Determination of 4-hydroxyandrostenedione in plasma and urine by extractive alkylation and electron-capture gas chromatography

A sensitive and specific quantitative assay has been developed for the determination of 4-hydroxyandrostenedione (4-OHA), a potent aromatase inhibitor used in the treatment of estrogen-dependent breast cancer. This steroid has a high first-pass metabolism and is extensively metabolized, mainly by glucuronidation. Plasma levels of unchanged 4-OHA are very low, even after high peroral doses. The analytical method is based on the addition of 17 alpha-ethinylestradiol (internal standard), liquid-liquid extraction from biological material followed by extractive alkylation with pentafluorobenzyl bromide and quantitation by gas chromatography. The method has been validated for sensitivity, accuracy and precision and was found to be suitable for application to pharmacokinetic and bioavailability studies of peroral formulations of 4-OHA.