Extraction of a Comprehensive Steroid Panel from Human Serum Using ISOLUTE® SLE+ Prior to LC/MS-MS Analysis

Significance of the Topic

Steroid hormone profiling in human serum is essential for clinical diagnostics and research applications. The complex matrix and low analyte concentrations require efficient sample preparation to ensure accurate sensitive quantitation by LC MS MS.

Objectives and Study Overview

This study presents a comprehensive workflow using ISOLUTE SLE+ supported liquid extraction plates to isolate a panel of over 20 steroid hormones from human serum. The goals include evaluating recoveries reproducibility linearity and phospholipid removal using two elution solvent systems.

Methodology

Sample pretreatment involves spiking serum with methanolic internal standards. Extracts are processed on ISOLUTE SLE+ 400 µL plates under low pressure equilibrated and eluted with ethyl acetate or ethyl acetate hexane (75:25). Eluates are dried at 40 °C under a stream of air or nitrogen and reconstituted in a 50:50 mixture of mobile phases.

• Sample loading volume 300 µL (up to 350 µL for increased sensitivity)
• Elution solvents EtOAc or EtOAc Hex 75/25 (2×500 µL)
• Drying 20 minutes at 40 °C
• Reconstitution volume 200 µL

Instrumentation

• ISOLUTE SLE+ 400 µL supported liquid extraction plate
• Biotage PRESSURE+96 positive pressure manifold
• Biotage SPE Dry 96 sample evaporator
• Shimadzu Nexera UHPLC with ACE C18 column (100×2.1 mm 1.7 µm)
• Shimadzu 8060 triple quadrupole mass spectrometer with ESI

Main Results and Discussion

The optimized protocols yielded analyte recoveries above 75% (above 80% for most steroids excluding DHEAS) with RSDs below 10%. Phospholipid interferences were effectively removed compared to protein precipitation. Linearity was demonstrated over 5–5000 pg/mL with r2 values above 0.99 and LOQs as low as <5 pg/mL for key analytes.

Benefits and Practical Applications

The SLE+ workflow simplifies sample preparation by eliminating emulsions delivering clean extracts and robust quantitation for bioanalytical laboratories. The method supports high throughput and reliable steroid hormone analysis for clinical research and quality control.

Future Trends and Opportunities

Further automation of the SLE+ procedure and integration with high resolution mass spectrometry may expand steroid panels and enhance sensitivity. Miniaturization of sample volumes and application to other biological matrices are promising directions.

Conclusion

The presented ISOLUTE SLE+ method offers a reproducible and efficient approach for extracting a broad steroid panel from human serum achieving high recoveries excellent linearity and effective matrix cleanup prior to LC MS MS analysis.

References

Biotage (2018) Extraction of a Comprehensive Steroid Panel from Human Serum Using ISOLUTE SLE+ Application Note AN890.