Simultaneous measurement of four testicular °4-3-ketosteroids by isocratic high-performance liquid chromatography with on-line ultraviolet absorbance detection

A method is described for the simultaneous measurement of testosterone, androstenedione, 17α-hydroxyprogesterone and progesterone in venous effluent from in vitro perfused rat testes. The assay uses a non-radioisotopic internal standard (11β-hydroxyandrostenedione), isocratic high-performance liquid chromatography (HPLC) and UV absorbance detection at 240 nm. Either of two isocratic HPLC systems described in this report (tetrahydrofuran—methanol—water, 16:28:56; methanol—acetonitrile—water, 9:36:55; v/v/v) may be used, and assay specificity is the same in each. The separation and measurement of all four steroids are completed in 25 min. Sensitivity of the method is 10 ng for testosterone, androstenedione and 17α-hydroxy-progesterone and 25 ng for progesterone. The linear range of the assay extends through 1600 ng which is the upper amount of each steroid tested. Average inter-assay coefficient of variation was 3.3% and average intra-assay coefficient of variation was 3.6%. This rapid, specific and reliable method requires minimal sample preparation and may be performed by inexperienced personnel.