Vortex-assisted dispersive liquid-liquid microextraction-gas chromatography (VADLLME-GC) determination of residual ketamine, nimetazepam, and xylazine from drug-spiked beverages appearing in liquid, droplet, and dry forms.

Presently, investigations of drug-facilitated crimes (DFCs) rely on the detection of substances extracted from biological samples following intake by the victim. However, such detection requires rapid sampling and analysis prior to metabolism and elimination of the drugs from the body. In cases of suspected DFCs, drug-spiked beverage samples, whether in liquid, droplet, or even dried form, can be tested for the presence of spike drugs and used as evidence for the occurrence of DFCs. This study aimed to quantitatively determine three sedative-hypnotics (ketamine, nimetazepam, and xylazine) from drug-spiked beverages using a vortex-assisted dispersive liquid-liquid microextraction-gas chromatography (VADLLME-GC) approach. In this study, a GC method was first developed and validated, followed by the optimization of the VADLLME protocol, which was then applied to quantify the target substances in simulated forensic case scenarios. The developed GC method was selective, sensitive (limit of detection: 0.08 µg/ml [ketamine]; 0.16 µg/ml [nimetazepam]; 0.08 µg/ml [xylazine]), linear (R2 > 0.99), precise (%RSD <7.2%), and accurate (% recovery: 92.8%-103.5%). Higher recoveries were achieved for the three drugs from beverage samples in liquid form (51%-97%) as compared to droplet (48%-96%) and dried (44%-93%) residues. The recovery was not hindered by very low volumes of spiked beverage and dried residues. In conclusion, the developed VADLLME-GC method successfully recovered ketamine, nimetazepam, and xylazine from spiked beverages that are likely to be encountered during forensic investigation of DFCs.

Rapid untargeted screening for drug residues in animal tissues with liquid microjunction surface sampling probe mass spectrometry.

An untargeted screening method for the rapid identification of veterinary drug residues in incurred animal tissues using liquid microjunction surface sampling probe mass spectrometry (LMJSSP-MS) was developed. Current analytical methods for veterinary drug residue screening involve lengthy sample preparation, extraction, and instrumental analysis steps. This method identifies veterinary drug residues in several different incurred animal tissues more quickly than conventional analytical methods. This LMJSSP-MS method uses an ambient ionization technology called liquid microjunction surface sampling probe along with a data dependent scan function of a quadrupole orbitrap mass spectrometer. Collected product ion spectra are searched against the mzCloud™ online mass spectral database to identify veterinary drug residues found in incurred animal tissue samples. Examples of veterinary drugs identified with this method include flunixin, tilmicosin, pentobarbital, xylazine, and ketamine. Optimization of method parameters is described and discussed. The limit of identification (LOI) of this method is estimated to be approximately 1 µg g-1 for xylazine and ketamine.

Highly Sensitive Voltammetric Sensor Using Carbon Nanotube and an Ionic Liquid Composite Electrode for Xylazine Hydrochloride.

Electrochemical techniques were used for estimating xylazine HCl (XLZ) in bulk powder, medicinal manufacturing and human serum. Electro-oxidation of XLZ at carbon multiwalled nanotube (MWCNT), 1-n-butyl-3-methylpyridinium hexafluorophosphate ion crystal (BMH) and sodium dodecyl sulfate (SDS) MWCNT-BMH-SDS electrode in 0.04 M Britton-Robinson buffer (BR) with pH 7.0, was studied in numerous buffer structures and at different pH values. The experimentation and instrumental parameters to assessable commitment of XLZ had been optimized, and a detection limit was observed as 4.80 nM. The precision and accuracy for the recognized method was tested by retrieval studies with good repeatability and reproducibility of the estimated method. The projected method was practiced successfully to the dosage form and spiked serum.

Toward Higher Sensitivity in Quantitative MALDI Imaging Mass Spectrometry of CNS Drugs Using a Nonpolar Matrix.

Tissue-specific ion suppression is an unavoidable matrix effect in MALDI mass spectrometry imaging (MALDI-MSI), the negative impact of which on precision and accuracy in quantitative MALDI-MSI can be reduced to some extent by applying isotope internal standards for normalization and matrix-matched calibration routines. The detection sensitivity still suffers, however, often resulting in significant loss of signal for the investigated analytes. An MSI application considerably affected by this phenomenon is the quantitative spatial analysis of central nervous system (CNS) drugs. Most of these drugs are low molecular weight, lipophilic compounds, which exhibit inefficient desorption and ionization during MALDI using conventional polar acidic matrices (CHCA, DHB). Here, we present the application of the (2-[(2 E)-3-(4- tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile) matrix for high sensitivity imaging of CNS drugs in mouse brain sections. Since DCTB is usually described as an electron-transfer matrix, we provide a rationale (i.e., computational calculations of gas-phase proton affinity and ionization energy) for an additional proton-transfer ionization mechanism with this matrix. Furthermore, we compare the extent of signal suppression for five different CNS drugs when employing DCTB versus CHCA matrices. The results showed that the signal suppression was not only several times lower with DCTB than with CHCA but also depended on the specific tissue investigated. Finally, we present the application of DCTB and ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry to quantitative MALDI imaging of the anesthetic drug xylazine in mouse brain sections based on a linear matrix-matched calibration curve. DCTB afforded up to 100-fold signal intensity improvement over CHCA when comparing representative single MSI pixels and >440-fold improvement for the averaged mass spectrum of the adjacent tissue sections.

Analysis of ketamine and xylazine in complex matrices using two-dimensional liquid chromatography/tandem mass spectrometry.

RATIONALE: Analyzing tissue samples is routinely performed when liquid biological samples are not available for replicate analysis. Preparing complex matrices, such as tissue, for analysis can be time-consuming. Traditional sample preparation methods typically begin with homogenization followed by a sample clean-up step such as liquid-liquid or solid-phase extraction. Samples are typically eluted, evaporated and reconstituted prior to instrumental analysis. The aim of this project was to evaluate the utility of multi-dimensional chromatography in reducing the amount of time from sample acquisition to analysis. METHODS: Tissue specimens were homogenized using a ceramic beads shaker. Homogenates were then diluted and loaded onto a mixed mode solid-phase sorbent. The sorbent was washed, and the final eluate was transferred directly to vials without evaporation or reconstitution steps. Analysis was performed using a two-dimensional (2D) ultra-performance liquid chromatography (UPLC) configuration with an At-column dilution option coupled to a triple quadrupole mass spectrometer. The target analytes (xylazine and ketamine) were quantified under multiple reaction monitoring (MRM) using electrospray ionization (ESI) in positive mode. RESULTS: The lowest limit of detection evaluated in this study was 0.01 ng/mL. The linear dynamic range utilized was 0.1 to 10 ng/mL. The concentrations for xylazine in their respective tissues ranged from 0 to 0.316 ng/mL. Ketamine concentrations ranged from 0 to 0.905 ng/mL. The overall time for sample preparation was reduced to 30 min. The total run time was 10 min. CONCLUSIONS: The use of multidimensional chromatography with At-column-dilution allows for significant reduction in sample preparation time. The concentrations determined in these samples highlight the need for sensitive analytical techniques with the ability to detect analyte concentrations at sub ng/mL.

Use of Xylazine in Drug-Facilitated Crimes.

Human xylazine poisoning is uncommon. This report describes the use of xylazine for intentional poisoning with criminal intent. Two incidents occurred within 3 weeks: the first involved one victim, and the second involved two victims. The clinical presentations were brief coma, bradycardia, hypotension, and hyperglycemia. The victims recalled having been given a drink from a stranger in a hospital waiting room before loss of consciousness. In the first case, general drug screening by gas chromatography/mass spectrometry (MS) revealed xylazine in the gastric contents, but liquid chromatography-tandem MS (LC-MS/MS) of serum did not. In the second incident, LC-MS/MS screening of both victims' urine and serum samples revealed an unknown peak in the total ion chromatograms, which a molecular mass database identified as morantel or xylazine. The latter was confirmed by comparison with a xylazine standard. Based on this report, we suggest that xylazine should be classified as a controlled drug.

Attempted Drug-facilitated Sexual Assault-Xylazine Intoxication in a Child.

There are only a few cases of drug-facilitated sexual assaults on children reported in the literature so far. Here, a case of a four-year-old boy is presented. He was unconscious, and the accompanying adults reported that the child had been at a playground on his own. Returning home, he complained of having been stung and collapsed immediately. Urine and serum samples of the child were investigated. In the toxicological analysis, xylazine, a sedative and muscle relaxant used in animals, was detected. Subsequent quantification by GC/MS after solid-phase extraction revealed 0.053 mg/L xylazine in serum and approximately 0.63 mg/L in urine. Furthermore, the child was examined by a forensic medical specialist. Police investigations revealed that the godfather, who had been previously accused of sexual abuse of children, had injected the child with the drug, possibly in preparation for a shared bath.

Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography-hybrid triple quadrupole-linear ion trap-mass spectrometry.

Xylazine is an α2 -adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100 × 2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 µL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05-50 µm. The precision (%CV) and accuracy (%NOM) observed were 2.3-7.2 and 88.2-96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (Km = 10.1 µm) and a low metabolic stability of xylazine with a half-life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N-(2,6-dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes.

Efeitos de detomidina e xilazina intravenosa sobre as variáveis basais e respostas comportamentais em bovinos / Effects of intravenous detomidine and xilazine on basal parameters and behavioral responses in bovine

Avaliaram-se, durante 60 minutos, 10 bovinos após administração intravenosa de 0,1mg.kg-1 de xilazina ou 10μg.kg-1 de detomidina, quanto às frequências cardíaca e respiratória, movimentos ruminais, pressão arterial média, temperatura retal e respostas comportamentais como ataxia ou decúbito, ptose palpebral, estado de alerta ou sedação e redução da altura da cabeça em relação ao solo, além da presença de salivação, micção e concentração sanguínea de glicose. Observou-se que a xilazina, via intravenosa, em bovinos, ao mesmo tempo que promove sedação mais intensa e prolongada que a detomidina, induz a uma maior quantidade de efeitos indesejáveis, como salivação e decúbito, e redução das frequências cardíaca e respiratória, da pressão arterial média, da motilidade ruminal e da temperatura, sendo estas alterações mais prolongadas. Conclui-se que a detomidina pode ser utilizada com segurança em bovinos na dose de 10μg.kg-1, promovendo sedação e permanência do animal em posição quadrupedal.

Ten bovine were evaluated after intravenous injection of 0,1mg.kg-1 of xylazine or 10μg.kg-1 of detomidine during 60 minutes for heart and respiratory rate, ruminal motility, mean arterial pressure, rectal temperature and behavioral responses like ataxia or recumbency, palpebral ptoses, state of sedation or alert and head drop, besides the measurement of salivation, urination and blood glucose concentration. It was observed that intravenous xylazine in bovine promotes more intense and prolonged sedation than detomidine, and at the same time induces a larger and more prolonged quantity of unwanted side effects such as salivation, recumbency, decrease of cardiac and respiratory rate, mean arterial pressure, ruminal motility and temperature. We concluded that detomidine can be used safely in bovines at 10μg.kg-1 dose, promoting sedation with standing position.

Beyond-use dating of extemporaneously compounded ketamine, acepromazine, and xylazine: safety, stability, and efficacy over time.

Ketamine-acepromazine-xylazine (KAX) has long been a popular combination of injectable anesthetics for use in laboratory rodents. These drugs are compounded extemporaneously at research facilities because a commercial mixture is not available. This study was designed to determine an appropriate period of use for this mixture by examining its safety, stability, and efficacy at 30-d intervals over an aging period of 270 d. For as long as 270 d after compounding, most of the data collected (chemical stability, sterility, pH, particulate formation, times to loss of righting reflex in injected mice and rats, and histopathology from these animals) supported the finding that the component drugs do not change or degrade. However, mice and rats did show significant differences in anesthetic responses after injection with KAX mixtures of different ages. In light of these findings, we suggest that KAX remains safe, stable, and efficacious for at least 180 d after mixing, and that 180 d constitutes an appropriate period of use for this drug combination when stored in a dark, room-temperature environment.

GC-MS confirmation of xylazine (Rompun), a veterinary sedative, in exchanged needles.

In order to assess the extent of xylazine (Xyz) injection in Puerto Rico, two waves of used-syringe collections were performed. In the first, syringes were gathered, anonymously and without additional information; in the second, a short interview, also anonymous, was administered. We found Xyz in 37.6% of the collected syringes; the majority of the Xyz-containing syringes came from ranching communities. Syringes containing Xyz more frequently also contained "speedball" than those without (90.6% and 66.7%, respectively). Self-reports of Xyz injection deviated markedly from actual detection: only 50% (self-described users) and 22% (self-described non-users) of the collected syringes contained the drug. With a high prevalence of skin ulcers (38.5% vs. 6.8%; p<0.001), Xyz users were more likely to be in poor health compared to non-users. Surprisingly, though a higher percentage of Xyz users than non-users had college-level educations (23.1% vs. 5.5%), they were more likely to be homeless (64.1% vs. 37%).

Concurrent detection of heroin, fentanyl, and xylazine in seven drug-related deaths reported from the Philadelphia Medical Examiner's Office.

Recreational drugs, such as cocaine and heroin, are often adulterated with other pharmacological agents to either enhance or diminish the drug effects. Between April 21, 2006 and August 8, 2006, the Philadelphia Medical Examiner's Office detected xylazine (a veterinary sedative) and fentanyl (a synthetic opioid) in specimens taken from seven cases. Initial immunoassay screening was performed on urine and blood for fentanyl, opiate, cocaine, phencyclidine (PCP), and benzodiazepines. All tests reported positive were confirmed by gas chromatography-mass spectrometry. All seven xylazine positive cases tested positive for fentanyl and six cases tested positive for 6-acetylmorphine (a metabolite and definitive marker for heroin). The seventh case was positive for morphine and had a history of heroin abuse. Xylazine was present in urine in all seven cases and blood levels were detected in three cases. The blood concentrations ranged from trace to 130 ng/mL. Fentanyl was present in the blood and urine in each case and blood concentrations ranged from 4.7 to 47 ng/mL. Adulteration of illicit drugs has become an epidemic health concern for drug users. Healthcare professionals need to be aware of this issue, so the patients can be treated in an effective, timely manner.

Clinicophysiological effects of spinally administered ketamine and its combination with xylazine and medetomidine in healthy goats.

The study was conducted in 9 healthy adult goats of either sex, weighing 15-20 kg, to evaluate and compare the clinicophysiological effects of spinally administered ketamine alone and in combination with xylazine and medetomidine. Nine trials each of the three treatments were conducted randomly by injecting ketamine (2.5 mg/kg) (n = 9), ketamine and xylazine (2.5 mg/kg and 0.05 mg/kg) (n = 9) and ketamine and medetomidine (2.5 mg/kg and 10 microg/kg) (n = 9). The drugs were administered at the lumbosacral subarachnoid space under strict aseptic conditions. The treatments were evaluated on the basis of clinicophysiological, haematological, biochemical and haemodynamic observations. Ketamine produced mild to moderate analgesia of the hindquarters. Its combination with either xylazine or medetomidine produced complete analgesia of the hindquarters for 45-60 min. Ataxia was moderate in the ketamine group, whereas animals attained sternal recumbency in the combination groups. A moderate degree of sedation was recorded in the combination groups. Heart rate and respiratory rate depression in the combination groups and heart rate and respiratory rate stimulation in ketamine group were recorded. Haematological parameters decreased in all the groups. Increase in serum glucose, creatinine and urea nitrogen was recorded in all the groups. Serum electrolytes did not show any significant change. The results showed that the combination of ketamine with xylazine or medetomidine at these dose rates produced a comparable degrees of analgesia of hindquarters with transient and minimal cardiopulmonary side effects.

[Use of hyaluronic acid cleaving enzymes for absorption acceleration. Results of an in vitro study with xylazine and ketamine]. / Einsatz von Hyaluronsäure spaltenden Enzymen zur Resorptionsbeschleunigung. Ergebnisse einer In-vitro-Studie mit Xylazin und Ketamin.

The so-called "Hellabrunner Mischung", (combination of xylazine and ketamine with hyaluronidase) is frequently used for the immobilisation of wildlife animals. The enzyme hyaluronidase shall improve the distribution of the intramuscularly or subcutaneously administered compounds in the tissue and enhance their absorption. These enhancing effects of two hyaluronate lyases of bacterial origin (Streptococcus agalactiae and Streptococcus equisimilis) and a testicular hyaluronidase were compared in an in vitro test. Using the isolated perfused bovine udder, 2 ml of a solution were administered subcutaneously containing 125 mg/ml xylazine and 100 mg/ml ketamine and one of the above mentioned enzymes (150 I.U.). All three enzymes enhanced the absorption rate of xylazine and ketamine determined by measurement of the concentration in the perfusate. The bacterial hyaluronate lyases were significantly more efficient, especially during the clinically important first minutes after administration.

Efeitos da administração epidural de amitraz, xilazina ou dimetil sulfóxido em vacas / Effects of epidural injection of amitraz, xylazine or dimethyl sulfoxide in cows

Avaliaram-se os efeitos da injeção epidural de amitraz (0,4mg/kg), xilazina (0,05mg/kg) ou dimetil sulfóxido 10 por cento (5,0ml) sobre a freqüência cardíaca (FC), pressão arterial sistólica (PAS), freqüência respiratória (FR), motilidade ruminal (MR), temperatura retal (TR), altura de cabeça (AC) e latência das respostas a estímulos nociceptivos nas regiões da coxa (LECC) e coroa do casco (LRRM) de vacas. Houve diminuição da FC e da MR nos grupos xilazina e amitraz. O tratamento com xilazina resultou em alterações na FR, PAS e AC. LECC e LRRM foram maiores nos tratamentos com agonistas alfa-2. Nas doses utilizadas, o amitraz aumentou a latência de resposta a estímulo nociceptivo em menor grau que a xilazina, sem induzir efeitos colaterais sistêmicos severos, em vacas.

Multiresidue method for the detection of tranquillisers, xylazine, and a beta-blocker in animal production by liquid chromatography-tandem mass spectrometry.

Tranquillisers are often used in animal production, especially in pigs to calm them before transport to the slaughterhouse. The use of certain substances (derived from phenothiazine) is totally prohibited, whilst other compounds (butyrophenone and beta-blockers) are regulated through the establishment of MRLs. A physico-chemical detection method based on liquid chromatography-tandem mass spectrometry is described. Validation was carried out according to the criteria laid down in Directive 2002/657/EC. The method was also used to detect and quantify these substances in treated animals.

The regulatory status of xylazine for use in food-producing animals in the United States.

Xylazine is commonly used in veterinary medicine as a tranquillizer or adjunct to surgical anaesthesia. Although its use is approved in companion animals and certain species of deer, xylazine remains unapproved for use in food-producing animals in the United States. This paper reviews existing toxicological and residue chemistry information on xylazine in food animals, particularly cattle, and discusses the regulatory status of the drug in the US, as well as the conclusions reached by the Joint FAO/WHO Expert Committee on Food Additives in its recent evaluation of xylazine.

Multi-residue analysis of tranquillizers in meat: confirmatory assays using mass spectrometry.

A rapid and sensitive multi-residue method was developed to attempt to confirm the presence of the beta-blocker carazolol and the tranquillizers acepromazine, azaperone, chlorpromazine, propionylpromazine and xylazine in pig muscle tissues. The procedure involves determination by liquid chromatography coupled with tandem mass spectrometry. The liquid chromatographic separation was performed on a Symmetry C18 column with gradient elution. A mixture of aqueous buffer, containing 0.01% m/v trifluoroacetic acid (pH 3.5), and acetonitrile at a flow rate of 0.4 ml min-1 was used as the mobile phase. The abundant parent ions [M+ H+] produced by positive electrospray ionisation were selected for collisional dissociation with argon. Fragment ions were recorded with daughter ion scan and multiple reaction monitoring. The analytes were identified unambiguously by assessing retention times and diagnostic ions in meat samples spiked from 50 micrograms kg-1 [maximum residue limit (MRL) for azaperone and azaperol] to 5 micrograms kg-1 (MRL for carazolol).