Analysis of norethindrone in plasma by high-performance liquid chromatography.

Abstract

The lack of specificity of some radioimmunological assays for the determination of norethindrone has been reported. This paper describes a high performance liquid chromatographic (HPLC) method that is sufficiently sensitive and specific for the determination of plasma samples containing 2 ng/ml norethindrone. Plasma samples were collected from 8 human volunteers. The solvents used for the mobile phase were methanol, HPLC grade acetonitrile, HPLC grade, and double glass-distilled water. The HPLC (Waters Associates, Milford, Massachusetts) was used at a nominal attenuation of .005 absorbance units full scale (AUFS) and fed into a 5 mV recorder, giving an overall response of 0.0025 AUFS. The HPLC was also fitted with an ultraviolet detector (254 nm). The organic solvent extract from plasma is chromatographed on a reversed phase column using the HPLC. Tritiated norethindrone was added to 2.0 ml of plasma containing from 2 to 20 ng/ml of norethindrone and was extracted using the procedure as described. The organic extract was then transferred into a scintillation vial and evaporated to dryness. A Beckman LS 150 scintillation counter with an automatic quench correction device was used to determine radioactivity. The results show that the extraction efficiency and reproducibility are comparable at plasma concentrations ranging between 2-20 ng/ml. No interfering compounds (metabolites and endogenous substances) were extracted using HPLC. Data analysis of a number of spiked plasma samples ranging from 2 ng/ml-20 ng/ml suggests the accuracy and precision of the method. In another experiment, 40 mg of norethindrone was dissolved in ethanolic saline and orally administered in a mini-pig. The plasma norethindrone concentration was readily detected. The experiments illustrate the stronger specificity of the new method compared to reported radioimmunoassays.