The detection and measurement of D-norgestrel in human milk using Sephadex LH 20 chromatography and radioimmunoassay

An accurate sensitive method for the assay of D-norgestrel in human milk is described. The steroid is isolated from an ether extract of milk by Sephadex LH 20 chromatography in the system iso-octane-benzene-methanol (70:20:10 v/v). The radioimmunoassay utilises a specific antibody produced in rabbits against D-'norgestrel 3-(O-carboxymethyl) - oxime coupled to bovine serum albumin with D-norgestrel 3-(0-carboxymethyl) -oxime/ [125I]-iodohistamine conjugate as radioligand. Accuracy, sensitivity and blank value are satisfactory. Milk samples were obtained from three subjects treated with 30 microgram/day D-norgestrel, treatment commencing two weeks following parturition. Significant amounts of D-norgestrel were found, ranging between 92-135 pg/ml milk at the end of the first two-week treatment regimen. In two of three subjects, lower, but significant concentrations (53 pg and 35 pg/ml respectively) of steroid were found at the end of four weeks treatment. In the third subject, D-norgestrel could not be detected at this time. As a check on the specificity of the assay, three samples were submitted to additional chromatographic purification on alumina thin layer in the system benzene-cyclohexane-ethanol (70:27:3 v/v). Although this additional chromatographic step yielded somewhat lower values, agreement between the respective sets of results was good. The significance and implications of these findings are discussed.