Screening of 152 Veterinary Drug Residues in Animal Source Foods by LC-MS/MS, Multilaboratory Validation Study: Final Action 2020.04.

BACKGROUND: The presence of veterinary drug residues in food-producing animals and animal products is regulated through the enforcement of maximum residue limits (MRLs). To answer the need of the food sector to monitor these substances in a wide range of food commodities, stakeholders at AOAC identified the need for a reliable confirmatory screening method. Such qualitative approach is required for compliance checking and to support product release in manufacturing. OBJECTIVE: Data were collected from 5 independent laboratories that applied the AOAC Official First Action Method AOAC 2020.04 to demonstrate adequate performance under reproducibility conditions. Probability of Detection (POD) was calculated in blank test samples and test samples spiked at the Screening Target Concentration (STC) level, with the objective to achieve PODs ≤ 10% and ≥ 90%, respectively. Additionally, the effectiveness of the screening method was assessed through participation to 92 proficiency test samples. METHODS: Four streams were optimized to screen for 152 veterinary drug residues by LC-MS/MS in a wide variety of food commodities including milk-based ingredients and related products (e.g., milk fractions, infant formula, infant cereals and baby foods), meat- and fish-based ingredients and related products (fresh, powdered, cooked, infant cereals and baby foods) and other ingredients such as eggs, animal fat and animal byproducts. The four streams covered 105 antibiotic residues, anti-inflammatory and antiparasitic agents (Stream A); 23 Beta-lactams (Stream B); 14 Aminoglycosides (Stream C) and 10 Tetracyclines (Stream D). RESULTS: The multi-laboratory validation led to PODs at the STC ≥ 94% and PODs in the blank ≤ 9%. Further application of the multi-laboratory validated method to 92 proficiency tests provided more than 99% satisfactory submitted results (n = 784). CONCLUSION: The inter-laboratory reproducibility determined for this method met the acceptance criteria defined in AOAC SMPR 2018.010. HIGHLIGHTS: AOAC has approved the method for Final Action Status.

Screening of 154 Veterinary Drug Residues in Foods of Animal Origin Using LC-MS/MS: First Action 2020.04.

BACKGROUND: Veterinary drug residues in food are substances (>200 compounds) exhibiting potential health risks for consumers, thus being regulated in national legislations and the Codex Alimentarius. Most of the compounds are regulated based upon a maximum residue limit (MRL) while a few of them are banned in food for humans. The food sector needs a reliable and consensus analytical platform able to monitor these substances in a wide range of food commodities. OBJECTIVE: Several confirmatory methods based on liquid chromatography-mass spectrometry are available in the literature for either screening or quantification of veterinary drug residues in food, but usually applicable to limited scope of matrices. The current work describes the single-laboratory validation (SLV) of a method for screening 154 veterinary drug residues in several food categories. METHODS: This work describes a streamlined platform making use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for screening 105 antibiotics, 41 antiparasitics, 5anti-inflammatory agents, and 3 tranquilizers in foods of animal origin. For the best performance across the commodities (dairy-, meat-, fish-, and egg-based materials), four method streams were established. As a screening tool, probabilities of detection (PODs) were assessed at the screening target concentration (STC < MRL) and the blank. RESULTS: The SLV led to PODs at the STC >94% and PODs in the blank < 4%. CONCLUSION: Performance is in agreement with the acceptance criteria defined in SMPR 2018.010. HIGHLIGHTS: The Expert Review Panel approved the present method as AOAC Official First Action 2020.04.

Levels of Alternaria Toxins in Selected Food Commodities Including Green Coffee.

Alternaria toxins are emerging mycotoxins, candidates for regulation by European Authorities. Therefore, highly sensitive, confirmatory, and reliable analytical methodologies are required for their monitoring in food. In that context, an isotope dilution LC-MS/MS method was developed for the analysis of five Alternaria toxins (Altenuene, Alternariol, Alternariol monomethylether, Tentoxin, and Tenuazonic Acid) in a broad range of commodities including cereals and cereal-based products, tomato-based products, tree nuts, vegetable oils, dried fruits, cocoa, green coffee, spices, herbs, and tea. Validation data collected in two different laboratories demonstrated the robustness of the method. Underestimation of Tenuazonic Acid level in dry samples such as cereals was reported when inappropriate extraction solvent mixtures were used as currently done in several published methodologies. An investigation survey performed on 216 food items evidenced large variations of Alternaria toxins levels, in line with data reported in the last EFSA safety assessment. The analysis of 78 green coffee samples collected from 21 producing countries demonstrated that coffee is a negligible source of exposure to Alternaria toxins. Its wide scope of application, adequate sample throughput, and high sensitivity make this method fit for purpose for the regular monitoring of Alternaria toxins in foods.

Development, validation and application of a LC-MS/MS method for quantification of 15 cannabinoids in food.

In this study, the occurrence of cannabinoids in hemp-based food products was investigated. For that purpose, a new liquid chromatography tandem mass spectrometry method for the quantification of fifteen cannabinoids was developed and validated for multiple matrices. Method performances were good, fulfilling the SANTE/11813/2017 requirements, and allowing for products compliance testing with various national legislations on cannabinoids levels in food products. The limit of quantification of each analyte was 0.15 mg/kg for hemp seed and hemp protein, 0.6 mg/kg for hemp seed oil, and 0.005 mg/kg for raw milk and milk powder. The applicability of the method was further demonstrated by conducting a limited survey on twenty hemp-based food products. The survey revealed that products from the same category can have very different cannabinoids profiles and levels. These results highlighted the importance of cannabinoids testing of food products in view of the current heterogeneous and fast evolving regulatory landscape worldwide.

A new high-throughput screening method to determine multiple dyes in herbs and spices.

The unauthorised addition of colours to herbs and spices is a recurrent issue affecting food business operators. Such a practice aims at improving food visual attractiveness, masking poor product quality, and/or compensating for natural colour variation with the ultimate goal to increase profits. To detect this fraud, a new LC-MS/MS method was developed for screening 58 dyes in both herbs and spices. This extended list of targets was established based on requirements from international spices organisations, past issues identified by web scouting and by notifications from the European Rapid Alert System for Food and Feed (RASFF). The method is intended to quickly detect fraudulent addition of dyes with Screening Target Concentrations ranging from 0.1 to 2.5 mg/kg. Validation was performed according to the European Community Reference Laboratories Residues Guidelines 20/1/2010. False positive and false negative rates were below 5% for all analytes and applicability of the method was further demonstrated by analysing 117 samples collected worldwide. None of the surveyed dyes was found in herbs (n = 28, 16 varieties) whereas 6% of spice samples (n = 89, 21 varieties) was found contaminated with one or two dyes at levels ranging from 0.12 to 255 mg/kg. Four out of the nine detected compounds have never been reported in the RASFF, thus demonstrating the usefulness of this analytical approach.

Determination of 14 aminoglycosides by LC-MS/MS using molecularly imprinted polymer solid phase extraction for clean-up.

An LC-MS/MS method for screening 14 aminoglycosides in foodstuffs of animal origin is presented. Its scope includes raw materials and processed ingredients but also finished products composed of milk, meat, fish, egg or fat. Aminoglycosides are extracted in an acidic aqueous solution, which is first recovered after centrifugation, then diluted with a basic buffer and finally purified by molecularly imprinted polymer-solid phase extraction (MIP-SPE). Analytes are detected within 8 min by ion-pair reversed phase LC-MS/MS. Due to the large range of foodstuffs involved, the variability of matrix effects led to significant MS signal variations. This was circumvented by systematically extracting each sample twice, i.e. 'unspiked' and 'spiked' at the screening target concentration of 50 µg kg-1. The method was validated according to the European Community Reference Laboratories Residues Guidelines giving false-negative and false-positive rates ≤3% for all compounds. Ruggedness of the method was further demonstrated in quality control operations by a second laboratory. The 14 aminoglycosides in water-based standard solutions were stable for up to 6 months when stored at either -80°C, -20°C or at 4°C storage temperatures.

Determination of five tetracyclines and their epimers by LC-MS/MS based on a liquid-liquid extraction with low temperature partitioning.

An LC-MS/MS method is presented for screening five tetracyclines and their epimers in a broad range of food products. The scope of matrices includes meat-, fish-, seafood-based products, various dairy ingredients, infant formulae and fats. The method principle is based on a liquid-liquid extraction with aqueous ethylenediaminetetraacetic acid (EDTA) and acetonitrile followed by a freezing step to promote phase separation at low temperature. After defatting with hexane, sample extracts were evaporated and reconstituted before injection onto the LC-MS/MS system. The addition of oxalic acid in the aqueous mobile phase was mandatory to maintain good peak shape and sensitivity over the run. The screening is based upon a double preparation of each sample, one 'as such' and a second one with the analytes spiked in the sample, in order to mitigate the risk of false negative response. The method was validated according to the European Community Reference Laboratories Residues Guidelines. A total of 93 samples were included in the validation by two independent laboratories giving both false-negative and false-positive rates at 0% for all compounds. Over the last two years, 2600 samples were analysed routinely and only one chicken sample was found above the regulatory limit.

Screening of 23 ß-lactams in foodstuffs by LC-MS/MS using an alkaline QuEChERS-like extraction.

A fast and robust high performance LC-MS/MS screening method was developed for the analysis of ß-lactam antibiotics in foods of animal origin: eggs, raw milk, processed dairy ingredients, infant formula, and meat- and fish-based products including baby foods. QuEChERS extraction with some adaptations enabled 23 drugs to be simultaneously monitored. Screening target concentrations were set at levels adequate to ensure compliance with current European, Chinese, US and Canadian regulations. The method was fully validated according to the European Community Reference Laboratories Residues Guidelines using 93 food samples of different composition. False-negative and false-positive rates were below 5% for all analytes. The method is adequate for use in high-routine laboratories. A 1-year study was additionally conducted to assess the stability of the 23 analytes in the working standard solution.

Determination of 105 antibiotic, anti-inflammatory, antiparasitic agents and tranquilizers by LC-MS/MS based on an acidic QuEChERS-like extraction.

A procedure for screening 105 veterinary drugs in foods by liquid chromatography tandem mass-spectrometry (LC-MS/MS) is presented. Its scope encompasses raw materials of animal origin (milk, meat, fish, egg and fat) but also related processed ingredients and finished products commonly used and manufactured by food business operators. Due to the complexity of the matrices considered and to efficiently deal with losses during extraction and matrix effects during MS source ionisation, each sample was analysed twice, that is 'unspiked' and 'spiked at the screening target concentration' using a QuEChERS-like extraction. The entire procedure was validated according to the European Community Reference Laboratories Residues Guidelines. False-negative and false-positive rates were below 5% for all veterinary drugs whatever the food matrix. Effectiveness of the procedure was further demonstrated through participation to five proficiency tests and its ruggedness demonstrated in quality control operations by a second laboratory.

Stability study of veterinary drugs in standard solutions for LC-MS/MS screening in food.

A study on stability of veterinary drugs in standard solutions stored at -80°C and at -20°C was conducted over 1 year. Data were acquired on 152 individual stock standard solutions and also on 15 family mixes and 2 working standard solutions. All solutions were prepared, stored and compared 1 year later against freshly prepared ones by LC-MS/MS. A statistical analysis was performed to set the acceptability criteria, taking into account the variability of standard preparations. In individual stock standard solutions stored at -80°C (12 months) and -20°C (9 months), stability was demonstrated for 141 and 140 out of 152 compounds, i.e. for 92% and 93% of compounds, respectively. Drugs were even more stable when solubilised in either diluted family mixes or working standard solutions, with more than 99% and 94% of compounds found unaltered when stored at -80°C and at -20°C, respectively. In mixes, beta-lactams from the cephalosporin (cefadroxil and cephalexin) and penicillin (amoxicillin and ampicillin) families were found to be the least stable compounds when stored at -20°C (6 months), necessitating storage at -80°C to achieve a 1-year shelf life. The study also evidenced solubility issues for two sulfonamides (sulfadiazine and sulfamerazine) in methanol-based solutions. An independent stability study conducted by a second laboratory confirmed the 1-year stability of 3 family mixes-quinolones, sulfonamides and tetracyclines.