High-Throughput LC-MS/MS Quantification of 17-Hydroxyprogesterone (17-OHP) in Human Blood Serum for Clinical Research Purposes

Importance of the Topic

17-hydroxyprogesterone (17-OHP) is a key intermediate in steroid biosynthesis, linking to corticosteroids, androgens, and estrogens.
Accurate quantification of 17-OHP in human serum is crucial for studying adrenal enzyme activity, diagnosing endocrine disorders, and conducting clinical research on steroid metabolism.

Objectives and Study Overview

The primary goal was to develop a fast, reliable, and high-throughput HPLC-MS/MS assay to measure 17-OHP in human serum over a concentration range of 10 to 1,000 ng/dL (0.3–30 nmol/L).
This method supports clinical research applications requiring robust quantitation with minimal sample preparation time.

Methodology

Sample Preparation:

• Serum aliquots (200 µL) were spiked with a deuterated internal standard (17-OHP-D4).
• Liquid-liquid extraction was performed with 1 mL methyl t-butyl ether (MTBE).
• Extracts were dried under heated nitrogen and reconstituted in water/methanol (1:1).

Used Instrumentation

• HPLC: Thermo Scientific Transcend LX-4 multichannel system with Accucore RP-MS column (2.6 µm, 50 × 2.1 mm).
• Mass spectrometer: Thermo Scientific TSQ Endura triple quadrupole with APCI source (positive mode).
• Data processing: Thermo Scientific TraceFinder software.

Main Results and Discussion

• Linearity: 10–1,000 ng/dL with r2 ≥ 0.999 (1/x weighting).
• Precision: intra- and inter-batch CVs < 5% and < 6%, respectively.
• Carryover: < 0.1% following the highest calibrator.
• Ion-ratio confirmation within ±20% of calibrator averages.
• Matrix effects: internal standard recovery averaged 87% with an RSD of 20%.
• Method comparison: 40 donor samples showed ≤ 20% difference versus a reference laboratory in 95% of cases.

Benefits and Practical Applications

• Simple, cost-effective sample preparation.
• Robust quantitation suitable for clinical and research laboratories.
• High-throughput capability (up to 56 injections per hour with multichannel HPLC).
• Applicable for enzyme-activity studies and steroid profiling.

Future Trends and Potential Applications

• Automation and integration into clinical workflows.
• Expansion to multiplexed steroid panels and high-resolution MS techniques.
• Application to dried blood spots and point-of-care testing.
• Implementation of AI-driven data analysis for enhanced interpretation.

Conclusion

The developed HPLC-MS/MS assay offers sensitive, precise, and high-throughput quantification of 17-OHP in human serum, meeting the demands of clinical research and supporting studies of steroid metabolism and endocrine disorders.