Evaluation of the ARK Diagnostics immunoassay for qualitative detection of xylazine in urine.

Xylazine exposure is common in some US cities, but a commercial assay for routine laboratory testing for xylazine is not currently available. We evaluated a pre-release version of the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxyxylazine in urine. Studies were conducted using either the semi-quantitative assay application (A. Roche Cobas 503 analyzer) or the qualitative assay application (B. Beckman Coulter AU480 analyzer). Study specimens consisted of deidentified patient urine samples submitted for routine drugs-of-abuse testing. Measurements of xylazine (X) were performed by LC-MS-MS to obtain X-NEGATIVE (X <10 ng/mL) and X-POSITIVE (X ≥10 ng/mL). The semi-quantitative ARK assay was calibrated with a 10 ng/mL cutoff for ARK-POSITVE. For (A): among 74 X-POSITIVE samples, there was 1 ARK-NEGATIVE result (false-negative rate = 1.4%); among 78 X-NEGATIVE samples by LC-MS-MS, there were 0% ARK-POSITIVE results (false-positive rate = 0%). For (B), among 74 X-POSITIVE samples, there were 0 ARK-NEGATIVE results (false-negative rate = 0%); among 78 X-NEGATIVE samples there was 1 ARK-POSITIVE sample (false-positive rate = 1.3%). Common sources of interferences were investigated without evidence of interference. The ARK xylazine/4-OH-xylazine immunoassay was found to be suitable for routine use in screening patient urine samples for presence of xylazine >10 ng/mL.

Plasma histamine concentrations in horses administered sodium penicillin, guaifenesin-xylazine-ketamine and isoflurane with morphine or butorphanol.

OBJECTIVE: Various drugs administered to horses undergoing surgical procedures can release histamine. Histamine concentrations were evaluated in horses prepared for surgery and administered butorphanol or morphine intraoperative infusions. STUDY DESIGN: Prospective studies with one randomized. ANIMALS: A total of 44 client-owned horses. METHODS: In one study, anesthesia was induced with xylazine followed by ketamine-diazepam. Anesthesia was maintained with guaifenesin-xylazine-ketamine (GXK) during surgical preparation. For surgery, isoflurane was administered with intravenous (IV) morphine (group M: 0.15 mg kg-1 and 0.1 mg kg-1 hour-1; 15 horses) or butorphanol (group B: 0.05 mg kg-1 and 0.01 mg kg-1 hour-1; 15 horses). Histamine and morphine concentrations were measured using enzyme-linked immunoassay before opioid injection (time 0), and after 1, 2, 5, 30, 60 and 90 minutes. In a subsequent study, plasma histamine concentrations were measured in 14 horses before drug administration (baseline), 15 minutes after IV sodium penicillin and 15 minutes after starting GXK IV infusion. Statistical comparison was performed using anova for repeated measures. Pearson correlation compared morphine and histamine concentrations. Data are presented as mean ± standard deviation. Significance was assumed when p ≤ 0.05. RESULTS: With histamine, differences occurred between baseline (3.2 ± 2.4 ng mL-1) and GXK (5.2 ± 7.1 ng mL-1) and between baseline and time 0 in group B (11.9 ± 13.4 ng mL-1) and group M (11.1 ± 12.4 ng mL-1). No differences occurred between baseline and after penicillin or between groups M and B. Morphine concentrations were higher at 1 minute following injection (8.1 ± 5.1 ng mL-1) than at 30 minutes (4.9 ± 3.1 ng mL-1) and 60 minutes (4.0 ± 2.5 ng mL-1). Histamine correlated with morphine at 2, 30 and 60 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: GXK increased histamine concentration, but concentrations were similar with morphine and butorphanol.

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.

Simultaneous Determination of Xylazine and 2,6-Xylidine in Blood and Urine by Auto Solid-Phase Extraction and Ultra High Performance Liquid Chromatography Coupled with Quadrupole-Time of Flight Mass Spectrometry.

Xylazine as veterinary medicine for sedation, but intoxication cases in humans were identified in the last few years. A highly sensitive method is required for analyzing xylazine and its metabolites in human blood and urine. This article presents an ultra high performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-QTOF) study for simultaneous determination of xylazine and 2,6-dimethylaniline (DMA) in human blood and urine. The samples were extracted and cleaned up by Oasis MCX solid-phase extraction. The analysis is performed using an UHPLC-QTOF. Analysis precision, accuracy, sensitivity, linear range, limit of detection (LOD) and limit of quantification (LOQ) were validated for the proposed method. In the blood and urine samples, the linear calibration curves with high linearity are obtained over the range of 2.0-1,000.0 ng/mL. The LOD for xylazine and DMA in blood are 0.2 and 0.1 ng/mL, in urine are 0.4 and 0.2 ng/mL; the LOQ for xylazine and DMA in blood are 0.6 and 0.3 ng/mL, in urine are 1.0 and 0.6 ng/mL, respectively. The intra- and interday precision is better than 8.6 and 11.9%. In conclusion, the proposed method is highly sensitive and reproducible, thus suitable for accurate quantification of xylazine and its metabolites in blood and urine.

Qualitative metabolism assessment and toxicological detection of xylazine, a veterinary tranquilizer and drug of abuse, in rat and human urine using GC-MS, LC-MSn, and LC-HR-MSn.

Xylazine is used in veterinary medicine for sedation, anesthesia, and analgesia. It has also been reported to be misused as a horse doping agent, a drug of abuse, a drug for attempted sexual assault, and as source of accidental or intended poisonings. So far, no data concerning human metabolism have been described. Such data are necessary for the development of toxicological detection methods for monitoring drug abuse, as in most cases the metabolites are the analytical targets. Therefore, the metabolism of xylazine was investigated in rat and human urine after several sample workup procedures. The metabolites were identified using gas chromatography (GC)-mass spectrometry (MS) and liquid chromatography (LC) coupled with linear ion trap high-resolution multistage MS (MS(n)). Xylazine was N-dealkylated and S-dealkylated, oxidized, and/or hydroxylated to 12 phase I metabolites. The phenolic metabolites were partly excreted as glucuronides or sulfates. All phase I and phase II metabolites identified in rat urine were also detected in human urine. In rat urine after a low dose as well as in human urine after an overdose, mainly the hydroxy metabolites were detected using the authors' standard urine screening approaches by GC-MS and LC-MS(n). Thus, it should be possible to monitor application of xylazine assuming similar toxicokinetics in humans.

Determination of xylazine and its metabolites by GC-MS in equine urine for doping analysis.

Xylazine and its main metabolites were detected in equine urine after a single-dose intravenous administration of 0.98 and 1.01 mg/kg body weight xylazine, respectively, in two horses, in order to be used for equine doping control routine analysis. The urine levels of the parent drug and its metabolites were determined using gas chromatography-mass spectrometry (GC-MS). Xylazine is metabolised rapidly, down to a concentration level of about 1.0 microg/ml after 1-3h administration. Seven metabolites were identified in urine. 4-Hydroxy-xylazine, the major metabolite, could be traced for 25 h and it is regarded as the long-term metabolite of xylazine in horse. 2,6-Dimethylaniline was, for the first time, reported as metabolite in equine.

Tissue distribution of xylazine in a suicide by hanging.

Xylazine (Rompun, Sedazine, AnaSed) is currently the most commonly used sedative-analgesic in veterinary medicine. There are nine published cases of xylazine's involvement in human drug-related deaths and impairment. However, blood concentrations were reported in only four of these cases. Three of these nine cases were fatalities involving xylazine, two of which involved xylazine alone but did not report blood concentrations because of extensive decomposition of the bodies. This report documents a case in which xylazine alone was identified in a suicide by hanging. The following xylazine concentrations were found: 2.3 mg/L in heart blood; 2.9 mg/L in peripheral (subclavian) blood; 6.3 mg/L in bile; 0.01 mg/L in urine; 6.1 mg/kg in liver; and 7.8 mg/kg in kidney.

Severe intoxication with the veterinary tranquilizer xylazine in humans.

Xylazine (Rompun, Proxylaz) is a veterinary tranquilizing agent. A case of self-injection of 1.5 g xylazine by a 27-year-old farmer is reported. He subsequently became comatose, hypotensive, bradycardic, and mildly glycemic. An intensive supportive therapy including intubation and ventilation was required. The patient made a full recovery over the next 30 h. The largest concentrations measured were 4.6 mg/L in plasma, 446 mg/L in gastric fluid, and 194 mg/L in urine. The calculated plasma half-life was 4.9 h. Kinetic data correlated with clinical symptoms. Qualitative and quantitative analyses of xylazine were done by thin-layer chromatography, gas chromatography-mass spectrometry, and high-performance liquid chromatography. These methods allow the detection of small amounts substance in stomach, plasma, and urine. Liquid-liquid extraction was used for the isolation of drug. The sensitvity is high, and with these methods, a rapid analysis is possible. Xylazine intoxications in humans are rare. We describe the management of acute poisoning and present a review of xylazine toxicity in humans.

Gas chromatographic-mass spectrometric analysis of veterinary tranquillizers in urine: evaluation of method performance.

A method for analysis of veterinary tranquillizers in urine using gas chromatography-mass spectrometry (GC-MS) is described. Detection limits are 5 microg/l for ketamine, azaperone and the phenothiazines (chlor-, aceto- and propionylpromazine), 10 microg/l for haloperidol, 20 microg/l for xylazine and 50 microg/l for azaperol, recoveries for all analytes were higher than 70%. Method performance in terms of within-batch, between-days and between-analysts reproducibility was studied and found to be acceptable. Compliance with European Union criteria for confirmation of GC-MS "positive" results is evaluated and discussed.