Selective and sensitive measurement of 17 steroids in human serum using a Stellar mass spectrometer

Importance of the topic

The accurate and simultaneous quantification of multiple steroid hormones in human serum is critical for clinical research, diagnostics, and monitoring of endocrine disorders. Steroid hormones regulate numerous physiological processes—reproduction, metabolism, immune function—and disruptions in their biosynthesis can lead to conditions such as congenital adrenal hyperplasia and Cushing’s syndrome. High-throughput, sensitive, and selective analytical methods are therefore essential to support reliable hormone profiling in complex biological matrices.

Objectives and overview of the study

This technical note describes the development and validation of a targeted LC–MS/MS method for the simultaneous measurement of 17 endogenous steroids in human serum. Leveraging the Thermo Scientific™ Stellar™ hybrid quadrupole linear ion trap mass spectrometer, the study demonstrates how complementary fragmentation modes (beam-type HCD and resonance-type CID) and rapid MS2/MS3 acquisition improve sensitivity and selectivity compared to conventional triple quadrupole instruments.

Methodology and used instrumentation

Sample preparation employed protein precipitation with ice-cold acetonitrile, centrifugation, and reconstitution steps to concentrate analytes from human serum. Calibration standards and internal standards were spiked into bovine serum albumin solutions across four orders of magnitude, enabling evaluation of limits of quantification (LOQs), linearity, and precision.
Used instrumentation:

• Vanquish™ Horizon UHPLC system with Kinetex™ C18 column (2.1×150 mm, 2.6 µm, 50 °C)
• Thermo Scientific™ Stellar™ mass spectrometer equipped with OptaMax™ Plus heated electrospray ionization source
• TraceFinder™ software for automated compound optimization and data processing

Chromatographic separation was achieved with a gradient of 0.05 mM ammonium fluoride in water (A) and methanol (B) at 300 µL/min. The method used fast polarity switching and narrow isolation windows (2 m/z) for targeted MS2 and MS3 scan events.

Main results and discussion

Compound optimization leveraged online LC-MS injection of standards and TraceFinder’s automated collision energy (CE) ramping to identify optimal MS2 HCD and CID CEs, as well as MS3 candidates for each steroid.
The Stellar MS demonstrated acquisition rates up to 140 Hz for MS2 and 40 Hz for MS3, enabling monitoring of 115 scan events per 1.2 s cycle. This performance yielded over seven data points across UHPLC peaks of 6–12 s width, even with a 5-minute total runtime.
Interferences in complex serum were addressed by using MS3 scans. For example, dihydrotestosterone qualifier ions showed high background in MS2-HCD but were substantially cleaner in MS3, improving qualifier-to-quantifier ratios and detection sensitivity.
LOQs ranged from 0.05 pg to 2 pg on-column for most analytes, with correlation coefficients (R2) > 0.99 and inter-day imprecision below 20% at low concentration levels. Internal standard peak area %RSD across 108 injections remained below 15%.

Benefits and practical applications

• Simultaneous quantification of a comprehensive steroid panel in a single, rapid (5 min) LC–MS run
• Improved sensitivity and selectivity through complementary HCD and CID fragmentation plus MS3
• High throughput enabled by fast scan speeds and automated optimization tools
• Robust performance in complex matrices such as human serum, suitable for clinical research laboratories

Conclusion

The developed method on the Stellar mass spectrometer delivers a high-throughput, sensitive, and selective platform for the targeted quantification of 17 steroids in human serum. By exploiting rapid MS2/MS3 acquisition and dual fragmentation strategies, the workflow addresses challenges posed by isobaric interferences and low analyte concentrations.

Future trends and potential applications

Further enhancements could include narrower scheduled retention time windows or single-scan event targeting to extend LOQs and precision. Expanding the panel to cover additional steroid metabolites and applying the method to other biofluids will broaden its clinical and research utility. Integration with advanced software workflows and multiplexed sample handling promises to accelerate large-scale studies in endocrinology and pharmaceutical monitoring.

References

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