UPLC-MS/MS Analysis of Dihydrotestosterone, Dehydroepiandrosterone, Testosterone, Androstenedione, 17-Hydroxyprogesterone, and Progesterone in Serum for Clinical Research

Significance of the topic

Steroid hormones such as dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), testosterone, androstenedione, 17-hydroxyprogesterone (17-OHP), and progesterone are critical regulators of metabolism, sexual development, blood pressure, and inflammatory responses
Traditional immunoassays suffer cross-reactivity and limited specificity, making sensitive and selective quantification challenging
UPLC-MS/MS offers high analytical selectivity, sensitivity, and precision, addressing these limitations in clinical research settings

Objectives and Study Overview

This study aimed to develop and validate a fully automated UPLC-MS/MS method for simultaneous quantification of six steroid hormones in human serum
The method integrates Oasis MAX µElution plate extraction with Tecan Freedom EVO automation, ACQUITY UPLC I-Class separation, and Xevo TQ-S micro detection
External Quality Assessment (EQA) samples were analyzed to assess bias and accuracy relative to established mass spectrometry methods

Methodology and Instrumentation

Sample preparation uses 100 µL serum spiked with stable isotope–labeled internal standards, protein precipitation with methanol, and mixed-mode anion exchange SPE on Oasis MAX µElution plates
Chromatography employs a CORTECS UPLC C18 column (1.6 µm, 2.1×50 mm), gradient elution (water/ammonium fluoride and methanol), 6.3-minute run time, and 50 °C column temperature
Mass spectrometry is performed on Xevo TQ-S micro in positive ESI mode with MRM acquisition for each analyte and its labeled analogue
Automation and data management are handled by Tecan Freedom EVO 100/4 for sample processing and Waters MassLynx v4.1 with TargetLynx and LIMS interface for integration

Main Results and Discussion

No significant interferences or carryover were observed; a 1:5 dilution maintained accuracy within ±13% and RSD ≤10% for high-concentration samples
Lower limits of quantification (LLOQs) with precision <20% and S/N>10 were achieved at 0.034 nmol/L for DHT, 0.17 nmol/L for DHEA, 0.007 nmol/L for testosterone, 0.035 nmol/L for androstenedione, 0.030 nmol/L for 17-OHP, and 0.016 nmol/L for progesterone
Total precision across low, mid, and high QC levels showed RSDs typically below 7% for all analytes
Matrix effects (n=6 individual sera) were compensated by internal standards, yielding normalized matrix factors close to unity with RSD ≤14%
Accuracy assessed by EQA samples demonstrated excellent Deming regression agreement (slope ~1, negligible constant bias, mean bias ≤5.6%) for testosterone, androstenedione, and 17-OHP
DHT accuracy compared to RCPAQAP target values and an independent LC-MS/MS method showed mean biases of 4.9% and –6.6%, respectively
Representative chromatograms at low analyte concentrations confirmed clear peak resolution and high signal-to-noise ratios

Benefits and Practical Applications

High chromatographic selectivity resolves isobaric interferences and structurally related compounds
Low sample volume (100 µL) is suitable for scarce clinical specimens
Automation reduces hands-on time, minimizes operator error, and supports high throughput in biomarker and clinical research studies

Instrumentation

• ACQUITY UPLC I-Class System with FTN and Column Heater
• CORTECS UPLC C18 Column (1.6 µm, 2.1×50 mm)
• Oasis MAX µElution Plate
• Xevo TQ-S micro Mass Spectrometer
• Tecan Freedom EVO 100/4 Liquid Handler
• MassLynx v4.1 with TargetLynx Application Manager
• MassLynx LIMS Interface v3.0 and Tecan File Converter v2.0

Future Trends and Applications

Further expansion to broader steroid panels and conjugated metabolites using high-resolution MS or ion mobility separation
Integration with advanced laboratory information systems and digital workflows for real-time data reporting
Miniaturization and microfluidic SPE to reduce sample and reagent consumption
Application in pharmacokinetic studies, endocrine disruptor monitoring, and personalized medicine biomarker discovery

Conclusion

The automated UPLC-MS/MS method demonstrates high sensitivity, specificity, and precision for six key serum steroids using minimal sample volume
Strong agreement with external quality schemes and independent methods validates its suitability for clinical research
Automation enhances laboratory efficiency and reliability, supporting large-scale studies and routine biomarker analysis

References

No primary literature references were provided in the source document