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Mass spectrometry methods have the potential to measure different hormones during the same analysis and have improved specificity and a wide analytical range compared with many immunoassay methods. Increasingly in clinical laboratories liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays are replacing immunoassays for the routine measurement of testosterone, 17-hydroxyprogesterone, and other steroid hormones. Reference LC-MS/MS methods for steroid, thyroid, and peptide hormones are being used for assessment of the performance and calibration of commercial immunoassays. In this chapter, the general principles of tandem mass spectrometry and examples of hormone assays are described.

A number of androgens are measured for clinical purposes. Most laboratories will run a testosterone assay but the requirement for other androgens may be too small for a laboratory to set up their own assay. In these cases samples would be sent to a specialized laboratory. In the routine laboratory testosterone is analyzed on automated systems but these lack the sensitivity to accurately measure the hormone in children and normal women. Many laboratories analyzed such samples by radioimmunoassay, but tandem mass spectrometry is now replacing these assays. This chapter provides simple methods for measuring the androgens in serum and saliva by radioimmunoassay and tandem mass spectrometry.

Background: The recent interest of clinical laboratories in developing serum testosterone assays based on isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) stems from the lack of accuracy of direct immunoassays. In this study, we assessed the accuracy and state of standardization (traceability) of 4 published ID-LC-MS/MS procedures in a method comparison with an ID-gas chromatography (GC)-MS reference measurement procedure listed in the database of the Joint Committee for Traceability in Laboratory Medicine., Methods: The study used 58 specimens from different patient categories. Each specimen was measured in triplicate (ID-LC-MS/MS) and quadruplicate (ID-GC-MS) in independent runs., Results: The testosterone concentrations by ID-GC-MS were 0.2-4.4 nmol/L (women), 0.2-2.0 nmol/L (hypogonadal man), and 10.1-31.3 nmol/L (normogonadal men). For ID-GC-MS, the CV was nearly constant, with a median of 1.0%; for ID-LC-MS/MS, it was concentration-dependent, with a median of up to 8%. Weighted Deming regression gave mean slopes, intercepts, and correlation coefficients of 0.90-1.11, -0.055-0.013 nmol/L, and 0.993-0.997, respectively. The % difference plot showed between 7% and 26% of the results outside a total error limit of 14%, with median deviations from ID-GC-MS between -9.6 and 0.4%., Conclusions: This study demonstrated fairly good accuracy and standardization of the tested ID-LC-MS/MS procedures. Performance differences between procedures were evident in some instances, due to improper calibration and between-run calibration control. This emphasizes the need for thorough validation, including traceability, of new ID-LC-MS/MS procedures.