UTILISING MIXED MODE μELUTION SPE FOR THE LC-MS/MS ANALYSIS OF STEROID HORMONES IN SERUM FOR CLINICAL RESEARCH

Significance of the Topic

Accurate quantification of testosterone and androstenedione in human serum is critical for investigating endocrine disorders, metabolic dysfunctions, and therapeutic monitoring. Highly sensitive and selective analytical methods are essential to differentiate structurally similar steroid hormones and to achieve reliable data for clinical research.

Study Objectives and Overview

This study evaluates a mixed-mode µElution solid phase extraction (SPE) in a 96-well plate format coupled with LC-MS/MS for the determination of testosterone and androstenedione at low physiological concentrations. The goal is to demonstrate improved sensitivity, precision, and robustness compared to traditional extraction techniques.

Methodology and Instrumentation

Sample Preparation:

• Serum samples spiked with certified reference materials (0.05–15 ng/mL).
• Pre-treatment with internal standards, ammonia, zinc sulfate, methanol, and water.
• Extraction using Waters Oasis MAX µElution SPE with washes (0.1% ammonia in 20% methanol) and elution (methanol then water).
• Automation performed on a Tecan Freedom Evo 100 liquid handler.

Chromatography and Detection:

• Waters ACQUITY UPLC I-Class system with HSS C18 SB column (2.1×50 mm).
• Gradient elution using water, methanol, and ammonium acetate.
• Detection on a Waters Xevo TQD mass spectrometer in MRM mode with optimized cone voltages and collision energies for each analyte.

Results and Discussion

Linearity and Sensitivity:

• Linear calibration from 0.05 to 15 ng/mL (r2 > 0.994, n=10).
• Limit of quantification at 0.025 ng/mL with <20% RSD (n=30) and signal-to-noise ratios >15:1.

Matrix Effects and Selectivity:

• Mean ion suppression of 33% for testosterone and 23% for androstenedione.
• Internal standards compensated effectively (normalized MF ~100%).
• Oasis MAX SPE removed co-eluting interferences better than protein precipitation, liquid–liquid extraction, and Oasis HLB, yielding higher signal-to-noise.

Precision and Accuracy:

• Total precision ≤4.7% RSD and repeatability ≤4.0% RSD across low, mid, and high QC levels.
• Method bias of 3.3% determined using CDC Hormone Standardization (HoSt) serum samples (n=40).
• Comparison to an independent LC-MS/MS method (n=35) yielded Deming regressions of y=1.07x+0.01 (testosterone) and y=0.96x+0.02 (androstenedione), with no constant bias for either analyte.

Benefits and Practical Applications

• High analytical selectivity minimizes interference and ensures integration reproducibility.
• Automation in 96-well format increases throughput for clinical and research laboratories.
• Robust performance at low concentrations supports studies in endocrinology, pharmacology, and toxicology.

Future Trends and Potential Applications

• Extension of µElution SPE workflows to broader steroid panels and other small molecule targets.
• Integration with high-resolution mass spectrometry for untargeted profiling.
• Advancements in microfluidic extraction and on-line SPE for more efficient sample handling.
• Personalized medicine applications through comprehensive endocrine profiling.

Conclusion

The mixed-mode µElution SPE coupled with LC-MS/MS on the Xevo TQD provides a sensitive, precise, and high-throughput method for quantifying testosterone and androstenedione in human serum, meeting the demands of clinical research and laboratory standardization programs.

Reference

• Häggström M, Richfield D (2014). Diagram of the pathways of human steroidogenesis. Wikiversity Journal of Medicine 1(1).