Rapid and harmonized analytical workflow for the determination of peptidic and non-peptidic doping agents in dried and liquid blood matrices.
Level 5 - mechanism / opinion, no new human data
Bench analytical chemistry method development and validation study without clinical outcomes (graded Level 5 by CEBM analogy).
PubMed 42328738 · doi:10.1039/d6an00455e
What was done
The authors developed and validated a unified analytical workflow to detect 54 prohibited peptidic and non-peptidic doping substances in dried blood spots (DBS), serum, and plasma. Sample preparation utilized a single microextraction step with 500 µL of methanol/water (8:2, v/v), followed by analysis using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). The protocol was validated for selectivity, detection limits, carry-over, matrix effects, extraction yield, and autosampler stability (72 h at 10 °C). Stability was assessed across matrices at -20 °C, 4 °C, and 22 °C, and the method was tested on samples containing sub-ng mL⁻¹ levels of ibutamoren.
What was found
Validation showed limits of detection between 0.05 and 1.25 ng mL⁻¹, matrix effects between 5% and 33%, extraction yields between 15% and 80%, zero carry-over, and no interfering background signals at analyte retention times. All 54 analytes showed <15% variation over two months at -20 °C in all matrices. However, at 4 °C and 22 °C in liquid matrices, alexamorelin, AOD9604, buserelin, hGH 176-191, kisspeptin-10, and LHRH extensively degraded within one week in serum and plasma, while BPC-157, TB500, vasopressin, lypressin, and terlipressin degraded completely in serum. In contrast, all analytes remained detectable in dried blood matrices at non-refrigerated temperatures throughout the study.
Why it matters
This method streamlines doping-control workflows by replacing multi-step preparations with a single extraction for both small molecules and peptides across multiple blood formats. It confirms dried blood spots as a viable, cost-effective transport medium that preserves otherwise heat- and enzyme-labile peptide doping agents.
Limits
The abstract does not state the number or demographic breadth of human blood donors evaluated. Extraction yields showed broad variability (15% to 80%), indicating low recovery for certain target analytes. In-use verification was limited to spiked or authentic sub-ng samples of a single drug (ibutamoren) rather than the complete 54-compound library.
Cited by
- supports BPC-157 is included on the World Anti-Doping Agency (WADA) prohibited list.