Which LC-MS/MS Platform is Most Appropriate for the Quantitative Analysis of Steroids in Serum for Clinical Research? WP129

Significance of the Topic

Endogenous steroids play crucial roles in metabolism, immune function, fluid balance and reproduction, making their accurate measurement essential for clinical research and diagnostics.

Objectives and Study Overview

This study compares three Thermo Scientific TSQ series tandem mass spectrometry platforms (Fortis, Quantis and Altis) to identify the most suitable system for quantitative analysis of a broad panel of serum steroids using a simple liquid-liquid extraction.

Methods and Sample Preparation

A one-step liquid-liquid extraction was performed on 200 µL serum samples spiked with deuterated internal standards. Separation was achieved on a Vanquish HPLC system with an Accucore C18 column using a gradient of 0.2 mM ammonium fluoride in water and methanol over an 11-minute run.

Instrumentation

• Mass Spectrometers: TSQ Fortis, TSQ Quantis and TSQ Altis triple quadrupole MS
• HPLC System: Vanquish Horizon binary pump, thermostatted column compartment
• Software: Xcalibur 3.1, TSQ Altis Tune 2.1 and TraceFinder 4.1

Key Results and Discussion

All platforms achieved baseline chromatographic separation of 18 steroids in 11 minutes with excellent linearity (R2>0.995) from 0.1 pg/mL to 1000 ng/mL. Limits of quantitation ranged from 1 to 5 pg/mL for most analytes and inter-assay precision and accuracy remained within 10% CV. The TSQ Altis demonstrated superior sensitivity for DHT and pregnenolone isomers, while TSQ Quantis and Fortis performed comparably for testosterone, corticosteroids and progesterone but showed less consistency for certain low-abundance steroids.

Benefits and Practical Applications

The validated method offers a robust, high-throughput approach for simultaneous quantitation of multiple steroids across different MS platforms, facilitating inter-laboratory comparability in clinical research and QA/QC environments.

Future Trends and Potential Applications

Integration of automated sample preparation, expansion of steroid panels with novel metabolites and application of high-resolution MS technologies may further enhance sensitivity and specificity. Adoption in clinical diagnostic workflows could support personalized medicine and endocrine disorder monitoring.

Conclusion

The comparative evaluation confirms that all TSQ series instruments reliably quantify a broad steroid panel with the TSQ Altis providing the greatest sensitivity for challenging analytes. The streamlined extraction and rapid HPLC method support versatile, high-throughput steroid profiling across research and clinical laboratories.

References

• Therese Koal et al Standardized LC–MS/MS based steroid hormone profile-analysis Journal of Steroid Biochemistry & Molecular Biology 2012 129 129–138
• Mikael Levi et al Fast Sensitive and Simultaneous Analysis of Multiple Steroids in Human Plasma by UHPLC–MS–MS LCGC March 1 2015 186