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Abstract INTRODUCTION: Clinical research of free testosterone in human serum has long been considered challenging, partly due to the inherent low concentrations (<3% of total testosterone is free). Furthermore, issues relating to variability in results obtained from equilibrium dialysis, the gold standard, have been noted due to technical challenges in addition to lack of control over temperature, pH and shifts in equilibrium. A calculation developed by Vermeulen et al has also been widely used, taking into account total testosterone serum albumin and sex hormone-binding globulin (SHBG) concentrations.
OBJECTIVES: The aim of this project was to develop a novel clinical research, method for the analysis of serum free testosterone using a short, reproducible and accurate equilibrium dialysis protocol and an analytically sensitive mass spectrometer.
METHODS: Testosterone certified reference material (Merck, UK) was used to create calibrators in 52.75 mM HEPES buffer adjusted to pH 7.4. In-house QC materials prepared in serum using individual, pooled and stripped serum matrices (Golden West Biologicals, USA and BioIVT, UK), were used to evaluate method precision. Serum samples (200 µL) were incubated at 37°C with 400 µL pH-adjusted 52.75 mM HEPES buffer for 2 hours, mixing at 800 r.p.m. with Rapid Equilibrium Analysis (RED) devices and plates (Thermo Fisher, UK). Diasylate was spiked with testosterone-13C3 (Merck, UK), then processed with a liquid-liquid extraction (LLE) procedure. Using an ACQUITY™ UPLC™ I-Class System, samples were injected onto an ACQUITY UPLC BEH™ C18, 1.7µm, 2.1 x 100 mm Column using a water/methanol/ammonium fluoride gradient elution profile and quantified with a Xevo™ TQ Absolute Mass Spectrometer. The standard procedure for achieving equilibrium in the incubation step is to incubate for 4 hours at 37°C, mixing at approximately 250 r.p.m. However, we have demonstrated that a relatively quick incubation step with a fast mix can achieve equilibrium as well. Additionally, it is essential to control the pH of the dialysis buffer and, particularly, the temperature of the chamber during incubation.
RESULTS: The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.5–650 pg/mL. Addition of high concentrations of endogenous substances (albumin, bilirubin, creatinine, cholesterol, triglycerides and uric acid) did not affect quantification. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (≤20%CV and ≤15% bias) at 0.50 pg/mL. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs at approximately 2.79, 8.80 and 134 pg/mL respectively were all ≤ 8.4% (n=25). Accuracy was assessed by analyzing 45 male external quality assurance (EQA) samples (NEQAS, UK) ranging in concentration from 50.1-268.6 pg/mL and the resulting Deming equation y=-2.869+0.947x demonstrated good agreement. When LC-MS/MS determined concentrations were processed against Vermeulen equation determined concentrations (total testosterone and SHBG concentrations were provided by the scheme, a concentration of 45 g/L albumin was assumed), again good agreement was shown with a Deming equation y=-0.2008+0.9325x.
CONCLUSIONS: We have successfully quantified free testosterone using a fast equilibrium dialysis procedure with LLE and LC-MS/MS analysis, for use in clinical research. The method demonstrates sub pg/mL analytical sensitivity, excellent linearity and precision, with minimal matrix effects and a strong agreement with an EQA scheme.
For Research Use Only, Not for use in diagnostic procedures.
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= Discovery stage. (16.60%, 2024)
= Translation stage. (37.02%, 2024)
= Clinically available. (46.38%, 2024)
