329. Competitive protein binding analysis of norethindrone in human plasma and urine

Abstract

A competitive protein binding method has been developed for the quantitative analysis of norethindrone (NE) in plasma and urine utilizaing the progesterone binding globulin of pregnant guinea pig serum. The method involves dilution of the sample with buffer and extraction with benzene. The ethinyl steroids are then converted to silver salts by extraction with 1N AgNO3. NH4C1 is added to dissociate the silver salt and the ethinyl steroids are back-extracted into benzene. This extract is evaporated to dryness under a stream of air and mixed with the binding solution (buffered pregnant guinea pig serum plus tritiated-progesterone). Following incubation at 45 degrees C for 5 minutes and then at 0 degrees C for 30 minutes the unbound steroids are removed by treatment with dextran-charcoal and an aliquot of the supernatant is taken for liquid scintillation counting. The resulting counts (converted to DPMs) are then compared with standards prepared with known concentrations of NE in blank plasma or urine. The standard curve is linear from 10 to 500 ng. Normal male and female volunteers receiving single 5-mg doses of NE showed peak plasma levels of 20 and 42 ng/ml respectively in 2-4 hours after dosing. The plasma half-life of NE was 4-6 hours. About 2% of the dose was recovered as NE (free and conjugated) in the urine within 48 hours.(FULL TEXT)