Simultaneous quantification of total and free testosterone in human serum by LC-MS/MS

Testosterone (TTe) and free testosterone (FTe) are clinically important indicators for the diagnosis of androgen disorders, so accurate quantitative determination of them in serum is clinically of paramount significance. Currently, there is no available method suitable for routine and simultaneous measurement of TTe and FTe. Here, we developed a new UPLC-MS/MS method to quantify serum TTe and FTe simultaneously and accurately. Rapid equilibrium dialysis was used to obtain FTe in serum followed by derivatization with hydroxylamine hydrochloride. With these strategies, TTe and FTe could be measured in single injection. After optimizing the extraction and derivatization conditions, the performance of LC-MS/MS was evaluated and applied to quantify the levels of TTe and FTe in clinical samples from 42 patients. The assays were linear for TTe within the range of 0.2-30 ng/mL and for FTe within the range of 1.5-1000 pg/mL. This improved method provided a limit of quantification for TTe of 0.2 ng/mL and for FTe of 1.5 pg/mL. The intra- and inter-run CVs were less than 4.3% and 3.6% for TTe and less than 8.2% and 6.7% for FTe, respectively. The intra- and inter-run accuracies for both TTe and FTe were in the range of 96.1-108.1%. Interference, carryover effect, and matrix effect were in acceptable range. In conclusion, our new LC-MS/MS method is simple to perform and can serve as a reliable method for simultaneous determination of TTe and FTe in clinical practice, providing important information for diagnosis, treatment, and monitoring of androgen-related diseases.