RAPID UPLC-MS/MS DRIED BLOOD SPOT ANALYSIS OF STEROID HORMONES USING THE XEVO TQ-S MICRO FOR CLINICAL RESEARCH

Importance of the Topic

Dried blood spot (DBS) sampling is a minimally invasive, cost-effective technique that simplifies collection, transport and storage of clinical specimens. In steroid hormone analysis, conventional ligand-binding assays lack the specificity to distinguish structurally similar analytes, often requiring confirmatory testing. A robust DBS-based UPLC-MS/MS panel provides detailed insights into enzyme activities and biomarker profiles, enhancing precision in clinical research.

Study Objectives and Overview

This study aimed to establish a rapid, high-throughput UPLC-MS/MS workflow for quantifying a panel of five steroid hormones—androstenedione, 17-hydroxyprogesterone (17-OHP), cortisol, 11-deoxycortisol and 21-deoxycortisol—from DBS samples. Key objectives included achieving baseline chromatographic resolution of isobaric compounds, ensuring linearity across clinically relevant ranges, and automating sample preparation to process large batches efficiently.

Methodology and Instrumentation

• Sample Preparation: Two 3 mm punches of DBS were extracted in 90% methanol containing stable-isotope internal standards, followed by aqueous dilution and automated SPE on Oasis™ MAX µElution plates. Elution was performed with 70% aqueous acetonitrile and samples were diluted prior to injection.
• Chromatography: Separation employed a Waters™ ACQUITY UPLC™ I-Class system with a CORTECS™ C18 2.7 µm column (2.1 × 50 mm) and VanGuard pre-column, using a methanol/ammonium fluoride gradient at high linear velocity.
• Mass Spectrometry: Detection utilized a Xevo™ TQ-S micro triple quadrupole in positive electrospray ionization with multiple reaction monitoring. Key MRM transitions and optimized cone voltages and collision energies were applied for each analyte and its ^13C3 internal standard.
• Automation: A Tecan Freedom Evo 100 liquid handler orchestrated SPE steps, enabling unattended processing of 384 samples in under 18 hours (2.3 min per injection).

Main Results and Discussion

• Chromatographic Performance: The CORTECS C18 column achieved baseline separation of all five steroids, including isobaric pairs, within a 2.3-minute cycle.
• Linearity and Sensitivity: Calibration curves were linear from 0.5 to 500 ng/mL (androstenedione, 11-deoxycortisol) and 1.0 to 500 ng/mL (17-OHP, cortisol, 21-deoxycortisol), with r² > 0.995. LLOQs were 0.5 ng/mL for androstenedione and 11-deoxycortisol; 1.0 ng/mL for the other steroids (S/N > 10).
• Precision and Accuracy: Across five days (n = 25), total precision and repeatability were ≤ 9.3%. QC accuracy ranged from 94.2% to 110.1% of nominal concentrations.

Benefits and Practical Applications

This method delivers high analytical specificity and throughput for simultaneous quantification of multiple steroids from minimal blood volume. It overcomes limitations of immunoassays by resolving isobaric interferences, and supports clinical research requiring detailed steroid profiling, such as studies of adrenal function and metabolic disorders.

Future Trends and Applications

Advances in microsampling and automation will expand DBS-MS/MS applications to broader biomarker panels and point-of-care diagnostics. Integration with digital workflows and machine learning for data interpretation can further enhance throughput and predictive power in clinical studies.

Conclusion

A rapid, automated UPLC-MS/MS assay using DBS samples and CORTECS C18 columns on the Waters ACQUITY UPLC I-Class with Xevo TQ-S micro offers sensitive, selective and reproducible quantification of key steroid hormones. The protocol supports high-volume clinical research workflows with excellent performance metrics.

Reference

• No external literature references were provided.