High-Throughput LC-MS/MS Quantification of Pregnenolone in Human Blood Serum for Clinical Research Purposes

Importance of the Topic

Pregnenolone serves as a key biosynthetic precursor to corticosteroids, androgens, and estrogens and its accurate quantification in human serum is critical for clinical research on steroidogenesis and related disorders. Robust, high-throughput assays support large-scale studies and enhance laboratory efficiency.

Objectives and Study Overview

The primary goal was to establish a reliable HPLC-MS/MS method for measuring pregnenolone concentrations from 10 to 500 ng/dL in human blood serum. The study evaluated sensitivity, linearity, precision, carryover, matrix effects, and comparability with a reference laboratory.

Methodology and Instrumentation

Sample preparation involved spiking 200 µL serum with a deuterated pregnenolone-D4 internal standard, liquid-liquid extraction with methyl t-butyl ether, evaporation, and derivatization to oxime with hydroxylamine. Reconstituted samples were analyzed by reversed-phase LC coupled to tandem MS. Key chromatographic and mass spectrometric conditions included:

• Thermo Scientific Transcend LX-4 multichannel HPLC system
• Accucore Phenyl-X column (2.6 µm, 50 × 2.1 mm) at 40 °C with a water/methanol gradient containing 0.1% formic acid
• Thermo Scientific TSQ Endura triple quadrupole MS with heated electrospray ionization (HESI) probe
• Selected reaction monitoring transitions for quantitation and confirmation of pregnenolone oxime and D4-oxime
• Data acquisition and processing using Thermo Scientific TraceFinder software

Key Results and Discussion

The assay exhibited excellent linearity (10–500 ng/dL) with r² ≥ 0.999 (1/X weighting). Intra- and inter-batch precision were below 5% and 8%, respectively, and carryover was under 0.2%. Internal standard peak areas demonstrated consistent recovery (~65%) and effectively compensated for matrix effects. Comparison of 40 donor samples with reference-lab values showed that 92.5% of results agreed within ±20%.

Benefits and Practical Applications

This method offers:

• Sensitive and specific quantitation over a clinically relevant range
• Simple, cost-effective sample preparation
• High throughput (up to 52 injections per hour on multichannel systems)
• Robust performance suitable for clinical research and QA/QC environments

Future Trends and Potential Applications

Advances may include integration with multiplex steroid panels, further automation of sample handling and derivatization, miniaturized flow-injection LC-MS/MS formats, and novel derivatization chemistries to expand analyte coverage. Such developments will streamline workflows and support precision medicine initiatives.

Conclusion

A rapid, sensitive, and high-throughput LC-MS/MS assay for pregnenolone in human serum was developed, demonstrating excellent linearity, precision, and consistency with reference methods. Its simplicity and robustness make it well-suited for clinical research applications.

Reference

1. Kushnir M.M. et al. Development and performance evaluation of a tandem mass spectrometry assay for four adrenal steroids. Clin Chem, 2006, 52, 1559–1567.