Chromatography of progesterone and its major metabolites in rat plasma using microbore high-performance liquid chromatography columns with conventional injection and detection systems.

A separation of progesterone and its metabolites 17 alpha-hydroxyprogesterone, 20 alpha-hydroxy-4-pregnen-3-one, androstenedione and testosterone by high-performance liquid chromatography with a ternary solvent system is demonstrated. It is found that using a 100 X 1 mm microbore column, higher sensitivity is obtained in the ultraviolet detection of these compounds by using conventionally sized flow cells despite the higher efficiency resulting from the use of a micro-flow cell. It is also shown that, when a non-eluting solvent is used for injection, large injection volumes do not reduce column efficiency, and a 14-fold increase in sensitivity is obtained with a 1-mm column in place of one 4.6 mm in diameter. Using solid-phase extraction and concentration of the sample as pretreatment, progesterone and its hydroxylated metabolites are determined in 0.5-ml samples of rat plasma. The progesterone levels are compared with those obtained by radioimmunoassay.