Quantification of endogenous hormonal steroids and their precursors is essential for diagnosing a wide range of endocrine disorders. Historically, these analyses have been carried out using immunoassay, but such methods are problematic, especially for low-concentration analytes, due to assay interference by other endogenous steroids. MS offers improved specificity over immunoassay and can be highly sensitive. GC–MS, with use of stable isotopically labeled internal standards, is considered the ‘gold standard’ method for serum steroid analysis. GC–MS is the method of choice for profiling steroid metabolites in urine, but these techniques are not appropriate for routine use in clinical laboratories owing to a need for extensive sample preparation, as well as analytical expertise. LC–MS/MS compares well to GC–MS in terms of accuracy, precision and sensitivity, but allows simplified sample preparation. While most publications have featured only one or a limited number of steroids, we consider that steroid paneling (which we propose as the preferred term for multitargeted steroid analysis) has great potential to enable clinicians to make a definitive diagnosis. It is adaptable for use in a number of matrices, including serum, saliva and dried blood spots. However, LC–MS/MS-based steroid analysis is not straightforward, and understanding the chemical and analytical processes involved is essential for implementation of a robust clinical service. This article discusses specific challenges in the measurement of endogenous steroids using LC–MS/MS, and provides examples of the benefits it offers. </jats:p
