Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Abstract

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.

Relationship between Serum Testosterone and Fracture Risk in Men: A Comparison of RIA and LC-MS/MS

“…At present, RIA and LC-MS/MS are commonly used for measuring TT, but it is not clear whether the discordance between the 2 methods could affect the classification of hypogonadism. In this study, we showed that the correlation between TT concentrations determined by RIA and LC-MS/MS was reasonably high, consistent with previous observations (5)(6)(7)(8). At the individual level, this correlation translated into some inconsistency in the classification of male hypogonadism between the 2 methods of measurement, regardless of the serum TT thresholds used.…”

“…The inconsistency between that study and ours could be a result of differences in the population studied, RIA methods used, or method of data analysis. The participants in the study of Wang et al (8 ) were much younger and less likely to have a low TT concentration than our participants. Wang et al (8 ) recruited 122 men, ages 18 -68 years; meanwhile, all 602 elderly men in our study were age Ն60 years old, with a mean age of 73 years.…”

“…The participants in the study of Wang et al (8 ) were much younger and less likely to have a low TT concentration than our participants. Wang et al (8 ) recruited 122 men, ages 18 -68 years; meanwhile, all 602 elderly men in our study were age Ն60 years old, with a mean age of 73 years. Aging is known to be associated with decline in serum testosterone (20,21 ).…”

“…Excellent agreement between TT RIA and TT LC-MS/MS has been reported (8 ), with correlation coefficients ranging from 0.92 for the automated multipurpose immunoassays from Bayer (Centaur) to 0.98 for a commercially available RIA kit (DPC-RIA, Core Endocrine Laboratory, Penn State University-Hershey Medical Center). All methods except the DPC-RIA, however, had an intercept of the Deming regression significantly different from that of LC-MS/MS, which is commonly considered a reference method.…”

Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Candidate Reference Method for Total Testosterone in Human Serum

“…The next steps in the sample preparation procedure were adapted from two published methods for total testosterone (26,30 ). In brief, testosterone was released from binding proteins with 600 L ammonium acetate (0.5 mol/L, pH 5.5) and equilibrated at room temperature (22°C) for 2 h. Liquid/liquid extraction (LLE) was performed 2 times with 2.5 mL ethyl acetate:hexane solution (3:2, vol:vol) to extract the analytes.…”