Comprehensive Quantitative Analysis of Multiclass Steroids in Serum

Significance of the Topic

The quantification of endogenous steroids in serum is critical for understanding hormonal regulation and diagnosing endocrine disorders. Traditional immunoassays suffer from cross reactivity and limited specificity when analytes share similar structures. Coupling liquid chromatography and tandem mass spectrometry has become the standard for achieving high specificity and sensitivity at trace levels, enabling reliable steroid profiling in clinical research settings.

Objectives and Overview of the Study

This study aimed to implement and optimize the Tecan Steroid Panel LC MS kit in a 96 well format on an Agilent 6495 triple quadrupole system. The goal was to streamline sample preparation, achieve efficient chromatographic separation of seventeen steroids plus dexamethasone in a single ten minute run, and to validate analytical performance in terms of precision, linearity and trueness using spiked human serum across multiple levels.

Methodology

Sample preparation was carried out using a semi automated positive pressure SPE workflow on a 96 well Tecan plate. An integrated evaporation function enabled direct drying without elevated temperatures. Chromatographic separation employed a rapid gradient on a C8 column, while mass detection was performed in both positive and negative ESI modes using dynamic multiple reaction monitoring. Calibration curves were constructed from kit standards and internal standards with appropriate weighting and quadratic fitting where needed.

Instrumentation Used

• Agilent 1290 Infinity III LC system with binary pump, multisampler and multicolumn thermostat
• Agilent 6495 triple quadrupole MS with Jet Stream electrospray ion source
• Tecan Steroid Panel LC MS kit consumables and reagents
• Tecan Resolvex A200 positive pressure SPE platform and dedicated evaporation plate
• Agilent MassHunter software version 12.2 for data acquisition and processing

Main Results and Discussion

The method delivered high precision with most coefficients of variation below 10 percent across six concentration levels, remaining within a 20 percent acceptance limit. Linearity was excellent for all analytes with coefficients of determination up to 0.999, applying quadratic models when required. Trueness assessed at high and low QC levels showed deviations generally within plus or minus fifteen percent. Critical isobaric pairs such as corticosterone and 21 deoxycortisol, cortisone and cortisol were baseline separated, facilitating confident quantification in a single rapid analysis.

Benefits and Practical Applications

This workflow offers robust and reproducible steroid profiling with minimal manual handling. The semi automated SPE increases throughput and reduces variability. The integration into a single ten minute run supports high sample throughput in clinical research laboratories and quality control environments where precise steroid quantification is required.

Future Trends and Potential Applications

Future developments may focus on expanding panels to include emerging steroid biomarkers and integrating automation with laboratory information management systems for fully traceable workflows. Advances in high resolution mass spectrometry could further enhance specificity and enable non targeted profiling of novel metabolites. There is also potential for adoption in therapeutic drug monitoring and personalized medicine research.

Conclusion

The optimized Tecan Steroid Panel LC MS kit on the Agilent 6495 system demonstrates a reliable, fast and accurate approach to multiclass steroid quantification in serum. High precision, excellent linearity and robust trueness were achieved in a streamlined 96 well workflow, supporting its applicability in advanced clinical and research settings. End users should perform laboratory specific validation to ensure compliance with research objectives.

References

• Vogeser M and Parhofer KG Liquid chromatography tandem mass spectrometry LC MS MS technique and applications in endocrinology Ann Lab Med 2007 27 401 410
• Keevil BG Novel LC MS MS methods for measuring steroids Best Pract Res Clin Endocrinol Metab 2016 27 663 674