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

Significance of the Topic

Dried blood spot (DBS) sampling combined with UPLC-MS/MS offers a minimally invasive, high-throughput approach for quantifying steroid hormones in clinical research. This approach reduces sample volume requirements, simplifies logistics, and enables reliable longitudinal monitoring of endocrinological markers.

Objectives and Study Overview

The study aimed to develop and validate a rapid method for simultaneous quantification of androstenedione, 17-hydroxyprogesterone, cortisol, 11-deoxycortisol, and 21-deoxycortisol in DBS samples. A workflow capable of processing 384 samples within 18 hours was designed to support large-scale clinical studies.

Methodology and Instrumentation

• Sample Preparation: Two 3 mm punches per DBS sample were transferred into 96-well plates. Internal standards in 90% methanol and water were added, followed by solid-phase extraction on Oasis MAX µElution plates.
• Automation: Tecan Freedom Evo 100 liquid handler executed extraction steps to enhance throughput and reproducibility.
• Chromatography: Waters ACQUITY UPLC I-Class system equipped with a 2.1×50 mm CORTECS C18 2.7 µm column and CORTECS C18 VanGuard pre-column, using a methanol and 0.05 mM ammonium fluoride gradient.
• Detection: Waters Xevo TQ-S micro tandem quadrupole detector in positive electrospray ionization mode, monitoring multiple reaction monitoring (MRM) transitions specific to each steroid.
• Run Time: Approximately 2.3 minutes injection-to-injection, enabling analysis of 384 samples in roughly 18 hours.

Main Results and Discussion

• Chromatographic Resolution: Baseline separation achieved for all steroid isobars, ensuring accurate quantification of structurally similar analytes.
• Calibration and Sensitivity: Linear response across 0.5–500 ng/mL for androstenedione and 11-deoxycortisol, and 1–500 ng/mL for 17-OHP, cortisol, and 21-deoxycortisol (r²>0.995). LLOQs were 0.5 ng/mL or 1.0 ng/mL (S/N>10).
• Precision and Accuracy: Total precision and repeatability ≤9.3% at four QC levels (2, 5, 50, 400 ng/mL). Accuracy ranged from 94.2% to 110.1% relative to nominal concentrations.
• Throughput: The integrated automated workflow allowed high sample throughput without compromising analytical performance.

Benefits and Practical Applications

This method delivers a robust platform for steroid profiling in pediatric, neonatal, and remote clinical settings where blood volume is limited. It supports large cohort studies, facilitates therapeutic monitoring, and can be adapted for additional small-molecule targets.

Future Trends and Opportunities

Integration of DBS-UPLC-MS/MS with remote sampling technologies, miniaturized devices, and expanded multiplex panels will further enhance point-of-care testing. Advances in data processing and automation could enable real-time monitoring and personalized endocrine profiling.

Conclusion

A fully validated, high-throughput DBS UPLC-MS/MS method using Xevo TQ-S micro and automated sample preparation provides excellent sensitivity, precision, and accuracy for steroid hormone analysis. This workflow meets the demands of clinical research requiring rapid, reliable, and low-volume sampling.

Reference

• Foley D, Calton LJ. Rapid UPLC-MS/MS Dried Blood Spot Analysis of Steroid Hormones Using the Xevo TQ-S Micro for Clinical Research. Waters Corporation; 2019.