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

Significance of the topic

The accurate measurement of steroid hormones in urine is essential for clinical diagnostics, therapeutic monitoring and anti-doping analyses. Efficient sample preparation and rapid chromatographic separation are key to high-throughput workflows in bioanalytical laboratories.

Purpose and study overview

This study presents a combined workflow using Thermo Scientific SOLA solid phase extraction (SPE) well plates for cleanup of urine samples, followed by fast LC–MS/MS analysis on an Accucore RP-MS column. Four steroids (hydrocortisone, cortisone, corticosterone and 11-α hydroxyprogesterone) are extracted, separated and quantified to demonstrate method performance.

Methodology and instrumentation

• Sample preparation: Urine aliquots spiked with internal standards are processed on a 96-well SOLA plate. Conditioning with methanol and water, sample application, wash (20:80 methanol/water) and elution with methanol are performed. Eluates are dried and reconstituted in water.
• Chromatography: Accucore RP-MS column (2.6 μm, 100 × 2.1 mm) operated at 25 °C. Mobile phases A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid). Gradient from 25% B to 75% B over 4 min, total run time 6 min, flow rate 0.6 mL/min.
• Mass spectrometry: Thermo Scientific TSQ Vantage with heated electrospray ionization in positive mode. Key parameters: spray voltage 3,000 V, vaporizer and capillary at 300 °C, collision pressure 1.5 mTorr. Quantitation using selected reaction monitoring transitions optimized for each analyte.
• Data processing: Thermo Scientific LC QUAN software for calibration, integration and reporting.

Main results and discussion

• Separation: Four steroids baseline-resolved in under 3 min.
• Linearity: Calibration curves from 1 to 1,000 ng/mL with r² ≥ 0.9937 for all compounds.
• Precision and accuracy: QC samples at low (15 ng/mL) and high (600 ng/mL) levels yielded precision ≤ 3% RSD and accuracy within ±4%.
• Endogenous measurements: Native levels of hydrocortisone and cortisone in urine averaged 17.3 ng/mL (11.1% RSD) and 55.6 ng/mL (7.4% RSD), respectively.
• Recovery and carryover: Overspike recoveries for corticosterone and 11-α hydroxyprogesterone were ~131%, carryover <14% of highest standard.

Benefits and practical applications

• High throughput: 96-well SPE format and fast chromatography accelerate sample processing.
• Reduced solvent use: Minimal wash and elution volumes lower operational costs.
• Robustness: Solid core column provides low backpressure and stable retention, enhancing reproducibility.
• Clean extracts: SOLA SPE removes phospholipids and proteins, improving MS sensitivity.

Future trends and opportunities

Further integration of automated liquid handling with SPE plates could streamline workflows. Expansion to additional steroid panels and conjugates, together with high-resolution MS detection and data analytics, will meet growing demands in clinical and sports testing. Miniaturized and on-line SPE–LC–MS approaches may further enhance throughput and reduce sample volumes.

Conclusion

The combination of SOLA SPE well plates and Accucore RP-MS chromatography enables rapid, sensitive and reproducible quantitation of multiple steroids in urine. This workflow is well suited for high-throughput bioanalytical applications, delivering excellent linearity, precision and cleanliness.

Reference

1. MacRitchie E., Phipps K. LC-MS/MS Method for the Determination of Steroids from Urine Using SOLA and Core Enhanced Technology Accucore HPLC Column. Application Note ANCCSSOLASTDS, Thermo Fisher Scientific, 2011.