Xylazine, a Veterinary Tranquilizer, Detected in 42 Accidental Fentanyl Intoxication Deaths.

ABSTRACT: Xylazine is an emerging adulterant with fentanyl in fatal drug intoxications, which has public health, safety, and criminal investigative implications. Xylazine is a nonnarcotic sedative used for analgesia and muscle relaxation exclusively in veterinary medicine. Its chemical structure is similar to clonidine and acts as a central α-2 agonist which may cause bradycardia and transient hypertension followed by hypotension. We report the detection of xylazine in 42 deaths in Connecticut from March to August 2019. Xylazine combined with an opioid or stimulant may affect the toxicity of these drugs. Detection of xylazine may help the forensic pathologist distinguish illicit from prescribed fentanyl, and law enforcement agents track the illicit drugs to a specific drug supplier. Because of its lack of response to naloxone, emergency medicine physicians need to be aware of its potential presence as it may affect therapy.

Pharmacokinetics of xylazine after 2-, 4-, and 6-hr durations of continuous rate infusions in horses.

Intravenous (i.v.) bolus administration of xylazine (XYL) (0.5 mg/kg) immediately followed by a continuous rate infusion (CRI) of 1 mg kg-1  hr-1 for 2, 4, and 6 hr produced immediate sedation, which lasted throughout the duration of the CRI. Heart rate decreased and blood pressure increased significantly (p > .05) in all horses during the first 15 min of infusion, both returned to and then remained at baseline during the duration of the infusion. Compartmental models were used to investigate the pharmacokinetics of XYL administration. Plasma concentration-time curves following bolus and CRI were best described by a one-compartment model. No differences were found between pharmacokinetic estimates of the CRIs for the fractional elimination rate constant (Ke ), half-life (t1/2e ), volume of distribution (Vd ), and clearance (Cl). Median and range were 0.42 (0.15-0.97)/hr, 1.68 (0.87-4.52) hr, 5.85 (2.10-19.34) L/kg, and 28.7 (19.6-39.5) ml min-1  kg-1 , respectively. Significant differences were seen for area under the curve ( AUC 0 ∞ ) (p < .0002) and maximum concentration (Cmax ) (p < .04). This indicates that with increasing duration of infusion, XYL may not accumulate in a clinically relevant way and hence no adjustments are required in a longer XYL CRI to maintain a constant level of sedation and a rapid recovery.

Re-evaluation of the pharmacokinetics of xylazine administered to Thoroughbred horses.

Xylazine is widely used worldwide as a short-acting sedative in general equine and racing practice. In the UK, although it has a legitimate use during training, equine anti-doping rules state it is a prohibited substance on race day. The aim of the study was to produce a detection time (DT) to better inform European veterinary surgeons so that xylazine can be used appropriately under regulatory rules. Previous publications have various limitations pertaining to analysis method, particularly for plasma and limited length of time of sample collection. In this study, pharmacokinetic data were produced for xylazine and 4-OH-xylazine in equine urine and plasma following a single intravenous xylazine dose of 0.4 mg/kg to six Thoroughbred horses. Pharmacokinetic parameters were generated from a 3-compartmental model with clearance = 15.8 ± 4.88 ml min-1  kg-1 , Vss = 1.44 ± 0.38 L/kg, terminal half-life = 29.8 ± 12.7 hr and a DT determined at 71 hr for the administration of xylazine (Chanazine® ) in plasma and urine. Urine screening should aim to detect the 4-OH-xylazine metabolite, which can act as an indicator for the xylazine plasma concentration. A DT of 72 hr has been agreed by the European Horserace Scientific Liaison Committee, to be implemented in June 2019.

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.

Quantitation of the anaesthetic xylazine in ovine plasma by LC-MS/MS.

Removal of the wool-bearing skin around a young lamb's rump (mulesing) provides long term health benefits for the animal, and the use of a sedative and analgesic agent such as xylazine may assist with pain relief to reduce discomfort and stress. Sensitive analytical methods are essential for monitoring pharmaceuticals and their metabolites in animals destined for human consumption. The following work reports a method that is 200 times more sensitive for xylazine detection than previously published methods, with lower limits of quantitation for xylazine and its primary metabolite in animals of 0.5pg and 2pg on-column, respectively. The use of a square wave solvent gradient immediately prior to analyte elution resulted in larger MS/MS peaks and a reduction in baseline noise, allowing reliable detection of lower analyte concentrations. The method uses as little as 1mL of plasma which allows replication within a sample if required, and requires simple sample preparation, minimising the introduction of matrix components into the MS/MS.

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.

Pharmacokinetics and pharmacodynamics of xylazine administered by the intravenous or intra-osseous route in adult horses.

In certain situations, an alternate route for parenteral drug administration in horses may be useful. The intra-osseous (IO) route may provide a safe alternative to the intravenous (i.v.) route for administration of sedatives to horses when the i.v. route is inaccessible or undesirable. Six adult horses were administered xylazine i.v. or IO in a block-randomized crossover design. For the i.v. trial, both jugular veins were catheterized, and one was used for xylazine administration, while the other was used for blood collection. For the IO trial, one jugular vein was catheterized for blood collection and an intra-osseous device was placed in the tuber coxae using a powered driver for xylazine administration. Heart rate, respiratory rate, and head position were measured, and concentration of sedation was assessed at various times up to 90 min. Xylazine concentrations were measured using high-performance liquid chromatography and noncompartmental analysis was performed. General linear mixed modeling and Wilcoxon signed-rank tests were used for statistical analysis, with P ≤ 0.05. There were no significant differences in heart rate, respiratory rate, head position, concentration of sedation, Cmax , Tmax , half-life, or AUC between the i.v. and the IO routes of drug administration. No complications were observed following placement of the intra-osseous device. Intra-osseous xylazine administration provides a useful option in emergent and other settings in which i.v. access is difficult or contraindicated.

Voltage changes in the lithium dilution cardiac output sensor after exposure to blood from horses given xylazine.

BACKGROUND: In a previous in vitro study using saline medium, the authors showed that certain drugs changed the voltages of lithium dilution cardiac output (LiDCO) sensors and also influenced their accuracy in measuring lithium concentrations. These two parameters correlated and so we examined whether such drug-sensor interaction exists when LiDCO sensor was exposed to xylazine in blood. METHODS: Five healthy adult warm-blood horses were injected with 0.5 mg kg(-1) xylazine i.v. Physiological saline solution and venous blood were consecutively sampled through the same LiDCO sensor at 60, 45, 30, 15, and 0 min before and then 5, 15, 30, 45, and 60 min after xylazine injection. Sensor voltages were recorded and the differences between saline- and blood-exposed sensor voltages were compared at each time point. RESULTS: Saline-exposed sensor voltages continuously increased in a non-linear pattern during the experiment. Blood-exposed sensor voltages also increased in a similar pattern, but it was interrupted by an abrupt increase in voltage after xylazine injection. The differences between saline- and blood-exposed sensor voltages were 7 (6.1-8) mV [median (range)] before xylazine but decreased significantly at 5 and 15 min after xylazine treatment. The highest drug-induced voltage change was 3.4 (1.6-7) mV. CONCLUSIONS: This study showed that exposure of a LiDCO sensor to blood after a single clinically relevant dose of xylazine in horses changed the voltages of the sensors for 15 min. Comparison of saline- and blood-exposed sensor voltages could become a tool to detect drug-sensor interactions.

Pharmacokinetics of ketamine and xylazine in young and old Sprague-Dawley rats.

To compare the pharmacokinetics of coadministered intraperitoneal ketamine and xylazine in young (8 to 10 wk; n = 6) and old rats (2 to 2.4 y; n = 6), blood samples obtained at 15 and 30 min and 1, 2, and 4 h after drug administration were analyzed by HPLC-tandem mass spectrometry. In both groups, the withdrawal reflex was absent during anesthesia and was present at 1.1 (± 0.2) and 2.6 (± 0.7) h after drug administration in young and old rats, respectively, with the first voluntary movement at 1.5 ± 0.2 and 4.9 ± 1.0 h. Drug availability of ketamine and xylazine was 6.0 and 6.7 times greater, respectively, in old than young rats. The rate constant of elimination of both drugs was greatly decreased and the elimination half-life was significantly greater in old compared with young rats. In conclusion, age and associated factors affect the availability of ketamine and xylazine when coadministered to attain clinical anesthesia, changing the pharmacokinetics of these drugs and prolonging anesthesia duration and recovery times with aging. Compared with their young counterparts, aged rats required much higher doses to attain a similar level of anesthesia. Finally, the long half-life of both ketamine and xylazine, when coadministered to old rats, may be a factor in research protocols because residual plasma concentrations could still be present for as long as 3 and 5 d, respectively, after administration.

Simultaneous determination of xylazine, free morphine, codeine, 6-acetylmorphine, cocaine and benzoylecgonine in postmortem blood by UPLC-MS-MS.

Xylazine, a veterinary sedative, has been found as an adulterant of heroin in street drugs in Puerto Rico. It was found in combination with free morphine and 6-acetylmorphine, codeine, cocaine and benzoylecgonine in postmortem cases at the Puerto Rico Institute of Forensic Sciences (PRIFS). Xylazine is not approved for human use because it has been proven harmful. Currently, three separate analyses are required to determine all the aforementioned drugs at the PRIFS's toxicology laboratory. To reduce analysis time consumption, sample volume, run time, sample preparation and cost, a high-throughput ultra-high-pressure liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of xylazine, free morphine, 6-acetylmorphine, codeine, cocaine and benzoylecgonine in 0.25 mL postmortem blood by protein precipitation, fulfilling confirmation criteria with three transitions for each compound with acceptable relative ion intensities. Linearity was established between 10-1,000 ng/mL. Total run time was 2.5 min. Limit of detection was 1 ng/mL for cocaine and xylazine, 2 ng/mL for 6-acetylmorphine and 10 ng/mL for free morphine, codeine and benzoylecgonine. The intra-day and inter-day precision and accuracy was less than 15.6%. Process efficiencies ranged from 35.9 to 123.4% and recoveries from 59.9 to 110.1%. The developed method was successfully applied to casework.

Qualitative and quantitative characteristics of the electroencephalogram in normal horses after sedation.

BACKGROUND: The administration of certain sedatives has been shown to promote sleep in humans. Related agents induce sleep-like behavior when administered to horses. Interpretation of electroencephalograms (EEGs) obtained from sedated horses should take into account background activity, presence of sleep-related EEG events, and the animal's behavior. HYPOTHESIS: Sedatives induce states of vigilance that are indistinguishable on EEGs from those that occur naturally. ANIMALS: Six healthy horses. METHODS: Digital EEG with video was recorded after administration of 1 of 4 sedatives (acepromazine, butorphanol, xylazine, or detomidine). Serum drug concentrations were measured. Recordings were reviewed, states were identified, and representative EEG samples were analysed. These data were compared with data previously obtained during a study of natural sleep. RESULTS: Butorphanol was associated with brief episodes resembling slow wave sleep in 1 horse. Acepromazine led to SWS in 3 horses, including 1 that also exhibited rapid eye movement sleep. Periods of SWS were observed in all horses afer xylazine or detomidine administration. Normal sleep-related EEG events and heart block, occurred in association with SWS regardless of which sedative was used. Spectral data varied primarily by state, but some differences were observed between sedative and natural data. CONCLUSIONS AND CLINICAL IMPORTANCE: Qualitatively, EEG findings appeared identical whether sedation-induced or naturally occurring. The startle response and heart block associated with some sedatives may be related to sleep. Alpha(2) agonists can be used to obtain high quality EEGs in horses, but acepromazine does not promote a relaxed state in all animals.

Simultaneous HPLC-UV determination of ketamine, xylazine, and midazolam in canine plasma.

An isocratic reversed-phase high-performance liquid chromatography method with UV detection is developed and validated for the simultaneous determination of ketamine, xylazine, and midazolam in canine plasma. Analytes are extracted from alkalinized samples into diethyl ether-methylene chloride (7:3, v:v) using single-step liquid-liquid extraction. Chromatographic separation is performed on a C(18) column using a mobile phase containing an acetonitrile-methanol-10 mM sodium heptanesulfonate buffer adjusted to pH 3, with glacial acetic acid (44:10:46, v:v) at a detection wavelength of 210 nm, with a total runtime of 10 min. The calibration is linear over the range of 78.125-5000 ng/mL for ketamine and 15.625-1000 ng/mL for xylazine and midazolam. The limits of detection are 17.8, 10.3, and 15.1 ng/mL for ketamine, xylazine, and midazolam, respectively. The extraction recoveries are 76.1% for ketamine, 91.0% for midazolam, and 78.2% for xylazine. The method is successfully used for clinical and pharmacokinetic studies of the three-drug fixed dose combination formulations.

Development of a xylazine constant rate infusion with or without butorphanol for standing sedation of horses.

OBJECTIVE: To elaborate constant rate infusion (CRI) protocols for xylazine (X) and xylazine/butorphanol (XB) which will result in constant sedation and steady xylazine plasma concentrations. STUDY DESIGN: Blinded randomized experimental study. ANIMALS: Ten adult research horses. METHODS: Part I: After normal height of head above ground (HHAG = 100%) was determined, a loading dose of xylazine (1 mg kg(-1) ) with butorphanol (XB: 18 µg kg(-1) ) or saline (X: equal volume) was given slowly intravenously (IV). Immediately afterwards, a CRI of butorphanol (XB: 25 µg kg(-1) hour(-1)) or saline (X) was administered for 2 hours. The HHAG was used as a marker of depth of sedation. Sedation was maintained for 2 hours by additional boluses of xylazine (0.3 mg kg(-1)) whenever HHAG >50%. The dose of xylazine (mg kg(-1) hour(-1)) required to maintain sedation was calculated for both groups. Part II: After the initial loading dose, the calculated xylazine infusion rates were administered in parallel to butorphanol (XB) or saline (X) and sedation evaluated. Xylazine plasma concentrations were measured by HPLC-MS-MS at time points 0, 5, 30, 45, 60, 90, and 120 minutes. Data were analyzed using paired t-test, Wilcoxon signed rank test and a 2-way anova for repeated measures (p < 0.05). RESULTS: There was no significant difference in xylazine requirements (X: 0.69, XB: 0.65 mg kg(-1) hour(-1)) between groups. With treatment X, a CRI leading to prolonged sedation was developed. With XB, five horses (part I: two, part II: three) fell down and during part II four horses appeared insufficiently sedated. Xylazine plasma concentrations were constant after 45 minutes in both groups. CONCLUSION: Xylazine bolus, followed by CRI, provided constant sedation. Additional butorphanol was ineffective in reducing xylazine requirements and increased ataxia and apparent early recovery from sedation in unstimulated horses. CLINICAL RELEVANCE: Data were obtained on unstimulated healthy horses and extrapolation to clinical conditions requires caution.

Pharmacokinetics and physiologic effects of intramuscularly administered xylazine hydrochloride-ketamine hydrochloride-butorphanol tartrate alone or in combination with orally administered sodium salicylate on biomarkers of pain in Holstein calves following castration and dehorning.

OBJECTIVE: To determine the pharmacokinetic parameters of xylazine, ketamine, and butorphanol (XKB) administered IM and sodium salicylate (SAL) administered PO to calves and to compare drug effects on biomarkers of pain and distress following sham and actual castration and dehorning. ANIMALS: 40 Holstein bull calves from 3 farms. PROCEDURES: Calves weighing 108 to 235 kg (n = 10 calves/group) received one of the following treatments prior to sham (period 1) and actual (period 2) castration and dehorning: saline (0.9% NaCl) solution IM (placebo); SAL administered PO through drinking water at concentrations from 2.5 to 5 mg/mL from 24 hours prior to period 1 to 48 hours after period 2; butorphanol (0.025 mg/kg), xylazine (0.05 mg/kg), and ketamine (0.1 mg/kg) coadministered IM immediately prior to both periods; and a combination of SAL and XKB (SAL+XKB). Plasma drug concentrations, average daily gain (ADG), chute exit velocity, serum cortisol concentrations, and electrodermal activity were evaluated. RESULTS: ADG (days 0 to 13) was significantly greater in the SAL and SAL+XKB groups than in the other 2 groups. Calves receiving XKB had reduced chute exit velocity in both periods. Serum cortisol concentrations increased in all groups from period 1 to period 2. However, XKB attenuated the cortisol response for the first hour after castration and dehorning and oral SAL administration reduced the response from 1 to 6 hours. Administration of XKB decreased electrodermal activity scores in both periods. CONCLUSIONS AND CLINICAL RELEVANCE: SAL administered PO through drinking water decreased cortisol concentrations and reduced the decrease in ADG associated with castration and dehorning in calves.

Solid-phase extraction and gas chromatographic- mass spectrometric determination of the veterinary drug xylazine in human blood.

This paper presents a method for the determination of xylazine in whole blood using solid-phase extraction and gas chromatography-mass spectrometry. This technique required only 0.5 mL of sample, and protriptyline was used as internal standard (IS). Limits of detection and quantitation (LOQ) were 2 and 10 ng/mL, respectively. The method was found to be linear between the LOQ and 3.50 microg/mL, with correlation coefficients higher than 0.9922. Precision (intra- and interday) and accuracy were in conformity with the criteria normally accepted in bioanalytical method validation. The analyte was stable in the matrix for at least 18 h at room temperature and for at least three freeze/thaw cycles. Mean recovery, calculated at three concentration levels, was 87%. To the best of our knowledge, this is the first time that solid-phase extraction is used as sample preparation technique for the determination of this compound in biological media. Because of its simplicity and speed when compared to other extraction techniques, the herein described method can be successfully applied in the diagnosis of intoxications by xylazine.

Determination of acepromazine, ketamine, medetomidine, and xylazine in serum: multi-residue screening by liquid chromatography-mass spectrometry.

A large variety of drugs are administered to large and small animals by veterinary clinicians for sedation, anesthesia, muscle relaxation, and analgesia. The present paper reports a simple and rapid multi-residue detection and quantitation method for four chemically different drugs: medetomidine, xylazine, ketamine, and acepromazine. Chromatographic separation was carried out on a liquid chromatography-mass spectrometry instrument with a C18-reversed-phase column. Fragmentation patterns were determined with atmospheric pressure chemical ionization mass spectrometry set to operate in a positive selective ion monitoring mode. The method was determined to be linear over the range of concentrations tested (2.0-100.0 ng/mL). Accuracy, precision, and specificity were evaluated and the method was determined to be applicable to detection of medetomidine, xylazine, ketamine, and acepromazine in serum samples of multiple animal species (canine, equine, and bovine). Matrix limits of quantitation were determined to be 5.0 ng/mL for all four analytes, and recoveries ranged between 82.0 and 118%, with a 3.0-18.3% relative standard deviation.

Pharmacokinetics of xylazine, 2,6-dimethylaniline, and tolazoline in tissues from yearling cattle and milk from mature dairy cows after sedation with xylazine hydrochloride and reversal with tolazoline hydrochloride.

Xylazine hydrochloride was administered i.m. at 0.35 mg/kg to 13 steers and 10 lactating dairy cows at Time 0. Ten minutes later, tolazoline hydrochloride was given i.v. at 4 mg/kg. Tissue and milk samples were analyzed using gas chromatography with nitrogen and phosphorous detection to determine concentrations of xylazine, 2,6-dimethylaniline (a toxic metabolite of xylazine), and tolazoline (at various intervals). Concentrations of xylazine and 2,6- dimethylaniline were below the limit of quantitation (10 microg/kg) by 72 hours in tissues and 12 hours in milk. The concentration of tolazoline was below 10 microg/kg by 96 hours in tissues and 48 hours in milk. Based on the results of these residue studies submitted by the sponsoring agency to the Ministry of Agriculture and Forestry in New Zealand, withholding periods for both xylazine hydrochloride and tolazoline hydrochloride injection were established.

A reported case involving impaired driving following self-administration of xylazine.

A case of suspected drug-impaired driving involving self-administration of xylazine (Xyla-Ject), a veterinary tranquilizing agent, and paroxetine is presented. Qualitative and quantitative analysis of xylazine and paroxetine were performed by gas chromatography with a flame-ionization detector (GC-FID) and gas chromatography/mass spectrometry (GC/MS). Whole blood xylazine and paroxetine concentrations were 0.57 and 0.02 microg/ml, respectively.

Simultaneous determination of ketamine and xylazine in canine plasma by liquid chromatography with ultraviolet absorbance detection.

An isocratic reversed-phase high-performance liquid chromatographic method for the simultaneous determination of ketamine and xylazine in canine plasma is described. Plasma samples (500 microl) are cleaned up via liquid-liquid extraction. The analytes and the internal standard clonidine are separated on a cyano (CN) column using a mobile phase containing acetonitrile-0.005 M phosphate buffer adjusted to pH 5.5 (3:2) at a detection wavelength of 215 nm. The method was validated according to specificity, sensitivity, accuracy and reproducibility and was used to determine the plasma concentrations of both compounds in dogs after intramuscular injection.