Overcoming Girard's P Reagent Incompatibility: A Single Injection Method for the Analysis of a Mixed Steroid Ester Panel in DBS

Abstract

This study describes a dried blood spot (DBS) screening method for a panel of four anabolic steroid esters using a single injection for a dual analysis. The method combines Girard's Reagent P (GRP) derivatization for testosterone cypionate (TC), testosterone undecanoate (TU), and nandrolone phenylpropionate (NPP), while simultaneously monitoring the underivatized form of boldenone cypionate (BC). This approach resolves the known chemical incompatibility of BC's A-ring with GRP. A direct comparison of sampling devices under identical, enhanced conditions confirmed that Volumetric Absorptive Micro-sampling (VAMS™) Mitra provides better chromatographic performance to the Tasso-M20 device. A separate evaluation of a rapid, non-enhanced protocol highlighted the risk of severe matrix effects and instrument carryover associated with improperly prepared DBS samples. The paper validates a combined method that provides full screening coverage and offers a reliable, practical solution for anti-doping analysis. This method represents the first proof of concept for a dual GRP underivatized single-injection approach, improving analytical efficiency for doping control laboratories.