MSACL 2023 Abstract

Validation of Bioavailable Testosterone by Mass Spectrometry

Jennifer Kemp(1), Robert Taylor(1), Anthony Maus(1), Ravinder J. Singh(1) and Stefan K. Grebe(1,2)
(1) Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN(2) Department of Medicine, Division of Endocrinology, Mayo Clinic, Rochester, MN

Jennifer Kemp (Presenter) Mayo Clinic

Relevant Financial Disclosures (within past 24 months, reported on Jul 11, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

OBJECTIVE: Our established bioavailable testosterone assay utilized differential precipitation of sex hormone binding globulin (SHBG) followed by scintillation counting. This method relied on antiquated equipment and necessitated the use of radioactive-labeled testosterone. Therefore, we sought to overcome these shortcomings by elimination of scintillation counting and transitioning to performing these measurements by liquid chromatography-tandem mass spectrometry.

METHODS: The method is based on the differential precipitation of SHBG by ammonium sulfate. SHBG bound testosterone is precipitated in patient sample using saturated ammonium sulfate, leaving the bioavailable testosterone in the supernatant. After addition of 13C labelled testosterone IS, the bioavailable testosterone and IS are extracted from the supernatant using isopropanol. The bioavailable testosterone and IS are then derivatized and analyzed by LC-MS/MS on a Sciex 7500 coupled to a ThermoFisher Scientific TLX-4 equipped with Vanquish pumps. Samples were first loaded onto a Phenomenex Cartridge Security Guard (C12, 4 x 2mm) and then separated using a Waters XBridge (C18, 2.1x50mm) analytical column.

RESULTS: To ensure assay validity the following metrics were assessed: intra and inter precision, accuracy, recovery, linearity, carryover, limit of detection (LOD), ion suppression, and interferences. Three levels were assessed (n=20) for intra assay precision, which yielded the following: 6.8% CV at a bioavailable testosterone concentration of 0.82 ng/dL, 4.1 % CV at a concentration of 6.5 ng/dL and 3.8% CV at a concentration of 259 ng/dL. Three levels were assessed for inter assay precision, which yielded 12.5 % CV at 0.90 ng/dL, 8.9% CV at 15 ng/dL and 13.1% CV at 402 ng/dL. When using linear regression analysis to compare results with the current assay, the resulting slope was 1.04 with an r2 of 0.96. Spike and recovery of five samples yielded a mean average recovery of 110.6%. For linearity 4 samples were diluted and the mean %difference from expected was -2.6%. Blanks were injected following the high standard and carryover was negligible. LOD was determined by running a low sample pool along with a blank over 20 assays. The LOD was determined to be less than the LLOQ. 58 common drugs/vitamins along with varying levels of hemolysis, lipemia, and bilirubin were also tested to ensure no interferences were seen. Ion suppression was performed to ensure the IS was compensating for ion suppression or enhancement.

CONCLUSION: The LC-MS/MS measurements met or exceeded the performance of the historical scintillation counting method and the acceptance criteria for all metrics assessed. Therefore, we can eliminate the use of harmful radioactive molecules and antiquated equipment, while providing superior quality testing to our patients.