Simultaneous Determination of Xylazine, Free Morphine, Codeine, 6-Acetylmorphine, Cocaine and Benzoylecgonine in Postmortem Blood by UPLC-MS-MS

Abstract

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.

Toxic effects of xylazine on endothelial cells in combination with cocaine and 6-monoacetylmorphine

“…As xylazine is substituting heroin as a drug of abuse and also been the main adulterant of heroin, the current mixture modality is now among cocaine and xylazine or a mixture of cocaine, xylazine and heroin. This information was obtained from the chemical analysis of seized drug, performed by the Controlled Substance Section at the Puerto Rico Institute of Forensic Science (PRIFS) [13]. …”

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

“…Most xylazine LC-MS/MS methods rely on the lighter product ion (m/z 89.7) for quantitation of XYL [6,9,10,13] or the precursor ion (m/z 221) alone [1,8]. However, the current study indicates that sensitivity for XYL can be improved by approximately 40% by using both m/z 89.7 (XYL1) and 163.7 (XYL2) product ions cumulatively.…”

“…The instrumental LLoQ for DMA was 0.2 ng/mL (2 pg on-column) on the LC-MS/MS and 0.4 ng/mL in the original, untreated plasma. In a study using human blood, the reported LLoQ for XYL was 10 ng/mL in blood, which equates to 100 pg on-column [10], and included a 2.5-fold concentration step during the extraction process. We speculate that this 200-fold difference in XYL LLoQs between methods may in part be due to the low organic solvent concentration, and the very short run time (2.5 mins) in the literature method, resulting in matrix suppression of the analyte.…”

“…HPLC-UV [4,7], LC-MS [8] and LC-MS/MS [9,10] have all been utilised for the determination of XYL. Sample preparation methods include solvent extraction and concentration (in order to improve the lower limit of quantitation (LLoQ) [6,10]), as well as simple protein precipitation [8]. However, these procedures increase the time and cost of sample processing and analysis.…”

“…However, these procedures increase the time and cost of sample processing and analysis. Some studies have reported LLoQs of xylazine in plasma as 105 ng/mL by GC-MS [3], 25 ng/mL by HPLC-UV [4] and 10 ng/mL by LC-MS/MS [10]. However, these limits may not be adequate for determining the pharmacokinetics of XYL in plasma when the reported half-life of XYL can be as brief as 22 min in sheep [11].…”