Comparison of Sample Preparation Options for the Extraction of a Panel of Endogenous Steroids from Serum Prior to UHPLC-MS/MS Analysis

Significance of the Topic

This study addresses the critical challenge of extracting low-level endogenous steroids from human serum for sensitive and reliable UHPLC-MS/MS analysis. Efficient sample preparation reduces matrix effects, improves reproducibility, and ensures accurate quantification of steroid hormones in clinical and research laboratories.

Aims and Overview

The objective was to compare supported liquid extraction (SLE+) with polymer-based solid phase extraction (SPE) using ABN and CX sorbents. Key performance metrics included extraction recoveries, ion suppression, phospholipid removal, calibration linearity, and limits of quantification (LOQs) for a panel of 19 steroids.

Used Instrumentation

• UHPLC: Shimadzu Nexera with ACE C18 1.7 µm 100 × 2.1 mm column at 40 °C
• Mass spectrometer: Shimadzu 8060 triple quadrupole with ESI interface, operated in MRM mode
• Mobile phases: 0.2 mM NH4F (aq) and methanol; flow rate 0.4 mL/min; gradient from 50:50 to 90% methanol

Methodology and Sample Preparation

• SLE+ protocol: 400 µL serum loaded on 400 µL capacity plate, two washes, elution with 75:25 ethyl acetate/hexane or pure ethyl acetate
• SPE protocols: 10 mg ABN and CX cartridges with formic acid equilibration, water and water/methanol washes, elution with methanol or ethyl acetate
• Evaporation at 40 °C, reconstitution in 200 µL 50:50 mobile phase A:B
• Elution solvent choice based on presence of DHEAS to minimize phospholipid co-extraction

Main Results and Discussion

• Wash solvent optimization: ABN tolerated up to 40% methanol, CX optimized around 30%; higher organic content improved cleanup without sacrificing recovery
• Elution solvents: ethyl acetate favored for clean extracts; 75:25 ethyl acetate/hexane gave highest recoveries for SLE+
• Evaporation losses were negligible when reconstituted with 50% methanol; no need for glycol additives
• Phospholipid removal: both SLE+ and ABN protocols achieved marked reduction in lyso- and glycerophospholipids, with SLE+ slightly outperforming in overall cleanliness
• Calibration and sensitivity: LOQs ranged from <5 pg/mL for many steroids to <500 pg/mL for pregnenolone and DHEA; correlation coefficients >0.994 across the panel

Benefits and Practical Applications

• High-throughput 96-well formats enable processing of large sample batches efficiently
• Robust recoveries and low LOQs support clinical and research demands for steroid profiling
• Effective matrix cleanup enhances assay precision and extends instrument uptime
• Flexible solvent selection accommodates panels including sulfate conjugates like DHEAS

Future Trends and Applications

Advances may include integration with automated liquid handling, development of greener sorbents and solvents, and application of machine learning to optimize extraction parameters. Expanding to broader steroid metabolomes and coupling with high-resolution MS could further enhance diagnostic and pharmacokinetic studies.

Conclusion

SLE+ using 75:25 ethyl acetate/hexane and SPE with ABN eluted in methanol deliver reproducible, clean, and sensitive extraction of endogenous steroids from serum. Both approaches meet stringent requirements for clinical and industrial UHPLC-MS/MS workflows.

Reference

1. Teehan KJ, Williams L, Senior A, Edgington A, Jones R, Lodder H, Davies G, Jordan S, Desbrow C, Roberts P, Marin S, Menasco D, Gairloch E. Comparison of Sample Preparation Options for the Extraction of a Panel of Endogenous Steroids from Serum Prior to UHPLC-MS/MS Analysis. Biotage; 2018.