High Sensitivity Analysis of Steroid Hormones with modified ESI to improve desolvation efficiency

Significance of the topic

Steroid hormones play a central role in metabolism, reproduction, intracellular signaling and disease mechanisms. Reliable quantification at low concentration levels is critical in clinical research and diagnostics, particularly for pediatric and post-menopausal samples where hormone levels are minimal and sample volumes may be limited.

Objectives and study overview

This work aimed to establish a highly sensitive LC-MS/MS method for a panel of twelve steroid hormones in human serum. By integrating a newly developed IonFocus ESI unit on a Shimadzu LCMS-8060NX, the study evaluated enhancements in desolvation efficiency and assessed their impact on analytical sensitivity.

Methodology

Sample preparation involved protein precipitation by adding methanol to serum (800 µL to 200 µL), vortex mixing, centrifugation at 14 000 g, evaporation to dryness and reconstitution in 50% methanol. Analytical separation was performed on a Shim-pack Velox Biphenyl column (50 mm × 3.0 mm, 2.7 µm) with mobile phases of 0.2 mmol/L ammonium fluoride in water (A) and acetonitrile (B). The flow rate was 0.8 mL/min for method optimization and 1.5 mL/min for sensitivity evaluation. Injection volume was 30 µL at 30 °C.
Mass spectrometry used the LCMS-8060NX with IonFocus ESI in positive/negative modes. Key operating parameters included interface temperatures of 200–400 °C, heating gas flows of 10–25 L/min, drying gas at 10 L/min and nebulizer gas at 3 L/min. MRM transitions were optimized for each hormone.

Instrumentation

• UHPLC: Shimadzu Nexera X2
• Mass spectrometer: Shimadzu LCMS-8060NX with IonFocus ESI unit
• Column: Shim-pack Velox Biphenyl, 50 × 3.0 mm I.D., 2.7 µm
• Ion source: Modified ESI probe with mesh heater
• Gas supplies: Heating gas (10–25 L/min), drying gas (10 L/min), nebulizing gas (3 L/min)

Results and discussion

Increasing interface temperature and heating gas flow significantly enhanced desolvation and signal intensity for eleven of twelve steroids. DHEA displayed the most dramatic improvement, with up to 3-fold higher peak intensities at 1.5 mL/min LC flow compared to conventional conditions. MRM chromatograms of pooled human serum confirmed clear detection of nine hormones, including low-level DHEA, androstenedione, cortisol, cortisone, 11-deoxycorticosterone, estrone, 17α-hydroxypregnenolone, progesterone and testosterone, without complex sample cleanup.

Practical benefits and applications

The enhanced sensitivity supports clinical and pharmaceutical research where sample volume is restricted and hormone levels are low. Applications include endocrine disorder studies, cancer research and regenerative medicine, enabling direct serum analysis without time-consuming extraction steps.

Future trends and applications

Continued developments may integrate high-resolution mass analyzers, further miniaturize ion sources, and automate workflows for routine clinical laboratories. Expansion of this approach to other challenging biomolecules and coupling with microflow LC could broaden its utility in metabolomics and clinical diagnostics.

Conclusion

By improving heat transfer and increasing heating gas flow, the IonFocus ESI unit significantly enhances desolvation efficiency and analytical sensitivity for steroid hormones. This approach enables reliable detection of low-abundance analytes in serum, facilitating advanced clinical and research applications.