Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample...

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Endocrinology

Background:

• Steroid analysis in clinical labs relies heavily on immunoassays (IAs), which face limitations in accuracy, precision, and sensitivity despite high sample throughput.
• Liquid chromatography coupled to mass spectrometry (LC-MS/MS) presents a more advanced alternative, offering improved sensitivity, specificity, and the capability for simultaneous analysis of multiple steroids and metabolites.

Purpose of the Study:

• To develop and validate a rapid LC-MS/MS assay for quantifying multiple steroid hormones in human serum.
• To compare the performance of the developed LC-MS/MS assay against conventional immunoassays (IAs).

Main Methods:

• A novel LC-MS/MS assay was developed featuring an eight-minute gradient elution and a simplified one-step sample preparation (protein precipitation with phospholipid removal).
• Assay validation included assessment of interday imprecision, trueness using certified reference materials and proficiency testing, and direct comparison with IVD-CE-certified immunoassays using patient samples.

Main Results:

• The LC-MS/MS assay demonstrated interday imprecision generally better than 15% and validated trueness.
• Comparison revealed that routine immunoassays failed to meet the standards of the LC-MS/MS assay, with significant bias observed for androstenedione and 17-OH-progesterone (>65%).
• Immunoassays also showed accuracy limitations at lower concentrations for progesterone, estradiol, and testosterone.

Conclusions:

• The developed LC-MS/MS method provides a reliable and comprehensive approach for steroid hormone quantification in clinical diagnostics.
• LC-MS/MS significantly outperforms conventional immunoassays in terms of accuracy, precision, and sensitivity, leading to more dependable diagnostic information for endocrinology.