Detection of 20 endogenous anabolic steroid esters with Girard's Reagent P derivatization in dried blood spots using UPLC-Q-Orbitrap-MS

The esters of endogenous anabolic steroids are the most frequently used doping agents for prolonging the half-life of exogenously ingested endogenous anabolic steroids. As a cost-and time-saving matrix, dried blood spots (DBSs) are valuable for directly detecting endogenous anabolic steroid esters in blood and for providing conclusive evidence of their abuse. In this study, a method for simultaneous detection of 20 endogenous anabolic steroid esters in DBSs based on ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) with parallel reaction monitoring (PRM) was developed and validated, and 10 of these esters were analyzed in DBSs for the first time. This method analyzes the greatest number and types of endogenous anabolic steroid esters of any current method using DBSs. Girard's Reagent P (GRP) was used for the derivatization of endogenous anabolic steroid esters in a DBS matrix for the first time, and the conditions of the derivatization reaction were optimized to achieve a higher sensitivity compared to previous methods. Selectivity, limit of detection (LOD), extraction recovery, precision (intra-and inter-), matrix effects, and carry-over were analyzed to validate the method. The LODs were lower and the recoveries were higher than those of previous studies. The relative standard deviation of the intraday precision was below 20% and the interday precision was below 35%. A product ion analysis of GRP nandrolone ester, GRP boldenone ester, GRP dehydroepiandrosterone acetate, and GRP androstanolone ester derivatives was performed, and the structures of the fragment ions were proposed for the first time.

Automated summary

In this study, a method based on ultra-high performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UPLC-Q-Orbitrap-MS) with parallel reaction monitoring (PRM) was developed to simultaneously detect 20 endogenous anabolic steroid esters in dried blood spots (DBSs), and 10 of these esters were analyzed for the first time. Girard's Reagent P (GRP) was used for the derivatization of the esters, resulting in higher sensitivity compared to previous methods. The method demonstrated good selectivity, low limit of detection, high extraction recovery, and precision.