Multi-mycotoxin analysis method using liquid chromatography with tandem mass spectrometry and fluorescence detection in Indian medicinal herbs: Development and validation.

While medicinal plants are in high demand worldwide for their therapeutic properties, they can constitute a health concern to consumers when contaminated with mycotoxins. The unavailability of standardised methods for multiclass mycotoxin analysis to assess health risks has thus been realised. This study reports a simple, robust and precise method to estimate nine regulated mycotoxins in a range of Indian medicinal plant matrices including giloy (Tinospora cordifolia), ashwagandha (Withania somnifera), safed musli (Chlorophytum borivilianum), satavari (Asparagus racemosus) and tulsi (Ocimum sanctum). The sample preparation method involved extraction of homogenised matrices (12.5 g) using methanol:water (8:2, 100 mL) followed by cleanup through a multi-mycotoxin immunoaffinity column (IAC), which significantly reduced matrix interferences. The method was initially developed and validated using liquid chromatography tandem mass spectrometry (LC-MS/MS) for the simultaneous analysis of aflatoxins (B1, B2, G1, G2), ochratoxin A, zearalenone, deoxynivalenol, T-2 and HT-2 toxin. Later, it was validated using LC-fluorescence (LC-FLD) for aflatoxins, ochratoxin A and zearalenone. The optimised sample preparation protocol and analytical method provided acceptable results. Compared to LC-FLD, it was possible to attain a lower limit of quantification (LOQ) with LC-MS/MS for all the tested analytes except aflatoxins. However, LOQs of both instruments were lower than the maximum limits (MLs), with recoveries ranging between 71 and 110% and precision (RSD) of ≤10% across matrices. Despite matrix-induced signal suppressions in LC-MS/MS analysis, the matrix-matched calibrations corrected all recoveries. Considering its accuracy, reliability, robustness and time-effectiveness, this method is recommended for regulatory testing purposes.

Analysis of aflatoxins and ochratoxin a in chilli powder using ultrahigh performance liquid chromatography with fluorescence detection and tandem mass spectrometry.

Chilli powder, a popular spice, is predominantly contaminated with aflatoxins (AFs) and ochratoxin A (OTA), posing a menace to public health. As no validated method exists for the simultaneous and direct analysis of AFs and OTA in chilli powder, it was imperative to develop one to ensure their effective monitoring and promote trade. In this research, we developed and validated a multi-mycotoxin analysis method that allows the simultaneous determination of AFs (AFB1, AFB2, AFG1 and AFG2) and OTA in chilli powder with high sensitivity, accuracy and precision. The optimised sample preparation workflow started with the extraction of chilli powder (25 g) with methanol-water (100 mL, 80:20). An aliquot (3 mL) was cleaned on a multi-mycotoxin, immunoaffinity column (AFLAOCHRA PREP®) and analysed using ultrahigh performance liquid chromatography with fluorescence (UHPLC-FLD) and tandem mass spectrometric (LC-MS/MS) detection in a single chromatographic run. The method performance was evaluated through intra- and inter-laboratory validation (ILV) studies, and also by analysing a certified reference material. A direct analysis using UHPLC-FLD (without derivatisation) provided the limits of quantification (LOQ) of 0.25 and 1 ng/g for AFs and OTA, respectively, while the LOQ for all these mycotoxins in LC-MS/MS was 0.5 ng/g. These LOQs are much lower than the maximum levels (MLs) specified by the European Commission. The recoveries of these analytes at LOQ and higher levels were above 75% (RSDr < 12%). The ILV study demonstrated satisfactory method-reproducibility (RSDR < 25%). The analysis of the certified reference material provided accuracies of AFs and OTA in the range of 83-101%. The analysis by UHPLC-FLD and LC-MS/MS provided very similar results. The incurred levels of B1 in market samples were estimated with a precision-RSD of < 6%. Considering its efficiency and alignment with the regulatory requirements, this method can be implemented for the routine analysis of AFs and OTA in chilli powder.

Rapid and Quantitative Analysis of Aflatoxin M1 From Milk Using Atmospheric Pressure-Matrix Assisted Laser Desorption/Ionization (AP-MALDI)-Triple Quadrupole Selected Reaction Monitoring.

BACKGROUND: Aflatoxin M1 (AFM1) is a carcinogenic hydroxylated metabolite commonly found in milk. It is relatively stable toward decontamination procedures posing a major health risk, and it requires an international regulatory mandate of detection at trace levels. OBJECTIVE: To develop a high-throughput, reliable, and compliant method for the identification of AFM1 in milk samples using atmospheric pressure-matrix assisted laser desorption/ionization (AP-MALDI) selected reaction monitoring (SRM) quantitation. METHOD: The milk sample was diluted in water and cleaned with immunoaffinity chromatography (IAC), followed by analysis using AP-MALDI hyphenated with a triple quadrupole mass spectrometer for SRM. RESULTS: A fast and reliable AP-MALDI SRM quantitative method was developed for the determination of AFM1 with analysis time of 1 min per sample. The diagnostic product ions of AFM1 at 273.1 u and 229.2 u were monitored during the SRM. The calibration curves yielded excellent linearity (R2 = 0.99) with good recoveries for quality control samples (97-106%). The ion ratios of the qualifier to quantifier displayed excellent RSD (1-7.8%) for n = 3. CONCLUSIONS: The developed method provided rapid quantification for AFM1. The fast AP-MALDI SRM method can allow analysis of AFM1 in a large number of milk samples. Given the time required for analysis, cost-effectiveness, and superior analytical performance, this method can be adopted in commercial food testing laboratories. HIGHLIGHTS: Aflatoxins (AF) are a major health risk. Speedy analysis of large sample sizes from food is a risk mitigation strategy but remains an unmet need. Quantitative, chromatography-free, and internal standard-free AP-MALDI SRM based analysis of AF is a high-throughput and cost-efficient alternative. Satisfactory performance was achieved for quantitative AP-MALDI SRM analysis of AFM1 in milk subsequent to a simple sample clean-up step.

Decontamination of aflatoxin B1 in peanuts using various cooking methods.

Peanut and its processed products are recurrently contaminated with aflatoxins (AFs) which are of potential public health concern. Among the different types of AFs, Aflatoxin B1 (B1) is the most frequently detected in peanuts over the maximum level (ML), and thus has warranted considerable research interest in the domain of food safety. In this study, we investigated the decontamination of B1 in three naturally-incurred lots (4, 12, and 40 µg/kg) of peanuts by a range of cooking treatments, including frying, pressure cooking, and roasting. B1 concentrations were determined by ultra-high performance liquid chromatography- fluorescence detection. The method provided a limit of quantification of 0.25 µg/kg for B1, which was much lower than any of its national and international MLs. The recoveries of B1 in fresh and cooked peanuts (positive-control) were in the range of 90-100%. Overall, all the cooking methods demonstrated a significant reduction in B1 loads. The degree to which the processing methods reduced the B1 content followed the pattern: roasting with a combination of NaCl and citric acid > pressure-cooking with a combination of NaCl and citric acid > frying. As the cooking procedures did not involve any complicated steps or sophisticated equipment, these could be readily adopted for decontamination or reduction in the level of B1 for a safer consumption of peanuts at the household level without affecting the organoleptic properties.

Development and validation of an analytical method for the multiresidue analysis of pesticides in sesame seeds using liquid- and gas chromatography with tandem mass spectrometry.

For the first time, an analytical method for the multiresidue analysis of multiclass pesticides in sesame seeds using liquid- and gas chromatography with tandem mass spectrometry (LC-MS/MS and GC-MS/MS) was developed and validated. At first, the sample was comminuted after adding water (1:2 w/v). The sample preparation workflow included acetonitrile extraction, followed by freeze-out of the extract at -80°C with a subsequent cleanup by dispersive solid phase extraction (dSPE) (100 mg of C18 + 150 mg of MgSO4 for LC-MS/MS and 100 mg of C18 + 25 mg florisil + 150 mg of MgSO4 for GC-MS/MS). As noted, these cleanup steps were quite effective in removing the fatty co-extractives. The optimised sample preparation method effectively minimised the matrix effects and offered a limit of quantification (LOQ) of 0.01 mg/kg for most compounds. The LC-MS/MS and GC-MS/MS methods were validated at three levels (0.01, 0.02 and 0.05 mg/kg) for 222 and 220 compounds respectively. The method accuracy and precision complied with the performance criteria of the SANTE/12682/2019 analytical quality control procedure. The results of the intra-laboratory (involving six analysts) and inter-laboratory studies (involving eight accredited laboratories) were comparable for all pesticides. Considering its performance efficiency and alignment with the regulatory guidelines, this method can be implemented across the food testing laboratories for the monitoring of pesticide residues in sesame seeds.

High-Throughput Analysis of Aflatoxins in Cereals, Nuts, and Processed Products Involving Automated Immunoaffinity Cleanup and Inline HPLC-Fluorescence Detection.

BACKGROUND: The testing of aflatoxins (AFs) in fresh and processed foods is highly in demand to comply with trade regulations. Consequently, commercial laboratories face huge AF sample loads in food consignments. Worldwide, there is a rising interest in implementing automation to increase sample throughput in AF analysis. OBJECTIVE: This study sought to evaluate the performance of an automated cleanup and HPLC analysis system for determination of regulated AFs (B1, B2, G1, G2) in rice, flattened rice, sorghum, raw and processed peanut, almond, peanut butter, and wheat-based cookies. METHODS: The samples were extracted with methanol-water (80:20), diluted with Triton X-100, and subjected to automated analysis, where the cleanup step through immunoaffinity column (IAC) and HPLC-fluorescence analyses (involving postcolumn bromination-derivatization) were performed in 10 and 11 min, respectively. The method was validated in all test matrices at the LOQ and higher levels. The method performance was also evaluated against a conventional workflow where cleanup and HPLC analysis were manually performed. RESULTS: The LOQ for peanut, sorghum, rice, and flattened rice was 0.125 ng/g, while it was 0.5 ng/g for peanut butter, almond, and wheat-based cookies. In all matrices, the recoveries at LOQ and higher levels were satisfactory. The double-cartridge exchange system completed the analysis of ∼96 injections in 18 h. Each IAC could be reused 15 times without incurring any recovery loss. The automated system provided a better precision (RSD < 9%) than the conventional workflow (RSD = 12-15%). CONCLUSIONS: Because of its high-throughput nature, this method is recommended for routine analysis of AFs. HIGHLIGHTS: A high-throughput method is reported where cleanup and HPLC analysis of AFs were automatically performed. Each IAC could be used 15 times without any loss of recovery. The method performance was better than the conventional approach and complied with the analytical quality control guidelines.

Development and validation of a liquid chromatographic tandem mass spectrometric method for the analysis of patulin in apple and apple juice.

This study reports a robust and sensitive method for rapid testing of patulin in apple and apple juice. The method involved extraction of homogenised samples (10 g) with ethyl acetate (10 mL) and clean up by dispersive-solid phase extraction using primary secondary amine (25 mg/mL). Prior to the LC-MS/MS analysis, the cleaned extract was reconstituted in methanol/water (2:8). The optimised LC-MS condition provided a symmetric peak of patulin within a short LC-runtime of 5 min. The recoveries at the limit of quantification (0.005 mg/kg) and higher levels were satisfactory (> 80%), with the precision-RSDr (< 11%). In an inter-laboratory comparison study involving 13 accredited laboratories, the reproducibility-RSDR and HorRat values ranged between 4.80 and 6.08% and between 0.18 and 0.23 respectively, indicating a satisfactory method-precision. The z-scores of the participating laboratories were within ± 2. When the method was applied to incurred samples, the contamination range was 0.008-0.225 mg/kg and 0.018-0.034 mg/kg for apple and juice respectively, demonstrating a satisfactory performance in terms of precision. Based on the solvent standard, matrix-matched standard and standard-addition approaches, the calibration graphs provided similar quantitative performances. Because of its reliability, robustness and time-effectiveness, the method can be recommended for regulatory testing purposes.

Development and Validation of a Method for Direct Analysis of Aflatoxins in Animal Feeds by Ultra-High-Performance Liquid Chromatography with Fluorescence Detection.

BACKGROUND AND OBJECTIVE: Aflatoxin (AF) contamination is one of the major regulatory concerns for animal feed. As feed is a complex analytical matrix, validated methods on AFs in feed are scanty. The available methods involve a derivatization step before AF analysis by high-performance liquid chromatography (HPLC) with fluorescence detection (FLD). The aim of this study was thus to develop and validate a simple and rapid method for direct analysis of AFs (AFB1, AFB2, AFG1, AFG2) in a range of animal feed matrices. METHODS: Feed samples were extracted with 80% methanol, followed by dilution with water and immmunoaffinity column cleanup. AFs were estimated using an ultra-high performance liquid chromatography (UHPLC) instrument. Use of a large volume flow cell in FLD allowed direct analysis of all AFs with high sensitivity. The method was thoroughly validated in a range of feed matrices. RESULTS: This sample preparation workflow minimized co-extractives, along with matrix interferences. In pigeon pea husk feed, the method provided a limit of quantification (LOQ) of 0.5 ng/g for each AF with recoveries of AF- B1, B2, G1, and G2 as 71.5, 75.6, 82.4, and 78.2%, respectively. The precision (relative standard deviation, RSD) was below 5%. A similar method performance was also recorded in other matrices, including wheat bran feed and poultry feed. CONCLUSIONS: The optimized method is suitable for regulatory testing because it is simple, robust, cost-effective, and high throughput in nature, with high sensitivity and selectivity. HIGHLIGHTS: Our workflow has provided a straightforward method for the analysis of AFs in a wide range of animal feed matrices with high sensitivity, selectivity, throughput, and cost-effectiveness. The method allowed a direct analysis of AFs by UHPLC-FLD without a step of derivatization.

Development and validation of a multiresidue method for pesticides and selected veterinary drugs in animal feed using liquid- and gas chromatography with tandem mass spectrometry.

Animal feeds are often reported to be contaminated with chemical residues, and when present above the maximum legal limit, these compounds can cause harmful effects to consumers of animal produce. Thus, animal feed safety is an important regulatory concern. The aim of this study was to optimise a multiresidue method for the simultaneous analysis of multi-class pesticides and a number of frequently used veterinary drugs using LC-MS/MS and GC-MS/MS. The method was validated in a range of feed matrices, including maize feed, poultry feed and mixed feed concentrate. The optimised sample preparation workflow involved extraction of feeds (5 g) with ethyl acetate (10 mL), followed by a freezing step (at -20°C) used for eliminating the matrix co-extractives. The extract was further cleaned by dispersive solid phase extraction with a combination of primary secondary amine, C18 and florisil sorbents. From the cleaned-extract, an aliquot was analysed by GC-MS/MS, while another portion of it was solvent-exchanged to acetonitrile:water (50:50) and then analysed by LC-MS/MS. This method effectively minimised the matrix interferences. A total of 192 pesticides was analysed by GC-MS/MS within a runtime of 22 min. The LC-MS/MS method was validated for 187 compounds including 17 veterinary drugs. For most of the compounds, the limit of quantification (LOQ) was 0.01 mg/kg. The recoveries at LOQ and higher levels ranged between 70% and 120%, with precision-RSDs of < 20%. The method provided a precise analysis in a wide range of market-feed samples. As shown, the method is suitable for regulatory and commercial testing purposes.

Simultaneous Direct Analysis of Aflatoxins and Ochratoxin A in Cereals and Their Processed Products by Ultra-High Performance Liquid Chromatography with Fluorescence Detection.

Background: Mycotoxins such as aflatoxins (AFs) and ochratoxin A (OTA) can pose severe health hazards because of their toxicity. Given a wide range of food matrices susceptible to fungal infections and possible cooccurrence of mycotoxins at different concentrations, validated multimycotoxin and multimatrix methods are strongly warranted. Objective: The aim of this research was to develop a simple and fast ultra-high performance LC (UHPLC) fluorescence detection (FLD)-based method to simultaneously determine AFs (B1, G1, B2, and G2) and OTA and, furthermore, to carry out single-laboratory validation in a range of cereals and processed product matrices. Methods: The sample preparation involved homogenization and extraction with methanol-water (80 + 20). For cleanup, an aliquot (3 mL) was diluted with phosphate-buffered saline, loaded on an immunoaffinity column (AFLAOCHRA PREP®), and eluted with methanol (1 mL). The cleaned extract was diluted with 0.2% acetic acid (at a 1:1 ratio) before injection into an ultra-high performance liquid chromatograph. To perform simultaneous analysis of AFs and OTA, the FLD program was developed by switching the excitation wavelength in a single chromatographic run. Results: The method provided LOQs of 0.25 and 1 ng/g for AFs and OTA, respectively, without involving any derivatization. In rice, the recoveries of AFs ranged from 84 to 106%, whereas OTA had a recovery above 72%, with the repeatability relative SDs <12% for both analytes. The method was successfully applied to a range of naturally contaminated market samples. Conclusions: The method is suitable for regulatory testing because of its significant time and cost effectiveness and sensitivity in compliance with the regulatory maximum levels. Highlights: The study achieves high-throughput analysis of AFs and OTA in raw and processed cereals using simultaneous extraction, cleanup, and UHPLC-FLD. Method sensitivity complies with the regulatory maximum levels. Single-laboratory validation results meet analytical QC requirements.

High-sensitivity direct analysis of aflatoxins in peanuts and cereal matrices by ultra-performance liquid chromatography with fluorescence detection involving a large volume flow cell.

This paper reports a sensitive and cost effective method of analysis for aflatoxins B1, B2, G1 and G2. The sample preparation method was primarily optimised in peanuts, followed by its validation in a range of peanut-processed products and cereal (rice, corn, millets) matrices. Peanut slurry [12.5 g peanut + 12.5 mL water] was extracted with methanol: water (8:2, 100 mL), cleaned through an immunoaffinity column and thereafter measured directly by ultra-performance liquid chromatography-fluorescence (UPLC-FLD) detection, within a chromatographic runtime of 5 minutes. The use of a large volume flow cell in the FLD nullified the requirement of any post-column derivatisation and provided the lowest ever reported limits of quantification of 0.025 for B1 and G1 and 0.01 µg/kg for B2 and G2. The single laboratory validation of the method provided acceptable selectivity, linearity, recovery and precision for reliable quantifications in all the test matrices as well as demonstrated compliance with the EC 401/2006 guidelines for analytical quality control of aflatoxins in foodstuffs.