LC-MS/MS Method for the Determination of Steroids from Urine Using SOLA and Core Enhanced Technology Accucore HPLC Column

Importance of the Topic

Urinary steroid profiling is vital in clinical diagnostics, endocrinology and anti-doping analysis. Rapid, reliable quantification of corticosteroids such as hydrocortisone, cortisone, corticosterone and 11-α hydroxyprogesterone helps monitor hormone therapy, detect metabolic disorders and enforce fair sports practices.

Objectives and Study Overview

This study demonstrates a streamlined workflow combining Thermo Scientific SOLA SPE 96-well plates for sample cleanup with fast LC-MS/MS separation on an Accucore RP-MS core-shell HPLC column. The aim is to achieve high throughput, excellent reproducibility and low solvent consumption while maintaining sensitivity and robustness.

Methodology and Instrumentation

Sample Preparation

• Matrix: human urine spiked with internal standards for corticosterone and 11-α hydroxyprogesterone
• SPE: SOLA 96-well plate conditioned with methanol and water, sample application, wash with 20:80 methanol/water and elution in methanol
• Evaporation: nitrogen drying, reconstitution in water

Chromatography

• Column: Accucore RP-MS 2.6 µm, 100×2.1 mm at 25 °C
• Mobile phases: A = water + 0.1% formic acid, B = acetonitrile + 0.1% formic acid
• Gradient: 25% B to 75% B in 4 min, total run time 6 min, flow 0.6 mL/min, injection volume 2.5 µL

Mass Spectrometry

• Instrument: TSQ Vantage with HESI source, positive mode
• Key settings: spray voltage 3000 V, vaporizer and capillary at 300 °C, collision pressure 1.5 mTorr

Main Results and Discussion

The four steroids were baseline-separated in under 3 minutes. Calibration curves from 1 to 1000 ng/mL showed linearity (r2 ≥ 0.9937). Endogenous levels in urine:

• Hydrocortisone: 17.3 ng/mL (RSD 11.1%)
• Cortisone: 55.6 ng/mL (RSD 7.4%)

Quality control for corticosterone and 11-α hydroxyprogesterone at 15 and 600 ng/mL yielded RSDs < 3% and recoveries of 131% and 130.8% respectively. Carryover was negligible (< 14% for corticosterone, < 8% for 11-α hydroxyprogesterone).

Benefits and Practical Applications

• High throughput SPE reduces processing time and solvent use
• Core-shell column delivers sharp peaks, low backpressure and fast runs
• Robust method suitable for routine clinical or anti-doping laboratories

Future Trends and Opportunities

Advancements may include integration of automated SPE platforms, expansion to broader steroid panels, coupling with high-resolution mass spectrometry for untargeted profiling and further miniaturization to reduce cost and sample volume.

Conclusion

The combination of SOLA SPE well plates and Accucore RP-MS columns provides a fast, reproducible and sensitive approach for quantitative analysis of urinary steroids. This workflow meets the demands of high-throughput clinical and bioanalytical settings without sacrificing data quality.

Reference

Thermo Fisher Scientific. Application Note ANCCSSOLASTDS: LC-MS/MS Method for the Determination of Steroids from Urine Using SOLA and Core Enhanced Technology Accucore HPLC Column (2011).