Automated Sample Preparation Enables LC-MS/MS as a Routine Diagnostic Analysis for Serum Testosterone

Abstract

Abstract Background The advantage of LC-MS/MS for analyzing serum testosterone in female, pediatric, and hypogonadal male patient samples is well accepted (J Clin Endocrinol Metab 2010;95:4542–8). However, many clinical laboratories still use testosterone immunoassays because of the technical challenges of LC-MS/MS (Clin Chem 2010;56:1234–44). Although LC-MS/MS has been shown to have better accuracy and specificity than immunoassay, better reproducibility has been more elusive because of the complexities of sample preparation (Clin Chem 2008;54:1290–7; Rev Endocr Metab Disord 2013;14:185–205). Methods We evaluated replacing automated immunoassay with LC-MS/MS for all testosterone analyses at the University of California San Diego Health System Center for Advanced Laboratory Medicine. We used a novel extraction media, AC Extraction Plate™ (AC Plate), from Tecan Schweiz with automated liquid handling for LC-MS/MS sample preparation. We modified the existing vendor application and validated the method for matrix effect, recovery, precision, trueness [accuracy relative to certified reference material (CRM)], specificity, reportable range, sample stability, and correlation with other methods. Results Method performance was excellent, with a reportable range of 4–1560 ng/dL (0.14–54.13 nmol/L), between-day CV <6%, mean accuracy for CRM of <4.0% bias, no interference from hemolysis, icterus, lipemia, serum separator tube gel, or common steroids/metabolites, and mean bias of 1.3% vs 4 other LC-MS/MS testosterone methods. A retrospective analysis of calibration stability suggests that sparse and/or historical calibration is feasible for routine use. Conclusions We conclude that automated extraction with the AC Plate and a focus on robustness during method development delivered ease of use and method performance consistent with adopting LC-MS/MS for all testosterone testing.