Practical application of blood microsampling: an effective VAMS-based workflow for anabolic androgenic steroid analysis

Volumetric absorptive microsampling (VAMS) has emerged as a promising alternative to traditional biological sampling methods,offering a high-reliability, field-deployable solution for steroid analysis with practical applications in anti-doping, forensic science and clinical testing. This study presents the optimisation of a VAMS-based workflow for the determination of anabolic androgenic steroids (AAS) in whole blood using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The research primarily focuses on refining microsampling and pretreatment strategies and addressing critical parameters such as volumetric accuracy, extraction efficiency,haematocrit effect mitigation and matrix interference. The workflow assesses both endogenous and exogenous steroids, overcoming limitations associated with traditional venous blood sampling. Experimental evaluations included assessments of sample homogeneity, recovery rates (87-95%), stability over time (up to 30 days) and the impact of haematocrit variability (-7% -+9%) on sampled volume. Strategies for internal standard (IS) addition to VAMS were also optimised to further enhance analytical accuracy. By integrating microsampling with the high reliability of mass spectrometric analysis, this study bridges the gap between laboratory research and practical applications in anti-doping testing, forensic science and clinical bioanalysis. The study provides a validated, cost-effective alternative to traditional sampling methods, confirming that VAMS is a promising minimally invasive tool for steroid detection in whole blood and highlighting its potential for broader applications in translational chemistry and personalised medicine.Keywords:Microsampling; volumetric absorptive microsampling; anabolic androgenic steroids; HPLC-MS/MS; anti-doping testing.