|Evrim Erdogan1, Mark M. Kushnir2, Bingfang Yue2, William L. Roberts1, Wayne A. Meikle1,3, Alan L. Rockwood1,2. |
Departments of 1Pathology and 3Medicine, University of Utah, Salt Lake City, UT
2ARUP Institute for Clinical & Experimental Pathology, Salt Lake City, UT.
|Testosterone (Te), a major androgen in males, is involved in the development and maintenance of the male phenotype. Te, also the predominant bioactive androgen in women, circulates at smaller concentrations compared to those in men. Te is important for muscle mass, bone metabolism, cognitive function, and libido. In men, testosterone concentrations are typically measured to evaluate testicular endocrine activity. In women, Te concentration is measured when investigating hyperandrogenism. In children, Te concentration is measured for gender assignment of newborns or infants with ambiguous genitalia, for disorders of puberty, inborn errors of sex-steroid metabolism, and in the follow-up of patients with congenital adrenal hyperplasia. |
Te circulates bound to sex hormone binding globulin (SHBG), free hormone, and albumin bound. Free and albumin bound forms are biologically active. Medical conditions that alter the serum concentrations of SHBG or albumin may affect the total Te level.
Measuring free Te (FTe) would more accurately reflect a patient’s Te status.
We compared the concentrations of FTe calculated from:
(1) LC-MS/MS measurement of total testosterone followed by mathematical modeling, and
(2) dialysis (to physically separate the FTe) followed by LC-MS-MS.
200 µL aliquots of serum samples or quality controls were dialyzed against 200 µL of dialysis buffer (PBS) at 37 °C for approximately 20 h. The dialysate was spiked with stable isotope-labeled internal standards and extracted into methyl-tert-butyl ether. The solvent was evaporated and steroids derivatized with hydroxylamine. Instrumental analysis was performed on an API 4000 (Applied Biosystems/ MDS SCIEX) using multiple reaction monitoring (MRM) mode with MRM transitions of 304 to 124, and 304 to 112 (m/z). Two-dimensional chromatographic separation was performed using a Gemini Phenyl cartridge as the first dimension, and a Gemini C18 column as the second dimension. LC-MS/MS method reportable range was 6-2500 pg/mL.
We analyzed 45 male samples (0.2-193 pg/mL) whose total Te concentrations were determined by the LC-MS/MS method, and whose FTe concentrations were calculated using a mathematical algorithm. The same samples were dialyzed and analyzed by LC-MS/MS (ED-LC-MS/MS). The method comparison yielded the following linear regression equation:
LC/MS-MS (mathematical algorithm) = 0.95*ED-LC-MS/MS – 3.37 (r=0.98)
These results validate that the mathematical modeling that we use routinely to determine FTe concentrations is comparable to dialysis. Our intention is to develop this ED-LC-MS/MS method further so it will be suitable for FTe analysis in females and children.