Accepted Manuscript Title: Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by liquid chromatography-tandem mass spectrometry Author: Shufang Wang Ling Cheng Shen Ji Ke Wang PII: DOI: Reference:
S0731-7085(14)00277-5 http://dx.doi.org/doi:10.1016/j.jpba.2014.05.037 PBA 9600
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-2-2014 22-5-2014 23-5-2014
Please cite this article as: S. Wang, L. Cheng, S. Ji, K. Wang, Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by liquid chromatographytandem mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.05.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by
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liquid chromatography-tandem mass spectrometry
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Shufang Wang a,*, Ling Chenga, Shen Ji b,*, Ke Wang b
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College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China
Department of Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control,
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1500 Zhangheng Road, Shanghai, 201203, China
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Corresponding authors: Dr. Shufang Wang, Pharmaceutical Informatics Institute, Zhejiang
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University, Zijingang Campus, No. 866 Yuhangtang Road, Hangzhou 310058, P.R. China. Email:
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[email protected]; Tel: +86 571 88208426; Fax: +86 571 88208426; Dr. Shen Ji, Department of
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Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control, 1500 Zhangheng
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Road, Shanghai, 201203, China. [email protected], Tel: +86 21 50798195; Fax: +86 21
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50798195.
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ABSTRACT This work reported an efficient and accurate liquid chromatography tandem mass
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spectrometry (LC–MS/MS) method for simultaneous determination of seventeen mycotoxins in
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Puerariae lobatae radix, a frequently used traditional Chinese medicine (TCM). The effects of
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four different clean-up methods, including TC-M160, TC-T220, Mycosep 227, and QuEChERS
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method, on the recoveries of mycotoxins were investigated and compared. Finally, TC-M160 was
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chosen for better recovery and repeatability for mycotoxins analysis. The analytes were separated
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on an Agilent ZORBAX SB C18 column (4.6 × 250 mm, 5 μm particle size), and eluted with a
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mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing
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0.1% formic acid at a flow rate of 0.6 ml/min. The separated compounds were detected by a triple
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quadrupole mass spectrometer operating in positive electrospray ionization with multiple reaction
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monitoring (MRM) mode. The results of method validation accorded with the requirement of
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analytical method for mycotoxins in COMMISSION REGULATION (EC) No 401/2006. The
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developed method was successfully applied for determination of mycotoxins in seventeen batches
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of Puerariae lobatae radix collected from different provinces of China. Three batches of them were found with contamination of mycotoxins AFB1 at (0.751±0.176) μg/kg, T-2 at (1.10±0.01) μg/kg, and T-2 at (0.853±0.044) μg/kg, respectively. The results demonstrated that the proposed method was suitable for monitoring mycotoxins residues in Puerariae lobatae radix.
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Keywords: Mycotoxins; Liquid chromatography-tandem mass spectrometry (LC-MS/MS);
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Puerariae lobatae radix; TC-M160
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1. Introduction 2
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Traditional Chinese medicine (TCM) has been used for prevention and treatment of various
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diseases. But it may mildew during storage and transportation, which would result in the
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contamination of mycotoxins. Mycotoxins are toxic secondary metabolites produced by some
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filamentous fungi, such as Fusarium, Aspergillus, Penicillium, and Alternaria species.
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Surveillance studies demonstrated that mycotoxin contamination is a world-wide problem. Their
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occurrence in food, beverages and feed has been recognized as potential threat to human and
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animal health. Aflatoxins (AFs), a group of secondary metabolites produced by Aspergillus flavus
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and Aspergillus parasiticus, mainly include AFB1, AFB2, AFG1, AFG2, which have hepatotoxicity
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and carcinogenicity. Especially, AFB1 has been defined as a Category A carcinogen by the
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International Agency for Research on Cancer (IARC) of World Health Organization (WHO) in
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1993[1]. Trichothecene (TCT) mycotoxins, mainly generated from the secondary metabolism of
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Fusarium, can result in immunosuppression, the necrosis of myeloid tissue and hemorrhage of
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visceral organs. Zearalenone (ZEN) mycotoxins, generated from Fusarium graminearum and
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other Fusarium molds, may damage the reproduction of mammals because of its forged estrogenic
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effects. Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins of Alternaria fungi. The extraction of A. alternata was considered as a suspected cancer-causing factor for human oesophageal cancer [2]. Considering their serious toxicity, maximum residue levels (MRLs) were set for some mycotoxins, and MRLs were different for
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same mycotoxin in different foodstuffs in European Union (EU) as described in Commission
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Regulation (EC) No. 1881/2006. MRLs of AFB1 are 0.1-8.0 µg/kg in seventeen kinds of
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foodstuffs, and 20-400 µg/kg for ZEN in ten types of foodstuffs. In Chinese pharmacopeia of 2010
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edition, MRL of AFB1 in traditional Chinese medicines is 5 µg/kg. Therefore, it is necessary to 3
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develop robust and sensitive methods for monitoring mycotoxins in TCMs to ensure providing
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consumers with safe TCM. Current analytical techniques of mycotoxins mainly include liquid chromatography with
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fluorescence detection (LC-FLD) [3], liquid chromatography with tandem mass spectrometry
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(LC-MS/MS) [4], gas chromatography with tandem mass spectrometry (GC-MS/MS) [5], and
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enzyme-linked immunosorbent assay (ELISA) [6]. Although ELISA is a fast and sensitive method,
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it is a semi-quantitative analysis, and false positive result might be obtained [6]. Sample
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derivatization procedure is needed for GC-MS and LC-FD method because many mycotoxins are
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not volatile and have no fluorescence property. The most frequently used method for mycotoxins
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analysis is LC-MS/MS, which has been extensively studied in various food and matrices [7]. The
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introduction of MS/MS detection also leads to a significant improvement for mycotoxins analysis
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because of its high sensitivity, selectivity and specificity [8-10]. Compared with other analytical
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technique, LC-MS/MS can be used not only for the quantitative analysis, but also for the
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confirmation of existence of mycotoxins. Up to now, most of the published works on mycotxoins
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analysis were about food samples; only several reports were concerned with TCMs [4, 11-16]. Considering the low residue level of mycotoxins (generally in µg/kg level) and complex
chemical composition of TCM and food samples, cleanup procedure was necessary before
analysis by LC-MS/MS. Zhang et al. used TC-M160 MultiPurification column as cleanup method
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for analysis of AFs, trichothecenes, and ZEAs mycotoxins in grains [17]. Yue et al. used TC-T220
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Tricothecene column to analyze deoxynivalenol and nivalenol in TCMs [16]. Razzazi-Fazeli et al.
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analyzed several type A trichothecenes in grains by using Mycosep 227 Trich+ Multifunctional
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cartridge as cleanup method [18]. QuEChERS method based on primary secondary amine (PSA) 4
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sorbent was also applied for mycotoxins analysis in cereals [19]. However, there has been no work
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to compare the effect of these cleanup methods systematically. In this study, a reliable LC–MS/MS method was developed to simultaneously determine
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seventeen mycotoxins, including four AFs (AFB1, AFB2, AFG1, and AFG2), five trichothecenes
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mycotoxins (T-2, HT-2, diacetoxyscirpenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol),
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six ZEAs (zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, and β-zearalanol),
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and two Alternaria toxins (alternariol and alternariol monomethyl ether). The extraction solvent
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was optimized. And, the effects of different clean-up methods, including TC-M160, TC-T220,
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Mycosep 227, and QuEChERS methods, on the recovery of mycotoxins were investigated and
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compared. Then, the developed method was successfully used to monitor the mycotoxins residues
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in seventeen batches of Puerariae lobatae radix, a frequently used TCM in China.
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2. Material and methods
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2.1. Chemicals, reagents and materials
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The standards of mycotoxins 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol
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(15-ADON), and diacetoxyscirpenol (DAS) were obtained from Fermentek (Jerusalem, Israel). AFs (AFB1, AFB2, AFG1, and AFG2), zearalenone (ZEN), α-zearalenol (α-ZEL), β-zearalenol
(β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), alternariol (AOH), alternariol monomethyl ether (AME), T-2, and HT-2 toxin were obtained from Sigma–Aldrich (St.
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Louis, MO, USA). The purity of mycotoxins was ≥ 97%. Acetonitrile and methanol of HPLC
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grade were obtained from Merck (Darmstadt, Germany). Other chemicals and solvents were of
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HPLC or analytical grade. Deionized water was purified using Milli-Q® Synthesis (Millipore
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SAS 67120 Molsheim, France). Mycosep® 227 Trich+ Multifunctional cartridge was from Romer 5
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Labs (Washington, MO, USA). PuriToxSR TC-M160 MultiPurification Column and PuriToxSR
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TC-T220 Tricothecene Column were purchased from Trilogy Labs (Darmstadt, Germany).
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Primary secondary amine (PSA) sorbent was purchased from Varian (California, USA), and
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magnesium sulfate anhydrous (MgSO4) was from Shanghai ANPEL Scientific Instrument Co.,
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Ltd.. A total of seventeen batches of Puerariae lobatae radix were collected from the local
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regulated medical material markets in different provinces of China. The samples were kept in 4 °C
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until analysis.
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2.2. Apparatus
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LC analysis was performed on a 1200 Series HPLC System (Agilent, Waldbronn, Germany).
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Chromatographic separation was achieved on an Agilent ZORBAX SB C18 column (4.6 × 250
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mm, 5 μm particle size) with a mobile-phase flow rate of 0.6 mL/min. The mobile phase consisted
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of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. A
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linear gradient elution program was applied as follows: 0 min 10% B, 6 min 40% B, 25 min 55%
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B, 45 min 75% B, 46 min 100% B, 55 min 100% B. The injection volume was 10 μL. The column
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temperature was kept at 25 °C.
Detection was performed on an Applied Biosystems API 4000 triple quadrupole mass
spectrometry (Foster City, CA, USA) with electrospray source in multiple reaction monitoring (MRM) positive ionization mode (ESI+). The ionization source conditions were set as follows:
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ionspray voltage 4 kV, source temperature 550 °C, cone gas flow 50 psi, and desolvation gas flows
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50 psi. The parameters of declustering potential (DP) and collision energy (CE) were optimized
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for each mycotoxin to obtain the optimum MS responses.
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2.3. Optimization of extraction solvent 6
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80% acetonitrile-water (80:20, v/v), 90% acetonitrile-water (90:10, v/v), and 100%
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acetonitrile was tested as the extraction solvent. The ultrasonic extraction (250W, 40 kHz) of
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Puerariae lobatae radix (5 g) was carried out with 20 mL extraction solvent for 20 min. And then,
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the extraction was repeated for another time. The extracts were combined and evaporated by a
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rotary evaporator at 40 °C. Then the residue was redissolved by 0.5 mL 90% (90:10, v/v)
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acetonitrile-water and used for LC-MS/MS analysis after centrifugation. The extraction efficiency
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was calculated by the ratio of area of mycotoxins after extraction to that of mycotoxins in pure
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solvent.
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2.4. Sample preparation
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The ground Puerariae lobatae radix (5 g) was homogenized with 20 mL of 90% (90:10, v/v)
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acetonitrile-water by shaking for 1 min on a vortex mixer (model XW-80A, Shanghai, China) and
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extracted by ultrasonic extraction (250W, 40 kHz) for 20 min. And then, the extraction was
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repeated for another time. The two extracts were combined and centrifuged at 4000 rpm for 10
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min (Eppendorf Centifuge 5810R, Hamburg, Germany). Then, the total extract solution (about 35
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mL) was passed through PuriToxSR TC-M160 MultiPurification Column and eluted with additional 8 mL of 90% (90:10, v/v) acetonitrile-water. The eluate was evaporated by a rotary evaporator at 40 °C. Then the residue was redissolved by 0.5 mL 90% (90:10, v/v) acetonitrile-water. The solution was centrifuged at 100,000 rpm for 10 min (Eppendorf Minispan AG22331, Hamburg, Germany), and the supernatants was used for LC–MS/MS analysis.
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The cleanup procedures for Mycosep® 227 Trich+ Multifunctional cartridge and PuriToxSR
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TC-T220 Tricothecene Column were same as those for PuriToxSR TC-M160 MultiPurification
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Column. QuEChERS methods: The extract of Puerariae lobatae radix was added with 60 mg PSA 7
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and 180 mg MgSO4, and shaked for 1 min on a vortex mixer. Then, the solution was centrifuged at
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100,000 rpm for 10 min, and the supernatant was evaporated by a rotary evaporator at 40 °C. The
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residue was redissolved by 0.5 mL 90% (90:10, v/v) acetonitrile-water. Finally, the solution was
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used for LC–MS/MS analysis after centrifugation.
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2.5. Method validation
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Linearity
Accurately weighed solid portions of each mycotoxin standard were dissolved in
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acetonitrile to prepare 0.1 mg/mL of stock solutions and stored at -20 °C in the darkness. Spanjer
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et al. reported that mycotoxins solution was stable under this conditions for at least two years [20].
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A serious of concentrations of standards mixture solutions was prepared from the stock solutions
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and diluted with acetonitrile. The calibration solutions were obtained by adding 10 µL standards
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mixture solution to 90 µL mycotoxin-free Puerariae lobatae radix that had been treated with
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TC-M160 clean-up method as described in section 2.3..
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Accuracy and intra-day precision
Mycotoxin-free Puerariae lobatae radix spiked with
low, intermediate, and high levels of 17 mycotoxins were treated with TC-M160 clean-up method
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as described in section 2.3, respectively, and the experiments were repeated at six times. Then the samples were analyzed by LC-MS/MS and the recoveries and intra-day precision of 17 mycotoxins were calculated. Inter-day precision
Two samples of mycotoxin-free Puerariae lobatae radix spiked with
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intermediate levels of 17 mycotoxins, respectively, were treated with TC-M160 clean-up method
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as described in section 2.3. And the experiments were repeated in six days. Then the samples were
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analyzed by LC-MS/MS to calculate the inter-day precision.
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2.6. Requirements of method validation 8
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COMMISSION REGULATION (EC) NO 401/2006 is a guidance document established by
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the Commission of the European Community for the methods of sampling and analysis for the
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official control of mycotoxins in foodstuffs. The related requirements for the method validation
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were listed in Table 1.
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3. Results and discussion
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3.1. LC–MS/MS method development
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3.1.1 Selection of mobile phase
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The selection of the mobile phase is important as it has significant effect on the separation
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and ionization efficiency of MS detection. Water-acetonitrile system was selected as the mobile
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phase. In order to improve the separation and of mycotoxins, the addition of 10 mM ammonium
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formate, 0.01% formic acid, and 0.1% formic acid into the mobile phase were tested, respectively.
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It was found that the separation was better with the addition of formic acid. And, because the MS
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response of mycotoxins was better in positive-ion mode than in negative-ion mode, the addition of
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formic acid into mobile phase was in favor of increasing the MS responses. In addition, the effects
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of column temperature and elution program on the separation were also investigated. Finally,
water-acetonitrile containing 0.1% formic was selected as mobile phase, and the elution program and column were chosen as described in section 2.2.
3.1.2
Optimization of MS parameters
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The MS parameters of DP and CE have great impact on the MS response of mycotoxins, so
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they were optimized to obtain better sensitivity for mycotoxins. The effects of CE on the MS
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response of mycotoxins were shown in Fig. 1. A detailed list of the optimum parameters for
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mycotoxins was shown in Table 2. In the optimum LC-MS/MS conditions, MRM chromatograms 9
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of the 17 mycotoxins in standard solutions were shown in Fig. 2.
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3.1.3. Optimization of sample pretreatment In order to get high extraction efficiency and less coextraction of interfering compounds,
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different concentrations of acetonitrile were tested as the extraction solvent. As shown in Fig. 3,
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the best extraction efficiency was obtained by using acetonitrile-water (90:10, v/v), which was
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selected as the extraction solvent.
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Considering the low maximum residue levels established by the legislations and the presence
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of matrix interferences, it is necessary to conduct a pre-concentration and purification step for the
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TCM extract. In this study, four kinds of pre-condition methods i.e., TC-M160, Mycosep 227,
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TC-T220, and QuEChERS methods, were tested for their recovery to mycotoxins and applicability
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for cleanup of sample extract. The recoveries of spiked mycotoxins in Puerariae lobatae radix
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matrix by different clean-up method were listed in Table 3. It was found that QuEChERS method
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had poor ability to remove pigment, and the recovery was poor. TC-M160 and TC-T220 methods
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both had better recoveries, but the repeatability of the latter was worse than that of the former.
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Finally, TC-M160 method was chosen for better repeatability. 3.1.4. Evaluation of matrix effects
In order to evaluate matrix effects on the MS responses of mycotoxins, the stock solution of
mycotoxins was diluted with mycotoxins-free Puerariae lobatae radix (blank matrix) solution or
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acetonitrile–water (90:10, v/v) to get three levels of mycotoxins mixture solution. The signal
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suppression/enhancement (SSE) was calculated according to Eq. (1). The results were shown in
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Table 4, which showed that matrix effect had significant impact on the MS response of some
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mycotoxins. To guarantee reliable quantification of mycotoxins, matrix calibration was utilized in 10
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the following calibration curves experiment.
SSE =
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3.2. LC–MS/MS method validation
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3.2.1. Linearity
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Peak area blank matrix × 100 % Peak area solvent
The calibration curves were performed in blank matrix spiked with a series of concentrations
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of mycotoxins. Then the samples were pretreated and analyzed in the optimum conditions. The
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results of linearity experiment (Table 5) showed that the calibration curves of seventeen
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mycotoxins exhibited good linearity in the concentration ranges. Limit of detections (LODs,
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S/N=3) and limit of quantifications (LOQs, S/N≥10) concentration of mycotoxins were also
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shown in Table 5. The sensitivity of the developed method could completely satisfy mycotoxins
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anlaysis in samples according to the restrict levels for mycotoxins in food and feed defined by the
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legislations of EU and other developed countries. Compared with literatures, LOQ of some
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mycotoxins (for example, 0.0488-0.099 µg/kg for AFs) in our work were higher than those
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(0.02-0.09 µg/kg for AFs) reported in literatures [11, 14], but lower than those (0.10-0.35 µg/kg for AFs) in literature [13]. However, LOD of almost all mycotoxins (for example, 0.002-0.01µg/kg for AFs) were lower than those (0.01-0.08 µg/kg for AFs) in literatures [11, 13, 14].
3.2.2. Accuracy and precision
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Recovery experiment was performed to evaluate the accuracy of method by standard addition.
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Standards of mycotoxins were added to Puerariae lobatae radix at high, intermediate, and low
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levels in six parallel. The results of recovery of mycotoxins were summarized in Table 6. The
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recovery of AFB1 (56%) at intermediate level was a little out of range, but the good repeatability 11
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(RSD = 5.2%) could compensate for this to some extent. The sample solution spiked with intermediate concentration of mycotoxins was injected
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repeatedly to evaluate instrument precision. Good injection precision was obtained with RSD of
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peak area of mycotoxins less than 6.0% (n=6). Intra-day precision experiments were performed by
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injecting six pretreated sample solutions spiked with intermediate concentration of mycotoxins
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within a day. For the evaluation of inter-day precision of method, six sample solutions was
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prepared and analyzed in six different days. The detailed precision results were listed in Table 7.
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According to the requirement to analytical method for mycotoxins in COMMISSION
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REGULATION (EC) NO 401/2006, the intra-day and inter-day precision (less than 16.9% and
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31.8%, respectively) of the method were acceptable. The precision result was comparable to that
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in literatures.
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3.3. Method application
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The validated method was applied to determine the residues of mycotoxins in seventeen
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batches of Puerariae lobatae radix. It was found that three Puerariae lobatae radix samples
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showed mycotoxin contamination. Batch no. 17 sample was detected with contamination of AFB1 at (0.751±0.176) μg/kg. Batch no. 6 and no. 9 were found with contamination of T-2 toxin at (1.10±0.01) μg/kg and (0.853±0.044) μg/kg, respectively. The MRM chromatograms of these
samples were shown in Fig. 4. The peaks of two mycotoxins in chromatograms of samples were
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confirmed by comparing the retention times, the intensity ratios of two pairs of ions (m/z 315/287
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and m/z 315/259 for AFB1, m/z 484/305 and m/z 484/215 for T-2), and spiking the standards to the
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samples.
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4. Conclusions 12
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An efficient and accurate LC–MS/MS method was developed for simultaneous determination
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of seventeen mycotoxins in Puerariae lobatae radix. All analytes could be completely separated in
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less than 35 min, providing good peaks shapes. Cleanup procedure was one of key points for
260
successful detection of mycotoxins in TCM. The effects of four different cleanup methods were
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compared, and PuriToxSR M160 MultiPurification Column was chosen for better recovery and
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repeatability for mycotoxins analysis. The established method was successfully employed to
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analyze a total number of seventeen batches of Puerariae lobatae radix collected from different
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provinces of China. And, three batches of samples were found with contamination of AFB1 or T-2
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mycotoxins. The proposed method can be used to monitor the mycotoxins residues in Puerariae
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lobatae radix.
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This
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(2009ZX09502-024).
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Acknowledgments
financially
supported
by
the
National
S&T
Major
Project
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299
[9] Y. Ren, Y. Zhang, S. Shao, Z. Cai, L. Feng, H. Pan, Z. Wang, Simultaneous determination of
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multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid 14
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chromatography tandem mass spectrometry, J. Chromatogr. A 1143 (2007) 48-64.
302
[10] J. Rubert, C. Soler, J. Manes, Evaluation of matrix solid-phase dispersion (MSPD) extraction
303
for multi-mycotoxin determination in different flours using LC-MS/MS, Talanta 85 (2011)
304
206-215.
305
[11] Z. Han, X. Liu, Y. Ren, L. Luan, Y. Wu, A rapid method with ultra-high-performance liquid
306
chromatography-tandem mass spectrometry for simultaneous determination of five type B
307
trichothecenes in traditional Chinese medicines, J. Sep. Sci. 33 (2010) 1923-1932.
308
[12] Z. Han, Y. Ren, X. Liu, L. Luan, Y. Wu, A reliable isotope dilution method for simultaneous
309
determination of fumonisins B1, B2 and B3 in traditional Chinese medicines by
310
ultra-high-performance liquid chromatography-tandem mass spectrometry, J. Sep. Sci. 33 (2010)
311
2723-2733.
312
[13] Z. Han, Y. Zheng, L. Luan, Z. Cai, Y. Ren, Y. Wu, An ultra-high-performance liquid
313
chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins
314
B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines, Anal. Chim. Acta 664 (2010)
316 317 318
cr
us
an
M
d
te
Ac ce p
315
ip t
301
165-171.
[14] Z. Han, Y. Ren, J. Zhu, Z. Cai, Y. Chen, L. Luan, Y. Wu, Multianalysis of 35 mycotoxins in traditional Chinese medicines by ultra-high-performance liquid chromatography-tandem mass
spectrometry coupled with accelerated solvent extraction, J. Agric. Food Chem. 60 (2012)
319
8233-8247.
320
[15] B.C. Liau, T.T. Jong, M.R. Lee, C.M. Chang, Supercritical fluid extraction and quantification
321
of aflatoxins in Zizyphi Fructus by liquid chromatography/atmospheric pressure chemical
322
ionization tandem mass spectrometry, Rapid Commun. Mass Spectrom. 21 (2007) 667-673. 15
Page 15 of 30
[16] Y.T. Yue, X.F. Zhang, M.H. Yang, O.Y. Zhen, H.B. Liu, Simultaneous Determination of
324
Deoxynivalenol and Nivalenol in Traditional Chinese Medicine by SPE and LC, Chromatographia
325
72 (2010) 551-555.
326
[17] Y. Zhang, J. Caupert, P.M. Imerman, J.L. Richard, G.C. Shurson, The occurrence and
327
concentration of mycotoxins in U.S. distillers dried grains with solubles, J. Agric. Food Chem. 57
328
(2009) 9828-9837.
329
[18] E. Razzazi-Fazeli, B. Rabus, B. Cecon, J. Bohm, Simultaneous quantification of
330
A-trichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical
331
ionisation mass spectrometry, J. Chromatogr. A 968 (2002) 129-142.
332
[19] L. Vaclavik, M. Zachariasova, V. Hrbek, J. Hajslova, Analysis of multiple mycotoxins in
333
cereals under ambient conditions using direct analysis in real time (DART) ionization coupled to
334
high resolution mass spectrometry, Talanta 82 (2010) 1950-1957.
335
[20] M.C. Spanjer, P.M. Rensen, J.M. Scholten, LC-MS/MS multi-method for mycotoxins after
336
single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and
338
cr us
an
M
d
te
Ac ce p
337
ip t
323
figs, Food Addit. Contam. Part A 25 (2008) 472-489.
16
Page 16 of 30
338
Figure legends:
339
Fig. 1.
340
Fig. 2. MRM chromatograms of 17 mycotoxins in the standard solutions. The concentrations of
341
mycotoxins were all 1 μg/mL. Symbols: 1. 15-ADON; 2. 3-ADON; 3. AFG2; 4. AFB2; 5. AFG1;
342
6. DAS; 7. HT-2; 8. AFB1; 9. AOH; 10. α-ZAL; 11. β-ZAL; 12. β-ZEL; 13. α-ZEL; 14. T-2; 15.
343
ZAN; 16. AME; 17. ZEN.
us
cr
ip t
Effects of collision energy (CE) on the MS response of mycotoxins.
Fig. 3.
Comparison of extraction efficiency of mycotoxins with different extraction solvents.
345
Fig. 4.
MRM chromatograms of AFB1 and T-2 toxin in standard solution and the residues of
346
AFB1 and T-2 toxin detected in three batches of Puerariae lobatae radix.
an
344
Ac ce p
te
d
M
347
17
Page 17 of 30
Table 1 The requirement to analytical method for mycotoxins in COMMISSION REGULATION (EC) NO 401/2006.
Aflatoxins B1, B2, G1, G2
Precision
Concentration (µg/kg)
Recovery
10
80-110%
>100 - ≤500
60-110%
>500
70-120%
RSDR Recommended: as derived from Horwitz Equation* ; Maximum permitted: 2 × value derived from Horwitz Equation ≤40%
us
Deoxynivalenol ≤50 Zearalenone
60-130% 50-250 60-130%
M
>250 100-200 HT-2
0.66 × RSDR
≤20%
≤50%
≤40%
≤40%
≤25%
≤60%
≤40%
≤50%
≤30%
≤60%
≤40%
≤50%
≤30%
an
>50 T-2
RSDr
ip t
Mycotoxins
cr
347 348
60-130%
d
>200
The calculation of precision values from Horwitz equation is RSDR = 2
(1− 0.5log C )
*
350 351
is the relative standard deviation of the results generated under reproducibility conditions, C is the concentration ratio (i.e., 1 µg/kg=1×10-9).
353 354
Ac ce p
352
te
349
, where: RSDR
18
Page 18 of 30
354 355
Table 2
LC–MS/MS parameters of seventeen mycotoxins.
m/z Abbreviation
DP(V)
tR(min) precursor ion
product ion
CE(V)
15-ADON
12.4
339.3 [M+H]+
231.2*,213.2
40
20
3-Acetyldeoxynivalenol
3-ADON
12.7
339.3 [M+H]+
231.2*,213.2
40
20
Aflatoxin G2
AFG2
15.3
331.0 [M+H]+
313.0,245.4*
Aflatoxin B2
AFB2
16.7
315.1 [M+H]+
Aflatoxin G1
AFG1
16.8
329.0 [M+H]+
Diacetoxyscirpenol
DAS
16.8
HT-2 toxin
HT-2
17.8
Aflatoxin B1
AFB1
Alternariol
AOH
cr
ip t
15-Acetyldeoxynivalenol
an
Mycotoxins
m/z
45
287.2,259.1*
80
43
243.4*,311.0
80
37
us
80
307.3*,247.5
60
15
425.3 [M+H]+
263.0*,157.4
50
15
18.3
313.0 [M+H]+
285.1,269.2*
80
47
19.7
259.2 [M+H]+
185.3*,213.3
50
45
te
d
M
367.1 [M+H]+
α-ZAL
20.4
323.1 [M+H]+
305.3*,189.3
40
13
β-ZAL
23.7
323.1 [M+H]+
305.3*,163.2
40
13
β-Zearalenol
β-ZEL
20.8
321.3 [M+H]+
285.0*,189.0
40
17
α-Zearalenol
α-ZEL
24.5
321.3 [M+H]+
285.0*,189.0
40
17
T-2 toxin
T-2
26.6
484.2 [M+NH4]+
305.3,215.4*
50
23
Zearalanone
ZAN
31.1
321.3 [M+H]+
303.1*,189.0
40
20
AME
31.4
273.2 [M+H]+
230.0*,199.3
60
43
ZEN
31.6
319.1 [M+H]+
283.0*,301.2
40
13
α-Zearalanol
Ac ce p
α-Zearalanol
Alternariol monomethyl ether Zearalenone 356 357
*
For quantitative analysis.
19
Page 19 of 30
359 360 361
Table 3
Comparison of the recoveries (mean and RSD, %, n=3) of spiked mycotoxins in Puerariae lobatae radix by different clean-up methods.
QuEChERS
TC-M160
TC-T220
Mycosep 227
RSD
Recovery
RSD
Recovery
RSD
15-ADON
20.5
31.7
66.0
6.9
70.8
5.7
3-ADON
19.1
31.4
44.8
15.2
44.8
6.5
AFG2
56.3
27.8
79.5
4.4
88.9
AFB2
43.5
22.6
71.5
5.6
AFG1
49.9
21.6
80.3
5.4
DAS
51.9
21.0
93.5
HT-2
38.3
19.3
64.2
AFB1
42.1
24.6
59.4
AOH
43.3
20.2
α-ZAL
114.8
RSD 3.5
33.1
5.2
12.4
us
73.7
4.6
72.4
13.0
64.0
1.8
84.0
10.8
71.6
2.1
94.5
12.0
74.4
7.9
5.4
61.6
16.9
54.3
4.8
3.2
61.5
15.6
55.4
5.7
62.7
6.2
69.4
18.8
58.2
5.9
96.6
19.3
93.6
25.2
75.1
9.0
an
64.3
d
M
6.6
te
Ac ce p
26.1
Recovery
ip t
Recovery
cr
Mycotoxins
β-ZAL
57.6
22.9
84.7
7.4
90.5
16.3
80.3
9.1
β-ZEL
41.4
21.5
72.8
6.1
75.5
18.3
67.4
8.4
α-ZEL
45.8
24.5
76.3
4.0
72.1
16.3
63.2
7.9
T-2
52.2
19.9
78.3
8.8
85.3
18.8
73.9
5.6
ZAN
46.3
21.1
72.9
3.7
61.7
13.1
54.0
3.9
AME
54.1
19.4
76.9
5.8
66.3
16.2
60.1
7.6
ZEN
46.8
19.8
76.3
3.6
75.9
19.5
66.7
7.7
362 363
21
Page 20 of 30
363
Table 4
SSEs of mycotoxins in matrix of Puerariae lobatae radix.
SSE* (%)
Mycotoxins
15-ADON
58.8
α-ZAL
SSE* (%) 365 366 367 95.0
3-ADON
65.9
β-ZAL
87.9
368
AFG2
38.4
β-ZEL
94.4
369
AFB2
72.7
α-ZEL
92.7
370
AFG1
70.8
T-2
89.9
371
DAS
99.9
ZAN
89.8
372
HT-2
92.7
AME
91.5
373
AFB1
41.0
ZEN
89.3
374
AOH
87.5
375 376
te
d
M
an
us
cr
Mycotoxins
ip t
364
SSE =
378
Ac ce p
Peak area blank matrix × 100 % Peak area solvent
377
22
Page 21 of 30
378 379 380
Table 5 Linearity, limit of quantification (LOQ) and limit of detection (LOD) of seventeen mycotoxins.
Linearity range (μg/kg)
linearity equations
correlation coefficients (r)
LOQ
LOD
(μg/kg)
(μg/kg)
15-ADON
5.00-400
y=108243x-184.43
0.9950
2.50
0.537
3-ADON
4.99-401
y=402115x+11692
0.9919
2.50
0.242
AFG2
0.502-40.5
y=6143657x+57914
0.9962
0.0994
0.0994
AFB2
0.501-40.8
y=8937279x+278758
0.9939
0.0499
0.00499
AFG1
0.499-38.7
y=13953935x+460373
0.9916
DAS
4.99-401
y=403762x+59765
0.9962
HT-2
5.02-400
y=595428x+32120
AFB1
0.499-41.4
y=9294161x+158623
AOH
5.00-401
y=1401287x+199127
α-ZAL
2.49-202
y=4032287x+179121
β-ZAL
2.50-200
β-ZEL
0.00506
1.00
0.150
0.9974
1.00
0.176
0.9968
0.0488
0.00203
0.9945
0.998
0.214
0.9977
2.49
0.574
y=4664606x+57462
0.9974
2.50
0.417
2.49-200
y=8968281x+435292
0.494
0.0309
α-ZEL
2.49-199
y=4210924 x+81083
0.9983
0.825
0.206
T-2
5.01-400
M
0.9968
y=1491763x+160609
0.9966
0.488
0.0775
ZAN
2.49-200
y=20083677x+1156770
0.9959
0.617
0.0502
AME
4.98-400
y=129173x+10708
0.9970
4.97
1.06
ZEN
2.49-200
y=5292209x+227667
0.9963
0.488
0.183
an
te
Ac ce p
381 382
us
0.0506
d
cr
ip t
Mycotoxins
23
Page 22 of 30
382 383 384
Table 6 The results of recoveries with their RSDs (n = 6) of seventeen mycotoxins at high, intermediate, and low levels.
Recovery in
RSD
Recovery in
RSD
Recovery in
RSD
high level (%)
(%)
intermediate level (%)
(%)
low level (%)
(%)
15-ADON a
86.3
11.6
70.5
13.5
3-ADON a
84.5
7.5
77.1
16.6
AFG2 b
76.0
12.0
73.8
16.9
AFB2 b
90.3
10.9
78.1
AFG1 b
91.8
9.3
80.8
DAS a
115.2
7.1
103.0
HT-2 a
110.0
8.2
AFB1 b
83.5
11.3
AOH a
106.7
α-ZAL c
113.0
13.8
56.9
5.2
53.3
5.9 6.5
11.3
51.2
7.8
8.3
84.5
11.3
78.4
5.5
79.0
6.4
56.0
5.2
58.3
5.3
5.5
89.9
5.2
82.4
5.3
3.7
104.4
6.8
te
d
M
51.1
an
us
cr
62.0
9.8
95.8
β-ZAL c
104.6
6.5
88.3
5.5
77.3
7.3
β-ZEL c
101.7
7.4
93.2
9.6
77.2
4.8
α-ZEL c
112.9
6.6
91.2
4.4
85.7
3.1
T-2 a
99.8
10.1
92.6
3.3
86.2
12.4
ZAN c
101.6
4.5
87.0
4.3
82.6
3.1
AME a
103.0
6.0
88.3
5.8
87.8
2.8
ZEN c
103.5
5.2
90.1
4.3
84.0
7.4
6.3
Ac ce p 385 386 387 388
ip t
Mycotoxins
Notes: a 240, 50 and 10 μg/kg for 3-ADON, 15-ADON, DAS, AOH, AME, T-2 and HT-2 toxin; b 24, 5 and 1 μg/kg for AFB1, AFB2, AFG1 and AFG2; c 120, 25 and 5μg/kg for ZAN, ZEN, α-ZAL, β-ZAL, α-ZEL and β-ZEL 24
Page 23 of 30
The results of precision (n = 6) of seventeen mycotoxins in the optimum conditions.
Interday precision (RSD, %)
4.8
13.5
AFG2
4.4
16.9
3-ADON
4.7
16.6
AFB2
2.7
9.8
AFG1
2.7
11.3
DAS
4.3
8.3
HT-2
4.6
AFB1
2.4
AOH
2.1
α-ZAL
1.4
31.8 22.9
21.8
24.4
an
15-ADON
Intraday precision (RSD, %)
ip t
Precision (RSD, %)
cr
Mycotoxins
us
Table 7
6.4 11.8 15.6
5.2
8.7
5.2
14.3
3.7
9.1
2.6
5.5
14.0
2.8
9.6
13.4
α-ZEL
3.5
4.4
11.8
T-2
6.0
3.3
11.7
ZAN
4.5
4.3
9.2
AME
3.7
5.8
9.1
ZEN
4.9
4.3
8.6
β-ZAL
d
Ac ce p
β-ZEL
M
5.5
te
388 389
390 391
25
Page 24 of 30
391
Highlights A LC–MS/MS method for determination of seventeen mycotoxins was developed.
393
Four different clean-up approaches were investigated and compared.
394
The method was used to determine mycotoxins residues in Puerariae lobatae radix.
395
Three batches of TCM were found with contamination of AFB1 or T-2 mycotoxins.
ip t
392
396
Ac ce p
te
d
M
an
us
cr
397
26
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
*Graphical Abstract
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 29 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 30 of 30
S0731-7085(14)00277-5 http://dx.doi.org/doi:10.1016/j.jpba.2014.05.037 PBA 9600
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-2-2014 22-5-2014 23-5-2014
Please cite this article as: S. Wang, L. Cheng, S. Ji, K. Wang, Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by liquid chromatographytandem mass spectrometry, Journal of Pharmaceutical and Biomedical Analysis (2014), http://dx.doi.org/10.1016/j.jpba.2014.05.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Simultaneous determination of seventeen mycotoxins residues in Puerariae lobatae radix by
2
liquid chromatography-tandem mass spectrometry
3
Shufang Wang a,*, Ling Chenga, Shen Ji b,*, Ke Wang b
4
a
College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China
Department of Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control,
us
b
cr
5 6
ip t
1
7
1500 Zhangheng Road, Shanghai, 201203, China
an
8
Corresponding authors: Dr. Shufang Wang, Pharmaceutical Informatics Institute, Zhejiang
10
University, Zijingang Campus, No. 866 Yuhangtang Road, Hangzhou 310058, P.R. China. Email:
11
[email protected]; Tel: +86 571 88208426; Fax: +86 571 88208426; Dr. Shen Ji, Department of
12
Traditional Chinese Medicine, Shanghai Institute for Food and Drug Control, 1500 Zhangheng
13
Road, Shanghai, 201203, China. [email protected], Tel: +86 21 50798195; Fax: +86 21
14
50798195.
d
te
Ac ce p
15
M
9
1
Page 1 of 30
15
ABSTRACT This work reported an efficient and accurate liquid chromatography tandem mass
17
spectrometry (LC–MS/MS) method for simultaneous determination of seventeen mycotoxins in
18
Puerariae lobatae radix, a frequently used traditional Chinese medicine (TCM). The effects of
19
four different clean-up methods, including TC-M160, TC-T220, Mycosep 227, and QuEChERS
20
method, on the recoveries of mycotoxins were investigated and compared. Finally, TC-M160 was
21
chosen for better recovery and repeatability for mycotoxins analysis. The analytes were separated
22
on an Agilent ZORBAX SB C18 column (4.6 × 250 mm, 5 μm particle size), and eluted with a
23
mobile phase consisting of (A) water containing 0.1% formic acid and (B) acetonitrile containing
24
0.1% formic acid at a flow rate of 0.6 ml/min. The separated compounds were detected by a triple
25
quadrupole mass spectrometer operating in positive electrospray ionization with multiple reaction
26
monitoring (MRM) mode. The results of method validation accorded with the requirement of
27
analytical method for mycotoxins in COMMISSION REGULATION (EC) No 401/2006. The
28
developed method was successfully applied for determination of mycotoxins in seventeen batches
30 31 32
cr
us
an
M
d
te
Ac ce p
29
ip t
16
of Puerariae lobatae radix collected from different provinces of China. Three batches of them were found with contamination of mycotoxins AFB1 at (0.751±0.176) μg/kg, T-2 at (1.10±0.01) μg/kg, and T-2 at (0.853±0.044) μg/kg, respectively. The results demonstrated that the proposed method was suitable for monitoring mycotoxins residues in Puerariae lobatae radix.
33 34
Keywords: Mycotoxins; Liquid chromatography-tandem mass spectrometry (LC-MS/MS);
35
Puerariae lobatae radix; TC-M160
36
1. Introduction 2
Page 2 of 30
Traditional Chinese medicine (TCM) has been used for prevention and treatment of various
38
diseases. But it may mildew during storage and transportation, which would result in the
39
contamination of mycotoxins. Mycotoxins are toxic secondary metabolites produced by some
40
filamentous fungi, such as Fusarium, Aspergillus, Penicillium, and Alternaria species.
41
Surveillance studies demonstrated that mycotoxin contamination is a world-wide problem. Their
42
occurrence in food, beverages and feed has been recognized as potential threat to human and
43
animal health. Aflatoxins (AFs), a group of secondary metabolites produced by Aspergillus flavus
44
and Aspergillus parasiticus, mainly include AFB1, AFB2, AFG1, AFG2, which have hepatotoxicity
45
and carcinogenicity. Especially, AFB1 has been defined as a Category A carcinogen by the
46
International Agency for Research on Cancer (IARC) of World Health Organization (WHO) in
47
1993[1]. Trichothecene (TCT) mycotoxins, mainly generated from the secondary metabolism of
48
Fusarium, can result in immunosuppression, the necrosis of myeloid tissue and hemorrhage of
49
visceral organs. Zearalenone (ZEN) mycotoxins, generated from Fusarium graminearum and
50
other Fusarium molds, may damage the reproduction of mammals because of its forged estrogenic
52 53 54
cr
us
an
M
d
te
Ac ce p
51
ip t
37
effects. Alternariol (AOH) and alternariol monomethyl ether (AME) are among the main mycotoxins of Alternaria fungi. The extraction of A. alternata was considered as a suspected cancer-causing factor for human oesophageal cancer [2]. Considering their serious toxicity, maximum residue levels (MRLs) were set for some mycotoxins, and MRLs were different for
55
same mycotoxin in different foodstuffs in European Union (EU) as described in Commission
56
Regulation (EC) No. 1881/2006. MRLs of AFB1 are 0.1-8.0 µg/kg in seventeen kinds of
57
foodstuffs, and 20-400 µg/kg for ZEN in ten types of foodstuffs. In Chinese pharmacopeia of 2010
58
edition, MRL of AFB1 in traditional Chinese medicines is 5 µg/kg. Therefore, it is necessary to 3
Page 3 of 30
59
develop robust and sensitive methods for monitoring mycotoxins in TCMs to ensure providing
60
consumers with safe TCM. Current analytical techniques of mycotoxins mainly include liquid chromatography with
62
fluorescence detection (LC-FLD) [3], liquid chromatography with tandem mass spectrometry
63
(LC-MS/MS) [4], gas chromatography with tandem mass spectrometry (GC-MS/MS) [5], and
64
enzyme-linked immunosorbent assay (ELISA) [6]. Although ELISA is a fast and sensitive method,
65
it is a semi-quantitative analysis, and false positive result might be obtained [6]. Sample
66
derivatization procedure is needed for GC-MS and LC-FD method because many mycotoxins are
67
not volatile and have no fluorescence property. The most frequently used method for mycotoxins
68
analysis is LC-MS/MS, which has been extensively studied in various food and matrices [7]. The
69
introduction of MS/MS detection also leads to a significant improvement for mycotoxins analysis
70
because of its high sensitivity, selectivity and specificity [8-10]. Compared with other analytical
71
technique, LC-MS/MS can be used not only for the quantitative analysis, but also for the
72
confirmation of existence of mycotoxins. Up to now, most of the published works on mycotxoins
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analysis were about food samples; only several reports were concerned with TCMs [4, 11-16]. Considering the low residue level of mycotoxins (generally in µg/kg level) and complex
chemical composition of TCM and food samples, cleanup procedure was necessary before
analysis by LC-MS/MS. Zhang et al. used TC-M160 MultiPurification column as cleanup method
77
for analysis of AFs, trichothecenes, and ZEAs mycotoxins in grains [17]. Yue et al. used TC-T220
78
Tricothecene column to analyze deoxynivalenol and nivalenol in TCMs [16]. Razzazi-Fazeli et al.
79
analyzed several type A trichothecenes in grains by using Mycosep 227 Trich+ Multifunctional
80
cartridge as cleanup method [18]. QuEChERS method based on primary secondary amine (PSA) 4
Page 4 of 30
81
sorbent was also applied for mycotoxins analysis in cereals [19]. However, there has been no work
82
to compare the effect of these cleanup methods systematically. In this study, a reliable LC–MS/MS method was developed to simultaneously determine
84
seventeen mycotoxins, including four AFs (AFB1, AFB2, AFG1, and AFG2), five trichothecenes
85
mycotoxins (T-2, HT-2, diacetoxyscirpenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol),
86
six ZEAs (zearalenone, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, and β-zearalanol),
87
and two Alternaria toxins (alternariol and alternariol monomethyl ether). The extraction solvent
88
was optimized. And, the effects of different clean-up methods, including TC-M160, TC-T220,
89
Mycosep 227, and QuEChERS methods, on the recovery of mycotoxins were investigated and
90
compared. Then, the developed method was successfully used to monitor the mycotoxins residues
91
in seventeen batches of Puerariae lobatae radix, a frequently used TCM in China.
92
2. Material and methods
93
2.1. Chemicals, reagents and materials
95 96 97 98
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The standards of mycotoxins 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol
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(15-ADON), and diacetoxyscirpenol (DAS) were obtained from Fermentek (Jerusalem, Israel). AFs (AFB1, AFB2, AFG1, and AFG2), zearalenone (ZEN), α-zearalenol (α-ZEL), β-zearalenol
(β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), alternariol (AOH), alternariol monomethyl ether (AME), T-2, and HT-2 toxin were obtained from Sigma–Aldrich (St.
99
Louis, MO, USA). The purity of mycotoxins was ≥ 97%. Acetonitrile and methanol of HPLC
100
grade were obtained from Merck (Darmstadt, Germany). Other chemicals and solvents were of
101
HPLC or analytical grade. Deionized water was purified using Milli-Q® Synthesis (Millipore
102
SAS 67120 Molsheim, France). Mycosep® 227 Trich+ Multifunctional cartridge was from Romer 5
Page 5 of 30
Labs (Washington, MO, USA). PuriToxSR TC-M160 MultiPurification Column and PuriToxSR
104
TC-T220 Tricothecene Column were purchased from Trilogy Labs (Darmstadt, Germany).
105
Primary secondary amine (PSA) sorbent was purchased from Varian (California, USA), and
106
magnesium sulfate anhydrous (MgSO4) was from Shanghai ANPEL Scientific Instrument Co.,
107
Ltd.. A total of seventeen batches of Puerariae lobatae radix were collected from the local
108
regulated medical material markets in different provinces of China. The samples were kept in 4 °C
109
until analysis.
110
2.2. Apparatus
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LC analysis was performed on a 1200 Series HPLC System (Agilent, Waldbronn, Germany).
112
Chromatographic separation was achieved on an Agilent ZORBAX SB C18 column (4.6 × 250
113
mm, 5 μm particle size) with a mobile-phase flow rate of 0.6 mL/min. The mobile phase consisted
114
of (A) water containing 0.1% formic acid and (B) acetonitrile containing 0.1% formic acid. A
115
linear gradient elution program was applied as follows: 0 min 10% B, 6 min 40% B, 25 min 55%
116
B, 45 min 75% B, 46 min 100% B, 55 min 100% B. The injection volume was 10 μL. The column
118 119 120
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M
111
temperature was kept at 25 °C.
Detection was performed on an Applied Biosystems API 4000 triple quadrupole mass
spectrometry (Foster City, CA, USA) with electrospray source in multiple reaction monitoring (MRM) positive ionization mode (ESI+). The ionization source conditions were set as follows:
121
ionspray voltage 4 kV, source temperature 550 °C, cone gas flow 50 psi, and desolvation gas flows
122
50 psi. The parameters of declustering potential (DP) and collision energy (CE) were optimized
123
for each mycotoxin to obtain the optimum MS responses.
124
2.3. Optimization of extraction solvent 6
Page 6 of 30
80% acetonitrile-water (80:20, v/v), 90% acetonitrile-water (90:10, v/v), and 100%
126
acetonitrile was tested as the extraction solvent. The ultrasonic extraction (250W, 40 kHz) of
127
Puerariae lobatae radix (5 g) was carried out with 20 mL extraction solvent for 20 min. And then,
128
the extraction was repeated for another time. The extracts were combined and evaporated by a
129
rotary evaporator at 40 °C. Then the residue was redissolved by 0.5 mL 90% (90:10, v/v)
130
acetonitrile-water and used for LC-MS/MS analysis after centrifugation. The extraction efficiency
131
was calculated by the ratio of area of mycotoxins after extraction to that of mycotoxins in pure
132
solvent.
133
2.4. Sample preparation
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The ground Puerariae lobatae radix (5 g) was homogenized with 20 mL of 90% (90:10, v/v)
135
acetonitrile-water by shaking for 1 min on a vortex mixer (model XW-80A, Shanghai, China) and
136
extracted by ultrasonic extraction (250W, 40 kHz) for 20 min. And then, the extraction was
137
repeated for another time. The two extracts were combined and centrifuged at 4000 rpm for 10
138
min (Eppendorf Centifuge 5810R, Hamburg, Germany). Then, the total extract solution (about 35
140 141 142 143
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134
mL) was passed through PuriToxSR TC-M160 MultiPurification Column and eluted with additional 8 mL of 90% (90:10, v/v) acetonitrile-water. The eluate was evaporated by a rotary evaporator at 40 °C. Then the residue was redissolved by 0.5 mL 90% (90:10, v/v) acetonitrile-water. The solution was centrifuged at 100,000 rpm for 10 min (Eppendorf Minispan AG22331, Hamburg, Germany), and the supernatants was used for LC–MS/MS analysis.
144
The cleanup procedures for Mycosep® 227 Trich+ Multifunctional cartridge and PuriToxSR
145
TC-T220 Tricothecene Column were same as those for PuriToxSR TC-M160 MultiPurification
146
Column. QuEChERS methods: The extract of Puerariae lobatae radix was added with 60 mg PSA 7
Page 7 of 30
and 180 mg MgSO4, and shaked for 1 min on a vortex mixer. Then, the solution was centrifuged at
148
100,000 rpm for 10 min, and the supernatant was evaporated by a rotary evaporator at 40 °C. The
149
residue was redissolved by 0.5 mL 90% (90:10, v/v) acetonitrile-water. Finally, the solution was
150
used for LC–MS/MS analysis after centrifugation.
151
2.5. Method validation
cr
Linearity
Accurately weighed solid portions of each mycotoxin standard were dissolved in
us
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147
acetonitrile to prepare 0.1 mg/mL of stock solutions and stored at -20 °C in the darkness. Spanjer
154
et al. reported that mycotoxins solution was stable under this conditions for at least two years [20].
155
A serious of concentrations of standards mixture solutions was prepared from the stock solutions
156
and diluted with acetonitrile. The calibration solutions were obtained by adding 10 µL standards
157
mixture solution to 90 µL mycotoxin-free Puerariae lobatae radix that had been treated with
158
TC-M160 clean-up method as described in section 2.3..
161 162 163 164
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Accuracy and intra-day precision
Mycotoxin-free Puerariae lobatae radix spiked with
low, intermediate, and high levels of 17 mycotoxins were treated with TC-M160 clean-up method
Ac ce p
159
an
153
as described in section 2.3, respectively, and the experiments were repeated at six times. Then the samples were analyzed by LC-MS/MS and the recoveries and intra-day precision of 17 mycotoxins were calculated. Inter-day precision
Two samples of mycotoxin-free Puerariae lobatae radix spiked with
165
intermediate levels of 17 mycotoxins, respectively, were treated with TC-M160 clean-up method
166
as described in section 2.3. And the experiments were repeated in six days. Then the samples were
167
analyzed by LC-MS/MS to calculate the inter-day precision.
168
2.6. Requirements of method validation 8
Page 8 of 30
COMMISSION REGULATION (EC) NO 401/2006 is a guidance document established by
170
the Commission of the European Community for the methods of sampling and analysis for the
171
official control of mycotoxins in foodstuffs. The related requirements for the method validation
172
were listed in Table 1.
173
3. Results and discussion
174
3.1. LC–MS/MS method development
175
3.1.1 Selection of mobile phase
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169
The selection of the mobile phase is important as it has significant effect on the separation
177
and ionization efficiency of MS detection. Water-acetonitrile system was selected as the mobile
178
phase. In order to improve the separation and of mycotoxins, the addition of 10 mM ammonium
179
formate, 0.01% formic acid, and 0.1% formic acid into the mobile phase were tested, respectively.
180
It was found that the separation was better with the addition of formic acid. And, because the MS
181
response of mycotoxins was better in positive-ion mode than in negative-ion mode, the addition of
182
formic acid into mobile phase was in favor of increasing the MS responses. In addition, the effects
184 185 186
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an
176
of column temperature and elution program on the separation were also investigated. Finally,
water-acetonitrile containing 0.1% formic was selected as mobile phase, and the elution program and column were chosen as described in section 2.2.
3.1.2
Optimization of MS parameters
187
The MS parameters of DP and CE have great impact on the MS response of mycotoxins, so
188
they were optimized to obtain better sensitivity for mycotoxins. The effects of CE on the MS
189
response of mycotoxins were shown in Fig. 1. A detailed list of the optimum parameters for
190
mycotoxins was shown in Table 2. In the optimum LC-MS/MS conditions, MRM chromatograms 9
Page 9 of 30
191
of the 17 mycotoxins in standard solutions were shown in Fig. 2.
192
3.1.3. Optimization of sample pretreatment In order to get high extraction efficiency and less coextraction of interfering compounds,
194
different concentrations of acetonitrile were tested as the extraction solvent. As shown in Fig. 3,
195
the best extraction efficiency was obtained by using acetonitrile-water (90:10, v/v), which was
196
selected as the extraction solvent.
us
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193
Considering the low maximum residue levels established by the legislations and the presence
198
of matrix interferences, it is necessary to conduct a pre-concentration and purification step for the
199
TCM extract. In this study, four kinds of pre-condition methods i.e., TC-M160, Mycosep 227,
200
TC-T220, and QuEChERS methods, were tested for their recovery to mycotoxins and applicability
201
for cleanup of sample extract. The recoveries of spiked mycotoxins in Puerariae lobatae radix
202
matrix by different clean-up method were listed in Table 3. It was found that QuEChERS method
203
had poor ability to remove pigment, and the recovery was poor. TC-M160 and TC-T220 methods
204
both had better recoveries, but the repeatability of the latter was worse than that of the former.
206 207 208
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an
197
Finally, TC-M160 method was chosen for better repeatability. 3.1.4. Evaluation of matrix effects
In order to evaluate matrix effects on the MS responses of mycotoxins, the stock solution of
mycotoxins was diluted with mycotoxins-free Puerariae lobatae radix (blank matrix) solution or
209
acetonitrile–water (90:10, v/v) to get three levels of mycotoxins mixture solution. The signal
210
suppression/enhancement (SSE) was calculated according to Eq. (1). The results were shown in
211
Table 4, which showed that matrix effect had significant impact on the MS response of some
212
mycotoxins. To guarantee reliable quantification of mycotoxins, matrix calibration was utilized in 10
Page 10 of 30
213
the following calibration curves experiment.
SSE =
214
3.2. LC–MS/MS method validation
216
3.2.1. Linearity
(1)
ip t
215
Peak area blank matrix × 100 % Peak area solvent
The calibration curves were performed in blank matrix spiked with a series of concentrations
218
of mycotoxins. Then the samples were pretreated and analyzed in the optimum conditions. The
219
results of linearity experiment (Table 5) showed that the calibration curves of seventeen
220
mycotoxins exhibited good linearity in the concentration ranges. Limit of detections (LODs,
221
S/N=3) and limit of quantifications (LOQs, S/N≥10) concentration of mycotoxins were also
222
shown in Table 5. The sensitivity of the developed method could completely satisfy mycotoxins
223
anlaysis in samples according to the restrict levels for mycotoxins in food and feed defined by the
224
legislations of EU and other developed countries. Compared with literatures, LOQ of some
225
mycotoxins (for example, 0.0488-0.099 µg/kg for AFs) in our work were higher than those
227 228 229 230
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(0.02-0.09 µg/kg for AFs) reported in literatures [11, 14], but lower than those (0.10-0.35 µg/kg for AFs) in literature [13]. However, LOD of almost all mycotoxins (for example, 0.002-0.01µg/kg for AFs) were lower than those (0.01-0.08 µg/kg for AFs) in literatures [11, 13, 14].
3.2.2. Accuracy and precision
231
Recovery experiment was performed to evaluate the accuracy of method by standard addition.
232
Standards of mycotoxins were added to Puerariae lobatae radix at high, intermediate, and low
233
levels in six parallel. The results of recovery of mycotoxins were summarized in Table 6. The
234
recovery of AFB1 (56%) at intermediate level was a little out of range, but the good repeatability 11
Page 11 of 30
235
(RSD = 5.2%) could compensate for this to some extent. The sample solution spiked with intermediate concentration of mycotoxins was injected
237
repeatedly to evaluate instrument precision. Good injection precision was obtained with RSD of
238
peak area of mycotoxins less than 6.0% (n=6). Intra-day precision experiments were performed by
239
injecting six pretreated sample solutions spiked with intermediate concentration of mycotoxins
240
within a day. For the evaluation of inter-day precision of method, six sample solutions was
241
prepared and analyzed in six different days. The detailed precision results were listed in Table 7.
242
According to the requirement to analytical method for mycotoxins in COMMISSION
243
REGULATION (EC) NO 401/2006, the intra-day and inter-day precision (less than 16.9% and
244
31.8%, respectively) of the method were acceptable. The precision result was comparable to that
245
in literatures.
246
3.3. Method application
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The validated method was applied to determine the residues of mycotoxins in seventeen
248
batches of Puerariae lobatae radix. It was found that three Puerariae lobatae radix samples
249 250 251 252
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showed mycotoxin contamination. Batch no. 17 sample was detected with contamination of AFB1 at (0.751±0.176) μg/kg. Batch no. 6 and no. 9 were found with contamination of T-2 toxin at (1.10±0.01) μg/kg and (0.853±0.044) μg/kg, respectively. The MRM chromatograms of these
samples were shown in Fig. 4. The peaks of two mycotoxins in chromatograms of samples were
253
confirmed by comparing the retention times, the intensity ratios of two pairs of ions (m/z 315/287
254
and m/z 315/259 for AFB1, m/z 484/305 and m/z 484/215 for T-2), and spiking the standards to the
255
samples.
256
4. Conclusions 12
Page 12 of 30
An efficient and accurate LC–MS/MS method was developed for simultaneous determination
258
of seventeen mycotoxins in Puerariae lobatae radix. All analytes could be completely separated in
259
less than 35 min, providing good peaks shapes. Cleanup procedure was one of key points for
260
successful detection of mycotoxins in TCM. The effects of four different cleanup methods were
261
compared, and PuriToxSR M160 MultiPurification Column was chosen for better recovery and
262
repeatability for mycotoxins analysis. The established method was successfully employed to
263
analyze a total number of seventeen batches of Puerariae lobatae radix collected from different
264
provinces of China. And, three batches of samples were found with contamination of AFB1 or T-2
265
mycotoxins. The proposed method can be used to monitor the mycotoxins residues in Puerariae
266
lobatae radix.
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This
work
d was
(2009ZX09502-024).
te
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Acknowledgments
financially
supported
by
the
National
S&T
Major
Project
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268
References
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determination of fumonisins B1, B2 and B3 in traditional Chinese medicines by
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chromatography-tandem mass spectrometry method for simultaneous determination of aflatoxins
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B1, B2, G1, G2, M1 and M2 in traditional Chinese medicines, Anal. Chim. Acta 664 (2010)
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Deoxynivalenol and Nivalenol in Traditional Chinese Medicine by SPE and LC, Chromatographia
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[17] Y. Zhang, J. Caupert, P.M. Imerman, J.L. Richard, G.C. Shurson, The occurrence and
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[18] E. Razzazi-Fazeli, B. Rabus, B. Cecon, J. Bohm, Simultaneous quantification of
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A-trichothecene mycotoxins in grains using liquid chromatography-atmospheric pressure chemical
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ionisation mass spectrometry, J. Chromatogr. A 968 (2002) 129-142.
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[19] L. Vaclavik, M. Zachariasova, V. Hrbek, J. Hajslova, Analysis of multiple mycotoxins in
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[20] M.C. Spanjer, P.M. Rensen, J.M. Scholten, LC-MS/MS multi-method for mycotoxins after
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Page 16 of 30
338
Figure legends:
339
Fig. 1.
340
Fig. 2. MRM chromatograms of 17 mycotoxins in the standard solutions. The concentrations of
341
mycotoxins were all 1 μg/mL. Symbols: 1. 15-ADON; 2. 3-ADON; 3. AFG2; 4. AFB2; 5. AFG1;
342
6. DAS; 7. HT-2; 8. AFB1; 9. AOH; 10. α-ZAL; 11. β-ZAL; 12. β-ZEL; 13. α-ZEL; 14. T-2; 15.
343
ZAN; 16. AME; 17. ZEN.
us
cr
ip t
Effects of collision energy (CE) on the MS response of mycotoxins.
Fig. 3.
Comparison of extraction efficiency of mycotoxins with different extraction solvents.
345
Fig. 4.
MRM chromatograms of AFB1 and T-2 toxin in standard solution and the residues of
346
AFB1 and T-2 toxin detected in three batches of Puerariae lobatae radix.
an
344
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17
Page 17 of 30
Table 1 The requirement to analytical method for mycotoxins in COMMISSION REGULATION (EC) NO 401/2006.
Aflatoxins B1, B2, G1, G2
Precision
Concentration (µg/kg)
Recovery
10
80-110%
>100 - ≤500
60-110%
>500
70-120%
RSDR Recommended: as derived from Horwitz Equation* ; Maximum permitted: 2 × value derived from Horwitz Equation ≤40%
us
Deoxynivalenol ≤50 Zearalenone
60-130% 50-250 60-130%
M
>250 100-200 HT-2
0.66 × RSDR
≤20%
≤50%
≤40%
≤40%
≤25%
≤60%
≤40%
≤50%
≤30%
≤60%
≤40%
≤50%
≤30%
an
>50 T-2
RSDr
ip t
Mycotoxins
cr
347 348
60-130%
d
>200
The calculation of precision values from Horwitz equation is RSDR = 2
(1− 0.5log C )
*
350 351
is the relative standard deviation of the results generated under reproducibility conditions, C is the concentration ratio (i.e., 1 µg/kg=1×10-9).
353 354
Ac ce p
352
te
349
, where: RSDR
18
Page 18 of 30
354 355
Table 2
LC–MS/MS parameters of seventeen mycotoxins.
m/z Abbreviation
DP(V)
tR(min) precursor ion
product ion
CE(V)
15-ADON
12.4
339.3 [M+H]+
231.2*,213.2
40
20
3-Acetyldeoxynivalenol
3-ADON
12.7
339.3 [M+H]+
231.2*,213.2
40
20
Aflatoxin G2
AFG2
15.3
331.0 [M+H]+
313.0,245.4*
Aflatoxin B2
AFB2
16.7
315.1 [M+H]+
Aflatoxin G1
AFG1
16.8
329.0 [M+H]+
Diacetoxyscirpenol
DAS
16.8
HT-2 toxin
HT-2
17.8
Aflatoxin B1
AFB1
Alternariol
AOH
cr
ip t
15-Acetyldeoxynivalenol
an
Mycotoxins
m/z
45
287.2,259.1*
80
43
243.4*,311.0
80
37
us
80
307.3*,247.5
60
15
425.3 [M+H]+
263.0*,157.4
50
15
18.3
313.0 [M+H]+
285.1,269.2*
80
47
19.7
259.2 [M+H]+
185.3*,213.3
50
45
te
d
M
367.1 [M+H]+
α-ZAL
20.4
323.1 [M+H]+
305.3*,189.3
40
13
β-ZAL
23.7
323.1 [M+H]+
305.3*,163.2
40
13
β-Zearalenol
β-ZEL
20.8
321.3 [M+H]+
285.0*,189.0
40
17
α-Zearalenol
α-ZEL
24.5
321.3 [M+H]+
285.0*,189.0
40
17
T-2 toxin
T-2
26.6
484.2 [M+NH4]+
305.3,215.4*
50
23
Zearalanone
ZAN
31.1
321.3 [M+H]+
303.1*,189.0
40
20
AME
31.4
273.2 [M+H]+
230.0*,199.3
60
43
ZEN
31.6
319.1 [M+H]+
283.0*,301.2
40
13
α-Zearalanol
Ac ce p
α-Zearalanol
Alternariol monomethyl ether Zearalenone 356 357
*
For quantitative analysis.
19
Page 19 of 30
359 360 361
Table 3
Comparison of the recoveries (mean and RSD, %, n=3) of spiked mycotoxins in Puerariae lobatae radix by different clean-up methods.
QuEChERS
TC-M160
TC-T220
Mycosep 227
RSD
Recovery
RSD
Recovery
RSD
15-ADON
20.5
31.7
66.0
6.9
70.8
5.7
3-ADON
19.1
31.4
44.8
15.2
44.8
6.5
AFG2
56.3
27.8
79.5
4.4
88.9
AFB2
43.5
22.6
71.5
5.6
AFG1
49.9
21.6
80.3
5.4
DAS
51.9
21.0
93.5
HT-2
38.3
19.3
64.2
AFB1
42.1
24.6
59.4
AOH
43.3
20.2
α-ZAL
114.8
RSD 3.5
33.1
5.2
12.4
us
73.7
4.6
72.4
13.0
64.0
1.8
84.0
10.8
71.6
2.1
94.5
12.0
74.4
7.9
5.4
61.6
16.9
54.3
4.8
3.2
61.5
15.6
55.4
5.7
62.7
6.2
69.4
18.8
58.2
5.9
96.6
19.3
93.6
25.2
75.1
9.0
an
64.3
d
M
6.6
te
Ac ce p
26.1
Recovery
ip t
Recovery
cr
Mycotoxins
β-ZAL
57.6
22.9
84.7
7.4
90.5
16.3
80.3
9.1
β-ZEL
41.4
21.5
72.8
6.1
75.5
18.3
67.4
8.4
α-ZEL
45.8
24.5
76.3
4.0
72.1
16.3
63.2
7.9
T-2
52.2
19.9
78.3
8.8
85.3
18.8
73.9
5.6
ZAN
46.3
21.1
72.9
3.7
61.7
13.1
54.0
3.9
AME
54.1
19.4
76.9
5.8
66.3
16.2
60.1
7.6
ZEN
46.8
19.8
76.3
3.6
75.9
19.5
66.7
7.7
362 363
21
Page 20 of 30
363
Table 4
SSEs of mycotoxins in matrix of Puerariae lobatae radix.
SSE* (%)
Mycotoxins
15-ADON
58.8
α-ZAL
SSE* (%) 365 366 367 95.0
3-ADON
65.9
β-ZAL
87.9
368
AFG2
38.4
β-ZEL
94.4
369
AFB2
72.7
α-ZEL
92.7
370
AFG1
70.8
T-2
89.9
371
DAS
99.9
ZAN
89.8
372
HT-2
92.7
AME
91.5
373
AFB1
41.0
ZEN
89.3
374
AOH
87.5
375 376
te
d
M
an
us
cr
Mycotoxins
ip t
364
SSE =
378
Ac ce p
Peak area blank matrix × 100 % Peak area solvent
377
22
Page 21 of 30
378 379 380
Table 5 Linearity, limit of quantification (LOQ) and limit of detection (LOD) of seventeen mycotoxins.
Linearity range (μg/kg)
linearity equations
correlation coefficients (r)
LOQ
LOD
(μg/kg)
(μg/kg)
15-ADON
5.00-400
y=108243x-184.43
0.9950
2.50
0.537
3-ADON
4.99-401
y=402115x+11692
0.9919
2.50
0.242
AFG2
0.502-40.5
y=6143657x+57914
0.9962
0.0994
0.0994
AFB2
0.501-40.8
y=8937279x+278758
0.9939
0.0499
0.00499
AFG1
0.499-38.7
y=13953935x+460373
0.9916
DAS
4.99-401
y=403762x+59765
0.9962
HT-2
5.02-400
y=595428x+32120
AFB1
0.499-41.4
y=9294161x+158623
AOH
5.00-401
y=1401287x+199127
α-ZAL
2.49-202
y=4032287x+179121
β-ZAL
2.50-200
β-ZEL
0.00506
1.00
0.150
0.9974
1.00
0.176
0.9968
0.0488
0.00203
0.9945
0.998
0.214
0.9977
2.49
0.574
y=4664606x+57462
0.9974
2.50
0.417
2.49-200
y=8968281x+435292
0.494
0.0309
α-ZEL
2.49-199
y=4210924 x+81083
0.9983
0.825
0.206
T-2
5.01-400
M
0.9968
y=1491763x+160609
0.9966
0.488
0.0775
ZAN
2.49-200
y=20083677x+1156770
0.9959
0.617
0.0502
AME
4.98-400
y=129173x+10708
0.9970
4.97
1.06
ZEN
2.49-200
y=5292209x+227667
0.9963
0.488
0.183
an
te
Ac ce p
381 382
us
0.0506
d
cr
ip t
Mycotoxins
23
Page 22 of 30
382 383 384
Table 6 The results of recoveries with their RSDs (n = 6) of seventeen mycotoxins at high, intermediate, and low levels.
Recovery in
RSD
Recovery in
RSD
Recovery in
RSD
high level (%)
(%)
intermediate level (%)
(%)
low level (%)
(%)
15-ADON a
86.3
11.6
70.5
13.5
3-ADON a
84.5
7.5
77.1
16.6
AFG2 b
76.0
12.0
73.8
16.9
AFB2 b
90.3
10.9
78.1
AFG1 b
91.8
9.3
80.8
DAS a
115.2
7.1
103.0
HT-2 a
110.0
8.2
AFB1 b
83.5
11.3
AOH a
106.7
α-ZAL c
113.0
13.8
56.9
5.2
53.3
5.9 6.5
11.3
51.2
7.8
8.3
84.5
11.3
78.4
5.5
79.0
6.4
56.0
5.2
58.3
5.3
5.5
89.9
5.2
82.4
5.3
3.7
104.4
6.8
te
d
M
51.1
an
us
cr
62.0
9.8
95.8
β-ZAL c
104.6
6.5
88.3
5.5
77.3
7.3
β-ZEL c
101.7
7.4
93.2
9.6
77.2
4.8
α-ZEL c
112.9
6.6
91.2
4.4
85.7
3.1
T-2 a
99.8
10.1
92.6
3.3
86.2
12.4
ZAN c
101.6
4.5
87.0
4.3
82.6
3.1
AME a
103.0
6.0
88.3
5.8
87.8
2.8
ZEN c
103.5
5.2
90.1
4.3
84.0
7.4
6.3
Ac ce p 385 386 387 388
ip t
Mycotoxins
Notes: a 240, 50 and 10 μg/kg for 3-ADON, 15-ADON, DAS, AOH, AME, T-2 and HT-2 toxin; b 24, 5 and 1 μg/kg for AFB1, AFB2, AFG1 and AFG2; c 120, 25 and 5μg/kg for ZAN, ZEN, α-ZAL, β-ZAL, α-ZEL and β-ZEL 24
Page 23 of 30
The results of precision (n = 6) of seventeen mycotoxins in the optimum conditions.
Interday precision (RSD, %)
4.8
13.5
AFG2
4.4
16.9
3-ADON
4.7
16.6
AFB2
2.7
9.8
AFG1
2.7
11.3
DAS
4.3
8.3
HT-2
4.6
AFB1
2.4
AOH
2.1
α-ZAL
1.4
31.8 22.9
21.8
24.4
an
15-ADON
Intraday precision (RSD, %)
ip t
Precision (RSD, %)
cr
Mycotoxins
us
Table 7
6.4 11.8 15.6
5.2
8.7
5.2
14.3
3.7
9.1
2.6
5.5
14.0
2.8
9.6
13.4
α-ZEL
3.5
4.4
11.8
T-2
6.0
3.3
11.7
ZAN
4.5
4.3
9.2
AME
3.7
5.8
9.1
ZEN
4.9
4.3
8.6
β-ZAL
d
Ac ce p
β-ZEL
M
5.5
te
388 389
390 391
25
Page 24 of 30
391
Highlights A LC–MS/MS method for determination of seventeen mycotoxins was developed.
393
Four different clean-up approaches were investigated and compared.
394
The method was used to determine mycotoxins residues in Puerariae lobatae radix.
395
Three batches of TCM were found with contamination of AFB1 or T-2 mycotoxins.
ip t
392
396
Ac ce p
te
d
M
an
us
cr
397
26
Page 25 of 30
Ac
ce
pt
ed
M
an
us
cr
i
*Graphical Abstract
Page 26 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 1
Page 27 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 2
Page 28 of 30
Ac
ce
pt
ed
M
an
us
cr
i
Figure 3
Page 29 of 30
Ac ce p
te
d
M
an
us
cr
ip t
Figure 4
Page 30 of 30
