment. AFB1, DON and ZEA standards were from FERMENTEK Ltd. (purity ≥ 99%, Israel). Isotope internal standards 13 C17-AFB1, 13C15-DON, and 13C18-ZEA were obtained from ROMER. Ultrapure water was obtained from a MilliQ Gradient System (Merck Millipore, China). Acetonitrile, methanol, ammonium acetate, and formic acid were of high performance liquid chromatography grade and purchased from Fisher Scientific (USA). The commonly used agents such as pepsin, trypsin, porcine bile salt, sodium chloride of analytical pure grade were purchased from Sinopharm Chemical Reagent Co. Ltd. (China). The eight montmorillonite adsorbents were purchased from Zhejiang Sanding Technology Co., Ltd. (China), Panzhihua Xingjiahuanbao Technology Co., Ltd. (China), Chifeng Hezhengmei Chemical Co., Ltd. (China), Chifeng Wuhuatianbao Mineral Material Co., Ltd. (China), Zhejiang Fenghong New Material Co., Ltd. (China), Shouguangzhonglian Fine Montmorillonite Co., Ltd. (China), Inner Mongolia Tianyuan Montmorillonite Development Co., Ltd. (China), and R&l Chemical Inner Mongolia Co., Ltd. (China), respectively. The five yeast cell wall adsorbents were obtained from Guangdong Jiangmen Center for Biotech Development Co., Ltd. (China), Beijing Youlibao Biotechnology Co., Ltd. (China), ICC Brazil (Brazil), Shanghai of Dinghu Biological Technology Co., Ltd. (China), and Angel Yeast Co., Ltd. (China), respectively. The formula feed for growing pigs was kindly provided by China National Feed Quality Control Center (Beijing). 2.2 2.2.1
Preparation of solutions Preparation of mixed standard solution of mycotoxins
Mixed standard stock solutions (5, 50 and 20 mg L–1) of AFB1, DON and ZEA were prepared by pipetting 50, 500, and 200 μL aliquots from 1000 mg L–1 stock solutions of AFB1, DON, and ZEA respectively, and then diluting to 10 mL with acetonitrile. The mixed stock solution was kept at −20 °C for further use. 2.2.2
Preparation of mixed isotope internal standard solution
Experimental Apparatus and reagents
TQD UPLC-tandem mass spectrometer (Waters, USA), RVC 2-18 desktop concentrator (CHRIST Inc., Germany),
Aliquots of 500 μg L–1 13C17-AFB1 (400 μL), 25 mg L–1 C15-DON (400 μL), and 25 mg L–1 13C18-ZEA (200 μL) were placed into a 2-mL centrifuge tube, dried in a concentrator at 1500 rpm under 40 °C, and then redissolved with the injection solution (0.2 mM aqueous ammonium acetate-acetonitrile-
13
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formic acid (95:4.9:0.1, V/V)) and subjected to ultrasonic treatment for 2 min. The resulting solutions were diluted to 100 mL with the injection solution, and stored at 4 °C. 2.2.3
Preparation of artificial porcine gastric juice
Sodium chloride (2 g) and pepsin (3.2 g) were initially dissolved with sufficient amount of water, then 7 mL of concentrated hydrochloric acid (36.5%) was added, and the obtained mixture was diluted to 1000 mL with water to prepare the artificial gastric juice. Certain amount of formula feed for growing pigs was weighed into a conical flask, and the artificial gastric juice was added in a ratio of 1:3 (w/V). The pH of the resulting solution was adjusted to 2.0 ± 0.5 with diluted hydrochloric acid, and then the solution was incubated in a constant temperature shaking incubator at 39.0 °C ± 0.5 °C and 220 rpm for 1 h, and left to stand for 1 min. The supernatant was transferred and centrifuged at 10000 rpm for 10 min. The supernatant was collected into a 1000 mL reagent bottle and kept at 4 °C for further use. 2.2.4 Preparation of artificial porcine small intestinal juice Monopotassium phosphate (6.8 g) was dissolved in 500 mL water, and the pH of the resulting solution was adjusted to 6.8 with 0.1 M sodium hydroxide. Trypsin (10 g) was dissolved in water, mixed with the monopotassium phosphate solution, and then 3 g of porcine bile salt was added and diluted with water to 1000 mL to obtain an artificial porcine small intestinal juice. Three volumes of artificial small intestinal juice were added to the residual feed precipitate when preparing artificial porcine gastric digested juice. The resulting artificial gastric juice was incubated in a constant temperature shaking incubator at 220 rpm under 39.0 °C ± 0.5 °C for 1 h and then left to stand for 1 min. The supernatant was centrifuged at 10000 rpm for 10 min. Then the supernatant was collected into a 1000 mL reagent bottle and kept at 4 °C.
swirling. The concentrations of free toxins in the balanced system were determined by LC-MS/MS. The adsorption rate of each adsorbent was determined in triplicate by using the samples without the mycotoxin adsorbent as matrix calibration group. The concentrations of the free toxins in the artificial small intestinal juice were determined by the same methods as mentioned above. The adsorption rates of the mycotoxin adsorbents (%) were calculated as follows: Y = (1 − Ceq/C0) × 100% (1) where, Y is the adsorption rate, C0 is the initial concentration of toxin (matrix calibration group), and Ceq is the concentration of free toxin in the balanced system. 2.4 Chromatographic and mass spectrometric conditions An Acquity UPLC BEH C18 chromatographic column (2.1 mm × 100 mm, 1.7 µm; Waters, USA) was used to analyze the mycotoxins. A column temperature of 40 °C, a flow rate of 0.40 mL min–1, and an injection volume of 5 μL were used for the analysis. The mycotoxins were eluted with 0.2 mM ammonium acetate aqueous solution (mobile phase A) and 0.1% formic acid–methanol (mobile phase B) following the gradient elution program as follows: 0–0.5 min, 90% A; 0.5–1.5 min, 70% A; 1.5–2.5 min, 40% A; 2.5–3.5 min, 30% A; 3.5–4.0 min, 20% A; 4.0–4.2 min, 90% A; 4.2–5.0 min, 90% A. The mycotoxins were analyzed using an electrospray ionization (ESI) mass spectrometry after separation by UPLC. The ion source temperature was set at 150 °C and desolvation agent temperature at 450 °C with N2 as desolvation and cone gas at flow rate of 900 and 20 L h–1, respectively. Positive ion mode was used to detect AFB1 and DON and negative ion mode was used to detect ZEA. A 0.75 kV capillary voltage was applied. Multiple-reaction monitoring (MRM) mode was adopted for the detection, and the parameters for detection ion, collision energy, cone voltage are presented in Table 1.
3 2.3
Results and discussion
Sample treatment 3.1
An aliquot of 40 μL mixed standard stock solution of the mycotoxins was placed into a 50-mL screw-capped plastic centrifuge tubes, dried under nitrogen flow at 40 °C, and redissolved with 5 mL artificial porcine gastric juice. The resulting solution was added with an accurately measured mycotoxin adsorbent (4.0 mg ± 0.1 mg), vortexed for 1 min, and then immediately heated to 39.0 °C ± 0.5 °C in a water bath. The mixtures were incubated in a constant temperature shaking incubator at 39.0 °C ± 0.5 °C and 220 rpm for 1 h and then immediately cooled down. Aliquots (1 mL) of the mixtures were placed into 2-mL centrifuge tubes and centrifuged at 13000 rpm for 5 min. The supernatant (50 µL) and isotope internal standard solution (450 μL) were mixed by
Optimization of conditions for mass spectrometry
The mass spectrometric conditions for the analysis of AFB1, DON, and ZEA and their respective isotopic internal standards were optimized using methanol-water (50:50, V/V) as mobile phase with different sampling mode combinations. Full scan was performed under positive- and negative-ion modes to select the appropriate quasi-molecular ion peaks and ionization modes. The quasi-molecular ions of AFB1 and DON ([M + H]+) were obtained under positive ionization mode, whereas the quasi-molecular ion of ZEA ([M ‒ H]‒) was obtained under negative ionization mode because it contains a phenolic hydroxyl group. The ionization mode for DON with the addition of acetate ions[20] or removal of
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hydrogen[21] had been reported. However, our study showed that a good response for DON could be obtained with the addition of hydrogen. The MS parameters including the characteristic ions of the three toxins (Table 1) were optimized based on the ion scan diagrams of the blank and standard solutions. 3.2
standard methods were calculated (Table 3). In quantitative determination of the three mycotoxins, a stronger matrix effect and poor precision were observed using the external standard method, whereas less matrix effect and good precision were obtained using the internal standard method. Although the quantification of the target analytes could be achieved with calibration curve using matrix spiked with standards, considering that the matrix effect might have negative influences on the linearity, accuracy and precision of the method[22], and the differences in matrix effects of various adsorbents might exist, in the present study, the internal standard method was adopted to eliminate the interference of matrix effect.
Optimization of liquid chromatography conditions
The composition and proportion of the mobile phase not only affect the chromatographic behavior of the target compounds, but also affect the ionization efficiency and sensitivity. The effects of four mobile phases, including 0.2 mM ammonium acetate/0.1% formic acid-methanol (A), 0.2 mM ammonium acetate/acetonitrile (B), 0.1% formic acid/ 0.1% formic acid-methanol (C), and 0.1% formic acid/ acetonitrile (D), on the separation and peak signal intensities of the three mycotoxins were investigated. The highest signal peak response value for the three mycotoxins with good peak separation was achieved using mobile phase A (Table 2). By further optimizing mobile phase conditions, sample detections were completed within 5 min using gradient elution. Figure 1 shows the separation of three mycotoxins and their isotopic internal standards. 3.3
3.4
Linear equation, correlation coefficient, limit of quantification and precision
Aliquots (100, 40, 20, 10, 5 and 2.5 μL) of the mixed standard stock solutions of the mycotoxins were accurately pipetted, dried at room temperature under nitrogen flow, and dissolved with 5 mL of the artificial gastric juice and small intestinal juice to prepare a series of matrix calibration standard solutions. The solutions were diluted 10 folds, added with the isotopic internal standard solutions, and analyzed by LC-MS/MS. The calibration curve was constructed using the ratios of target compound peak area and internal standard peak area at different concentration levels. The limit of detection (LOD) was calculated as a signal to noise ratio of 3:1 using blank sample. The linear equations and LODs for each analyte in the matrixes are shown in Table 4. The mixed standard stock solution of the mycotoxins (40 µL) was analyzed in parallel for six times. RSDs of 0.11%, 0.23% and 0.24% were obtained for AFB1, DON, and ZEA in artificial gastric juice matrix, and 0.24%, 0.16%, and 0.07% for AFB1, DON and
Matrix effect
Given that 10-fold diluted samples were injected into the system and no purification was conducted, a strong matrix effect was observed in the LC-MS/MS analysis. The peak areas of the porcine artificial gastric and small intestinal juices spiked with high, moderate, and low levels of mycotoxin were compared with those of the standard solutions with the same concentrations, and the matrix effects in external and internal
Table 1 Optimized MS/MS parameters of AFB1, DON and ZEA and their isotopic internal standards Scan mode
Analyte
Parent ion (m/z)
AFB1
2.67
313.10
13
2.67
330.09
DON
2.02
297.22
13
2.02
312.43
ZEA
3.28
317.22
13
3.28
335.07
Positive ion
Negative ion
Retention time (min)
C17-AFB1
C15-DON
C18-ZEA
Product ion (m/z)
Cone voltage (V)
Collision energy (eV)
Dwell time (s)
241.04* 284.86 255.10 249.06* 231.07 262.80
46 46 46 20 20 25
36 22 36 12 10 12
0.106 0.106 0.106 0.106 0.106 0.106
175.06* 187.12 140.04
38 38 40
26 22 30
0.106 0.106 0.106
*Quantification ions.
Table 2 Effect of mobile phase systems on the signal responses of AFB1, DON and ZEA Mobile phase system Analyte
0.2 mM ammonium acetate/0.1% formic acid-methanol (%)
0.2 mM ammonium acetate/acetonitrile (%)
0.1% formic acid/0.1% formic acid-methanol (%)
0.1% formic acid/acetonitrile (%)
AFB1 DON ZEA
100 100 100
78.5 24.7 57.3
22.5 36.4 18.2
47.3 30.7 15.3
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standard peak area after incubation for 1 h was set as 100%, and the ratios at other time points were found to be 95.9%–111.3%, indicating that no degradation was detected after incubation at 39 °C ± 0.5 °C for 10 h and the mycotoxins had high stability under the experimental conditions. 3.6
Fig.1
The practical samples were treated as described in section 3.2 in a ratio of pig gastrointestinal fluid to feed of 3:1 (w/w), corresponding to the addition of 0.24% of the mycotoxin adsorbent in the formula feed contaminated with mycotoxins at the level of AFB1 120 μg kg–1, DON 1200 μg kg–1 and ZEA 480 μg kg–1, which was close to the conditions in practical productions. The adsorption efficacies of eight montmorillonite adsorbents and five yeast cell wall adsorbents were evaluated by the proposed method. The mineral binders demonstrated binding efficacies of 85.13%–96.50%, 8.11%–14.71%, and 13.67%–29.97% for AFB1, DON, and ZEA in gastric digestive juice, as well as 76.15%–92.96%, 12.29%–31.31%, and 0%–23.16% in intestinal digestive juice, respectively. The organic adsorbents exhibited binding efficacies of 7.42%–16.65%, 6.68%–16.24%, and 18.56%–38.96% for AFB1, DON, and ZEA in gastric digestive juice, as well as 8.65%–13.42%, 3.83%–23.49%, and 24.88%–4.76% in intestinal digestive juice, respectively. The measured results were in accordance with the adsorption properties of the two types of adsorbents for different toxins, and were consistent with the results in literatures[4–6]. The experiment results indicated that the proposed method can be used to determine the adsorption rates of mycotoxin adsorbents.
Chromatograms of quantification ions of AFB1, DON and ZEA and their isotopic internal standards in artificial gastric juices (40 μg L–1 for AFB1, 400 μg L–1 for DON, 160 μg L–1 for ZEA)
ZEA in small intestinal juice, respectively, which satisfied the precision requirement of the instrument. 3.5
Analysis of practical samples
Stability
The stability of the mycotoxins in the detection system is an important factor in evaluating the adsorption efficiency of the adsorbents. In the present study, the stability of the three mycotoxins incubated in artificial porcine gastric and small intestinal juices at 39.0 °C ± 0.5 °C for 10 h were investigated. The samples were treated following the procedure in section 2.3 without the addition of mycotoxin adsorbents, and then incubated in the shaking incubator for 10 h. The samples after incubation for 1, 2, 5 and 10 h were analyzed by the proposed method. The ratio of mycotoxin peak area and internal
Table 3 Matrix effects of external calibration (EC) and isotope dilution (ID) methods (n = 5) Analyte
Addition concentration (μg L–1)
Injection concentration (μg L–1)
100 50 20 1000 500 200 400 200 80
10 5 2 100 50 20 40 20 8
AFB1
DON
ZEA
Artificial gastric juice
Artificial intestinal juice
ECa MEc/RSDd
IDb ME/RSD
EC ME/RSD
ID ME/RSD
45.0%/7.2% 49.2%/5.1% 48.4%/12.7% 58.4%/5.4% 59.5%10.1% 64.3%13.0% 13.6%/7.3% 10.4%/9.1% 8.7%/16.5%
106.9%/1.6% 116.4%/1.3% 110.3%/1.9% 107.0%/1.7% 105.3%/1.9% 111.2%/2.8% 110.2%/2.2% 107.6%/0.9% 93.9%/2.5%
55.0%/9.7% 58.7%11.2% 60.2%/13.1% 48.1%/5.5% 42.0%/9.6% 37.8%/10.1% 20.2%/6.3% 23.9%/14.8% 25.1%/21.3%
102.2%/1.6% 114.7%/1.8% 114.0%/2.5% 102.1%/0.8% 104.8%/1.6% 108.0%/1.2% 103.5%/1.0% 115.4%/2.4% 110.5%/2.6%
a: External calibration (EC); b: isotope dilution (ID); c: Matrix effect (ME); d: Relative standard deviation (RSD).
Table 4 Calibration equations and LODs of AFB1, DON and ZEA Solution system Artificial gastric juice
Artificial small intestinal juice
Analyte
Linear range (μg L–1)
Calibration equation
Correlation coefficient (R2)
LOD (μg L–1)
AFB1 DON ZEA AFB1 DON ZEA
2.5–100 50–1000 40–400 2.5–100 50–1000 20–400
y = 0.072x + 0.039 y = 0.003x + 0.253 y = 0.007x − 0.113 y = 0.067x + 0.085 y = 0.007x − 0.368 y = 0.006x − 0.014
0.9991 0.9996 0.9994 0.9989 0.9983 0.9992
1.0 50.0 40.0 0.3 50.0 20.0
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