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

Importance of the Topic

Steroid hormones such as hydrocortisone, cortisone, corticosterone and 11-α hydroxyprogesterone play critical roles in clinical diagnostics, endocrinology research and doping control. Urinary steroid profiling requires fast, reproducible and sensitive methods that deliver clean extracts with minimal solvent consumption, supporting high throughput bioanalytical laboratories.

Objectives and Study Overview

This study demonstrates an LC-MS/MS workflow combining Thermo Scientific SOLA solid phase extraction (SPE) 96 well plates and an Accucore RP-MS core-enhanced HPLC column to extract, separate and quantify four progesterone-class steroids from urine. Betamethasone was used as internal standard to ensure accurate quantification.

Methodology and Used Instrumentation

Sample preparation involved:

• Conditioning SOLA SPE wells with methanol and water
• Loading 1 mL urine spiked with internal standard
• Washing with 20:80 methanol/water
• Elution with methanol, evaporation under nitrogen and reconstitution in water

Chromatographic conditions:

• Column: Accucore RP-MS 100 × 2.1 mm, 2.6 µm core-shell particles
• Mobile phases: A = water + 0.1% formic acid; B = acetonitrile + 0.1% formic acid
• Gradient: 25% B (0 min) to 75% B (4 min), return to 25% B by 4.51 min; total run 6 min
• Flow rate: 0.6 mL/min; temperature: 25 °C; injection: 2.5 µL

Mass spectrometry settings:

• Instrument: Thermo Scientific TSQ Vantage with HESI positive ionization
• Spray voltage: 3000 V; vaporizer and capillary: 300 °C; sheath gas: 60; auxiliary gas: 30
• Collision pressure: 1.5 mTorr; scan time: 0.02 s; Q1/Q3 resolution: 0.7 FWHM

Data processing was performed using Thermo Scientific LC QUAN software.

Main Results and Discussion

All four steroids were baseline separated in under 3 minutes. Calibration curves prepared in water showed linearity from 1 to 1000 ng/mL with R² values above 0.9937. Endogenous levels in urine were determined with acceptable precision: hydrocortisone 17.3 ng/mL (11.1% RSD), cortisone 55.6 ng/mL (7.4% RSD). Quality control samples for corticosterone and 11-α hydroxyprogesterone at 15 and 600 ng/mL exhibited RSDs below 3%. Recovery for corticosterone and 11-α hydroxyprogesterone exceeded 130%, with negligible carryover observed.

Benefits and Practical Applications

This workflow offers rapid throughput with minimal solvent use and high extract purity. The core-shell column reduces backpressure and peak tailing, enhancing robustness. The method is well suited for clinical, bioanalytical and doping control laboratories requiring accurate, high-volume steroid analysis.

Future Trends and Applications

Expansion to a broader steroid panel and other bioactive metabolites could provide more comprehensive endocrine profiling. Advances in SPE media and core-shell column designs will further reduce analysis times and improve sensitivity. Automation and integration with high resolution mass spectrometry may enable detailed metabolomic investigations.

Conclusion

The combination of SOLA SPE well plates and an Accucore RP-MS column delivers a fast, reproducible and sensitive LC-MS/MS method for urinary steroid quantification, improving laboratory efficiency and data confidence.