Fast and Reliable Method for the Analysis of Testosterone, Androstenedione, and 17-hydroxy Progesterone from Human Plasma

Importance of the Topic

Steroid hormones play a central role in endocrine signaling and disease diagnosis. Accurate measurement of testosterone, androstenedione, and 17-hydroxyprogesterone in human plasma is essential for clinical assessment of adrenal and gonadal function. Fast, sensitive, and reliable analytical techniques are required to handle high endogenous levels and limited sample volumes.

Objectives and Study Overview

The main goal of the study was to develop a high-throughput, robust analytical method combining micro-scale solid-phase extraction (SPE) with liquid chromatography–tandem mass spectrometry (LC-MS/MS). The method aims to quantify three key steroids over a broad dynamic range, using a surrogate matrix for calibration standards and verifying performance against human plasma.

Methodology and Instrumentation

The workflow integrates Thermo Scientific products and automated hardware for streamlined sample processing and analysis:

• Sample preparation: Micro-scale SPE was performed on SOLAµ HRP plates. Calibration standards were prepared in phosphate-buffered saline (PBS) over 0.5–500 ng/mL, and quality control (QC) samples were spiked into both PBS and human plasma.
• SPE protocol: Conditioning with methanol, equilibration with 0.1% formic acid in water, sample loading, two wash steps, and low-volume methanol elution. Extracts were diluted with dilute formic acid before injection.
• Chromatography: Separation on a Syncronis C18 1.7 µm, 100 × 2.1 mm column with Hypersil GOLD guard at 45 °C. Mobile phases were 0.1% formic acid in water (A) and acetonitrile (B), using a rapid gradient to achieve baseline separation in under four minutes. Flow rate was 0.30 mL/min; injection volume 15 µL.
• Detection: A Thermo Scientific TSQ Quantiva triple quadrupole mass spectrometer with atmospheric pressure chemical ionization (APCI) in positive mode. Multiple reaction monitoring transitions were optimized for each analyte and an internal standard (corticosteroid-d8).

Key Results and Discussion

The method demonstrated excellent analytical performance:

• Linearity: Calibration curves in PBS were linear (0.5–500 ng/mL) with correlation coefficients (r2) >0.99 using 1/x² weighting.
• Accuracy and precision: Bias for standards and QCs was within ±15%; precision (RSD) remained below 11% at low and high QC levels in both PBS and plasma.
• Recovery and matrix effects: Absolute recoveries ranged from 92% to 111%. Matrix effect values were low (<15% suppression or enhancement) for both surrogate and plasma matrices.
• Endogenous levels: Mean endogenous concentrations in blank plasma were determined (e.g., testosterone ~3.7 ng/mL), confirming the need for surrogate calibration while monitoring batch-to-batch consistency.

Benefits and Practical Applications

This protocol offers:

• Rapid throughput: SPE microplates and fast LC gradient reduce total analysis time per sample to minutes.
• High sensitivity: Low elution volumes and optimized APCI improve limits of detection.
• Robustness: Consistent recoveries and minimal matrix interference support reliable quantification in clinical and research laboratories.
• Flexibility: Surrogate matrix calibration enables analysis across normal and pathological concentration ranges.

Future Trends and Potential Applications

Emerging developments may include:

• Expansion of the steroid panel to include cortisol, estradiol, and other metabolites for comprehensive profiling.
• Integration with automated SPE‐LC systems for unattended, high-throughput screening.
• Use of alternative ionization techniques (e.g., electrospray ionization) to broaden applicability.
• Miniaturized LC-MS platforms for point-of-care diagnostics and therapeutic drug monitoring.

Conclusion

The presented micro-scale SPE LC-MS/MS method delivers fast, precise, and accurate quantification of testosterone, androstenedione, and 17-hydroxyprogesterone in human plasma. High recoveries, low matrix effects, and robust linearity support its use in clinical, research, and quality control environments.

Reference

No external literature references were provided in the source document.