Automated high‐throughput analysis of tramadol and O‐desmethyltramadol in dried blood spots

Abstract

The World Anti‐Doping Agency (WADA) and the International Testing Agency (ITA) recently announced the development and implementation of dried blood spot (DBS) testing for routine analysis in time for the 2022 Winter Olympic and Paralympic Games in Beijing. Following the introduction of a ban on the use of tramadol in competition in March 2019, the Union Cycliste International (UCI) started a pilot study for the manual analysis of tramadol in DBS for antidoping purposes. In this context, we present a fully automated LC–MS/MS‐based method with automated sample preparation using a CAMAG DBS‐MS 500 for the analysis of tramadol and its metabolite O‐desmethyltramadol in DBS. The presented approach reduces manual handling in the laboratory to an absolute minimum, only requiring the preparation of calibration and quality control DBS cards. The method was developed, optimized, and validated before performing cross‐validation with a liquid blood‐based analysis method using authentic samples from forensic cases. During the validation process, the method showed an extraction efficiency of 62%, linearity r2 > 0.99, accuracy and precision (within ± 15% and ± 20% at the LLOQ) for the determination of tramadol and O‐desmethyltramadol. Method comparison in liquid blood with 26 samples showed good agreement (90 ± 19% for tramadol and 94 ± 14% for O‐desmethyltramadol). In conclusion, automated analysis of tramadol and O‐desmethyltramadol in DBS provides a fast and accurate solution for antidoping screening. It is suited for high‐throughput analysis, having a run time of about 4 min per sample. Furthermore, with the automated approach, manual sample extraction becomes obsolete.

An Update on the Implications of New Psychoactive Substances in Public Health

“…Opioids cause anesthetic and depressant effects; however, their recreational use has been of public health concern [247]. According to the UNODC report from 2013, up until this date, the only opioid in the spotlight among NPS was O-desmethyltramadol (the main active metabolite of tramadol) that was consumed together with kratom [12,248]. Novel synthetic opioids, or simply synthetic opioids, include fentanyl and analogues, but compounds such as tramadol, methadone, pethidine, ketobemidone, levorphanol, dextromoramide, dipipanone, and others are also included in this category.…”

Evaluation of dried blood spots as an alternative sample matrix for equine antidoping analysis

“…As with current approaches for routine post‐race and out‐of‐competition samples, complete coverage for all classes of molecules will likely require several different extraction and instrumental analysis approaches to reach the desired levels of coverage. Several of these classes of agents have been evaluated in DBS for human antidoping efforts though further evaluation of the use of DBS in the horse should be undertaken if coverage for these drug classes is desired 7,8,17–19 …”

Dried blood spots for anti‐doping: Why just going volumetric may not be sufficient

“…Different HCT levels may have a positive or negative impact on the recovery of a target analyte leading to undesirable bias, known as hematocrit extraction bias. [35][36][37] For some analytes, such as naproxen, high HCT levels are expected to trap the drug within the matrix buildup, leading to the observation of lower desorption from the spot, resulting in a lower recovery. 37 For volumetric absorptive microsampling (VAMS), two studies reported that the drug recovery levels of analytes were inversely correlated with HCT levels.…”