Determination of Testosterone in Human Peripheral Blood Using Gas-Liquid Chromatography with Electron Capture Detection

Abstract

A method for the estimation of testosterone in human peripheral plasma is described. Using sensitive electron capture detection after gas-liquid chromatography of testosterone chloroacetate, it is possible to quantitate nanogram amounts of testosterone. After addition of testosterone-l,2-3H to the plasma, testosterone is extracted and purified by thin-layer chromatography. The chloroacetate of testosterone is prepared and subsequently purified by thin-layer chromatography. The testosterone chloroacetate is subjected to gas chromatography after addition of cholesterol chloroacetate as an internal standard. The sensitivity of the method for steroid detection is such that the presence of 0.001 μg pure testosterone chloroacetate can be demonstrated. The results of replicate (10) analysis on 5 ml samples of a male plasma pool gave a mean value of 0.021 μg (standard deviation ±0.006) testosterone/5 ml. Replicate (15) analysis on 10 ml samples of a female plasma pool gave a mean value of 0.006 (standard deviation ±0.003) μg testosterone/10 ml. None of the normally occurring plasma steroids interfere with the estimation of testosterone; the chloroacetate of 17-epitestosterone and 19-nortestosterone are, however, not completely separated from testosterone chloroacetate upon gas chromatography.