Development and Validation of a Serum Total Testosterone LC-MS/MS assay Certified by the CDC Hormone Standardization Program.

Objective: We aimed to develop and validate a serum testosterone liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay and sought certification by the CDC Hormone Standardization (HoSt) Program.

Methods: Serum samples mixed with internal standards were extracted with hexane: ethyl acetate. An Agilent Poroshell 120 EC-C18 column and an Agilent 1260 Infinity HPLC system were used for liquid chromatography. An Agilent 6460C QQQ triple quadrupole mass spectrometer was used for multiple reaction monitoring. The assay was validated for matrix effect, lower limit of quantification, analytical measurement range, precision, accuracy, dilution, specificity, interference, and carryover.

Results: The LC-MS/MS testosterone assay demonstrated an analytical measurement range (AMR) of 2.9-2330.4 ng/dl with a mean bias of 0.4% (95% CI: -2.8% to 3.6%) for the certified reference materials from the HoSt Program. The total CVs were 2.4-4.7%. The correlation between this LC-MS/MS (y) and a reference laboratory LC-MS/MS (x) was excellent: y=0.939x-8.3 (R=0.9978). The method was certified by the CDC HoSt Program.

Conclusion: An accurate and sensitive LC-MS/MS assay to measure serum testosterone is developed, validated, and certified by the HoSt Program. The assay can be used for serum testosterone testing in pediatric, female, and hypogonadal male populations.