Simultaneous Determination Of Aflatoxin B1 Research Articles

Optimization and Validation of Dispersive Liquid-Liquid Microextraction for Simultaneous Determination of Aflatoxins B1, B2, G1, and G2 in Senna Leaves and Pods Using HPLC-FLD with Pre-Column Derivatization.

Dispersive liquid-liquid microextraction (DLLME) was optimized for the simultaneous extraction of aflatoxins (AFB1, AFB2, AFG1, and AFG2) from powdered senna leaves and pods. Detection was performed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) and pre-column derivatization. The parameters affecting the DLLME extraction efficiency were evaluated. Chloroform (200 µL) was used as an extraction solvent, 500 µL of distilled water was used as a dispersive solvent, and the extraction was performed at pH 5.6 with no salt added. The optimized method was validated using leaves and pods according to the European Commission guidelines. The linear range for all aflatoxins was 2-50 µg/kg, with values for regression coefficients of determination exceeding 0.995. The recoveries of spiked senna leaves and pods were in the ranges of 91.77-108.71% and 83.50-102.73%, respectively. The RSD values for intra-day and inter-day precisions were in the ranges of 2.30-7.93% and 3.13-10.59%, respectively. The limits of detection and quantification varied in the ranges of 0.70-1.27 µg/kg and 2.13-3.84 µg/kg, respectively. The validated method was successfully applied for the quantification of aflatoxins in 60 real samples of dried senna leaves and pods.

Simultaneous determination of aflatoxins B1, B2, G1 and G2 in commercial rices using immunoaffinity column clean-up and HPLC-MS/MS

Rice is frequently contaminated with aflatoxins, that are highly toxic fungal substances and strongly involved on hepatic cancer. In this work, different extraction and clean-up methods were evaluated for the simultaneous extraction and clean-up of aflatoxins B1, B2, G1 and G2 from rice. Favourable results were obtained by using methanol – water (80:20, v/v) extraction followed by immunoaffinity columns for clean-up, with recoveries of 86–92%, standard deviations between 5 and 11%, LOD ranged between 0.09 and 0.32 µg/kg, and LOQ between 0.31 and 1.06 µg/kg. Method validation and sample analysis were performed by using HPLC-MS/MS. Nine rice samples from different origin, varieties and specific characteristics, acquired in Spanish supermarkets were analysed. In two basmati samples from the same batch aflatoxin B1 was detected at (1.62 ± 0.08) µg/kg and (0.77 ± 0.03) µg/kg, both lower than the levels established by European Regulation for aflatoxin B1 in cereals.

Evaluation of Extraction Procedures Efficiency for Determination Of Aflatoxins B1 and M1 in Raw Cow's Milk Collected from Dairy Farms in Qassim Region, Saudi Arabia

A comparison between the efficiency of the solid-phase extraction (SPE) and dispersive-solid phase extraction (d-SPE) was conducted using ultra-high-performance liquid chromatography equipped with tandem mass spectrometry UPLC-MS/MS, in order to have a simple and sensitive method for the simultaneous determination of aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1) in cow’s milk samples. This method was used to analyze 61 of cow’s milk samples collected from different dairy farms in Qassim region, Saudi Arabia. It was found that the concentrations of AFM1 in 5 (8.2 %) samples were higher than the International levels as recommended by European Commission (EC, 2006). Seven (11.5%) of the samples were contaminated by AFM1 with a concentration lower than the international limits, while only one sample (1.6%) was contaminated with AFB1. The d-SPE procedure has many advantages, such as high recovery and lower value of Limit of Quantitation (LOQ). Therefore, the d-SPE procedure is recommended for analyzing the AFB1 and AFM1 in milk.

Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label-free monolithically integrated optoelectronic biosensor.

A label-free optical biosensor for the fast simultaneous determination of three mycotoxins, aflatoxin B1 (AFB1), fumonisin B1 (FB1) and deoxynivalenol (DON), in beer samples is presented. The biosensor is based on an array of ten Mach-Zehnder interferometers (MZIs) monolithically integrated along with their respective broad-band silicon light sources onto a single chip. Multi-analyte determination is accomplished by functionalizing the sensing arms of individual MZIs with mycotoxin-protein conjugates. Assay is performed by pumping over the chip mixtures of calibrators or samples with a mixture of specific monoclonal antibodies, followed by reaction with a secondary anti-mouse IgG antibody. Reactions are monitored in real-time by continuously recording the MZI output spectra, which are then subjected to Discrete Fourier Transform to convert spectrum shifts to phase shifts. The detection limits achieved for AFB1, FB1 and DON were 0.8, 5.6 and 24 ng/ml, respectively, while the assay duration was 12 min. Recovery values ranging from 85 to 115% were determined in beer samples spiked with known concentrations of the three mycotoxins. In addition, beers of different types and origin were analysed with the biosensor developed and the results were compared with those provided by established laboratory methods, further supporting the accuracy of the proposed device.

An On-Site Simultaneous Semi-quantification of Aflatoxin B1, Zearalenone, and T-2 Toxin in Maize- and Cereal-based Feed via Multicolor Immunochromatographic Assay.

Multiple-mycotoxin contamination has been frequently found in the agro-food monitoring due to the coexistence of fungi. However, many determination methods focused on a single mycotoxin, highlighting the demand for on-site determination of multiple mycotoxins in a single run. We develop a multicolor-based immunochromatographic strip (ICS) for simultaneous determination of aflatoxin B1 (AFB1), zearalenone (ZEN) and T-2 toxin in maize- and cereal-based animal feeds. The nanoparticles with different colors are conjugated with three monoclonal antibodies, which serve as the immunoassay probes. The decrease in color intensity is observed by the naked eyes, providing simultaneous quantification of three mycotoxins. The visible limits of detection for AFB1, ZEN and T-2 are estimated to be 0.5, 2, and 30 ng/mL, respectively. The cut-off values are 1, 10, and 50 ng/mL, respectively. Considerable specificity and stability are found using real samples. The results are in excellent agreement with those from high-performance liquid chromatography/tandem mass spectrometry. The multi-color ICS boasts sensitive and rapid visual differentiation and simultaneous semi-quantification of aflatoxin B1, zearalenone and T-2 toxin in maize- and cereal-based feed samples within 20 min.

Non-destructive and rapid evaluation of aflatoxins in brown rice by using near-infrared and mid-infrared spectroscopic techniques.

The applicability of near-infrared (NIR) and mid-infrared (MIR) spectroscopy combined with chemometrics was explored in this study to develop rapid, low-cost and non-destructive spectroscopic methods for classification and quantification of aflatoxins in brown rice. A total of 132 brown rice samples within the aflatoxin concentration range of 0-2435.8μg/kg were prepared by artificially inoculated with A. flavus and A. parasiticus strains of fungus. For the classification of samples at varying levels of aflatoxin B1, the linear discriminant analysis model obtained correct classification rate of 96.9 and 90.6% for NIR and MIR spectroscopy, respectively. For the simultaneous determination of aflatoxins B1, B2, G1, G2 and the total aflatoxins, partial least squares regression also showed good predictive accuracy for both NIR (rv =0.936-0.973, RPD=2.5-4.0) and MIR spectroscopy (rv =0.922-0.970, RPD=2.5-4.0). The overall results indicated that the two spectroscopic techniques offered the feasibility to be used as alternative tools for rapid detection of various aflatoxin contaminations in grain.

Simultaneous determination of aflatoxin B1 and zearalenone by magnetic nanoparticle filled amino-modified multi-walled carbon nanotubes

We report a novel extractant based MSPE-HPLC-DAD method for simultaneous determination of aflatoxin B1 and zearalenone.

Simultaneous Determination of Aflatoxin B1, Bisphenol A, and 4-Nonylphenol in Peanut Oils by Liquid-Liquid Extraction Combined with Solid-Phase Extraction and Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

An analytical method based on liquid-liquid extraction combined with solid-phase extraction and isotope dilution-ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was well developed for simultaneous determination of aflatoxin B1 (AFB1), bisphenol A (BPA), and 4-nonylphenol (4-NP) in peanut oil. After adding isotope internal standards, the samples were firstly diluted by normal hexane and then extracted by acetonitrile and Carb/PSA solid-phase extraction cartridge in sequence to obtain the extracted solution. All the extracted solution was merged and was subsequently dried to near dryness by a mild nitrogen stream. Three target analytes were separated on a Phenomenex Luna C18 chromatographic column, quantified by an internal standard method and detected by ESI positive (ESI+) and negative (ESI−) subsection acquisition modes under multi-reaction monitoring (MRM) conditions. Results demonstrated that the three target analytes exhibited excellent linearity in their corresponding concentration ranges of 0.1–100.0 μg/L with correlation coefficients all greater than 0.998. The corresponding method limits of quantitation (MLOQ, S/N = 10) of AFB1, BPA, and 4-NP were 0.2, 1.0, and 2.0 μg/kg, respectively. Moreover, the mean recoveries for negative samples spiked at three concentration levels were calculated between 87.7 and 105.1% with relative standard deviation (RSD, n = 6) ranging from 2.2 to 7.9% and the interday precision (n = 5) ranging from 5.0 to 8.7%. Finally, the method was successfully applied to analyze 52 peanut oil samples, and AFB1 and 4-NP were detected in 43 samples with the concentrations in the ranges of 0.5–69.4 and 9.3–77.8 μg/kg, respectively. None of BPA was detected in any samples.

Broad-spectrum immunoaffinity cleanup for the determination of aflatoxins B1, B2, G1, G2, M1, M2 in Ophiocordyceps sinensis and its pharmaceutical preparations by ultra performance liquid chromatography tandem mass spectrometry.

An ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of aflatoxins B1, B2, G1, G2, M1 and M2 (AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2) in Ophiocordyceps sinensis and its pharmaceutical preparations. A rapid and reliable immunoaffinity column containing a broad-spectrum monoclonal antibody for six aflatoxins was used for sample cleanup. Under the optimized conditions, the home-made immunoaffinity column capacity were about 315, 319, 292, 102, 444 and 369ng/mL gel for AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2, respectively. Recoveries for all tested aflatoxins ranged from 79.28% to 103.42% with relative standard deviation less than 8%. The limits of quantitation were in the range of 0.008-0.045μg/kg. Among 31 real samples analyzed, one sample was contaminated with AFB1, AFB2 and AFM1 at levels of 0.483, 0.068 and 0.104μg/kg, respectively. The established method is simple, accurate, and can be effectively used to determine the aflatoxins in Ophiocordyceps sinensis and its pharmaceutical preparations.

A Rapid Single-Extraction Method for the Simultaneous Determination of Aflatoxins B1, B2, G1, G2, Fumonisin B1, and Zearalenone in Corn Meal by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

A single-extraction method to simultaneously determine aflatoxins (B1, B2, G1, G2), fumonisin B1, and zearalenone in corn meal by ultra performance liquid chromatography tandem mass spectrometry, using a triple quadrupole, was optimized, validated, and applied in an occurrence study. Different extraction solutions were tested, with better performance for methanol/acetonitrile/water (60:20:20, v/v/v). Linearity was observed from 0.25 to 1.50 ng/mL for aflatoxins, from 20 to 120 ng/mL for fumonisin, and from 7.00 to 42.00 ng/mL for zearalenone. Significant matrix effects were shown for all groups. Selectivity was demonstrated, as matrix or spectral interferences were not observed at the predicted retention time window of the target analytes. Average recoveries of 87.57, 93.18, 93.35, 94.20, 78.76, and 95.98% were obtained for aflatoxins (B1, B2, G1, and G2) fumonisin and zearalenone, respectively. A z-score of 0.19 was estimated in a corn certified reference material for fumonisin B1. Maximum relative standard deviation values under repeatability and intermediate precision conditions were determined to be 13.6 and 13.6% for aflatoxins, 3.7 and 6.3% for fumonisin, and 3.5 and 4.0% for zearalenone, respectively. In the occurrence study, 50 samples were analyzed and 44% had measurable levels of fumonisin. Zearalenone was detected in 18%. The proposed method showed considerable advantages, considering environmental impacts, efficiency, and reliability.

Development and validation of a simple and fast method for simultaneous determination of aflatoxin B1 and sterigmatocystin in grains.

In this study, a simple, fast, sensitive and reliable method was developed for the simultaneous determination of aflatoxin B1 (AFB1) and sterigmatocystin (STC) in rice, wheat, maize, oat, rye and barley grains based on a modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). AFB1 and STC were extracted using 95% acetonitrile in water and cleaned up using N-Propylethane-1,2-diamine (PSA). The method was validated according to the SANCO/12571/2013 regulation of the European Union (EU). Recoveries at three spiked levels of 0.5, 2.5 and 5.0μg/kg ranged from 77.7% to 119.7% and the relative standard deviations (RSDs) were between 1.3% and 12.8%. Limits of detection (LOD) were 0.03 and 0.02μg/kg for AFB1 and STC, respectively. The limit of quantification (LOQ) was 0.5μg/kg for both AFB1 and STC. The proposed method was successfully applied to determine AFB1 and STC levels in ten non-processed rice samples from Guangdong province in China.

Detection and quantitation of mycotoxins in infant cereals in the U.S. market by LC-MS/MS using a stable isotope dilution assay

The aim of this study was to develop an analytical method for the simultaneous determination of aflatoxins B1, B2, G1, G2, ochratoxin A, fumonisins B1 and B2, zearalenone, deoxynivalenol, T-2 toxin, and HT-2 toxin in infant cereals using rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS). Another goal was to survey infant cereals purchased in the United States for the presence and amounts of 11 mycotoxins. Separation of the mycotoxins was accomplished using ultra-high performance liquid chromatography (UHPLC) with <10 min analysis time. The toxins were then detected by dynamic multiple reaction monitoring (dMRM) in positive electrospray ionization mode. Due to matrix effects, [13C]-uniformly labeled mycotoxins were added to the sample extracts prior to LC-MS/MS analysis. Overall recoveries of mycotoxins were 81–130% in rice, 70–119% in barley, 87–123% in oat, and 82–127% in mixed-grain cereals with lower recoveries for fumonisins B1 and B2, (33–67%) at three spiking levels (12.5, 25 and 50 ng/g). The relative standard deviation was <20% for all analytes in the infant cereals. When this method was applied to measure mycotoxin concentrations in infant cereals (n = 64) purchased in the U.S. in 2012, 78% of infant cereals were found to be contaminated with at least one of these mycotoxins. None of the infant cereals exceeded the established regulatory limits in the United States for any of the analyzed mycotoxins. However, a total of 21 samples exceeded the European Union maximum limits for three of the mycotoxins including aflatoxin B1, ochratoxin A and zearalenone. Results of this study suggest that cereals intended for infants should be routinely monitored for mycotoxin content.

Occurrence and exposure assessment of multiple mycotoxins in dried fruits based on liquid chromatography-tandem mass spectrometry

A reliable analytical method based on liquid chromatography-tandem mass spectrometry was developed for simultaneous determination of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1, aflatoxin G2, ochratoxin A (OTA), deoxynivalenol, T-2 and HT-2 toxin, and zearalenone (ZEA) in various dried fruits. A simple one-step sample extraction without using clean-up cartridges made the established method less labour consuming and less expensive, while optimisation of the several important MS/MS parameters, i.e. the scan time and run segments, ensured its sensitivity and selectivity. After careful validation of the method by determining the linearity (R2&gt;0.99), recovery (77.8-115.9%), precision (relative standard deviation ≤19.5%) and sensitivity (limits of quantification in the range of 0.1-10 μg/kg), a survey of 125 dried fruit samples including 25 pistachios, 28 dried longans, 32 raisins and 40 dried dates randomly collected from different markets in Shanghai, China, was performed. Results revealed that 32.0% of samples were contaminated with different mycotoxins, among which, OTA was the most frequent contaminant with the incidence of 29.6% attaining the concentration levels in the range of 0.4-212.6 μg/kg. ZEA was positively found in 2 pistachio samples with the concentrations of 84.9 μg/kg and 426.9 μg/kg. Trace amounts of AFB1 (0.8 μg/kg) and AFB2 (0.2 μg/kg) were also observed in one pistachio sample and one dried longan sample, respectively. To the best of our knowledge, this is the first report to reveal the real situations of mycotoxin contaminations in various dried fruits in China.

Simultaneous determination of aflatoxin B1 and M1 in milk, fresh milk and milk powder by LC-MS/MS utilising online turbulent flow chromatography

A novel, fully automated method based on dual-column switching using online turbulent flow chromatography followed by LC-MS/MS was developed for the determination of aflatoxin B1 and M1 in milk, fresh milk and milk powder samples. After ultrasound-assisted extraction, samples were directly injected into the chromatographic system and the analytes were concentrated on the clean-up loading column. Through purge switch, analytes were transferred to the analytical column for subsequent detection by mass spectrometry. Different types of TurboFlowTM columns, transfer flow rates and transfer times were optimised. Method limits of detection obtained for AFB1 and AFM1 were 0.05 μg kg–1, and limits of quantification were 0.1 μg kg–1. Recoveries of aflatoxin B1 and M1 were in range of 81.1–102.1% for all samples. Matrix effects of aflatoxin B1 and M1 were in range of 63.1–94.3%. The developed method was successfully used for the analysis of aflatoxin B1 and M1 in real samples.

Ultra-high performance liquid chromatography tandem mass spectrometry for simultaneous analysis of aflatoxins B1, G1, B2, G2, zearalenone and its metabolites in eggs using a QuEChERS-based extraction procedure

A reliable UPLC-MS/MS method for the simultaneous determination of aflatoxins B1, G1, B2, G2, zearalenone and its metabolites α-zearalanol, β-zearalanol was developed.

Development and validation of a high-performance liquid chromatography method with post-column derivatization for the detection of aflatoxins in cereals and grains.

A novel, reliable and rapid high-performance liquid chromatography (HPLC) method with post-column derivatization was developed and validated. The HPLC method was used for the simultaneous determination of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) in various cereals and grains. Samples were extracted with 80:20 (v/v) methanol:water and purified using C18 (40-63 μm) solid-phase extraction cartridges. AFs were separated using a LiChroCART-RP-18 (5 μm, 250 × 4.0 mm(2)) column. The mobile phase consisted of methanol:acetonitrile:buffer (17.5:17.5:65 v/v) (pH 7.4) delivered at the flow rate of 1.0 mL min(-1) The fluorescence of each AF was detected at λex = 365 nm and λem = 435 nm. All four AFs were properly resolved within the total run time of 20 min. The established method was extensively validated as a final verification of the method development by the evaluation of selectivity (AFB1, AFB2, AFG1 and AFG2), linearity (R(2) ≥ 0.9994), precision (average SD ≤ 2.79), accuracy (relative mean error ≤ -5.51), robustness (p < 0.0080), ruggedness (p < 0.0100) and average recoveries (89.2-97.8%). The limits of quantification of AFB1, AFB2, AFG1 and AFG2 were 0.080, 0.073, 0.062 and 0.066 ng g(-1), respectively. Finally, the developed method was applied for the analysis of AFs in 45 samples comprising rice (n = 20), wheat (n = 15) and maize (n = 10). The results showed that 65% of rice, 20% of wheat and 80% of maize samples were found contaminated with AFs. Thus, according to the achieved results, it is suggested that the newly developed HPLC method could be effectively applied for the routine analysis of the AFs in different cereals and grains.

Simultaneous determination of aflatoxins B₁, B₂, G₁, G₂ in Fructus Bruceae by high-performance liquid chromatography with online postcolumn photochemical derivatization.

A simple, reliable, and highly sensitive method for the simultaneous determination of aflatoxin B1 , B2 , G1 , G2 in Fructus Bruceae was developed using high-performance liquid chromatography coupled to online postcolumn photochemical derivatization and fluorescence detection. Aflatoxins were first extracted by a methanol/water mixture and then cleaned up with an AflaTest™ immunoaffinity column. Different clean-up and derivatization methods were compared and optimized. The established method was extensively validated to show satisfactory performance of linearity (R(2) ≥ 0.9997), recovery (74.3-100.8%), and precision (RSDs ≤ 2.8%) for the investigated aflatoxins. This proposed method was also applied to 11 F. Bruceae samples and the results showed that 10 out of 11 were contaminated with aflatoxins ranging from 0.26 to 27.52 μg/kg and the occurrence of aflatoxin B1 , the most toxic one, was as high as 91% in all the samples, highlighting the severe contamination and the necessity to set legal limits for aflatoxins in F. Bruceae.

Simultaneous determination of aflatoxin B1, B2, G1, G2, ochratoxin A, and sterigmatocystin in traditional Chinese medicines by LC–MS–MS

In this paper we describe a rapid, simple, and costeffective liquid chromatography–tandem mass spectrometric (LC–MS–MS) method for simultaneous analysis of aflatoxin B1, B2, G1, and G2, ochratoxin A, and sterigmatocystin in 25 traditional Chinese medicines (TCMs). The method is based on single extraction with 84:16 (v/v) acetonitrile–water then analysis of the diluted crude extract without further clean-up. Chromatographic separation was achieved on a C18 column, with a mobile phase gradient prepared from aqueous 4 mmol L−1 ammonium acetate–0.1 % formic acid and methanol. Quantification of the analytes was by selective reaction monitoring (SRM) on a triple-quadrupole mass spectrometer in positive-ionization mode. Special focus was on investigating and reducing matrix effects to improve accuracy. The established method was validated by determination of linearity (r>0.995), sensitivity (limits of quantification 1.6–25.0 ng L−1), apparent recovery (84.8–110.6 %), extraction recovery (83.6–106.1 %), and precision (relative standard deviation ≤9.9 %) for two representative TCMs, Semen Armeniacae Amarae and Radix Pseudostellariae. The applicability of the method to TCMs other than these was further investigated, and 23 other TCMs with acceptable matrix effects (80.2–118.6 %) were screened. The validated method was finally used to assess mycotoxin contamination of 244 samples of 25 TCMs collected from local hospitals and TCM pharmacies. Aflatoxin B1 and ochratoxin A were detected in 5.3 % of the samples. Sterigmatocystin, the most prevalent mycotoxin contaminant, was present in 26.2 % of the samples tested; this has not been reported previously. The results of this work imply greater attention should be devoted to evaluation of the potential hazard caused by sterigmatocystin in TCMs.

Multimycotoxin analysis of crude extracts of nuts with ultra-high performance liquid chromatography/tandem mass spectrometry

A reliable, fast and simple method using ultra-high performance liquid chromatography with heated electrospray ionization triple quadrupole mass spectrometry (UHPLC/HESI-MS/MS) was developed for the simultaneous determination of aflatoxins B1, G1, B2 and G2, ochratoxin A (OTA), zearalenone (ZEA), HT-2 toxin, T-2 toxin and fumonisins B1 and B2 in crude extracts of various types of nuts. The procedure is based on the simultaneous extraction of selected mycotoxins with a mixture of acetonitrile/water/acetic acid (79:20:1, v/v/v) and defatting the obtained extract with hexane in order to remove the lipids. The validation data indicated that the analysis of different types of crude extracts of nuts is feasible and sensitive enough for determination of the majority of the studied mycotoxins. The recoveries of various nuts matrices ranged between 71.25% and 140.11% with relative standard deviation lower than 12%. The satisfactory recoveries were obtained for the most of mycotoxins using walnut matrix-matched calibration curves indicating the multi-matrix feasibility of the method. The applicability of the method was successfully demonstrated on 17 samples of nuts collected in a region of northern Serbian province of Vojvodina. Total frequency of the occurrence of the selected mycotoxins was 12%.

Simultaneous determination of aflatoxin B1, aflatoxin B2, mycophenolic acid and sterigmatocystin in grape pomace by UHPLC-MS/MS

A reliable ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous determination of aflatoxin B1, aflatoxin B2, mycophenolic acid and sterigmatocystin in grape pomace. The samples were extracted by acetonitrile aqueous solution and further purified using a solid-phase extraction-based homemade clean-up cartridge. Next, the analytes were separated on a reversed-phase C18 column with a mobile phase consisting of water and acetonitrile. The separated compounds were detected with a tandem quadrupole mass spectrometer operating in positive electro-spray ionisation mode using multiple reaction monitoring. The established method was extensively validated by determining linearity (R2≯0.999), recovery (97.5-102.8%) and precision (relative standard deviation ≤7.0%). This method was then used for the simultaneous determination of the four mycotoxins in grape pomace samples.