DART mass spectrometry: a rapid tool for the identification of feed additives containing coccidiostats as active substances.

Feed additives require pre-market authorisation prior to their use in the EU. For the group of coccidiostats, the EU regulations authorising these products include specifications for these substances and the major components of the feed additive formulations. Feed business operators can use only feed additives that meet these criteria. The traceability of these products is supported by the allocation of specific identification numbers that need to be printed on the feed additive label along with other information. In the present study, Direct Analysis in Real Time mass spectrometry (DART-MS) was applied to investigate the correct characterisation of feed additives that contain coccidiostats as active substances. The results of the study demonstrated that this technique allows an unequivocal identification of the active substances in the feed additive formulations when combining DART with high-resolution mass spectrometry. In addition, two feed additives containing the same coccidiostat, but different excipients could be correctly classified according to their composition. The method protocol is very simple and comprises two steps, namely the extraction of the feed additives with an organic solvent and the subsequent measurement with DART-MS. For the evaluation of the MS spectra, chemometrics was applied offering an effective method for classification. Chemometric models were established with nominal masses obtained from the analysis of the samples, thus showing that these feed additives could be correctly classified even using low-resolution mass spectrometry. Moreover, we demonstrated that molecule-specific stable isotope patterns obtained with low-resolution mass spectrometry could be used as an alternative tool for the confirmation of the active substance.

Determination and confirmation of selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors in bovine and porcine urine using UHPLC-MS/MS.

Selective estrogen receptor modulators (SERMs), anti-estrogens and aromatase inhibitors are prohibited in human sports doping. However, they also present a risk of being used illegally in animal husbandry for fattening purposes. A method was developed and validated using UHPLC-MS/MS for the determination and confirmation of SERMs, anti-estrogens and aromatase inhibiters in bovine and porcine urine. This method was used in a survey of more than 200 bovine and porcine urine samples from Dutch farms. In 18 out of 103 porcine urine samples (17%) and two out of 114 bovine samples (2%) formestane, an aromatase inhibitor, was detected. None of the other compounds was detected. From human doping control it is known that formestane can, in some cases, be of natural origin. Analyses of reference samples from untreated bovine and porcine animals demonstrated the presence of formestane in bovine animals, but not yet in porcine animals. Future research will focus on whether the detected formestane in porcine and bovine urine is from endogenous or exogenous origin, using GC-c-IRMS.

High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory.

The occurrence of 67 pharmaceutical and antifungal residues in the Danube river on the Romanian territory was studied by using solid-phase extraction (SPE) and LC-Q Exactive Orbitrap high resolution MS in both full scan (FS) MS and targeted MS/MS modes. A single-laboratory validation procedure was carried out for the determination of 67 compounds in FSMS mode evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance. The evaluation of the recovery concluded that 75% of the compounds show recoveries between 85 and 115% and 10% of the compounds show recoveries between 85% and 65%. The level of detection was lower than 5 ng l(-1) for 66% of the compounds, between 5 and 10 ng l(-1) for 22% and between 10 and 25 ng l(-1) for 14% of the compounds. The coefficients of determination R(2) were higher than 0.99 for 79% of the compounds, over a linearity range of 2.5-50 ng l(-1). Targeted MS/MS analysis, performed in addition to the full scan acquisition was used for confirmatory purpose. Twenty samples of Danube water and three of the main tributaries were collected in May, July, August and October 2014. Analysis of the selected water samples revealed the occurrence of 23 compounds such as diclofenac, carbamazepine, sulfamethoxazole, tylosin, indomethacin, ketoprofen, piroxicam, together with antifungals like thiabendazole, and carbendazim. Carbamazepine was detected in 17 samples, the maximum concentration being 40 ng l(-1). The highest concentration reached was 166 ng l(-1) for diclofenac.

A multi-residue method for determination of 70 organic micropollutants in surface waters by solid-phase extraction followed by gas chromatography coupled to tandem mass spectrometry.

A multi-residue method, based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), has been developed for the determination of 70 organic micropollutants from various chemical classes (organochlorinated, organophosphorous, triazines, carbamate and urea, polycyclic aromatic hydrocarbons, polychlorinated biphenyls, pharmaceuticals, phenols, etc.) in surface waters. A single-step SPE extraction using OASIS HLB cartridges was employed for the recovery of target micropollutants. The method has been validated according to monitoring performance criteria of the Water Framework Directive, taking into account the approved guidelines on quality assurance and quality control. The recoveries ranged from 60 to 110 %, the coefficient of variation from 0.84 to 27.4 %, and the uncertainty from 6 to 37 %. The LOD varied from 6.0 to 40 ng/L. The limits of quantification for the priority pollutants anthracene, alachlor, atrazine, benzo(a)pyrene, chlorfenvinphos, diuron, isoproturon, nonylphenol, simazine, and terbutryn fulfill the criterion of <30 % of the relevant environmental standards. The method was employed to investigate the water quality in the basin of a transboundary river, Strymonas, in NE Greece during three sampling campaigns conducted in the year 2013. Thirty-nine compounds were detected in the river water. Metolachlor, diuron, isoproturon, salicylic acid, chlorfenvinphos, 1,2-benzanthracene, pyrene, diflubenzuron, and carbaryl exhibited the highest detection frequencies.

Multi-residue method for the detection of veterinary drugs in distillers grains by liquid chromatography-Orbitrap high resolution mass spectrometry.

Distillers Grain (DG) is an important by-product of ethanol production. The ethanol production process uses only the starch portion of the plant and all the remaining nutrients, protein, fat, minerals, and vitamins remain in DGs, a valuable feed material for livestock. The use of antimicrobial drugs is helpful to limit harmful bacterial growth during the early part of the fermentation process. This can lead to the possible presence of contaminants in the by-products that are used in the food and feed industries, resulting in a major concern for the development of bacterial resistance in both humans and animals. To facilitate the detection of antimicrobial and other commonly used veterinary drugs in DGs, a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method was developed targeting a wide range of 12 chemical classes of anti-bacterial substances and drugs, such as ionophore and non-ionophore authorized coccidiostats, banned coccidiostats, macrolides, tetracyclines, nitroimidazoles, amphenicols, quinolones, sulphonamides, tranquilizers, non-steroidal anti-inflammatory drugs and benzimidazoles. Following a simple and fast extraction step with a mixture of organic solvents, the extract was directly injected into the LC coupled to an Orbitrap mass analyzer. The identification of residues is based on accurate mass measurement. The high mass resolution of 50,000 full width at half maximum (FWHM) and corresponding narrow mass windows permitted a very selective and sensitive detection of the analytes in such a complex matrix. A single-laboratory validation procedure was carried out evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance for precision and trueness, and allowed the determination of the compounds at low concentration. The proposed multi-method demonstrated that liquid chromatography coupled to an Orbitrap mass spectrometer is a promising analytical technique, suitable for official residue control of a variety of veterinary drugs in DGs supporting feed safety policies.

Rapid multi-method for the determination of growth promoters in bovine milk by liquid chromatography-tandem mass spectrometry.

A rapid multi-method was developed for the determination of 21 growth promoters from different classes, including gestagens, corticosteroids, RALs, stilbenes, steroids in bovine milk by liquid chromatography-tandem mass spectrometry (LC-MS/MS). All compounds were eluted from the analytical column in less than 8.5min and were subsequently analyzed with atmospheric pressure chemical ionization (APCI) using both positive and negative mode. Sample preparation included extraction of the compounds with acetonitrile and purification with solid-phase extraction (SPE). The method was validated according to Commission Decision 2002/657/EC, at a validation level of 1ng/ml. The specificity, accuracy, precision, decision limit (CCα) and the detection capability (CCß) were satisfactory evaluated. The recoveries ranged from 80.7% to 118.8% and reproducibility represented as coefficient of variance (CV) was from 1.8% to 13.0%. The CCα and CCß values were in the ranges 0.06-0.10ng/ml and 0.11-0.17ng/ml, respectively. The developed method was applied in real samples proving its rapidness and sensitivity for the determination of the 21 growth promoters.

Determination of dapsone in muscle tissue and milk using high-performance liquid chromatography-tandem mass spectrometry.

A precise and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of dapsone in muscle tissue and milk has been developed. The sample preparation was based on extraction with organic solvent and automated solid-phase extraction (SPE) cleanup. At least three product ions were monitored for the analyte. The method was validated according to the European Decision 2002/657/EC. Estimated analytical limits were 0.0018 ng/g for CCα and 0.0031 ng/g for CCß in meat and milk. An excellent linear concentration range was observed for both matrices with a correlation coefficient better than 0.997. Recoveries were 105-117% in meat and 101-108% in milk, with satisfactory precision and coefficients of variance (CV) less than 8%. Additionally, a simplified quantification approach was successfully evaluated depending only on the response factor (F) without the use of calibration curve. The developed method provides reliable and sensitive identification and quantification of dapsone in meat and milk.

Determination of Carbadox and metabolites of Carbadox and Olaquindox in muscle tissue using high performance liquid chromatography-tandem mass spectrometry.

A sensitive and robust LC-APCI-MS/MS method has been developed for the unambiguous detection and quantitative determination of the antimicrobial agent Carbadox, its metabolite quinoxaline-2-carboxylic acid and methyl-3-quinoxaline-2-carboxylic acid the major metabolite of Olaquindox. The method was aimed for application in the assaying of muscle tissue so the developed sample preparation scheme subjected samples to enzymatic digestion prior to the application of solid phase extraction clean-up. Subsequently the purified extracts were analyzed by reversed-phase LC-MS/MS in positive APCI and multiple reaction monitoring mode. The method was validated at a level of 1 µg/kg. The decision limits CCα and detection capability CCß ranged from 0.09 µg/kg to 0.24 µg/kg and from 0.12 µg/kg to 0.41 µg/kg, respectively. The accuracy and precision of the method were satisfactory. The recoveries ranged from 92% to 101% for the metabolites and from 60% to 62% for Carbadox, with coefficient of variances (CVs) less than 12%. The developed method proved efficient and straightforward allowing positive identification and quantitation of the target banned analytes and is thus suitable for application in residue control programmes and metabolism studies.

Determination of anabolic steroids in bovine serum by liquid chromatography-tandem mass spectrometry.

In the present paper we report the LC-MS/MS determination of residues of 12 anabolic steroids in bovine serum, as an expansion of our work protocols for steroids determination in biological matrices. Steroids analyzed included α-zearalanol, ß-zearalanol, α-trenbolone, ß-trenbolone, methyltestosterone, α-estradiol, ß-estradiol, ethynylestradiol, α-boldenone, ß-boldenone, α-nortestosterone and ß-nortestosterone. Following protein precipitation, serum samples were cleaned up by solid-phase extraction using Oasis HLB and Amino cartridges. Atmospheric pressure chemical ionization (APCI) in both positive and negative ionization modes was used and mass spectrometry detection was carried out in multiple reaction monitoring mode following two or (in most cases) three product ions per precursor ion. The method was validated in accordance with the Commission Decision 2002/657/EC. The decision limit (CCα) values obtained, ranged from 0.01 to 0.07 ng/ml and the detection capability (CCß) values obtained ranged from 0.02 to 0.12 ng/ml. The recoveries ranged from 70.2% to 118.2%. The developed method is suitable for routine and confirmatory purposes such as control of illegal use in livestock production.

Gel permeation chromatography clean-up for the determination of gestagens in kidney fat by liquid chromatography-tandem mass spectrometry and validation according to 2002/657/EC.

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of gestagens in kidney fat (medroxyprogesterone acetate, megestrol acetate and melengestrol acetate). The procedure involved a clean-up procedure with gel permeation chromatography (GPC). The analytes were analyzed by reversed-phase LC-MS/MS, in positive multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from the chosen precursor for the unambiguous confirmation of the gestagens. The method was validated at the validation level of 1.0 ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCalpha ranged from 0.20 to 0.22 ng/g while the detection capabilities CCbeta ranged from 0.33 to 0.38 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate mean for residue analysis studies.

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry.

A specific and sensitive multi-method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of 20 anabolic steroids in muscle tissue (diethylstilbestrol, beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha/beta-nortestosterone, methyltestosterone, beta-trenbolone, triamcinolone acetonide, dexamethasone, flumethasone, alpha/beta-zearalenol, alpha/beta-zearalanol, zearalenone, melengestrol acetate, megestrol acetate and medroxyprogesterone acetate). The procedure involved hydrolysis, extraction with tert-butyl methyl ether, defattening and final clean-up with solid phase extraction (SPE) on Oasis HLB and Amino cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, in positive and negative multiple reaction monitoring (MRM) mode, acquiring two diagnostic product ions from each of the chosen precursor ions for the unambiguous confirmation of the hormones. The method was validated at the validation level of 0.5ng/g. The accuracy and precision of the method were satisfactory. The decision limits CCalpha ranged from 0.03 to 0.14ng/g while the detection capabilities CCbeta ranged from 0.05 to 0.24ng/g. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in muscle tissue and can be used for residue control programs.

Determination of anabolic steroids in bovine urine by liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric (LC-MS/MS) multi-method has been developed for the determination of 15 anabolic steroids in bovine urine (diethylstilbestrol, dienestrol, hexestrol, beta-estradiol, ethynylestradiol, alpha/beta-boldenone, alpha-nortestosterone, alpha/beta-zearalenol, alpha/beta-zaeralanol, zearalenone, stanozolol and 16beta-OH-stanozolol). The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, a washing step with hexane and final clean-up with SPE with Oasis HLB and Amino cartridges. The analytes were quantified by liquid chromatography coupled to a tandem mass spectrometer (LC-TSQ Quantum AM) operating in both positive and negative atmospheric pressure chemical ionisation (APCI). Data acquisition was performed in multiple reaction monitoring (MRM) mode quantifying two diagnostic product ions from a chosen precursor. The method was validated according to the Commission Decision 2002/657/EC, for the detection and confirmation of residues in products of animal origin. The method specificity, sensitivity, accuracy and precision were evaluated. The decision limits CCalpha ranged from 0.06 to 0.26 ng/ml and the detection capabilities CCbeta ranged from 0.11 to 0.49 ng/ml. The developed method is sensitive and useful for detection, quantification and confirmation of these anabolic steroids in bovine urine and can be used for residue control programs.

Determination of anabolic steroids in muscle tissue by liquid chromatography-tandem mass spectrometry.

A specific and sensitive method based on liquid chromatography-tandem mass spectrometry using atmospheric pressure chemical ionization (LC-APCI-MS/MS) has been developed for the determination of four anabolic steroids [trenbolone, methylboldenone, methyltestosterone, and norethandrolone] in bovine muscle. Methyltestosterone- d 3 was used as internal standard. The procedure involved enzymatic hydrolysis, extraction with tert-butyl methyl ether, defattening, and final cleanup with solid-phase extraction with Oasis HLB cartridges. The analytes were analyzed by reversed-phase LC-MS/MS, acquiring two diagnostic product ions from the chosen precursor [M + H] (+) for the unambiguous confirmation of hormones. The method was validated according to the European Commission Decision 2002/657/EC for the detection and confirmation of residues in products of animal origin. The limits of detection (LOD) and limits of quantitation (LOQ) were found to be 0.3 ng/g and 1.0 ng/g, respectively. The accuracy and precision have been determined, with recoveries ranging from 83% to 104% and the CV factor not exceeding the value of 7%. The decision limits CCalpha were calculated and ranged from 0.05 to 0.15 ng/g while the detection capabilities CCbeta ranged from 0.09 to 0.25 ng/g. The method proved to be sensitive and reliable and thus renders an appropriate means for residue analysis studies.