Fast and reliable method for the analysis of testosterone, androstenedione, and 17-hydroxy progesterone from human plasma

Importance of the Topic

The reliable quantification of steroid hormones such as testosterone, androstenedione, and 17-hydroxy progesterone in human plasma is critical for clinical diagnostics, sports anti‐doping, and endocrine research. Variability in endogenous levels and the challenge of obtaining true blank matrices necessitate a sensitive, high‐throughput method with robust matrix matching and minimal interference.

Goals and Overview

This application note outlines a fast, accurate, and precise workflow combining micro‐scale solid phase extraction (SPE) with LC‐MS/MS detection. The method was designed to process limited sample volumes, achieve full chromatographic separation in under four minutes, and support calibration in surrogate matrices while monitoring endogenous steroid levels.

Methodology and Used Instrumentation

Sample Preparation:

• Internal standard (corticosteroid-d8) spiked into plasma or PBS surrogate.
• Acidification with 0.1% formic acid, vortex mixing, and loading onto SPE.

SPE Protocol (SOLAµ HRP plate):

• Conditioning: methanol then 0.1% formic acid.
• Loading: entire pretreated sample.
• Washes: 0.1% formic acid, then 40% methanol.
• Elution: two 25 µL volumes of methanol, diluted with formic acid prior to injection.

LC-MS/MS Conditions:

• LC System: Thermo Scientific Dionex UltiMate 3000 RSLC, Syncronis C18 (1.7 µm, 100 × 2.1 mm) with Hypersil GOLD guard.
• Mobile Phases: 0.1% formic acid in water (A), acetonitrile (B); gradient from 50:50 to 0:100 in 3.5 min.
• Flow Rate: 0.3 mL/min, Column Temp: 45 °C, Injection: 15 µL.
• MS: Thermo Scientific TSQ Quantiva triple quadrupole, APCI positive mode; scan time 0.02 s; optimized transitions for each analyte.

Main Results and Discussion

Chromatography achieved baseline resolution of all analytes in under four minutes. Calibration in PBS exhibited linearity from 0.5 to 500 ng/mL (R2 > 0.99) with X2 weighting. Accuracy across ten levels showed biases within ±15%. QC samples at 1.5 and 400 ng/mL in PBS and plasma delivered accuracy within ±11% and precision (RSD) below 11%. Recovery ranged from 92% to 106%, with matrix effects under ±13% in PBS and ±9% in plasma. Endogenous levels measured in blank plasma were 3.73 ng/mL (testosterone), 1.29 ng/mL (androstenedione), and 2.63 ng/mL (17-hydroxy progesterone).

Benefits and Practical Applications

• High throughput: minimal elution volumes and rapid extraction shorten sample-to-result time.
• Low sample consumption: micro-scale SPE suitable for limited-volume specimens.
• Robust surrogate calibration: maintained accuracy despite high endogenous background.
• Enhanced sensitivity and reproducibility support clinical and research workflows.

Future Trends and Potential Uses

Advances may include further miniaturization of SPE formats, integration with fully automated platforms, and expansion to other steroid panels or biomarkers. Coupling micro-SPE with high-resolution MS could improve specificity and multiplexing in clinical diagnostics. Development of matched surrogate materials may also enhance reproducibility across laboratories.

Conclusion

The described micro-scale SPE coupled with LC-MS/MS on a SOLAµ HRP plate and Syncronis C18 column offers a fast, sensitive, and reproducible approach for steroid hormone analysis in human plasma. The method demonstrates excellent linearity, accuracy, precision, and minimal matrix effects, making it suitable for routine bioanalysis and clinical screening.

Reference

• Thermo Fisher Scientific. Fast and reliable method for the analysis of testosterone, androstenedione, and 17-hydroxy progesterone from human plasma. Application Note No. 21173.