Analysis of Corticosteroids and Androgens in Serum for Clinical Research

Significance of the Topic

The precise measurement of steroid hormones in serum underpins research into endocrine disorders, metabolic regulation and inflammatory response. Conventional immunoassays often suffer from cross-reactivity and limited specificity, while LC-MS/MS delivers enhanced selectivity, sensitivity and accuracy.

Study Objectives and Overview

This study presents an automated LC-MS/MS workflow for quantifying testosterone, androstenedione, 17-hydroxyprogesterone, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum. Key goals included establishing chromatographic separation of isobaric steroids, validating method performance against external quality assessment (EQA) samples, and streamlining sample preparation via automation.

Methodology and Instrumentation

Sample preparation employed Oasis PRiME HLB µElution plates and a Tecan Freedom EVO liquid handler. Internal standards for each analyte enabled isotope dilution quantification of 100 µL serum. Chromatography used an ACQUITY UPLC I-Class system with HSS T3 VanGuard pre-column and HSS T3 column at 50 °C under a 4.7 min gradient. Detection was carried out on a Xevo TQ-S micro tandem quadrupole mass spectrometer using ESI in positive and negative modes with MRM transitions optimized for each steroid. Data acquisition and processing were managed by MassLynx and TargetLynx, with LIMS connectivity through Tecan File Converter and MassLynx LIMS Interface.

Main Results and Discussion

Baseline chromatographic resolution was achieved for isobaric pairs (e.g., 11-deoxycortisol/corticosterone, testosterone/epitestosterone). Limits of quantification ranged from 0.09 nmol/L for androstenedione to 0.72 nmol/L for deoxycortisol isomers, with signal-to-noise ratios above 10:1. Total precision (n = 30) and repeatability across low, mid and high QC levels were ≤ 7.6% RSD. Calibration curves were linear (r2 > 0.994) across defined ranges. Matrix effect studies in six individual sera showed internal standards effectively compensated ion suppression. Deming regression and Bland-Altman analysis of EQA samples demonstrated excellent agreement with mass spectrometry consensus means, with mean biases within ±5.8%.

Benefits and Practical Applications

• High throughput:
  Automated SPE and 4.7 min run time facilitate large cohort studies.
• Low sample volume:
  Only 100 µL serum required, preserving precious samples.
• Enhanced selectivity:
  Isobaric steroids fully resolved, minimizing interference.
• Robust quantification:
  Isotope dilution ensures accuracy across broad concentration ranges.

Future Trends and Applications

Advances may include expansion to broader steroid panels, integration of microflow LC for sensitivity gains, high-resolution MS for structural elucidation, and further automation with AI-driven sample handling. These developments will support personalized medicine, doping control, and pediatric endocrinology research.

Conclusion

This automated LC-MS/MS method delivers sensitive, selective and reproducible quantification of key corticosteroids and androgens in serum. Excellent agreement with EQA standards and streamlined workflow improve laboratory efficiency and data reliability, making it highly suited for clinical research studies.