A-042 Optimized state-of-the-art direct equilibrium dialysis for free testosterone analysis by LC-MS/MS: Achieving accurate measurements in hours

Abstract Background Testosterone in circulation is mostly bound to sex hormone binding globulin (SHBG) and albumin, with free testosterone (FT) and the fraction bound to albumin collectively known as bioavailable testosterone. In cases where binding protein concentrations are altered, total testosterone is an unreliable measure of androgen status. Measuring FT can help evaluate androgen status in women and hypogonadal men with normal or borderline total testosterone and/or cases with altered binding proteins. Equilibrium dialysis (ED) is the preferred method for measuring FT, followed by ultrafiltration, but these methods are technically challenging and time-consuming. Calculation of FT from total testosterone and SHBG (with or without albumin) has limitations and may be inaccurate. There is a need for reliable, easy-to-use, and high throughput methods for clinical measurement of FT. Here we describe an LC-MS/MS assay for measuring FT directly from the dialysate at various ED timepoints. Methods ED was performed on the Rapid Equilibrium Dialysis (RED) 8K MWCO plates (Thermo Scientific, San Jose, CA) using serum and of HEPES buffer in a 1:1.82 ratio, according to the manufacturer’s protocol. Per the IFU, incubating for 4 hours is sufficient to reach equilibrium. Residual serum samples (2 from women (W), 2 from men (M), 1 W pool and 1 M pool) were analyzed in duplicate. The plate was incubated at 37 °C with gentle mixing for 4, 6, 8, and 12 hours before retrieving the dialysate. Dialysate, calibrators, and controls were mixed with the testosterone internal standard, followed by extraction using the ISOLUTE SLE+ plates (Biotage, Charlotte, NC), drying down, and reconstitution in methanol and water. The reconstituted samples (50 µL) were analyzed by LC-MS/MS (TSQ Altis triple quadrupole MS and Transcend II LX-2 UPLC and autosampler, Thermo Scientific). Statistical analyses were performed in GraphPad Prism v9. Outliers were evaluated using Grubb’s test. The sample and time-point results were evaluated for significance using one-way ANOVA with multiple comparisons. The performance of FT was assessed by linear correlation relative to comparable LC-MS/MS methods in two reference laboratories at one ED timepoint. Results The free testosterone concentrations in the six samples ranged from <0.1 to 6.53 ng/dL on average across all the timepoints. The mean concentrations (ng/dL) for each sample and % coefficient of variation (%CV) were: W1 0.14, 36%; W2 0.61, 7%; W pool 0.52, 12%; M1 3.26, 7%; M2 5.93, 9% and M pool 2.66, 9%. There was no statistical significance across the ED timepoints (p-value 0.0656), ANOVA). Multiple comparison analysis did not detect statistical significance across any paired time-point (e.g., 4 hours vs. 6 hours) (p-values 0.1888 to 0.3502, Tukey’s test). The FT method comparison correlation after ED for 8 hours was as follows: Method 1: y=1.03x-3.04, R2=0.9949, n=43 and Method 2: y=1.13x-0.08, R2=0.9278, n=55. Conclusion FT results by direct ED reached equilibrium after 4 hours of incubation and did not change significantly for up to 12 hours. These results indicate that accurate FT results are achievable within ED hours, allowing laboratories to optimize incubation workflows to maximize efficiencies.