Development of a new generic extraction method for the analysis of pesticides, mycotoxins, and polycyclic aromatic hydrocarbons in representative animal feed and food samples.

Various generic extraction methods have been used to determine pesticide residues, mycotoxins, and polycyclic aromatic hydrocarbons (PAHs) in food and animal feed to ensure consumer safety. However, these methods cannot extract all relevant compounds at an acceptable rate of recovery. This study presents a new extraction method. This new method facilitated the identification of 231 compounds, including 196 pesticides, 11 mycotoxins, and 24 PAHs over a broad range of polarities. These compounds were identified in various sample matrices, including those that are lipid-rich. The processed sample is first extracted with water, acetonitrile, formic acid, and heptane. The addition of ammonium formate results in separation into three phases and enables analysis of the aqueous phase. Solid-phase extraction clean-up procedures were performed as necessary followed by analysis by liquid or gas chromatography and mass spectrometry. Analyte recoveries were typically in the range of 70 - 120% with relative standard deviations below 20%.

Pesticide residues in European agricultural soils - A hidden reality unfolded.

Pesticide use is a major foundation of the agricultural intensification observed over the last few decades. As a result, soil contamination by pesticide residues has become an issue of increasing concern due to some pesticides' high soil persistence and toxicity to non-target species. In this study, the distribution of 76 pesticide residues was evaluated in 317 agricultural topsoil samples from across the European Union. The soils were collected in 2015 and originated from 11 EU Member States and 6 main cropping systems. Over 80% of the tested soils contained pesticide residues (25% of samples had 1 residue, 58% of samples had mixtures of two or more residues), in a total of 166 different pesticide combinations. Glyphosate and its metabolite AMPA, DDTs (DDT and its metabolites) and the broad-spectrum fungicides boscalid, epoxiconazole and tebuconazole were the compounds most frequently found in soil samples and the compounds found at the highest concentrations. These compounds occasionally exceeded their predicted environmental concentrations in soil but were below the respective toxic endpoints for standard in-soil organisms. Maximum individual pesticide content assessed in a soil sample was 2.05 mg kg-1 while maximum total pesticide content was 2.87 mg kg-1. This study reveals that the presence of mixtures of pesticide residues in soils are the rule rather than the exception, indicating that environmental risk assessment procedures should be adapted accordingly to minimize related risks to soil life and beyond. This information can be used to implement monitoring programs for pesticide residues in soil and to trigger toxicity assessments of mixtures of pesticide residues on a wider range of soil species in order to perform more comprehensive and accurate risk assessments.

Comparison of gas chromatography/quadrupole time-of-flight and quadrupole Orbitrap mass spectrometry in anti-doping analysis: I. Detection of anabolic-androgenic steroids.

RATIONALE: The World Anti-Doping Agency (WADA) encourages drug-testing laboratories to develop screening methods that can detect as many doping substances as possible in urine. The use of full-scan high-resolution acquisition (FS/HR) with gas chromatography/mass spectrometry (GC/MS) for the detection of known and unknown trimethylsilyl (TMS) derivatives of anabolic-androgenic steroids (AAS) provides anti-doping testing bodies with a new analytical tool. METHODS: The AAS were extracted from urine samples by generic liquid-liquid extraction, after enzymatic hydrolysis, and TMS derivatization. The extracted urine was analyzed by GC/Q-TOF and GC/Q-Orbitrap to compare the performance of the two instrument types for the detection of 46 AAS in human urine. The quantitation of endogenous anabolic steroids and the ability of the two analytical platforms to comply with the requirements for testing as part of the WADA Athlete Biological Passport (ABP) were also assessed. RESULTS: The data presented show that the analytical performance for both instruments complies with the WADA specifications. The limits of detection (LODs) for both instruments are well below the WADA 50% Minimum Required Performance Levels. The mass errors in the current study for the GC/Q-Orbitrap platform are lower than those obtained for the GC/Q-TOF instrument. CONCLUSIONS: The data presented herein proved that both molecular profiling platforms can be used for antidoping screening. The mass accuracies are excellent in both instruments; however, the GC/Q-Orbitrap performs better as it provides higher resolution than the GC/Q-TOF platform.

Application of Gas Chromatography Coupled to Quadrupole-Orbitrap Mass Spectrometry for Pesticide Residue Analysis in Cereals and Feed Ingredients.

A method for residue analysis of pesticides and polychlorinated biphenyls in cereals and feed ingredients based on QuEChERS extraction, programmed temperature vaporizer large-volume injection, and GC with electron ionization (EI) quadrupole Orbitrap full-scan high-resolution MS (60 000 full width at half-maximum at m/z 200) has been developed. In addition to full-scan acquisition, simultaneous full-scan and selected-ion monitoring acquisition was used to improve detectability in incidental cases in which analytes coeluted with intense signals from coextractants. The method was successfully validated down to 10 µg/kg for a single commodity (wheat) using matrix-matched calibration, and for multiple-feed matrixes using standard addition. Identification according to European Union requirements was achieved in >90% of the analyte/matrix combinations, and suggestions for further increasing identification rates have been made. Performance characteristics were compared to an existing method for residue analysis based on GC with EI tandem MS (triple quadrupole).

Evaluation of gas chromatography - electron ionization - full scan high resolution Orbitrap mass spectrometry for pesticide residue analysis.

Gas chromatography with electron ionization and full scan high resolution mass spectrometry with an Orbitrap mass analyzer (GC-EI-full scan Orbitrap HRMS) was evaluated for residue analysis. Pesticides in fruit and vegetables were taken as an example application. The relevant aspects for GC-MS based residue analysis, including the resolving power (15,000 to 120,000 FWHM at m/z 200), scan rate, dynamic range, selectivity, sensitivity, analyte identification, and utility of existing EI-libraries, are assessed and discussed in detail. The optimum acquisition conditions in full scan mode (m/z 50-500) were a resolving power of 60,000 and an automatic-gain-control target value of 3E6. These conditions provided (i) an optimum mass accuracy: within 2 ppm over a wide concentration range, with/without matrix, enabling the use of ±5 ppm mass extraction windows (ii) adequate scan speed: minimum 12 scans/peak, (iii) an intra-scan dynamic range sufficient to achieve LOD/LOQs ≤0.5 pg in fruit/vegetable matrices (corresponding to ≤0.5 µg kg(-1)) for most pesticides. EI-Orbitrap spectra were consistent over a very wide concentration range (5 orders) with good match values against NIST (EI-quadrupole) spectra. The applicability for quantitative residue analysis was verified by validation of 54 pesticides in three matrices (tomato, leek, orange) at 10 and 50 µg/kg. The method involved a QuEChERS-based extraction with a solvent switch into iso-octane, and 1 µL hot splitless injection into the GC-HRMS system. A recovery between 70 and 120% and a repeatability RSD <10% was obtained in most cases. Linearity was demonstrated for the range ≤5-250 µg kg(-1). The pesticides could be identified according to the applicable EU criteria for GC-HRMS (SANTE/11945/2015). GC-EI-full scan Orbitrap HRMS was found to be highly suited for quantitative pesticide residue analysis. The potential of qualitative screening to extend the scope makes it an attractive alternative to GC-triple quadrupole MS.

Validation of an automated screening method for persistent organic contaminants in fats and oils by GC×GC-ToFMS.

An screening method, comprised of straightforward sample treatment based on silica clean-up, GC×GC-ToFMS detection and automated data processing with the non-proprietary free downloadable software MetAlignID, has been successfully validated with respect to false negatives for the sum PCB 28, 52, 101, 138, 153 and 180), for the sum of BDE 28, 47, 99, 100, 153, 154 and 183, for the four markers of PAHs and for a number of emerging brominated flame retardants. A screening detection limit (SDL) equal to or lower than the maximum regulatory level was always achieved. MetAlignID considerably decreased the time needed for data treatment from 20 to 5min/file. Automated identification of the signature mass spectral patterns was applied to identify chlorinated- and brominated-containing substances with more than two halogen atoms, and PAH derivates. Although the success rate was variable and needs to be further improved, the tool was considered to be of added value.

Implementation of toxicokinetics in toxicity studies--Toxicokinetics of 4-methylanisole and its metabolites in juvenile and adult rats.

The current risk assessment of compounds is generally based on external exposure and effect relationships. External doses are often not representative for internal exposure concentrations. The aim of this study was to show how the implementation of toxicokinetics in a scheduled toxicity study contributes to improved data interpretation without additional use of animals and to the three goals of the 3R principles for animal testing. Toxicokinetic analyses were implemented in a rat developmental immunotoxicity study with 4-methylanisole without interfering with the outcome of the study and without the use of additional animals. 4-Methylanisole and its metabolites were analysed in plasma of adult rats and in pups at postnatal day 10. 4-Methylanisole has a short half-life in adult animals and the plasma concentrations increased more than proportional with increasing dose. The metabolic profile appeared to be different at low dose as compared to high dose. This information on the dose-proportionality of the internal exposure is crucial for the interpretation of the toxicity data and helps to identify the toxic agent and the appropriate dose metric. The metabolism was similar in adult and juvenile animals. Large inter-individual variability in adult animals, as observed for 4-methylanisole, may hamper dose-response analyses of the results. In addition, 4-metylanisole was excreted via milk, but concentrations in the juvenile animals appeared to be 20- to 100-fold lower than via direct gavage exposure. The toxicokinetic parameters support the data interpretation, among others by providing better insight into internal exposures. Subsequently, it will help to prevent testing of irrelevant exposure scenarios and exposure concentrations. Overall, implementation of kinetics with limited effort provides useful information to support the interpretation of toxicological data and can contribute to reduction and refinement of animal testing.

Urine metabolomics combined with the personalized diagnosis guided by Chinese medicine reveals subtypes of pre-diabetes.

The prevalence of type 2 diabetes continuously increases globally. A personalized strategy applied in the pre-diabetic stage is vital for diabetic prevention and management. The personalized diagnosis of Chinese Medicine (CM) may help to stratify the diabetics. Metabolomics is regarded as a potential platform to provide biomarkers for disease-subtypes. We designed an explorative study of 50 pre-diabetic males, combining GC-MS urine metabolomics with CM diagnosis in order to identify diagnostic biomarkers for pre-diabetic subtypes. Three CM physicians reached 85% diagnosis consistency resulting in the classification of 3 pre-diabetic groups. The urine metabolic patterns of groups 1 'Qi-Yin deficiency' and 2 'Qi-Yin deficiency with dampness' (subtype A) and group 3 'Qi-Yin deficiency with stagnation' (subtype B) were clearly discriminated. The majority of metabolites (51%), mainly sugars and amino acids, showed higher urine levels in subtype B compared with subtype A. This indicated more disturbances of carbohydrate metabolism and renal function in subtype B compared with subtype A. No differences were found for hematological and biochemical parameters except for levels of glucose and γ-glutamyltransferase that were significantly higher in subtype B compared with subtype A. This study proved that combining metabolomics with CM diagnosis can reveal metabolic signatures for pre-diabetic subtypes. The identified urinary metabolites may be of special clinical relevance for non-invasive screening for subtypes of pre-diabetes, which could lead to an improvement in personalized interventions for diabetics.