Robust extraction, separation, and quantitation of structural isomer steroids from human plasma by SPE-UHPLC-MS/MS

Significance of the topic

The accurate quantitation of steroid hormones in human plasma is critical for clinical research and diagnostic applications. Structural isomers among steroids pose analytical challenges, as they share identical mass transitions and can coelute, leading to misestimation of hormone levels. High-resolution chromatographic separation combined with sensitive mass spectrometric detection ensures reliable profiling of endogenous and exogenous steroids across a broad concentration range.

Objectives and study overview

This study aimed to develop and validate a solid-phase extraction (SPE)–UHPLC-MS/MS method for the robust extraction, separation, and quantitation of 12 clinically relevant steroids, including structural isomers. Key goals included:

• Achieve baseline separation of isomeric steroids using an Accucore Biphenyl phase.
• Compare chromatographic performance against a conventional C18 column.
• Demonstrate method precision, accuracy, retention time stability, and dynamic range in human plasma extracts.

Methodology and instrumentation

Sample preparation and chromatographic conditions were optimized for two workflows:

• SPE protocol: 400 µL of plasma or PBS spiked with analytes was mixed 1:1 with 2% ZnSO₄, centrifuged, and loaded onto a SOLAµ HRP 96-well plate. After a 20% MeOH wash, steroids were eluted with 80% ACN/20% MeOH, then diluted to injection volume.
• UHPLC system: Thermo Scientific Vanquish Horizon with active preheating, split sampler, binary pump, and Accucore Biphenyl column (100 × 2.1 mm, 2.6 µm). Method 1 used a fast ACN gradient (4.2 min) for common steroids; Method 2 applied a 12 min MeOH gradient for closely related isobars.
• Mass spectrometry: Thermo Scientific TSQ Quantiva triple quadrupole with HESI-II probe in positive mode. Source parameters included 3500 V spray voltage, 400 °C vaporizer, and optimized CID gas and RF lens settings. Data were acquired and processed in Chromeleon 7.2.8.

Main results and discussion

Chromatographic performance on the Biphenyl phase outperformed C18 in isomer resolution and peak capacity:

• Eleven steroids were separated in 4.2 min using ACN; extended 12 min MeOH gradient resolved 12 isobaric compounds.
• Resolution improvements for key isomer pairs rose from ~2.0 to 7.9 for 21-deoxycortisol/11-deoxycortisol and from ~1.4 to 4.97 for 11-deoxycorticosterone/17α-hydroxyprogesterone.
• Average peak widths decreased from 0.066 min (C18) to 0.058 min (Biphenyl), increasing peak capacity from 146 to 165 over a 9.5 min gradient.
• Retention time stability across 25 injections of plasma and PBS extracts showed ≤0.10% RSD for all analytes.
• Calibration curves spanning 50–50 000 pg/mL exhibited r² >0.98. QC accuracy ranged 95–107% with precision <6% RSD at low and high levels.
• Standard addition in plasma confirmed recoveries of 84–117%, demonstrating reliable endogenous steroid quantitation.

Benefits and practical applications

The validated SPE-UHPLC-MS/MS workflow offers:

• Accurate separation of steroid isomers to prevent analytical bias.
• Alternative selectivity to C18, enhancing structural differentiation.
• Robust retention time reproducibility in complex biological matrices.
• Wide dynamic range covering three orders of magnitude for clinical and research assays.

Future trends and opportunities

Advancements may include integration of automated SPE systems, expansion to larger steroid panels, and coupling with high-resolution mass spectrometry for untargeted profiling. Miniaturized columns and microflow LC could further reduce solvent consumption. The method’s robustness supports applications in endocrinology research, therapeutic drug monitoring, and personalized medicine.

Conclusion

The developed SPE-UHPLC-MS/MS method employing an Accucore Biphenyl column provides superior resolution of structural steroid isomers, stable retention times, and high quantitative performance across a wide concentration range. This approach is well suited for accurate steroid profiling in clinical research and diagnostic laboratories.