[Determination of 12 prohibited veterinary drug residues in pig urine by ultra high performance liquid chromatography-tandem mass spectrometry].

A method was established for the simultaneous detection of 12 prohibited veterinary drugs, including ß2-receptor agonists, nitrofuran metabolites, nitroimidazoles, chlorpromazine, and chloramphenicol, in pig urine. The sample was pretreated by enzymolysis, acid hydrolysis/derivatization, and liquid-liquid extraction combined with solid-phase extraction. Detection was performed using ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). Ammonium acetate solution (0.2 mol/L, 4.5 mL) and ß-glucuronidase/aryl sulfatase (40 µL) were added to the sample, which was subsequently enzymolized at 37 ℃ for 2 h. Then, 1.5 mL of 1.0 mol/L hydrochloric acid solution and 100 µL of 0.1 mol/L o-nitrobenzaldehyde solution were added to the sample. The mixture was incubated at 37 ℃ for 16 h, and the analytes were extracted with 8 mL of ethyl acetate by liquid-liquid extraction. The lower aqueous phase obtained after extraction was extracted and purified using a mixed cation-exchange solid-phase extraction column. The extracts were combined, the extraction solution was blow-dried with nitrogen, and the residue was redissolved for determination. The samples were analyzed under multiple-reaction monitoring mode with both positive and negative electrospray ionization, and quantified using an isotope internal standard method. The correlation coefficients (r) of the 12 compounds were >0.99. The limits of detection (LODs) and quantification (LOQs) of chloramphenicol were 0.05 and 0.1 µg/L, respectively, and the LODs and LOQs of the other compounds were 0.25 and 0.5 µg/L, respectively. The mean recoveries and RSDs at 1, 2, and 10 times the LOQ were 83.6%-115.3% and 2.20%-12.34%, respectively. The proposed method has the advantages of high sensitivity, good stability, and accurate quantification; thus, it is suitable for the simultaneous determination of the 12 prohibited veterinary drug residues in pig urine.

Development and validation of a multi-residue analytical method for veterinarian and human pharmaceuticals in livestock urine and blood using UHPLC-QTOF.

The aim of this work is to contribute to the assessment of multi-residue analysis of veterinarian and human pharmaceuticals using UHPLC-QTOF in livestock urine and blood (cattle, chicken, sheep and pig). Firstly, an in-house database including compound name, monoisotopic mass, chemical formula, retention time, chemical structure, and three CID MS-MS spectra of the 234 selected drugs were built for qualitative detection. Secondly, the method validation result showed that all the 234 drugs exhibited good linearity with determination coefficients (R2) higher than 0.999. Then, the distribution of the drugs recoveries, intra-day RSD and inter-day RSD results for all seven matrices were tested. Finally, after a carefully cross check, 150 veterinarian and human pharmaceuticals could meet the methodological requirements (recovery, 50-120%; intra-day RSD ≤ 15%, inter-day RSD ≤ 20%) in all seven matrices. Our results suggested that although the main applications of UHPLC-QTOF are directed towards detection and identification of the compounds, this method should be also applied for quantitative purposes.

The search for a microbiological inhibition method for the rapid, broad-spectrum and high-throughput screening of six kinds of antibiotic residues in swine urine.

In this study, a microbiological inhibition method for rapidly screening antibiotics in swine urine was established with an easy sample pre-treatment. The microbiological system consisted of an agar medium mixed with nutrients, sensitizers, a test bacterium (Geobacillus stearothermophilus ATCC12980) and pH indicator (bromocresol purple). It was observed that the detection limits of the test kit for twenty-eight common antimicrobial residues in urine, including ß-lactams, aminoglycosides, tetracyclines, sulfonamides, macrolides, and lincosamides, were less than or equal to the maximum residue limits of the kidney, as determined by the EU and China. Moreover, the false negative rate and the false positive rate, along with other performance indexes such as interassay coefficients of variation and shelf life of the kit, all met the standard requirements of the ISO13969:2003 guidelines. Additionally, our results were consistent with those using the gold-standard physical chemistry method, which suggest the proposed method is suitable for screening antibiotic residues.

Electrospray ionization rapid screening sans liquid chromatography column: A sensitive method for detection and quantification of chemicals in animal tissues and urine.

RATIONALE: Electrospray ionization mass spectrometry (ESI-MS) in conjunction with liquid chromatography (LC) can provide accurate quantitative data, but it is not well-suited for the rapid screening (RS) of analytes incurred into complex matrices. This study was designed to determine the usefulness of ESI for rapid detection and quantitation of veterinary drugs from complex biological matrices under near real-time conditions. METHODS: Nine veterinary drugs or metabolites, clenbuterol, erythromycin, flunixin, 5-hydroxyflunixin, meloxicam, ractopamine, salbutamol, tylosin and zilpaterol, present in cow urine, sheep urine, sheep tissues (kidney, muscle, liver and lung) or pig kidney, were simultaneously analyzed. A simple sample clean-up procedure, which included dilution with 10% sodium carbonate followed by extraction with ethyl acetate, was used. For tissues, an additional pre-extraction with hexane was performed to remove fat prior to MS analysis. Samples were introduced into the mass spectrometer through the LC autosampler, but no chromatographic separation was employed. A Sciex 5600+ triple time-of-flight mass spectrometer with a dual-spray source interfaced with a Shimadzu Nexera LC system was used. Samples were analyzed in positive ion mode. RESULTS: Sample extraction times were typically 10-30 min or less and instrumental analysis time was 1 min/sample. Regression coefficients of matrix-matched standard curves across all compounds ranged from 0.9701-0.9999 in urine (cow and sheep) and tissues (sheep kidney, liver, lung, muscle and pig kidney). Limits of detection ranged from 0.11 to 2.03 ng/mL across analytes in urine and 0.11 to 8.86 ng/g across tissues. Correlations between RS-ESI-MS and LC/MS/MS results were 0.956 to 0.998 for incurred residues of flunixin in cow urine, ractopamine in pig kidney and zilpaterol in sheep urine. CONCLUSIONS: RS-ESI-MS provided rapid, sensitive, and accurate analyses of nine veterinary drugs from complex matrices with very little sample preparation and produced quantitative data akin to LC/MS/MS.

A Method for the Determination of Veterinary Drugs from Different Therapeutic Classes in Animal Urine.

A rapid, precise and robust HPLC separation procedure has been developed and optimized for the determination of a series of drugs of different therapeutic classes: chlortetracycline, oxitetracycline, cefoperazone, diclofenac, tiamphenicol, marbofloxacin, ciprofloxacin, danofloxacin, enrofloxacin and flumequine. The chromatographic method used a monolithic C18 column and both diode array and fluorescence detection. This procedure was validated for the analysis of drugs in cow urine, using a simple and fast procedure with methanol/acetonitrile, allowing the simultaneous and efficient extraction of most of the studied drugs. The proposed method was successfully applied to the determination of enrofloxacin in cow urine, collected after the administration of this antibiotic.

Antibiotic exposure across three generations from Chinese families and cumulative health risk.

Extensive antibiotic exposure in the general population has been documented by bio-monitoring, but data regarding antibiotic burden across three generations in families living in the same household are lacking. We investigated the distribution of antibiotics and the potential health risk among the three generations by selecting 691 participants from 256 households in Fuyang city, China. A total of 45 antibiotics and two metabolites were screened in urine samples through liquid chromatography electrospray tandem mass spectrometry. In total, 34 antibiotics were found in the samples with an overall detection frequency of 92.0%, and the detection frequencies of individual antibiotic ranged from 0.3% to 28.7%. Specifically, the concentrations of seven antibiotics (azithromycin, amoxicillin, oxytetracycline, levofloxacin, norfloxacin, trimethoprim and sulfamethoxazole) were extremely high (i.e., above 10, 000 ng/mL). The detection rates of tetracyclines were significantly different among the three generations, with parents having the highest detection rate. Penicillin V, chlortetracycline, doxycycline, enrofloxacin, and ciprofloxacin showed a higher detection frequency in parents, whereas tetracycline, danofloxacin, and ofloxacin were more likely to be found in grandparents. The proportions of the sum of the daily exposure dose of VAs and PVAs more than 1 µg/kg/d in children, parents, and grandparents were 31.6%, 39.5%, and 26.5%, respectively. A hazard index (HI) greater than 1 was observed in 14.7% children, which was less than the 23.6% in parents and slightly higher than the 11.8% in grandparents. Ciprofloxacin was the biggest contributor to HI among the three generations. Collectively, these findings indicate that households are widely exposed to various antibiotics in Fuyang city, where parents had the highest health risk associated with the disturbance of gut microbiota.

Selective extraction and detection of ß-agonists in swine urine for monitoring illegal use in livestock breeding.

The illegal use of ß-agonists often endangers animal-derived food safety. In this study, a selective detection method for ß-agonists in swine urine was established via the combination of polymeric ionic liquid-molecularly imprinted graphene oxide-miniaturized pipette tip solid-phase extraction and high-performance liquid chromatography. It is worth noting that this method relied mainly on the designed adsorbent, which presented a rich adsorption mechanism, fast mass transfer rate, and high selectivity, and was successfully utilized in the selective extraction of ß-agonists from swine urine samples. The proposed method has low LOD (0.20-0.56 ng/mL), high recovery (94.9-107.9%), and high reusability (4 times, 91.9-108.8%), which indicates its high potential as a selective, sensitive, accurate, and nonfatal method for monitoring the illegal use of ß-agonists in the livestock breeding stage.

Rapid simultaneous determination of 160 drugs in urine and blood of livestock and poultry by ultra-high-performance liquid chromatography-tandem mass spectrometry.

In order to address the specific question of food safety in livestock and poultry, it is imperative to monitor veterinary drugs at every moment in the process of livestock and poultry breeding. Thus, multi-residue analysis of a wide variety of drugs using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) has become a tool of critical significance, especially for veterinary drug monitoring programs. A total of 160 compounds, belonging to 17 different families of veterinary drugs, were investigated in the urine and blood of livestock and poultry. Drug samples were extracted using a slightly acidic acetonitrile solution. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) preparation method, combined with dispersive solid phase extraction (d-SPE) was compared with the approach of solid phase extraction (SPE). In the end, the QuEChERS extraction procedure was selected to reduce matrix effects and efficiently extract target veterinary drugs, and d-SPE was applied as a cleanup step. Electrospray ionization coupled with positive dynamic multiple reaction monitoring (dMRM) was utilized for the analysis of 160 different drugs in a single chromatographic run of 24 min. The efficiency of this method was evaluated using 7 matrices (pig blood, cattle blood, sheep blood, chicken blood, pig urine, cattle urine, and sheep urine). Good linearity was obtained for the analytes in a concentration range of 1-100 ng/mL, with correlation coefficients higher than 0.990. Most of the 160 drugs studied gave estimated limits of detection (LOQs) of 1 ng/mL, with some LOQs reaching as much as 5 ng/mL. The mean recoveries at four spike-in levels of 1, 5, 10, and 50 ng/mL, ranged from 60% to 120%. The intra-day precision measurements had coefficients of variation (n = 6) <15%, and the inter-day precision measurements were below 25%. Our method was applied in real samples and proved to be adequate for routine analysis. The proposed method proved to be simple, rapid and reliable for monitoring 160 drugs in the urine and blood of livestock and poultry, and can also be used for food safety monitoring.

A reliable, simple and cost-efficient TLC-HPLC method for simultaneously determining florfenicol and florfenicol amine in porcine urine: application to residue surveillance.

Violative residues of florfenicol (FF) in porcine edible tissues pose a potential risk for human health. In this study, urine was selected as target matrix for routine residue monitoring of FF in pig, and a thin layer chromatography (TLC)-high-performance liquid chromatography (HPLC) method was developed for simultaneously determining FF and florfenicol amine (FFA) in porcine urine. The urine samples were extracted with ethyl acetate under alkaline environment. The extracts were enriched through evaporation, purified by TLC and analysed by HPLC at 225 nm. A Waters Symmetry C18 column was used for the separation of the two analytes. The mobile phase was acetonitrile-phosphate buffer mixtures (33.3: 66.7, v/v), and was pumped at 0.6 mL/min. The TLC-HPLC method was well validated and successfully applied to residue depletion study. Good analytical specificity was confirmed by the lack of interfering peaks at the retention times of FF and FFA. The standard curves showed good linearity (FF: y = 143064x - 1045.3, r= 0.9999; FFA: y = 275826x + 1888.8, r= 0.9999) over the range of 0.0625-8 µg/mL. The precision ranged from 0.83% to 11.66% and 2.19% to 8.75% for intraday and interday determination, respectively. The corresponding accuracy ranged from -13.38% to 10.78% and -12.15% to 7.14%, respectively. The limits of quantification (LOQs) for FF and FFA were 0.125 µg/mL. The residue depletion study showed that the concentrations of FF and FFA in urine were higher than those in edible tissues at three time points. This method was reliable, simple and cost efficient, and could be used to monitor FF residues in porcine edible tissue without slaughtering animals. TLC showed excellent purification efficiency and is expected to solve matrix interferences in veterinary drug residue analysis.

Sensitive and Matrix-Tolerant Lateral Flow Immunoassay Based on Fluorescent Magnetic Nanobeads for the Detection of Clenbuterol in Swine Urine.

The lack of sensitivity and poor matrix tolerance are the main bottlenecks of the lateral flow immunoassay (LFIA). Here, a sensitive and matrix-tolerant method that integrated immunomagnetic separation and fluorescent lateral flow immunoassay (IMS-FLFIA) based on fluorescent magnetic nanobeads was developed to detect the clenbuterol (CLE) residue in swine urine. The limit of detection (LOD) of IMS-FLFIA is 4 times lower than that of traditional colloidal gold LFIA. This method, which exhibits similar LOD and linearity range in both phosphate-buffered saline and urine swine, is highly correlated with liquid chromatography-tandem mass spectrometry for the detection of real swine urine samples. The result indicated that IMS-FLFIA has a universal resistance to the swine urine matrix. The merits of this assay, high sensitivity, matrix tolerance, accuracy, and specificity, ensure a promising future in detection of veterinary drug residues.

Rapid Detection of the Antibiotic Sulfamethazine in Pig Body Fluids by Paper Spray Mass Spectrometry.

We report herein a practical method for nonlethal detection of the antibiotic sulfamethazine in pig body fluids via the combination of simple extraction and paper spray mass spectrometry (PS-MS). This method requires minimal sample preparation while still providing high sensitivities and accuracies in complex matrices including pig whole blood (LOD = 7.9 µg/L; recovery = 95.4-103.7%), pig serum (LOD = 11.5 µg/L; recovery = 103.2-106.2%), and synthetic urine (LOD = 11.2 µg/L; recovery = 99.1-103.2%). Given a known correlation between the level of sulfamethazine in body fluids and edible tissues, this method shows great promise as a practical and nonlethal solution for rapid testing of the drug, which can substantially aid managerial decision in the livestock industry.

Collision cross section (CCS) as a complementary parameter to characterize human and veterinary drugs.

In the context of human and veterinary drugs identification, ion mobility spectrometry (IMS) in combination with mass spectrometry (MS) may provide a relevant complementary piece of information to mass-to-charge ratio (m/z), the so-called collision-cross-section (CCS). Up to now, however, the application of CCS as identification parameter has not been fully investigated due to the reduced number of these drugs that have being characterized in terms of CCS. This work proposes a CCS database for 92 human and veterinary drugs, including eighteen benzimidazoles, eleven 5-nitroimidazoles, eleven aminoglycosides, nineteen quinolones, eighteen ß-lactams, ten sulfonamides and five tetracyclines. Among them, 37 drugs have been characterized in terms of CCS for the first time. The CCS values of the other 55 compounds have been compared with those from a recently published database in order to evaluate inter-laboratory reproducibility, which is crucial for the implementation of the CCS as identification parameter. CCS values were measured by traveling wave ion mobility spectrometry (TWIMS) under positive ionization conditions. Nitrogen was used as drift gas in the ion mobility cell. The proposed database covers 173 ions including [M+H]+ and [M+Na]+ species. High correlation between m/z and CCS has been observed for [M+H]+ (R2 = 0.9518, n = 91) and [M+Na]+ (R2 = 0.9135, n = 82) ions. As expected, CCS values for sodium adducts are generally greater than for protonated molecules because they exhibit higher molecular weight. However, sodium adducts of aminoglycosides, ß-lactams, and of several quinolones and benzimidazoles, were characterized as more compact ions than their related protonated molecule. In addition, this work describes the fragmentation pattern observed for the studied molecules. For the first time, the main fragment ions for most of the compounds have also been characterized in terms of CCS, involving a total of 238 ions. As proof of concept, for the application of this database to biological matrices, eleven veterinary drugs in bovine urine samples were characterized in terms of CCS, showing that this parameter was not influenced by the matrix.

Supercritical fluid chromatography applied to the highly selective isolation of urinary steroid hormones prior to GC/MS analysis.

To assess the presence of prohibited anabolic substances used to promote growth in livestock, calf urine is the most relevant matrix. However, the sample preparation methods (required to remove unwanted matrix components and fractionate isobaric species that may be unresolved by gas chromatography- mass spectrometry GC/MS) are long and complex. In this context, semi-preparative supercritical fluid chromatography (SFC) was considered to possibly simplify the sample preparation in reducing the number of procedures. Fifteen stationary phases were screened with SFC combined with UV and evaporative light-scattering detection (ELSD), among which two columns (Cosmosil π-NAP and Princeton DIOL) were retained for their ability to isolate steroid hormones from other matrix components and, for the second column, for the additional possibility to fractionate steroid hormones into different families (estrogens, mono-hydroxylated and di-hydroxylated androgens). The fractions were further analysed with GC/MS showing the benefit of class fractionation. The final method allows for significant time, solvent and money savings compared to the previously widely used method (solid-phase extraction combined with semi-preparative high-performance liquid chromatography).

Veterinary antibiotics in food, drinking water, and the urine of preschool children in Hong Kong.

Due to the harmful effects of veterinary antibiotics (VAs) residues in food on children's health, urine samples from 31 preschool and primary school children were analyzed for 13 common VAs. Samples of raw and cooked pork, chicken, fish, milk and drinking water from the children's living areas were also analyzed for residual VAs. Urinalysis revealed one to four target antibiotics in 77.4% of the sample group, with concentrations as high as 0.36ng/mL. Norfloxacin and penicillin had the highest detection rates (48.4% and 35.5%, respectively), with median concentrations of 0.037 and 0.13ng/mL, respectively. The VA burden of children in HK was lower than that in Shanghai. Enrofloxacin, penicillin, and erythromycin were the most detected VAs in raw and cooked food. Only oxytetracycline was detected in terminal tap water, and none were detected in milk. Tetracycline and doxycycline hyclate were detected in organic eggs (up to 7.1ng/g) and regular eggs (up to 6.6ng/g), which were common in children's diets. Traditional Chinese cooking processes did not completely eliminate VAs, and the concentrations of some VAs increased, especially after frying and roasting. The estimated daily intake (EDI) results show that the contribution of dietary intake and that based on the urine concentrations of VAs were far below the acceptable daily intake (ADI). The EDIs from urine were significantly lower than those based on cooked foods. The highest level of achievement percentage (LAP) based on dietary consumption and urine concentrations were 39.7% and 1.79%, respectively, and thus current levels of exposure to VAs would not seem to pose a risk to children's health. However, harmful effects of residual VAs during developmental periods may occur with exposure to much lower doses than those considered harmful to adults, and further investigation of these emerging pollutants is urgently encouraged.

Carbon isotope ratios of endogenous steroids in Belgian Blue and Holstein cattle: Method development, reference population studies and application to steroid misuse control.

RATIONALE: The misuse of growth promoters in livestock and breeding animals is prohibited according to the laws of the European Union. Among these growth promoters, the detection of endogenous steroids like testosterone, estradiol or progesterone remains especially challenging as concentration-based urinary thresholds may not provide conclusive results due to large inter-individual variations. In addition to the detection of intact steroid esters in blood or hair, carbon isotope ratio (CIR) determination of urinary steroids has commonly been the method of choice. METHODS: A comprehensive sample clean-up procedure was developed and validated, which enables for the first time simultaneous CIR measurements of testosterone metabolites (17α-hydroxyandrost-4-en-3-one, 3α-hydroxy-5ß-androstan-17-one and 5α-androstane-3ß,17α-diol), the estradiol metabolite 17α-estradiol (ESTR) and the progesterone metabolite 5ß-pregnane-3α,20α-diol (PD) from a single urine specimen. As endogenous reference compounds 3ß-hydroxyandrost-5-en-17-one and 5-androstene-3ß,17α-diol (5EN) were chosen. The method was validated by means of linear mixing models and a reference population encompassing n = 53 Belgian Blue and Holstein cattle was investigated to enable the calculation of population-based Δ13 C thresholds. RESULTS: The combined measurement uncertainty determined for the Δ13 C-values of all steroids under investigation was found to be <0.8 ‰. Within the reference population studies, 5EN was demonstrated to be the most promising endogenous reference compound resulting in comparably low Δ-values and accompanying thresholds. For PD, a surprisingly high number of samples (n = 9) yielded significantly 13 C-depleted values and ESTR was only detectable in n = 13 samples. Proof-of-concept was accomplished by investigating two post-administration samples. CONCLUSIONS: This first comprehensive investigation on the CIRs of endogenous urinary steroids demonstrated once more the potential of isotope ratios in aiding discrimination between endogenously produced and exogenously administered steroids. By means of the reference population-derived CIRs, it is possible to apply cattle-specific thresholds to differentiate between treated and non-treated animals.

Pharmacokinetic and pharmacodynamics of xylazine administered to exercised thoroughbred horses.

There is limited data describing xylazine serum concentrations in the horse and no reports of concentrations beyond 24 hours. The primary goal of the study reported here was to update the pharmacokinetics of xylazine following intravenous (IV) administration in order to assess the applicability of current regulatory recommendations. Pharmacodynamic parameters were determined using PK-PD modeling. Sixteen exercised adult Thoroughbred horses received a single IV dose of 200 mg of xylazine. Blood and urine samples were collected at time 0 and at various times for up to 96 hours and analyzed using liquid chromatography tandem mass spectrometry. Xylazine serum concentrations were best fit by a 3-compartment model. Mean ± SEM systemic clearance, volume of distribution at steady state, beta half-life and gamma half-life were 12.7 ± 0.735 mL/min/kg, 0.660 ± 0.053 L/kg, 2.79 ± 0.105 hours and 26.0 ± 1.9, respectively. Immediately following administration, horses appeared sedate as noted by a decrease in chin-to-ground distance, decreased locomotion and decreased heart rate (HR). Sedation lasted approximately 45 minutes. Glucose concentrations were elevated for 1-hour post administration. The EC50 (IC50) was 636.1, 702.2, 314.1 and 325.7 ng/mL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. The Emax (Imax) was 27.3 beats per minute, 47.5%, 42.4 cm and 0.28 mg/dL for HR, atrioventricular block, chin-to-ground distance and glucose concentrations, respectively. Pharmacokinetic parameters differ from previous reports and a prolonged detection time suggests that an extended withdrawal time, beyond current regulatory recommendations, is warranted to avoid inadvertent positive regulatory findings in performance horses. Copyright © 2016 John Wiley & Sons, Ltd.

Distribution and Depletion of Ractopamine in Goat Plasma, Urine and Various Muscle Tissues.

This study investigated the ractopamine (RAC) distribution and depletion process in goat plasma, urine and various muscle tissues which were associated with a potential risk for consumer health. The experiment was carried out in 21 goats (18 treated and 3 controls). Treated animals were administered orally a dose of 1 mg/kg body mass per day for 28 consecutive days and randomly sacrificed on Days 0.25, 1, 3, 7, 14 and 21 of the withdrawal period. RAC in goat samples was analyzed by using ultra-high performance liquid chromatography-quadrupole-orbitrap high-resolution mass spectrometry. RAC was below the limits of detection (LOD = 0.15 ng/mL) in plasma while which was higher than the LOD in urine on withdrawal day 21. The residues in goat longissimus dorsi muscle, biceps femoris muscle and triceps surae muscle were differed significantly. These findings demonstrated that urine can be used as the target matrix for monitoring RAC abuse in goat.

A critical assessment of the performance criteria in confirmatory analysis for veterinary drug residue analysis using mass spectrometric detection in selected reaction monitoring mode.

Besides the identification point system to assure adequate set-up of instrumentation, European Commission Decision 2002/657/EC includes performance criteria regarding relative ion abundances in mass spectrometry and chromatographic retention time. In confirmatory analysis, the relative abundance of two product ions, acquired in selected reaction monitoring mode, the ion ratio should be within certain ranges for confirmation of the identity of a substance. The acceptable tolerance of the ion ratio varies with the relative abundance of the two product ions and for retention time, CD 2002/657/EC allows a tolerance of 5%. Because of rapid technical advances in analytical instruments and new approaches applied in the field of contaminant testing in food products (multi-compound and multi-class methods) a critical assessment of these criteria is justified. In this study a large number of representative, though challenging sample extracts were prepared, including muscle, urine, milk and liver, spiked with 100 registered and banned veterinary drugs at levels ranging from 0.5 to 100 µg/kg. These extracts were analysed using SRM mode using different chromatographic conditions and mass spectrometers from different vendors. In the initial study, robust data was collected using four different instrumental set-ups. Based on a unique and highly relevant data set, consisting of over 39 000 data points, the ion ratio and retention time criteria for applicability in confirmatory analysis were assessed. The outcomes were verified based on a collaborative trial including laboratories from all over the world. It was concluded that the ion ratio deviation is not related to the value of the ion ratio, but rather to the intensity of the lowest product ion. Therefore a fixed ion ratio deviation tolerance of 50% (relative) is proposed, which also is applicable for compounds present at sub-ppb levels or having poor ionisation efficiency. Furthermore, it was observed that retention time shifts, when using gradient elution, as is common practice nowadays, are mainly observed for early eluting compounds. Therefore a maximum retention time deviation of 0.2 min (absolute) is proposed. These findings should serve as input for discussions on the revision of currently applied criteria and the establishment of a new, globally accepted, criterion document for confirmatory analysis. Copyright © 2016 John Wiley & Sons, Ltd.

High throughput-screening of animal urine samples: It is fast but is it also reliable?

Advanced analytical technologies like ultra-high-performance liquid chromatography coupled to high resolution mass spectrometry can be used for veterinary drug screening of animal urine. The technique is sufficiently robust and reliable to detect veterinary drugs in urine samples of animals where the maximum residue limit of these compounds in organs like muscle, kidney, or liver has been exceeded. The limitations and possibilities of the technique are discussed. The most critical point is the variability of the drug concentration ratio between the tissue and urine. Ways to manage the false positive and false negatives are discussed. The capability to confirm findings and the possibility of semi-targeted analysis are also addressed. Copyright © 2016 John Wiley & Sons, Ltd.

Determination of veterinary antibiotics in bovine urine by liquid chromatography-tandem mass spectrometry.

A follow-up of antibiotics (tetracyclines, fluoroquinolones, cephalosporins, penicillins and amphenicols) in the bovine urine is important for two reasons: to understand if they are still present in organism, and whether their occurrence in urine might be considered as an environmental risk. A validated HPLC-MS/MS method (Decision 2002/657/EC) for antibiotics determination in bovine urine was developed. CCα and CCß were in the range of 0.58-0.83 and 0.55-1.1 ng mL(-1), respectively. Recoveries were 92-108%, with inter-day repeatability below 12%. Analysis of bovine urine revealed frequent presence of tetracyclines, which was related with animal's age. The cause, most presumably, might be found in different therapeutic protocols applied for veal calves and young bulls enrolled in this study. Most abundant was oxytetracycline with highest level in veal calves (1718 ng mL(-1)) vs. young bulls (2.8 ng mL(-1)). Our results indicate the necessity of antibiotics monitoring in bovine urine before animals undergo further processing in the food industry.