A bar adsorptive microextraction (BAµE) workflow has been validated for qualitative confirmation of cocaine and three metabolites in human urine, offering a lower-solvent alternative for anti-doping laboratories using gas chromatography-mass spectrometry (GC-MS).
Cocaine is frequently detected in anti-doping testing, but confirmatory analysis can be complicated by the low concentrations of cocaine and its metabolites in urine and by endogenous compounds that may interfere with their detection. Effective sample preparation is therefore needed to concentrate the target analytes and reduce matrix interference.
The study focuses on qualitative confirmation of cocaine and three urinary metabolites: benzoylecgonine, ecgonine methyl ester, and cocaethylene.
The BAµE step uses a small coated bar to selectively retain target analytes from the sample. After testing several polymeric and activated-carbon sorbents, the team selected a divinylbenzene polymer phase, which gave the best overall performance across cocaine and its more polar metabolites. The optimized workflow used a two-hour extraction from urine at pH 9, followed by micro-liquid desorption with acetonitrile, derivatization, and GC-MS analysis in selected ion monitoring mode.
Compared with conventional solid-phase or liquid-liquid extraction, BAµE is designed to reduce solvent use while still producing extracts suitable for chromatographic analysis. In the optimized procedure, the team used 150 µL of acetonitrile for back-extraction. Although the extraction step takes two hours, the authors note that the method is intended for confirmatory testing rather than high-throughput screening, where batches are typically small.
Validation was carried out in native urine samples against World Anti-Doping Agency (WADA) criteria. The method showed no false positives in blank urine samples and no false negatives in spiked samples. Limits of identification met the relevant WADA requirements, including 1.0 ng/mL for cocaine, and the team found no carryover after high-level injections. Final extracts remained stable for up to 144 hours.
The workflow was then tested on three proficiency urine samples and two authentic urine samples previously analyzed by the laboratory. Target compounds were identified in line with the expected results, extending the validation beyond spiked urine samples alone.
The authors also compared the method with existing laboratory procedures. Relative to an internal solid-phase extraction/GC-MS method, the BAµE-based workflow improved selectivity and sensitivity while retaining compatibility with single-quadrupole GC-MS. An AGREE assessment indicated a moderate green analytical profile, reflecting lower solvent use alongside the remaining demands of sample preparation and derivatization.
The authors suggest that BAµE could be extended to other prohibited substances or coupled with liquid chromatography-mass spectrometry (LC-MS) platforms, broadening its use beyond cocaine confirmation while preserving the lower-solvent sample-preparation format.
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