Journal of Chromatography A, 1345 (2014) 107–114

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Multi-mycotoxin analysis in dairy products by liquid chromatography coupled to quadrupole orbitrap mass spectrometry Wei Jia a,b , Xiaogang Chu a,b,∗ , Yun Ling b , Junrong Huang a , James Chang c a b c

College of Chemistry & Chemical Engineering, Shaanxi University of Science & Technology, Xi’an 710021, China Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134,USA

a r t i c l e

i n f o

Article history: Received 17 February 2014 Received in revised form 7 April 2014 Accepted 8 April 2014 Available online 15 April 2014 Keywords: QuEChERS Q-Orbitrap Dairy products Mycotoxins

a b s t r a c t A new analytical method was developed and validated for simultaneous analysis of 58 mycotoxins in dairy products. Response surface methodology was employed to optimize a quick, easy, cheap, effective, rugged, and safe (QuEChERS) sample preparation method. Ultrahigh-performance liquid chromatography and electrospray ionization quadrupole Orbitrap high-resolution mass spectrometry (UHPLC/ESI Q-Orbitrap) was used for the separation and detection of all the analytes. The method was validated by taking into consideration the guidelines specified in Commission Decision 2002/657/EC and 401/2006/EC. The extraction recoveries were in a range of 86.6–113.7%, with coefficient of variation 0.99. The limits of detection for the analytes are in the range 0.001–0.92 ␮g kg−1 . The repeatability was lower than 6.4%. This method has been successfully applied on screening of mycotoxins in commercial dairy product samples, and it is very useful for fast screening of different food contaminants. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Mycotoxins are secondary metabolites produced by many species of fungi. Mycotoxins have a wide range of adverse effects such as carcinogenic, mutagenic, estrogenic, nephrotoxic, hepatotoxic, neurotoxic and immunosuppressive effects, and hence may lead to great economic losses to farm husbandry [1–3]. In addition to mycotoxicoses which is caused by direct consumption of contaminated food and feed, the effect of “carry over” of mycotoxins and their metabolites into milk and animal tissues should not be neglected [4–6]. To avoid the risk of mycotoxin ingestion and intoxication, agencies around the world have established acceptable limits for aflatoxin concentration in milk and feeds. In the United States, the Food and Drug Administration (FDA) stipulated action levels for aflatoxin M1 in raw milk and lactating cow feeds are 0.5 and 20 ␮g kg−1 , respectively. The maximum allowable concentration set by the European Commission is 0.05 ␮g kg−1 of milk [7,8]. Moreover, epidemiological investigations in cattle have demonstrated

∗ Corresponding author at: Institute of Food Safety, Chinese Academy of Inspection and Quarantine, Beijing 100123, China. Tel.: +86 010 85778904; fax: +86 010 857707750. E-mail address: [email protected] (X. Chu). http://dx.doi.org/10.1016/j.chroma.2014.04.021 0021-9673/© 2014 Elsevier B.V. All rights reserved.

the interest of mycotoxin quantification in milk, when estimating feed contamination [9]. In addition, the assessment of mycotoxins in the mammary secretion could be of interest for studies in distribution, biotransformation and bioavailability [10]. For monitoring purposes, broad range analytical methods are needed to reduce analytical costs and allow for a more frequent monitoring of mycotoxins in dairy products. Then, appropriate methods based on simultaneous extraction procedures are highly required for the mycotoxins determination that can occur in foodstuffs [11–13]. Currently, more than 400 mycotoxins have been identified in the world and most of them can be categorized into Aflatoxins (e.g. aflatoxin B1, aflatoxin M1), Trichothecenes (e.g. HT-2 toxin, diacetoxyscrirpenol), Fumonisins (e.g. fumonisin B1, fumonisin B2), Ochratoxins (e.g. ochratoxin A, ochratoxin B), Ergot alkaloids (e.g. ergocorninine, ergocristinine), Zearalenone (e.g. zearalenone, zearalanone) and Alternaria toxins (e.g. alternariol, tenuazonic acid) [14–18]. Mycotoxins exhibit a wide range of different physicochemical properties in terms of solubility, pH stability, diversity of chemical structure and molecular weight. Therefore, they have a significant influence on extraction, separation and ionization techniques [19–21]. For the detection and quantification of mycotoxins, chromatographic techniques like high performance liquid chromatography (HPLC) with fluorescence detection (FLD) [22], gas chromatography (GC) and liquid chromatography (LC) coupled to mass spectrometry (MS) can be used [23,24]. LC–MS is a

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suitable technique for the analysis of polar substances like mycotoxins because no derivatization step is required as in GC–MS [25]. In this sense, LC–tandem quadrupole MS has been widely accepted as the main tool in the structural characterization, identification, and quantitative analysis of multi-mycotoxin owing to its efficiency, superior sensitivity and specificity [26]. However, this method is not suitable for simultaneous screening a large number of mycotoxins; in such targeted analyses, signals from all other compounds are ignored and the multiple isomers and structural analogs of analytes are difficult to separate [27]. From last year the role of UHPLC/ESI Q-Orbitrap and related techniques is increasingly built up as enabling tool in food safety analysis for it can provide detailed structural information. In spite of the potential value of the application, to the best of our knowledge, so far no one has reported the application of Q-Orbitrap mass spectrometry combined with high performance liquid chromatography for simultaneous determination for a group of mycotoxins in foods [28]. The analysis of mycotoxins in dairy products is a difficult task because these are complex matrix, and the chromatographic analysis requires the application of previous extraction and clean-up steps in order to remove lipids and proteins. A wide variety of sample preparation has been reported in literature for mycotoxins, such as liquid–liquid extraction (LLE), solid-phase extraction (SPE) and dilute-and-shoot (DAS) [29–31]. However, some of these methods still have some limitation, such as high variability in results and high requirement for clean sample, which make them inadequate for routine analyses. In addition, for multi-mycotoxin analysis, with LLE, SPE and DAS, it is difficult to obtain satisfactory recoveries for all compounds in one step. Hence, new straightforward approaches involving simpler and fewer steps would be welcome for a more effective clean-up of complex matrices such as dairy products samples. In this way, QuEChERS has been checked elsewhere for the extraction of pesticide and veterinary drug residues in food and feed, but to date, no work focused on the determination of mycotoxins in dairy products using QuEChERS has been published [32]. In this paper, we describe the development of a cost-effective, time-efficient and easy-to-use sample preparation method based on QuEChERS for the simultaneous extraction of the 58 most important mycotoxins from dairy products. Coupled with an optimized UHPLC/ESI Q-Orbitrap method, this method was successfully applied on screening of mycotoxins in dairy products samples from local market.

2. Experimental 2.1. Chemicals and reagents Acetic acid, formic acid (FAc), ammonium formate, sodium acetate, sodium chloride and anhydrous magnesium sulfate (MgSO4 ) were purchased from Sigma–Aldrich (Steinheim, Germany). HPLC-grade acetonitrile (MeCN) and methanol (MeOH) were sourced from J.T. Baker (Deventer, Holland). BAKERBOND® octadecyl (C18 ), bondesil primary secondary amine (PSA), and ceramic homogenizers obtained from Agilent Technologies (Harbor City, USA). Ultrafree-MC centrifugal filter devices (0.22 ␮m) of Millipore (Millipore, Brussels, Belgium) were used. Trifluoroacetic acid was obtained from Fluka (Buch, Switzerland). Ultrapure Water (resistivity, 18.2 M) was purified on a Milli-Q Plus apparatus (Millipore, Brussels, Belgium). Locally purchased milk, milk beverages and yogurt samples found to contain no response at the retention times of reference compounds or metabolite were selected for use as negative controls and stored at 4 ◦ C prior to analysis. Standards of Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2, Aflatoxin M1, Citrinin, Kojic acid, 15-Acetoxyscirpenol,

15-Acetyldeoxynivalenol, 3-Acetyldeoxynivalenol, Ergocornine, Ergocorninine, Ergocristine, Ergocristinine, Ergocryptinine and Ergosine were obtained from Fluka (Buchs, Switzerland). Reference srtandards of Agroclavine, Altermariolmethylether, Alternariol, Citreoviridin, Deepoxy-deoxynivalenol, Deoxynivalenol, Deoxynivalenol 3-glucoside, Diacetoxyscrirpenol, Dihydroergocristine, Fumagillin, Fumonisin B1, Fumonisin B2, Fumonisin B3, Fusarenon X, Gliotoxin, HT-2 toxin, Moniliformin, Mycophenolic acid, Neosolaniol, Nivalenol, Ochratoxin A, Ochratoxin B, Ochratoxin-␣, Patulin, Paxilline, Penicilic acid, Roquefortine C, Stachybotrylactam, Sterigmatocystin, Tentoxin, Trichothecenes, Zearalanone, Zearalenone, ␣-Zearalanol, ␣-Zearalenol, ␤-Zearalanol and ␤Zearalenol by Sigma–Aldrich (Steinheim, Germany) and Meleagrin, T-2 tetraol, T-2 triol, Verruculogen and Wortmannin were purchased from Dr. Ehrenstorfer (Augsburg, Germany). All reference compounds have a purified of >98.0%. Stock solutions of individual compounds were prepared in MeCN (1 mg mL−1 ) and stored at −20 ◦ C in the dark. Then, a multicompound working standard solution at a concentration of 1 mg L−1 of each compound was prepared by combining suitable aliquots of each individual standard stock solution and diluting them with appropriate amounts of MeCN and stored in screwcapped glass tubes at −20 ◦ C in the dark. 2.2. Analytical procedure 2.2.1. Sample preparation 15 g of each sample was weighed in polypropylene centrifuge tubes (50 mL). Method matrix fortified calibration curve stands were prepared by adding the analytes to blank samples at a concentration range of 0.001–100 ␮g kg−1 . 10 mL volume of a MeCN/water solution (84/16, v/v) with 1% acetic acid was added as an extraction solvent and the tube was tightly capped and vigorously mixed for 1 min using a vortex (Scientific Industries, New York, USA) mixer at maximum speed. MgSO4 (6 g), sodium acetate anhydrous (1.45 g) and ceramic homogenizers were added to the tube, to induce phase separation. After that, the tube was immediately shaken for 1 min, and then centrifuged for 5 min at 4000 rcf at 4 ◦ C (Beckman Couler, Brea, USA). Then the upper layer (8 mL) was submitted to a dispersive SPE clean up with a mixture of 1.2 g of MgSO4 , 108 mg of PSA and 405 mg of C18 . The tube was vortexed for 1 min and centrifuged for 5 min at 4000 rcf at 4 ◦ C. An aliquot of the final upper layer (200 ␮L) was transferred into a Mini-UniPrep vial, 300 ␮L MeOH and 500 ␮L 8 mM ammonium formate buffer were added. After the vial was capped, vortexed for 30 s. 1 mL of the sample extract was taken and filtered through a Millex-GN nylon filter (0.22 ␮m, Pall Corporation, Harbor, USA). The cleaned extract was collected in a vial for injection into the UHPLC/ESI Q-Orbitrap system. 2.2.2. Experimental design for response surface methodology (RSM) Response surface methodology (RSM) was employed to investigate the variations in recovery rates with respect to the preparation of conditions including extraction solvent volume, the amounts of Sodium Acetate, PSA, and C18 . The optimal composition of the 4 variables was determined by using a central composite design (CCD) approach. In this work, the full CCD consisted of (1) a complete two-factorial design; (2) n0 , center point (n0 > 1), and (3) two axial points on the axis of each design variable at a distance of ˛ = 2.000 from the design center. Hence, a total number of design points of N = 2k + 2k + n0 was used. The actual variable was coded to facilitate multiple regression analysis. The complete design consisted of 30 combinations including seven replicates of the center point with five degrees of freedom for calculation of errors in the experiments. The optimal values of response Y (individual recovery of interest compounds) were obtained by solving

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Table 1 Variables and levels evaluated in the central composite design to optimize the extraction conditions. Independent variable

Unit

Symbol

Extraction solvent volume Na Acetate quantity PSA quantity C18 quantity

mL g mg mg

X1 X2 X3 X4

the regression equation and by analyzing the response surface contour plots. Table 1 indicates the coded and CCD-processed variables for the optimization of the QuEChERS method for samples. The resulting 30 experiments were carried out randomly. The goodness of fit of the regression model and the significance of parameter estimates were determined through appropriate statistical methods. Design Expert trial version 8.0.6.0 was used (Stat-Ease Inc., Minneapolis, MN). 2.2.3. Instruments and analytical conditions The UHPLC/ESI Q-Orbitrap system consisted of an Accela 1250 LC pump and a CTC Analytics PAL open autosampler coupled with a Q Exactive mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). The system was controlled by Exactive Tune 1.1 and Xcalibur 2.2 software (Thermo Fisher Scientific, San Jose, USA). The analytical column used was a 100 mm × 2.1 mm, i.d., 2.6 ␮m, Thermo Accucore C-18 aQ connected to a 10 mm × 2.1 mm, Accucore C-18 aQ guard column (Thermo Fisher Scientific, San Jose, USA). A mobile phase consisting of eluent A (water, 0.1% FAc, 4 mM ammonium formate) and eluent B (MeOH, 0.1% FAc, 4 mM ammonium formate) was used at a flow rate of 0.3 mL min−1 . The following gradient was used: 0 min, 100% A; 1 min, 100% A; 7 min, 0% A; 12 min, 0% A; 13 min, 100% A; until the end of the run at 15 min. The sample injection volume was set at 5 ␮L. All the fiftyeight analytes eluting over 0–9 min while the last 6 min were used for column cleaning and re-equilibration. The mass spectrometer Q-Orbitrap was equipped with a Heated Electrospray Ionization (HESI) source. The optimized HESI temperature was set at 350 ◦ C, the capillary temperature at 320 ◦ C, the electrospray voltage at 3.5 kV and 3.0 kV for positive and negative modes, respectively. Sheath and auxiliary gas were 18 and 3 L min−1 . All quantitative data in this study were acquired using full MS scan mode. In full MS/dd-MS2 (TopN), which is used for confirmatory purpose, the Q-Orbitrap performs data-dependent scans. As long as the targeted compounds were detected, precursor ions that were selected by the quadrupole were sent to the HCD collision cell of the Q-Orbitrap mass spectrometer. Here, they were fragmented with normalized collision energy (NCE) to obtain product ion spectra. At this stage, the mass resolution was set at 17,500 FWHM (m/z 200) and NCE 35%. Using this strategy, coeluting matrix compounds from the matrix or noisy peaks can be easily excluded, facilitating the identification and quantification of known or unknown analytes in a single run analysis. 3. Results and discussion 3.1. Optimization of the LC-Q-Orbitrap conditions Chromatographic conditions were studied in order to achieve the best separation and retention for the compounds. First, several experiments were performed on different mobile phases consisting of MeCN or MeOH as organic phase and water as polar phase with different concentrations of acetic and formic acid (from 0.05 to 0.5%), ammonium formate, ammonium acetate (from 1 mM to 10 mM). MeOH was chosen as the organic phase because it achieved a better resolution and sensitivity than MeCN. On the other hand,

Coded levels −˛

−1

0

+1

+˛

2 0.5 0 0

6 1.0 50 200

10 1.5 100 400

14 2.0 150 600

18 2.5 200 800

the addition of FAc-ammonium formate provided better results than acetic acid-ammonium acetate and it was used to improve the ionization efficiency and achieve better chromatography. Moreover, the addition of FAc also helped in the separation of the Zearalenone isomers (␣-Zearalanol and ␤-Zearalanol, ␣-Zearalenol and ␤-Zearalenol), without which, these isomers overlapped and could not be quantified separately. Finally, the best results were obtained when MeOH was used as organic modifier and aqueous solution of FAc (0.1%) -ammonium formate (4 mM) was employed. We evaluated various HPLC columns (Thermo Scientific Accucore: aQ C18 , RP-MS, C18 , PFP, Phenyl-Hexyl) in order to optimize the chromatographic separation of the above mentioned fifty-eight analytes and the a Q C18 column yielded the best results. Several gradient profiles were studied, obtaining good response with the gradient described in Section 2. Under these conditions retention times of the analytes were constant, ranging from 0.75 (Moniliformin) to 8.17 (Paxilline) min. The optimum mass spectrometric parameters for the identification and quantification of the fifty-eight analytes were first obtained after analyzing the compounds by flow injection analysis respectively. Sensitivity of target analytes was checked by recording chromatograms in full scan method in both positive and negative ionization mode. Due to adduct formation with FAc/ammonium formate buffer, some analytes exhibit strong formic or ammonium adduct species ([M+FAc]− or [M+NH4 ]+ ) which appear to be the most predominant ions in the mass spectrum. Full MS scan mode allowed for screening and quantifying the analytes or retrospectively looking into unknowns, such as fragment identification. When operated in full MS/dd-MS2 mode, a product ion spectrum with accurate mass measurement is obtained automatically according to inclusion list (a list of targeted accurate masses), and this defined as a data dependent acquisition (dd-MS2 ). After full scan analysis, specific mass windows were extracted to screen the data for the presence of analytes. The effect of the mass extraction window on selectivity for analytes in milk, milk beverages and yogurt products at a concentration 1 ␮g kg−1 (2–10 ppm mass deviations) was tested. The best results were obtained when mass extraction windows of 2.5 ppm were employed. Table 2 summarizes the optimal parameters of the UHPLC/ESI Q-Orbitrap.

3.2. Optimisation of the extraction procedure Despite QuEChERS uses MeCN acidified with acetic acid, and bearing in mind that conventional extraction procedures of mycotoxins from different samples use a mixture of MeCN/water or MeOH/water, the extraction solvent was evaluated first, using the extraction procedure described in Section 2.3, except that the type of solvent was varied. Thus, different solvents such a mixture of MeCN/water (84:16, v/v), a mixture of MeCN/water (80:20, v/v), and a mixture of MeOH/water (84:16, v/v) were checked. The obtained results are shown in Fig. 1. It can be observed that the best results were obtained when a mixture of MeCN/water (84:16, v/v) was used, increasing the recovery of some compounds such as 3-acetyldeoxynivalenol, ergocorninine, and ochratoxin A. MeCN/water (84:16, v/v) was chosen as the extraction solvent

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Table 2 UHPLC/ESI Q-Orbitrap parameters of the 58 additional analytes. Peak

Compound

RT (min)

Elemental composition

Ionization mode

Theoretical (m/z)

Measured (m/z)

Accuracy (ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Moniliformin Patulin Kojic acid T-2 tetraol Nivalenol Deoxynivalenol Deoxynivalenol 3-glucoside Fusarenon X Deepoxy-deoxynivalenol Penicilic acid Neosolaniol 3-Acetyldeoxynivalenol 15-Acetyldeoxynivalenol Agroclavine Ochratoxin-␣ Aflatoxin M1 Aflatoxin G2 15-Acetoxyscirpenol Aflatoxin G1 Aflatoxin B2 Gliotoxin Diacetoxyscrirpenol Aflatoxin B1 Ergosine Wortmannin T-2 triol Citrinin Meleagrin Ergocorninine Ergocornine HT-2 toxin Dihydroergocristine Ergocryptinine ␤-Zearalanol Tentoxin Ochratoxin B Ergocristine Roquefortine C Mycophenolic acid Ergocristinine Fumonisin B1 ␤-Zearalenol Alternariol Trichothecenes ␣-Zearalanol Fumonisin B3 ␣-Zearalenol Ochratoxin A Citreoviridin Zearalanone Zearalenone Fumonisin B2 Sterigmatocystin Verruculogen Stachybotrylactam Altermariolmethylether Fumagillin Paxilline

0.75 0.85 1.62 3.81 3.84 4.32 4.37 4.84 4.89 4.89 5.00 5.35 5.35 5.47 5.75 5.75 5.76 5.78 5.91 6.08 6.20 6.20 6.25 6.31 6.31 6.33 6.34 6.41 6.63 6.65 6.69 6.75 6.82 6.85 6.85 6.89 6.89 6.90 6.92 6.93 6.96 7.01 7.01 7.01 7.18 7.21 7.27 7.28 7.32 7.34 7.37 7.44 7.58 7.60 7.71 7.75 7.76 8.17

C4 HNaO3 C7 H6 O4 C6 H6 O4 C15 H22 O6 C15 H20 O7 C15 H20 O6 C21 H30 O11 C17 H22 O8 C15 H20 O5 C8 H10 O4 C19 H26 O8 C17 H22 O7 C17 H22 O7 C16 H18 N2 C11 H9 O5 Cl C17 H12 O7 C17 H14 O7 C17 H24 O6 C17 H12 O7 C17 H14 O6 C13 H14 O4 N2 S2 C19 H26 O7 C17 H12 O6 C30 H37 N5 O5 C23 H24 O8 C20 H30 O7 C13 H14 O5 C23 H23 N5 O4 C31 H39 N5 O5 C31 H39 N5 O5 C22 H32 O8 C35 H41 N5 O5 C32 H41 N5 O5 C18 H26 O5 C22 H30 O4 N4 C20 H19 NO6 C35 H39 N5 O5 C22 H23 N5 O2 C17 H20 O6 C35 H39 N5 O5 C34 H59 NO15 C18 H24 O5 C14 H10 O5 C24 H34 O9 C18 H26 O5 C34 H59 NO14 C18 H24 O5 C20 H18 NO6 Cl C23 H30 O6 C18 H24 O5 C18 H22 O5 C34 H59 NO14 C18 H12 O6 C27 H33 O7 N3 C23 H31 NO4 C15 H12 O5 C26 H34 O7 C27 H33 O4 N

[M−Na]− [M+NH4 ]+ [M+H]+ [M+NH4 ]+ [M+FAc]− [M+H]+ [M+FAc]− [M+NH4 ]+ [M+H]+ [M+H]+ [M+NH4 ]+ [M+H]+ [M+NH4 ]+ [M+H]+ [M−H]− [M+H]+ [M+H]+ [M+NH4 ]+ [M+H]+ [M+H]+ [M+H]+ [M+NH4 ]+ [M+H]+ [M+H]+ [M+NH4 ]+ [M+NH4 ]+ [M−H]− [M−H]− [M−H]− [M+H]+ [M+NH4 ]+ [M+H]+ [M+H]+ [M+H]+ [M-H]− [M+H]+ [M+H]+ [M+H]+ [M−H]− [M+H]+ [M+H]+ [M+H]+ [M-H]− [M+NH4 ]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M+H]+ [M−H]− [M−H]− [M+H]+

96.99312 172.06043 143.03389 316.17546 357.11910 297.13326 503.17701 372.16529 281.13835 171.06519 400.19659 339.14383 356.17038 239.15428 255.00657 329.06558 331.08123 342.19111 329.06558 315.08631 327.04677 384.20168 313.07066 548.28675 446.18094 400.23298 249.07685 432.16772 560.28784 562.30240 442.24354 612.31805 576.31805 323.18530 413.21942 370.12851 610.30240 390.19245 319.11871 610.30240 722.39575 321.16965 257.04555 484.25411 323.18530 706.40083 321.16965 404.08954 403.21152 321.16965 319.15400 706.40083 325.07066 512.23913 386.23259 271.06120 457.22318 436.24824

96.99314 172.06001 143.03362 316.17493 357.11938 297.13293 503.17725 372.16476 281.13776 171.06477 400.19586 339.14349 356.16992 239.15399 255.00673 329.06491 331.08069 342.19031 329.06494 315.08569 327.04596 384.20102 313.06989 548.2865 446.17993 400.23264 249.07689 432.16791 560.28802 562.30206 442.24268 612.31781 576.31769 323.18497 413.2196 370.12775 610.30219 390.19189 319.1185 610.30249 722.39465 321.16931 257.04572 484.25381 323.18536 706.40039 321.16934 404.08893 403.21100 321.16922 319.15332 706.39954 325.07001 512.23828 386.23169 271.06152 457.22372 436.24744

0.21 2.44 1.89 1.68 0.78 1.11 0.48 1.42 2.10 2.46 1.82 1.00 1.29 1.21 0.61 2.04 1.63 2.34 1.94 1.97 2.48 1.72 2.46 0.46 2.26 0.85 0.17 0.42 0.32 0.60 1.94 0.39 0.62 1.02 0.41 2.05 0.34 1.44 0.66 0.15 1.52 1.06 0.67 0.62 0.19 0.62 0.97 1.51 1.29 1.34 2.13 1.83 2.00 1.66 2.33 1.19 1.19 1.83

because it good miscibility with water, and its ability to recover the analytes without extracting high quantities of lipophilic material. Critical factors which could have an influence on the analyte recovery and method sensitivity were investigated by a central composite design (CCD). In general, higher amount of extraction solvent leads to a higher analytical signal in almost all analytes, but there was no preliminary study performed testing different ratios of mass (g) of sample per volume (mL) of extraction solvent. Compared with those from a non-buffered QuEChERS method, the acetate buffered method improved stabilities and recoveries of certain pH-dependent compounds (e.g. patulin and zearalenone). Sodium acetate also has a dissolving effect on milk protein and fat globules, which could affect recovery rates. The PSA and C18

sorbent, as a dispersive medium, are used to retain those nondesirable components (organic acids, polar pigments, sugars, fatty acids, and lipid matrix) co-extracted from the matrix while target compounds remain in solution. Therefore, the factors included (1) volume of extraction solvent, (2) amount of sodium acetate, (3) amount of PSA, (4) amount of C18 . In order to verify if the models generated and explained a significant part of the variations in the experimental data, the F-test for significance was carried out. It can conclude that the model was well-fitted to the observations and the regression was statistically significant at the 99% confidence level. Among the linear, quadratic, and cross-product forms of the independent variables, the coefficient constant, X1 , X2 , X3 , X4 , X2 × X3 , X12 , X22 ,

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111

Fig. 1. Effect of type of solvent on the extraction recovery (n = 7 batches) of mycotoxins in milk.

X32 , and X42 were significant at the level of p < 0.05. The Model F-value of 2198.3 implies the model is significant. The “Lack of Fit F-value” of 0.215 implies the Lack of Fit is not significant relative to the pure error. Therefore, after the response recovery rates were experimentally determined under the 30 sets of conditions, the regression coefficients for the recoveries were calculated and a polynomial regression model equation was fitted as follow: Y = 99.12 + 7.82X1 + 0.52X2 + 6.40X3 + 7.73X4 + 0.15X1 × X2 − 0.15X1 × X3 − 0.02X1 × X4 − 0.85X2 × X3 + 0.27X2 × X4 + 0.23X3 × X4 − 9.42X12 − 17.42X22 − 13.80X32 − 10.92X42 . The individual recoveries of all analytes were introduced separately as the response in the statistical program. Fig. 2 shows the different response plots for zearalenone for different combinations of the parameters investigated. The optimum conditions were chosen by taking into consideration data obtained from the response surface pots and the regression coefficient plots. Therefore, the response surfaces generated suggest that the best extraction conditions for zearalenone were extraction volume 10 mL, sodium acetate 1.45 g, PSA 108 mg, C18 405 mg. While the optimized regression equations had been obtained and the predicted maximum recoveries of fifty-eight target analytes calculated, the simultaneous extraction of fifty-eight different mycotoxins with different properties from a complex matrix such as milk, milk beverages and yogurt requires a compromise between each individual extraction optimum conditions to perform simultaneous analysis of multiple compounds with a single pretreatment. For this reason, multi-mycotoxin methods for recovery were tested under the conditions optimized for zearalenone, which gave the highest observed recovery in the single recovery tests and were therefore used to test the multiresidual compound quantification. The results of the multiresidue methods recovery test and validation

information for each analyte are shown in Table 3. In summary, the resulting conditions allowed reliable simultaneous analysis of fifty-eight of the target analytes with recoveries in the range of 86.6–113.7%. 3.3. Validation of the proposed method Method validation was performed in terms of matrix-effect, specificity, linearity, trueness, precision, CC␣ and CC␤. Commission Decision 2002/657/EC and 401/2006/EC were used as guidelines for the validation studies. The matrix effect was tested by comparing the slopes of the matrix-free calibration curves to the matrix-matched calibration curves. Matrix effect was investigated by calculating the percentage (C%) of signal enhancement or suppression, according to Eq. (1). C% =



1−

ss sm



× 100

(1)

where ss is the slope of calibration plot with matrix-matched calibration solutions and sm is the slope of calibration plot with calibration solutions in solvent. In the fifty-eight analytes, fifty-five did not display significant changes in the slopes between standard calibration curves and matrix-matched calibration curves (−50% < C% < +50%). The remaining three compounds that had significant matrix effect were namely fumonisin B3, aflatoxin G1 and 3-acetyldeoxynivalenol (C% < −50% or C% > +50%). Apparently, QuEChERS was as effective for mycotoxins extraction and sample cleanup for milk, milk beverages and yogurt products. It is interesting to notice that the matrix effects from different types of dairy products showed a similar ion enhancement or suppression profile (Fig. 3). Therefore, any

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Fig. 2. Response surface plot for zearalenone.

dairy product may be chosen as a standard matrix to construct matrix-matched calibration curves for quantification in routine practice. Extraction recoveries were assessed by spiking blank milk, milk beverages and yogurt samples before and after extraction at three concentration levels (CC␤, 2 CC␤, 4 CC␤) with five replicates at each level. Linearity was assessed with spiked blank matrix at four concentration levels ranging between 0.001 and 100 ␮g kg−1 . For all analytes, r2 was greater than 0.99 and the deviation of each point from the calibration line was lower than 15%. Specificity was assessed by verifying the presence of interference at the retention time of analytes greater than a signal-to-noise ratio of three. Within-laboratory reproducibility was assessed by spiking blank milk, milk beverages and yogurt samples at four different concentrations on nine defferent days. Trueness was calculated as the percentage of error between spiked and found concentrations. CC␣ was estimated from the calibration curve prepared by spiking blank milk, milk beverages and yogurt matrices at four

concentration levels in the low concentration range. CC␣ is calculated as the concentration corresponding to the y-intercept plus 2.33 times its standard deviation. In the case of CC␤, the concentration corresponds to CC␣ + 1.64s, s being the standard deviation obtained at the CC␣ level. CC␣ ranged between 0.006 ␮g kg−1 and 0.55 ␮g kg−1 , and CC␤ ranged between 0.001 ␮g kg−1 and 0.92 ␮g kg−1 . The results of this validation are summarized in Table 3. Three consecutive injections on-column at 1 ␮g kg−1 were carried out to detect any decrease in mass accuracy. No significant decrease was observed and the maximum mass deviation ranged from 0.2 to 2.5 ppm. This minor deviation in mass accuracy demonstrated the wide dynamic range of Q-Orbitrap for qualitative analysis at a resolving power of 70,000 FWHM. The CC␣ values of the proposed Q-Orbitrap mass spectrometer method are comparable to the limits of the previously reported triple quadrupole mass spectrometer methods. Comparing with the detection limits reported in literatures [33–36], the detection sensitivity and accuracy was improved more than 5 times.

Fig. 3. UHPLC/ESI Q-Orbitrap matrix effects of 3 dairy products. The evaluation was done at 1 ␮g kg−1 equivalent in samples.

W. Jia et al. / J. Chromatogr. A 1345 (2014) 107–114

113

Table 3 Validation parameters of the developed method for milk sample. Compound

Moniliformin Patulin Kojic acid T-2 tetraol Nivalenol Deoxynivalenol Deoxynivalenol 3-glucoside Fusarenon X Deepoxy-deoxynivalenol Penicilic acid Neosolaniol 3-Acetyldeoxynivalenol 15-Acetyldeoxynivalenol Agroclavine Ochratoxin-␣ Aflatoxin M1 Aflatoxin G2 15-Acetoxyscirpenol Aflatoxin G1 Aflatoxin B2 Gliotoxin Diacetoxyscrirpenol Aflatoxin B1 Ergosine Wortmannin T-2 triol Citrinin Meleagrin Ergocorninine Ergocornine HT-2 toxin Dihydroergocristine Ergocryptinine ␤-Zearalanol Tentoxin Ochratoxin B Ergocristine Roquefortine C Mycophenolic acid Ergocristinine Fumonisin B1 ␤-Zearalenol Alternariol Trichothecenes ␣-Zearalanol Fumonisin B3 ␣-Zearalenol Ochratoxin A Citreoviridin Zearalanone Zearalenone Fumonisin B2 Sterigmatocystin Verruculogen Stachybotrylactam Altermariolmethylether Fumagillin Paxilline a b

Matrix effect C (%)

10.8 4.8 −15.0 15.4 −13.8 −17.1 −15.2 9.0 −11.2 15.9 1.4 −50.7 −19.9 10.8 −14.7 −13.0 −17.0 12.2 −51.4 −13.6 3.0 14.3 −14.5 −11.9 −15.4 16.8 −10.0 10.6 −16.9 −19.3 6.4 13.8 −19.3 −19.6 −15.6 −14.0 −21.7 8.3 15.2 −15.1 19.5 −15.6 17.0 18.9 −16.1 52.2 −17.9 −14.5 14.5 14.3 19.0 19.6 11.7 20.7 11.3 9.2 23.9 18.4

Extraction Recoveries%a

89.3 90.2 95.8 93.2 91.5 93.6 97.5 97.9 96.0 98.2 95.4 97.0 87.4 90.3 95.2 92.3 90.3 95.8 87.3 90.4 95.0 106.6 91.2 99.9 89.4 89.3 103.2 95.6 103.9 92.3 104.2 87.5 113.7 98.2 95.7 98.5 94.3 90.2 99.2 107.7 86.6 102.4 95.2 89.0 101.5 90.3 99.7 90.6 95.2 95.1 91.0 99.8 95.2 91.3 90.2 87.1 89.7 94.5

Calibration equation

Dynamic range

CC␣

CC␤

(y=)

(␮g kg−1 )

(␮g kg−1 )

(␮g kg−1 )

−3012 + 6513x −46890 + 8524x −2323 + 6891x −232 + 7762x −61279 + 5657x −76419 + 6032x −62089 + 5323x −81439 + 5146x −1112 + 6547x −3147 + 6358x −1268 + 5250x 81703 + 7098x −66819 + 7212x −99148 + 4657x −165 + 5308x −131 + 1461x −190 + 2867x −65703 + 4407x −111 + 5876x −314 + 8257x 713 + 1368x −812 + 2578x −178 + 2157x −945 + 4764x −921 + 2357x −176 + 4790x −912 + 7346x −96780 + 3768x −1890 + 1357x −845 + 2679x 932 + 2210x −8238 + 3458x −856 + 2659x −91179 + 1670x −86280 + 3417x −183 + 1890x −931 + 7785x −87519 + 1543x −823 + 9664x −629 + 6638x −757 + 2836x −86730 + 1116x −913 + 3573x 97301 + 7638x −81472 + 7957x −83084 + 7382x −92638 + 5159x −125 + 1813x 8789 + 3093x −82591 + 1937x −925 + 3193x −978 + 5347x −1625 + 1942x −192 + 1725x 61683 + 1937x 48346 + 2936x 60361 + 5944x −70234 + 3847x

0.02–5 1–100 0.1–10 0.005–1 1–100 1–100 1–100 1–100 0.1–10 0.02–5 0.1–10 1–100 1–100 1–100 0.002–0.5 0.001–0.5 0.01–5 1–100 0.01–5 0.01–5 0.1–10 0.1–10 0.01–5 0.1–10 0.1–10 0.01–5 0.1–10 1–100 0.02–5 0.1–10 0.1–10 1–100 0.1–10 1–100 1–100 0.01–5 0.1–10 1–100 0.1–10 0.1–10 0.1–10 1–100 0.1–10 1–100 1–100 1–100 1–100 0.01–5 1–100 1–100 0.1–10 0.1–10 0.02–5 0.01–5 1–100 1–100 1–100 1–100

0.01 0.49 0.04 0.003 0.07 0.09 0.28 0.06 0.01 0.006 0.03 0.07 0.07 0.55 0.0012 0.0006 0.004 0.11 0.003 0.005 0.03 0.01 0.005 0.02 0.05 0.003 0.04 0.35 0.01 0.01 0.05 0.28 0.03 0.07 0.19 0.06 0.02 0.33 0.03 0.03 0.04 0.25 0.01 0.52 0.18 0.32 0.19 0.03 0.09 0.13 0.04 0.05 0.01 0.004 0.09 0.38 0.05 0.25

0.02 0.82 0.08 0.005 0.12 0.15 0.47 0.11 0.03 0.01 0.06 0.12 0.13 0.92 0.002 0.001 0.007 0.19 0.005 0.009 0.06 0.03 0.009 0.04 0.09 0.006 0.08 0.59 0.02 0.03 0.09 0.47 0.05 0.12 0.32 0.01 0.04 0.56 0.06 0.06 0.08 0.42 0.03 0.88 0.31 0.54 0.32 0.009 0.16 0.22 0.08 0.09 0.02 0.008 0.15 0.64 0.09 0.43

r2

RSD% at three Levels(n = 9 days) b

0.9990 0.9974 0.9915 0.9969 0.9937 0.9964 0.9992 0.9978 0.9991 0.9987 0.9996 0.9963 0.9934 0.9927 0.9932 0.9946 0.9972 0.9952 0.9976 0.9945 0.9990 0.9983 0.9974 0.9975 0.9984 0.9971 0.9908 0.9933 0.9941 0.9999 0.9926 0.9975 0.9928 0.9904 0.9973 0.9952 0.9993 0.9978 0.9930 0.9922 0.9928 0.9957 0.9907 0.9990 0.9997 0.9946 0.9934 0.9975 0.9932 0.9991 0.9990 0.9947 0.9984 0.9995 0.9961 0.9950 0.9915 0.9993

Level 1

Level 2

Level 3

4.9 5.9 3.4 3.0 1.6 4.2 2.9 4.6 5.2 4.9 3.5 5.9 5.8 6.0 3.7 2.3 2.9 4.7 6.1 5.1 2.2 5.7 4.3 2.1 4.3 5.8 5.4 3.1 4.2 2.2 4.9 4.6 2.0 2.3 4.1 4.6 2.7 5.9 4.7 5.9 3.4 2.3 2.8 2.3 1.4 3.6 5.0 3.9 4.2 2.9 4.8 2.6 3.5 4.9 4.6 5.1 5.6 3.5

4.8 5.1 2.8 5.6 3.4 5.7 2.1 5.9 4.0 3.2 4.9 4.1 2.6 6.1 4.4 2.1 4.9 4.6 4.4 3.9 4.4 4.6 4.1 4.2 2.6 4.4 2.3 5.6 4.5 1.3 4.4 3.4 4.5 3.4 5.1 4.9 3.5 4.2 1.9 6.1 5.4 5.0 4.4 4.3 5.7 2.0 3.8 3.4 6.2 3.7 4.1 5.8 5.4 3.7 6.1 4.4 5.0 4.5

5.3 5.5 5.6 3.1 3.9 5.6 2.3 4.8 5.6 4.4 2.3 5.7 4.7 3.5 4.7 2.0 3.9 4.2 6.4 5.9 3.9 4.1 3.4 2.2 4.5 5.3 6.1 2.3 3.9 5.3 6.0 5.8 5.5 6.0 4.4 2.2 5.7 6.1 5.3 4.9 1.5 1.6 3.4 2.2 1.8 3.6 5.0 3.5 3.4 4.8 2.9 1.0 6.2 5.9 4.7 5.6 3.9 4.4

Average of three concentration levels-CC␤, 2 CC␤, 4 CC␤. Interday precision.

3.4. Sample analysis Once the proposed methods were optimized and validated, it was applied to investigate the occurrence of the fifty-eight mycotoxins in a total of thirty commercial milk, milk beverages and yogurt products. As shown in Table 4, only Aflatoxin M1, Ochratoxin A, Ochratoxin-␣ and Fumonisin B1 were detected in dairy

products. Fig. 4 shows the typical chromatograms and spectra from a full MS/dd-MS2 experiment of analytes detected in positive samples. In total, 13% (4/30) of the samples were contaminated with one or more mycotoxins. With this UHPLC/ESI Q-Orbitrap method, not only accuracy was enhanced, but also the low concentration preservative, this suggested that the UHPLC-ESI-Q-Orbitrap-MS method was appropriate for the screening of mycotoxins in foods.

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Table 4 Quantification results for target analytes in positive dairy products analyzed by UHPLC/ESI Q-Orbitrap. Sample a

Compound

Concentration (␮g kg−1 )

RSD (%) (n = 9)

No. 2

Aflatoxin M1 Ochratoxin AOchratoxin-␣ Fumonisin B1 Aflatoxin M1 Aflatoxin M1

0.002 0.010.009 0.11 0.002 0.005

0.9 2.93.1 2.3 1.3 2.6

No. 16 No. 29 No. 30 a

Type of dairy products are indicated herein: milk, No. 2; milk beverages, No. 16; yogurt, No. 29 and No. 30.

References

Fig. 4. Examples of typical UHPLC/ESI Q-Orbitrap MS chromatograms and spectra from a full MS/dd-MS2 experiment: (A1) extracted ion chromatogram (displayed as a stick per scan) of Aflatoxin M1 [M+H]+ m/z 329.06493 in sample No. 29; (A2) dd-MS2 total ion chromatogram of Aflatoxin M1 [M+H]+ m/z 329.06493 in sample No. 29.

4. Conclusions A new analytical method has been developed and applied in routine for screening and quantitation of mycotoxins in dairy products. In summary, by combining QuEChERS extraction procedure and UHPLC/ESI Q-Orbitrap, an accurate and highly sensitive method was developed to screen fifty-eight mycotoxins in foods. Compared with traditional methods, the sensitivity was enhanced, and the accuracy was improved by more than 5 times, leading to a powerful method for screening mycotoxins in foods. Acknowledgments We wish to thank ThermoFisher Scientific (San Jose, USA) for providing us with UHPLC/ESI Q-Orbitrap. We would also like to thank James Chang for the technical support. The present research was financially supported by the grants from the project of General Administration of Quality Supervision, Inspection and Quarantine of the People’s Republic of China (Project number: 2012104002; 2013IK198), and the Graduate Innovation Fund of Shaanxi University of Science and Technology.

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