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Abstract BACKGROUND:
Steroid hormones are essential regulators of metabolism, immune function, stress response, and reproduction. Accurate quantification of steroid hormones is crucial for clinical diagnostics and biomedical research, especially in the context of endocrine disorders and metabolic diseases. Traditional immunoassays, while widely used, are often limited by cross-reactivity and lack of multiplexing capability, which can compromise specificity and throughput. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become the gold standard for steroid quantification, offering superior specificity, sensitivity, and the ability to simultaneously measure multiple analytes in complex biological matrices (1,2).
OBJECTIVE:
The objective of this study was to assess the analytical performance of a research-use-only steroid quantification kit in conjunction with the Shimadzu Nexera X3 UHPLC and LCMS-8060 platform for the simultaneous determination of 17 steroids. Key evaluation criteria included intra-assay precision, and trueness across three sample levels, with the goal of establishing the suitability of this workflow for clinical research applications in the field of steroidomics.
METHODS:
Sample preparation followed the Tecan kit IFU (Cat. No. 30220266), which includes solid-phase extraction (SPE) to ensure optimal recovery and ionization efficiency for all target analytes. The workflow consists of two LC-MS/MS runs: the first run quantifies 15 steroids, while the second run quantifies two additional steroids, for a total of 17 steroids. The calibrators and quality control samples are lyophilized in serum and reconstituted with LC-MS grade water before analysis. Chromatographic separation was optimized to resolve analytes with similar structures and mass transitions, with particular attention to those known to be challenging, such as 11-deoxycortisol, 21-deoxycortisol, corticosterone, cortisol and cortisone, as well as 17-hydroxyprogesterone and 11-deoxycorticosterone(3). Proper chromatographic separation of these analytes is essential to avoid cross-talk and ensure accurate quantification. The Shimadzu LC-MS/MS system was operated in multiple reaction monitoring mode (MRM), with compound-specific transitions and collision energies. Data acquisition and quantification were performed using LabSolutions software, with internal standard correction applied to all analytes. Precision was evaluated as the relative standard deviation (%) of replicate measurements at each quality control level, with a classical acceptance criterion of less than 20%. Trueness was assessed by comparing the measured values to the corresponding target values.
RESULTS:
The Tecan kit demonstrated robust analytical performance across all tested analytes and quality control levels. Precision was assessed for all analytes at both high- and low-quality control levels using 15 replicates each. All analytes demonstrated %RSD values below 20%, indicating strong repeatability. For example, 17-OHP4 showed %RSD values of 3.9% (QC H) and 3.7% (QC L), testosterone 1.9% (QC H) and 3.2% (QC L), and cortisol 4.5% (QC H) and 8.7% (QC L) showed %RSDs well within this range at both QC levels. The method provides reliable and robust precision across the entire steroid panel.
Trueness was evaluated at three sample levels (S1, S2, S3) for each analyte. Most analytes showed trueness within the generally accepted range. For example, 17-OHP4 demonstrated trueness values of 0.2% (S1), 1.3% (S2), and 6.2% (S3); testosterone showed 13.0% (S1), 1.6% (S2), and -5.4% (S3); and cortisol showed 7.0% (S1), 7.2% (S2), and 4.5% (S3). All analytes were within ±20% of the target value across all levels, supporting the method’s suitability for clinical research applications.
Calibration curves for all analytes demonstrated good linearity, with R² values >0.99 for almost all analytes. For key clinical markers, R² values were 0.9978 for 17-OHP4, 0.9989 for testosterone, and 0.999 for cortisol. All analytes were fitted using a linear model with either 1/C or 1/C² weighting, as appropriate.
CONCLUSION:
The comprehensive evaluation of the Steroid Panel LC-MS kit in combination with the Shimadzu platform for high-throughput, multiplexed steroid analysis. The ability to reliably quantify a broad panel of steroids in just two runs offers significant advantages for research laboratories investigating complex endocrine pathways, disease mechanisms, or therapeutic interventions. The standardized workflow reduces operator variability and enhances reproducibility, while the high sensitivity and selectivity of the Shimadzu LC-MS/MS platform ensure confident detection and quantification even at low physiological concentrations. The method meets established acceptance criteria for precision across the vast majority of analytes and quality control levels. Trueness results further support the method’s reliability for clinical research applications.
References:
1. Taylor, A.E., Keevil, B.G., & Huhtaniemi, I.T. (2015). Mass spectrometry and immunoassay: how to measure steroid hormones today and tomorrow. European Journal of Endocrinology, 173(2), D1–D12.
2. Vogeser, M., & Parhofer, K.G. (2007). Liquid chromatography tandem-mass spectrometry (LC-MS/MS)–technique and applications in endocrinology. Experimental and Clinical Endocrinology & Diabetes, 115(09), 559-570.
3. Zhu, Y., Li, Y., Wang, Y., Wang, Y., & Wang, Y. (2022). Simultaneous determination of 11 endogenous corticosteroids in human plasma by liquid chromatography–tandem mass spectrometry for the diagnosis of adrenal diseases. Frontiers in Endocrinology, 13(9), 940314. https://doi.org/10.3389/fendo.2022.940314.
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= Discovery stage. (53.14%, 2025)
= Translation stage. (22.33%, 2025)
= Clinically available. (24.53%, 2025)
