Analysis of Serum Cortisol, Androstenedione, and 17-hydroxyprogesterone Using 2D UPLC-MS/MS for Clinical Research

Significance of the Topic

The accurate measurement of serum cortisol, androstenedione, and 17-hydroxyprogesterone is essential in clinical research and patient monitoring of endocrine disorders. High sensitivity and reproducibility are required to support diagnostic accuracy, therapeutic monitoring, and research into hormone-related pathologies.

Study Objectives and Overview

This study aimed to develop and validate a two-dimensional ultra-performance liquid chromatography coupled with tandem mass spectrometry (2D UPLC-MS/MS) method for simultaneous quantification of three steroid hormones in human serum. The method focuses on online sample clean-up, simplified preparation, and flexibility between multidimensional and direct chromatography modes.

Methodology and Instrumentation

Sample preparation involved protein precipitation of 100 µL serum using zinc sulfate and methanol, followed by centrifugation. The supernatant was injected onto a 2D UPLC system configured for trapping and back-flush:

• First dimension: ACQUITY UPLC BEH C18 SB VanGuard pre-column with water/methanol/formic acid loading gradient
• Second dimension: ACQUITY UPLC HSS T3 analytical column with ammonium acetate/formic acid gradient
• Detection: Xevo TQD mass spectrometer, electrospray ionization in positive mode, multiple reaction monitoring with specific transitions for each analyte and 13C-labeled internal standards

Used Instrumentation

• Waters ACQUITY UPLC I-Class System with 2D technology
• ACQUITY UPLC BEH C18 SB VanGuard Pre-column
• ACQUITY UPLC HSS T3 Column
• Waters Xevo TQD tandem quadrupole mass spectrometer

Main Results and Discussion

Calibration curves demonstrated excellent linearity (r²>0.99) over the ranges 0.17–1380 nmol/L. Total precision across low, mid, and high concentration levels was ≤4.7% coefficient of variation. Limits of quantification, defined by a signal-to-noise ratio >10:1 and <20% CV, were established at 1.9 nmol/L for cortisol, 0.16 nmol/L for androstenedione, and 0.37 nmol/L for 17-hydroxyprogesterone. External quality assessment against UK NEQAS samples yielded Deming regression slopes of 1.03–1.06, indicating close agreement with consensus values.

Benefits and Practical Applications

The proposed 2D UPLC-MS/MS workflow offers simplified sample preparation, minimizing manual handling and potential errors through online clean-up. Only 100 µL of serum is required, making it suitable for studies with limited sample volumes. High analytical sensitivity and robust precision support its application in clinical research, hormone profiling, and quality control laboratories.

Future Trends and Potential Applications

Emerging opportunities include expanding the panel to additional steroid hormones, integrating automated sample preparation modules, and applying the flexible 2D configuration to other bioanalytical targets. Advances in column chemistries and mass spectrometer sensitivity may further reduce sample requirements and analysis time, enhancing throughput in routine and research settings.

Conclusion

A versatile 2D UPLC-MS/MS method for quantifying serum cortisol, androstenedione, and 17-hydroxyprogesterone was developed and validated, demonstrating excellent sensitivity, precision, and agreement with external quality standards. The flexible system configuration allows seamless switching to direct UPLC-MS/MS without hardware changes, supporting diverse analytical demands.