Modified Fully Automated Dried Blood Spot Sample Preparation System and Its Application in Steroid Ester Detection

The detection of anabolic androgenic steroids (AAS) remains a significant challenge in antidoping, particularly in distinguishing endogenous steroids from their synthetic counterparts. Although isotope ratio mass spectrometry (IRMS) is a commonly used confirmatory method, its complexity, high equipment requirements, and susceptibility to interference from drug formulations, diet, and environmental factors limit its practical application. In recent years, the direct detection of steroid esters in dried blood spots (DBS) has proven to be a simple and efficient complementary approach. However, conventional DBS processing is labor-intensive, and existing fully automated systems generally follow fixed workflows that lack flexibility, thereby failing to meet the high-sensitivity requirements needed for multitarget detection. In this study, we optimized the existing fully automated DBS processing system by introducing an independent desorption pump to improve the sample flow path. Based on this optimized system, we developed a high-throughput screening method for 30 steroid esters and a confirmatory method for 26 targets, using high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS). Method validation showed that the method achieved a low limit of detection (as low as 0.05 ng/mL for most substances) and good reproducibility (RSD < 15%). Furthermore, blind sample testing, administration studies, and comparisons with traditional IRMS methods verified the practical performance of our approach. Overall, this study establishes an efficient and sensitive automated DBS analytical method, providing a viable technical solution for the screening and confirmation of steroid esters, and potentially other doping agents.