Physical separation of amphiprotic-polar aprotic solvents for simultaneous extraction and clean-up of clomiphene from plasma before liquid chromatographic analyzes

An efficient and quantitative two phase freezing (TPF) method coupled with high performance liquid chromatography and UV-Vis detector was developed for the extraction, clean up and determination of clomiphene citrate (CLC) in plasma samples. The separation of two miscible solvents by TPF method permits that the CLC was efficiently removed from proteins and transferred into the relative aprotic dipolar organic phase and in consequence, gave a higher recovery. The TPF method was compared to conventional liquid-liquid extraction and it gave more clean solution with better reproducibility. Linear range, limit of detection and limit of quantification for CLC in plasma were obtained in the range of 0.06-18, 0.02 and 0.06 µg ml-1, respectively. The intraday and interday reproducibility for concentration of 1.0 µg ml-1 (%RSD) were 3.2% and 4.6%, respectively. In addition, the trueness, ruggedness, and realistic of TPF were assessment. Finally, several real plasma samples were successfully analyzed using the developed method.