Confidence in Your Calibrators: MassTrak Endocrine Steroid Calibrators and Quality Control Sets for the LC-MS/MS Analysis of Steroid Hormones

Importance of the topic

The accurate quantification of steroid hormones is essential for diagnosing and monitoring conditions related to sexual development, inflammation and cardiovascular health. Mass spectrometry-based methods offer superior sensitivity and selectivity compared to traditional ligand-binding assays, supporting multi-analyte detection and improved clinical decision making.

Objectives and overview of the study

This application note evaluates the performance of the Waters MassTrak Endocrine Steroid Calibrator and Quality Control Sets (IVD) in conjunction with a validated LC-MS/MS workflow. The goal is to demonstrate metrologically traceable calibration, method precision, accuracy and compliance with ISO 15189 requirements.

Methodology and instrumentation

Sample preparation and analysis were performed as follows:

• Protein precipitation: 125 µL serum mixed with internal standard and methanol, followed by centrifugation.
• Solid phase extraction: Oasis MAX µElution plate with conditioning, loading, washing and elution steps.
• LC conditions: ACQUITY UPLC I-Class PLUS, CORTECS C8 column (2.1 × 100 mm, 2.7 µm), gradient elution with ammonium fluoride in water and methanol, 7.8 min run time.
• MS conditions: Xevo TQ-S micro triple quadrupole, positive/negative ESI, multiple reaction monitoring for 12 steroid analytes.
• Software: MassLynx v4.2 with TargetLynx XS for data acquisition and processing.

Main results and discussion

Key performance metrics included:

• Chromatographic resolution: Baseline separation of isomeric steroids such as 21-deoxycortisol, corticosterone, 11-deoxycortisol and 17-OHP.
• Linearity: Calibration curves with mean r² > 0.994 across all analytes.
• Sensitivity: Signal-to-noise ratios > 10 at the lowest calibrator levels (C1).
• Precision: Total precision and repeatability ≤ 7.7 % CV over five days (n = 25) for low, mid and high QC levels.
• Accuracy: QC recoveries between 91.0 % and 112.4 %.
• External quality assessment: Method bias within ± 7.8 % versus EQA scheme means for DHEA-S, cortisol, androstenedione, testosterone, 17-OHP, progesterone and DHT.

Benefits and practical applications of the method

The validated workflow delivers:

• Metrologically traceable calibrators and QCs supporting ISO 15189 compliance.
• Confidence in multi-analyte steroid quantification with robust accuracy and precision.
• Reduced sample preparation time through lyophilized reagents and µElution SPE plates.
• Capability to detect low-level steroid hormones using limited sample volumes (125 µL).

Future trends and potential applications

Advances likely to shape steroid analysis include:

• Broader standardization and harmonization of LC-MS/MS methods across clinical laboratories.
• Integration with automated sample handling and data processing for high-throughput workflows.
• Expansion of calibration sets to additional metabolites and conjugates for comprehensive steroid profiling.
• Application of high-resolution mass spectrometry and multi-omics approaches to deepen metabolic insights.

Conclusion

The Waters MassTrak Endocrine Steroid Calibrator and Quality Control Sets, combined with the ACQUITY UPLC I-Class PLUS and Xevo TQ-S micro system, enable precise, accurate and traceable quantification of 12 key steroid hormones. The workflow meets stringent regulatory requirements and supports robust clinical and research applications.

Reference

• Foley D, Calton LJ. Confidence in Your Calibrators: MassTrak Endocrine Steroid Calibrators and Quality Control Sets for the LC-MS/MS Analysis of Steroid Hormones. Waters Corporation; 2021.