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Abstract INTRODUCTION
Steroids are essential endogenous metabolites that regulate numerous physiological processes, making their accurate quantification critical for clinical research. Traditional immunoassays often lack the specificity required for structurally similar steroids, leading to cross-reactivity and limited analytical performance. Liquid chromatography-mass spectrometry (LC-MS) has become the gold standard for steroid analysis due to its superior specificity and sensitivity. However, achieving high precision and accuracy across a broad panel of analytes remains challenging. This study presents a robust workflow integrating the Tecan Steroid Panel LC-MS kit with the Agilent 6495 triple quadrupole LC-MS, enabling comprehensive and efficient quantification of 17 steroids and dexamethasone in serum.
METHODS
Chromatographic separation was performed using an Agilent 1290 Infinity III LC system with a C8 column (Tecan REF: 30215928), coupled to an Agilent 6495 triple quadrupole mass spectrometer. The system operated in both positive and negative ESI modes, with MRM transitions optimized using Agilent MassHunter Optimizer software. Data acquisition and analysis were conducted with Agilent MassHunter software.
Sample preparation utilized a semi-automated solid phase extraction (SPE) protocol on a Tecan Resolvex® A200 positive pressure manifold, using 96-well SPE plates provided in the kit. All solvents were LC-MS grade, and the kit supplied all necessary reagents, calibrators, controls, and internal standards. Chromatographic conditions were optimized for the resolution of closely related analytes. Calibration curves were constructed using six points and a zero calibrator for each analyte.
RESULTS
The workflow enabled robust separation and quantification of all target analytes, including challenging pairs such as corticosterone, 11-deoxycortisol, and 21-deoxycortisol, as well as cortisone and cortisol. Precision, expressed as coefficient of variation (CV%), was typically below 10% for most analytes, with all remaining within the 20% acceptance criterion even at low and high concentration levels. Linearity was good, with high coefficients of determination (R²) for all analytes; most were fit by linear regression, while some required quadratic models and appropriate weighting. Trueness, assessed by comparison to QC samples with metrological traceability, showed deviations within ±20% for all analytes, with most well below 15%. The semi-automated SPE protocol improved throughput and reduced manual handling errors compared to traditional methods.
CONCLUSION
The optimized Steroid Panel LC-MS workflow, combining the Tecan kit and Agilent 6495 LC-MS, provides a reliable, high-throughput solution for comprehensive steroid profiling in serum. The method demonstrates high precision, excellent linearity, and robust trueness across a wide concentration range. The semi-automated sample preparation using the Resolvex A200 enhances laboratory efficiency and consistency. While some analytes showed minor variability or bias, all results met established acceptance criteria. The kit is compatible with the Agilent platform and suitable for advanced research applications, though end users should perform validation in their own laboratories. Overall, this workflow represents a robust and efficient tool for multiclass steroid quantification in clinical research.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
