Development of highly sensitive quantification method for testosterone and dihydrotestosterone in human serum and prostate tissue by liquid chromatography–electrospray ionization tandem mass spectrometry

We developed highly sensitive detection of testosterone (T) and 5α-dihydrotestosterone (DHT) by liquid chromatography–electrospray ionization tandem mass spectrometry using high proton affinitive derivatization of 17β-hydroxyl group of T and DHT with picolinic acid, mobile phase consisting of MeCN–MeOH–H2O–formic acid and conventional octadecylsilica (ODS) column. Purification of the derivatives was carried out using solid-phase extraction with ODS cartridge. By this method, T and DHT were determined simultaneously with limits of quantification (LOQs) of 1 pg/0.2 ml in serum, and T and DHT with LOQs of 0.5 pg and 1 pg/3 mg in prostate tissue, respectively, under acceptable assay performance (intra-assay and inter-assay accuracy and precision). The present method provides reliable and reproducible results for quantification of T and DHT in small volumes of serum and prostate samples for diagnosis in prostatic disorders and male climacteric.