Quantification of Steroids in Human Plasma by Liquid Chromatography Coupled with the Affordable Q Exactive Focus HRMS

Importance of the Topic

Steroid hormones play critical roles in human physiology and are present at very low concentrations in plasma, often in the nanomolar to picomolar range. Accurate measurement is essential for diagnosing endocrine disorders, monitoring therapy, and supporting clinical research. Traditional immunoassays lack sufficient specificity and can be confounded by cross-reactivity, particularly in pediatric and female populations. High-resolution mass spectrometry coupled with liquid chromatography (LC-HRMS) offers enhanced selectivity, sensitivity, and the potential for non-targeted metabolite screening.

Objectives and Study Overview

This study aimed to develop and validate a rapid, robust LC-HRMS method using the Thermo Scientific Q Exactive Focus instrument for routine quantification of eight clinically relevant steroids in human plasma, meeting sensitivity and precision requirements of clinical research.

Used Instrumentation

• Rheos Allegro UHPLC pump and CTC PAL autosampler
• Thermo Scientific Accucore C18 column (2.1 × 50 mm, 2.6 μm)
• Thermo Scientific Q Exactive Focus high-resolution mass spectrometer with HESI II source

Methodology and Instrumentation

• Sample Preparation: 100 μL plasma mixed with 5% phosphoric acid containing deuterated internal standards, followed by mixed-mode cation exchange solid phase extraction. Eluates were dried under nitrogen and reconstituted in 50:50 water/methanol.
• Chromatography: Gradient elution from 10 to 100% methanol (0.005% formic acid) over 18 min at 600 μL/min, column temperature 60 °C, injection volume 40 μL.
• Mass Spectrometry: Positive-mode heated ESI, full scan m/z 200–400 at 70 000 resolution (FWHM), spray voltage 4000 V, sheath gas 60 AU, capillary 380 °C. Targeted SIM for testosterone (centered m/z 290) also evaluated.

Main Results and Discussion

All eight steroids were detected as singly charged [M+H]+ ions and baseline resolved within 14 min. Calibration curves were linear across a broad concentration range, with accuracy and precision within ±15% for calibrators and QCs, and ±20% at the lower limit of quantification (LLOQ). LLOQs ranged from 0.13 nM (testosterone) to 2.19 nM (cortisol). The testosterone LLOQ was 0.125 nM (1.45 pg on column) with full scan and targeted SIM modes yielding comparable sensitivity.

Benefits and Practical Applications

• High specificity and sensitivity rivaling triple-quadrupole MS without derivatization.
• Full scan capability enables retrospective screening of steroid metabolites.
• Rapid analysis (<20 min) supports high-throughput research workflows.
• Targeted SIM enhances detection of low-abundance analytes when required.

Future Trends and Potential Applications

• Optimizing ESI source conditions and increasing sample volume may further improve LLOQs.
• Coupling with automated sample preparation and two-dimensional LC could enhance isomer separation.
• Expanding the steroid panel and integrating non-targeted metabolomics analyses using HR full-scan data.
• Broader adoption in clinical diagnostics for populations where immunoassays are inadequate.

Conclusion

The developed LC-HRMS method on the Q Exactive Focus platform delivers a robust, sensitive, and high-throughput solution for quantitative plasma steroid analysis in clinical research, matching the performance of conventional triple-quadrupole systems and enabling comprehensive steroid profiling.

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