Why Use Mass Spectrometry instead of immunoassays for steroid measurement?

Steroid hormones are commonly measured using immunoassays such as ELISA or radioimmunoassay. While these methods are widely used, they can suffer from limited specificity and sensitivity when measuring low-abundance hormones yielding highly-inaccurate results.  LC-MS/MS provides a highly specific and sensitive alternative for accurate quantification of steroid hormones in biological samples.

Immunoassays are susceptible to cross-reactivity. Because many steroid hormones share nearly identical chemical structures, antibodies can bind to structurally related steroids, leading to inaccurate measurements.

Direct immunoassays often exhibit significant measurement bias. Studies evaluating estradiol assays have reported positive bias ranging from ~6% to 74%, indicating systematic overestimation of hormone concentrations compared with LC-MS/MS measurements (PMID 24334824).

Accuracy of immunoassays can be extremely poor in experimental models. In mouse serum samples, testosterone immunoassays produced accurate measurements in only ~54% of male samples and ~9% of female samples when compared with LC-MS/MS reference methods (PMID 25365769).

For these reasons, major endocrine journals and professional societies recommend mass spectrometry–based methods for accurate steroid hormone measurement in research and clinical endocrinology.