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.

Quantification of Chlorate and Perchlorate in a Broad Range of Food Commodities, Including Baby Food, Nutritional Formulas, and Ingredients by LC-MS/MS: First Action AOAC 2022.06.

BACKGROUND: Chlorate is an effective herbicide, but also a byproduct of chlorinating agents used to disinfect water, which is one of the reasons why it is regularly found in food. Perchlorate is a ubiquitous contaminant, which is naturally occurring in the environment but also released from anthropogenic sources such as the industrial use of certain natural fertilizers. Chlorate affects the hematological system, and perchlorate the thyroid. OBJECTIVE: Implement and validate a simple and robust analytical method for the accurate determination of chlorate and perchlorate in baby food, infant and adult formulas, and ingredients thereof, which is suited for its application in routine environments where a broad variety of food commodities must be analyzed simultaneously. METHOD: Typically, analytes are extracted with a mixture of water, acidified methanol, and dichloromethane. Optionally, for dairy products and byproducts, extraction can be performed with water, acidified methanol, and EDTA, followed by two steps of cleanup (freezing out and dispersive solid-phase extraction with C18 in acetonitrile). Quantitative determination is carried out by isotopic dilution liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: The method was single-laboratory validated in five Nestlé Quality Assurance Centers (NQACs) in a comprehensive range of representative matrixes of different categories such as baby foods, infant/adult formulas, and ingredients, with results generally in agreement with the acceptance criteria of the Standard Method Performance Requirement (SMPR®) 2021.001 defined by AOAC INTERNATIONAL, in terms of representative matrixes validated, LOQs, trueness, and precision.The data generated during validation show that the method proposed is simple, accurate and robust enough to be implemented and applied in routine environments. CONCLUSION: The data generated during validation show that the method proposed is simple, accurate and robust enough to be implemented and applied in routine environments. HIGHLIGHTS: The AOAC Expert Review Panel approved the present method as AOAC Official First Action 2022.06.

An LC-MS/MS method for the quantitative determination of 57 per- and polyfluoroalkyl substances at ng/kg levels in different food matrices.

To enable the monitoring of a wide scope of per- and polyfluoroalkyl substances (PFAS) in the ng/kg level in foodstuffs, an LC-MS/MS method comprising 57 analytes was developed and validated in seven different matrices (milk powder, milk-based infant formula, meat-based baby food puree, fish and fish oil, fresh egg, and soluble coffee). The analytical approach was based on an acetonitrile:water extraction followed by solid phase extraction clean-up with subsequent quantification of the extracted analytes either by isotope dilution (55 compounds) or by standard addition (2 compounds) mass spectrometry. The validation criteria followed the guidance document for the analysis of PFAS issued by the European Union Reference Laboratory for Halogenated Persistent Organic Pollutants. The lowest limits of quantification (LOQs) for the four recently regulated compounds (L-PFOS, PFOA, PFNA, L-PFHxS) were set at 0.010 µg/kg in baby and infant foods (as sold) but also in dairy ingredients. Exception was for PFOA in milk powder due to too large variability in the repeatability. Applicability of the method was further demonstrated in 37 commodity check matrices. Overall validation data demonstrated the robustness of the method for most of the compounds and the LOQs achieved were low enough to ensure compliance with Commission Regulation EU 2022/2388 but also to support future collection of occurrence data in ng/kg level in food.

Inadequate definition of the limit of quantification used for the analysis of perfluoroalkyl substances in food by liquid chromatography-tandem mass spectrometry may compromise the reliability of the data requested by the European regulation.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a widespread technology used for the quantitative determination of per- and polyfluoroalkyl substances (PFAS) in foodstuff. Specifically, LC-MS/MS offers an attractive performance by combining the sensitivity and selectivity required by the European Union for testing perfluorooctane sulfonic acid, perfluorooctanoic acid, perfluorononanoic acid, and perfluorohexane sulfonic acid with maximum limits of quantification (LOQ) in the sub-parts-per-billion (µg/kg) or the parts-per-trillion (ng/kg) domains. In this article, we highlight the important diversity in LOQ definitions applied in LC-MS/MS methods described in the literature that raise concerns about the capability of some of those to generate reliable data requested by the European regulation. Here, we point out the risk of false response or misquantification if the criteria for assessing LOQ suffer from a lack of rigor. We emphasize the need to use PFAS-free samples spiked with the analyte(s) of interest and the application of identification criteria according to official documents for a sound measurement of the LOQ.

Confirmation of the full conversion of ethylene oxide to 2-chloroethanol in fumigated foodstuffs: possible implications for risk assessment.

This study describes the extension of a gas chromatography mass spectrometry (GC-MS) method, initially devoted to the analysis of ethylene oxide (EO) in ice cream, to a larger range of food items including herbs, spices, vegetables, inorganic salts, food supplements, thickeners, etc. Results are reported as EOTotal according to EC 2015/868 definition (expressed as EO equivalents as the sum of native EO and 2-chloroethanol (2-CE) after acidic hydrolysis) with a limit of quantification at 0.01 mg/kg regardless of the food item. Its ruggedness was demonstrated through fortification experiments on hundreds of samples. Re-analysis of 146 positive food samples without hydrolysis demonstrated that not EO but 2-CE is the predominant analyte detected in the different processed ingredients suspected to have been previously treated with EO. A series of eight contaminated dried herbs and spices were also re-analysed by four ISO 17025 accredited commercial laboratories making use of different analytical strategies for EO determination in foods. Each laboratory reported EOTotal levels within the same concentration range, but the resulting reproducibility ranged from 23% to 41% depending on the sample. Additionally, we show that results of free EO from methods based on conversion to 2-iodoethanol may lead to artefactual detection of native EO (false positive). An official method of analysis applicable for different food matrices would be useful to avoid discrepancies of results. Altogether, these data re-enforce the fact that in absence of native EO in food items, risk assessment of EO in foodstuffs should consider the predominance of 2-CE. A toxicological risk assessment using the food additive xanthan gum as a case study is discussed.

Rapid and Reliable Data Treatment for the Control of Food Chemical Contaminants by LC-HRMS.

Liquid chromatography-high resolution mass spectrometry (LC-HRMS) is considered an unavoidable extension of low-resolution LC-MS/MS that stretches the capabilities of multi-residue analysis of chemical contaminants in food. However, LC-HRMS acquisitions generate a massive amount of information available for data processing with supplier software that still miss critical calculation features and adapted reporting tools. Consequently, routine laboratories are still reluctant to switch from LC-MS/MS to LC-HRMS, the latter is still perceived as a cumbersome and demanding technology. In that context, we propose a four-step LC-HRMS workflow to speed-up data processing in situations of multi-residue multi-matrix analysis with the goal to maximize the time spent on data interpretation rather than on data formatting. The first three steps of the workflow imply specific settings on the Orbitrap HRMS associated software (TraceFinderTM) while the fourth step is the novelty i.e. a newly coded R-script capable to translate a raw export file into a comprehensive .xlsx report file in a few seconds. As recommended by various international guidelines and in some official methods, standard addition-based applications are fully embedded in this reporting tool whilst still being the main bottleneck of supplier's software. The reporting tool also allows appropriate data formatting, filtering, and color-coding options to provide a clear picture of compounds being detected or not, and those requiring specific attention due to unmet quality control criteria as required by European legislation (European Commission SANTE 11312/2021). It is hoped that additional functionalities compatible with R scripts will be soon fully embedded in the supplier's software for easier data interpretation and reporting.

Analysis of ethylene oxide in ice creams manufactured with contaminated carob bean gum (E410).

Residues of ethylene oxide (EO), a banned fumigant in the EU, were found at amounts above the maximum residue limit (MRL) in carob (locust) bean gum (additive E410). The pesticide entered the food chain via stabiliser blends that are used as minor ingredients in the manufacture of ice cream. Consequently, all products that contained the non-compliant ingredient were withdrawn or recalled in several countries across the EU, in most cases irrespective of whether the pesticide residue was detectable or not in the final product. This is the first report of a reliable method to determine EO and its metabolite/marker compound 2-chloroethanol (2-CE), either together or independently in ice cream, with a limit of quantification at 0.01 mg EO/kg and recovery in the range of 87-104% across the levels investigated (0.01, 0.02 and 0.06 mg EO/kg). The method applies QuEChERS extraction and isotope dilution gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). High resolution mass spectrometry (HRMS) confirmed the specificity of low mass ions. Data on the stability of EO and 2-CE under thermal conditions revealed that 2-CE is relatively stable in an ice cream matrix (ca. 80% recovery of spiked material). Importantly, this study also demonstrates that not EO, but 2-CE is the predominant analyte detected in the contaminated samples, which is new information of significance in terms of the overall risk assessment of EO in foodstuffs.

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.

Multiple Mycotoxins Determination in Food by LC-MS/MS: An International Collaborative Study.

An intercollaborative study was organized to evaluate the performance characteristics of a liquid chromatography tandem mass spectrometry procedure for the simultaneous determination of 12 mycotoxins in food, which were ochratoxin A, aflatoxins B1, B2, G1, G2, and M1, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins. The method combined the simplicity of the QuEChERS (Quick, Easy, Cheap, Efficient, Rugged and Safe) approach with the efficiency of immunoaffinity column cleanup (the step used to enhance sensitivity and sample cleanup for some matrices only). Twenty-three entities were enrolled and were European reference laboratories for mycotoxin analysis, U.S. and European service laboratories, and Nestlé laboratories. Each participant analyzed 28 incurred and/or spiked blind samples composed of spices, nuts, milk powder, dried fruits, cereals, and baby food using the protocol given. Method performances were assessed according to ISO 5725-2. Relative standard deviations of repeatability and reproducibility and trueness values for each of the 115 mycotoxin/sample combinations ranged from 5% to 23%, 7% to 26%, and 85% to 129%, respectively, in line with requirements defined in EC 401/2006. The overall set of data gathered demonstrated that the method offered a unique platform to ensure compliance with EC 1881/2006 and EC 165/2013 regulations setting maximum limits for mycotoxins in food samples, even at low regulated levels for foods intended for infants and young children. The method was applicable regardless of the food, the regulated mycotoxin, and the concentration level, and thus is an excellent candidate for future standardization.

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.

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.

Process-induced formation of imidazoles in selected foods.

The presence of 4-methylimidazole (4-MEI), 2-methylimidazole (2-MEI) and 2-acetyl-4-tetrahydroxybutylimidazole (THI) in some foods may result from the usage of caramel colorants E150c and E150d as food additives. This study demonstrates that alkylimidazoles are also byproducts formed from natural constituents in foods during thermal processes. A range of heat-processed foods that are known not to contain caramel colorants were analyzed by isotope dilution LC-MS/MS to determine the contamination levels. Highest 4-MEI concentrations (up to 466µg/kg) were observed in roasted barley, roasted malt and cocoa powders, with the concomitant presence of 2-MEI and/or THI in some cases, albeit at significantly lower levels. Low amounts of 4-MEI (<20µg/kg) were also detected in cereal-based foods such as breakfast cereals and bread toasted to a brown color (medium toasted). The occurrence of 4-MEI in certain processed foods is therefore not a reliable indicator of the presence of the additives E150c or E150d.

Determination of steroid hormones in bovine milk by LC-MS/MS and their levels in Swiss Holstein cow milk.

Synthetic and natural steroid hormones have attracted some attention in recent years as endocrine active substances (EAS) that interact or interfere with the endocrine system. Endogenous hormones occur naturally in food of animal origin, among which bovine milk represents an important source. This study was conducted to determine the occurrence of steroid hormones (oestrogens, androgens, progestogens and glucocorticoids) in cow's milk samples from three farms in Switzerland. An isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of 12 hormones in milk. Some hormonal levels from individual cows showed large variations. The average levels of the hormones analysed (17α-estradiol = 31 ng kg(-)(1), 17ß-estradiol = 6 ng kg(-)(1), estrone = 159 ng kg(-)(1), 4-androstenedione = 684 ng kg(-)(1), progesterone = 15486 ng kg(-)(1), 17-hydroxyprogesterone = 214 ng kg(-)(1), cortisone = 112 ng kg(-)(1), and cortisol = 235 ng kg(-)(1)) were comparable with literature data. Estriol, testosterone and androstenediols were not detected at their respective limit of quantification. No significant differences of hormonal content among milk from cows at different lactation/calving numbers were evidenced, except for progesterone and 4-androstenedione. Due to confounding parameters linked to the physiological stage of the animal, like pregnancy and gestational stage (pregnancy trimester), the causal correlation between the variation of the levels for these two hormones and the lactation/calving number could not be unambiguously demonstrated.

Quantitative determination of sodium monofluoroacetate "1080" in infant formulas and dairy products by isotope dilution LC-MS/MS.

A fast and easy-to-use confirmatory liquid-chromatography tandem mass-spectrometry (LC-MS/MS) based-method was developed for the analysis of the pesticide sodium monofluoroacetate (MFA, also called "1080") in infant formulas and related dairy products. Extraction of the compound encompassed sample reconstitution and liquid-liquid extraction under acidic conditions. Time-consuming solid-phase extraction steps for clean-up and enrichment and tedious derivatisation were thus avoided. Resulting sample extracts were analysed by electrospray ionisation (ESI) in negative mode. Quantification was performed by the isotopic dilution approach using (13)C-labelled MFA as internal standard. The procedure was validated according to the European document SANCO/12571/2013 and performance parameters such as linearity (r(2) > 0.99), precision (RSD(r) ≤ 9%, RSD(iR) ≤ 11%) and recovery (96-117%) fulfilled its requirements. Limit of quantifications (LOQ) was 1 µg kg(-1) for infant formulas and related dairy products except for whey proteins powders with a LOQ of 5 µg kg(-1). Method ruggedness was further assessed in another laboratory devoted to routine testing for quality control.