Analysis of Plasma 17-Hydroxyprogesterone, Androstenedione, and Cortisol Using a Novel Solid-Phase Extraction (SPE) Sorbent, Oasis PRiME HLB, for UPLC-MS/MS Analysis in Clinical Research

Importance of the Topic

Accurate measurement of plasma corticosteroids such as cortisol, androstenedione and 17α-hydroxyprogesterone is critical in clinical research and diagnostic settings. Traditional immunoassays may suffer from cross-reactivity and require separate analyses. The adoption of UPLC-MS/MS with efficient sample cleanup enhances selectivity, sensitivity and throughput.

Study Objectives and Overview

This work evaluates a novel solid-phase extraction sorbent, Oasis PRiME HLB, in a μElution plate format for the rapid and reliable extraction of three steroids from human plasma. Key aims include streamlining sample preparation, maximizing phospholipid removal, and achieving accurate, multiplexed quantification by UPLC-MS/MS.

Methodology and Instrumentation

Sample preparation combines protein precipitation with ZnSO₄/methanol followed by direct loading onto the PRiME HLB μElution plate without conditioning. After two washes with 25% methanol, analytes are eluted with 90:10 acetonitrile:methanol and diluted prior to injection.

Instrumentation Used

• UPLC System: ACQUITY UPLC I-Class with HSS T3 column (2.1×50 mm, 1.8 μm) at 40 °C.
• Mass Spectrometer: Xevo TQ-S in ESI positive mode with optimized MRM transitions for each steroid and corresponding internal standards.
• μElution Plate: Oasis PRiME HLB (p/n 186008052) and ACQUITY 96-well collection plate (p/n 186005837).

Results and Discussion

Chromatographic separation achieved all target analytes within 2 minutes with enhanced retention and minimal solvent effects. Extraction recoveries were consistent at 71–73% with RSDs below 5%. Matrix effects were low (–19% for cortisol; <10% for androstenedione and 17-OHP) and reproducible (SD<3.1%). Calibration curves were linear (R²>0.989) over clinically relevant ranges. Lower limits of quantification reached 1 ng/mL for cortisol and 0.05 ng/mL for the other steroids, meeting regulatory criteria. QC samples showed accuracy within 8.4% of nominal values and precision below 6% CV.

Phospholipid analysis demonstrated >97% removal compared to protein precipitation alone, reducing potential matrix interferences and instrument fouling.

Benefits and Practical Applications

• Streamlined workflow without sorbent conditioning or solvent evaporation.
• High throughput μElution format enabling direct injection of concentrated extracts.
• Multiplexed steroid quantification with improved specificity and robustness.
• Extended column lifetime and reduced MS source maintenance due to phospholipid minimization.

Future Trends and Applications

The integration of advanced SPE formats with UPLC-MS/MS is expected to expand steroid panels in clinical diagnostics and research. Future developments may include automated sample processing, coupling with high-resolution mass spectrometry, and application to broader classes of bioactive lipids and metabolites.

Conclusion

Oasis PRiME HLB μElution plates offer a fast, reliable and reproducible approach for the analysis of key plasma steroids by UPLC-MS/MS. The simplified workflow, excellent cleanup and strong analytical performance support its adoption in high-volume clinical and research laboratories.

References

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• Chambers E. et al. Systematic strategy for reducing matrix effects in LC-MS/MS analyses. J Chrom B. 2007;852(1–2):22–34.
• Xia Y., Jemal M. Phospholipids in LC/MS bioanalysis: impact on matrix effects. Rapid Commun Mass Spectrom. 2009;23(14):2125–2138.