BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION OF HCG (HUMAN CHORIONIC GONADOTROPIN)
Keywords:RP-HPLC, HCG, BSA, Bioanalytical, Validation
Objective: To develop and validate simple, rapid, specific, accurate and precise bioanalytical method for determination of the HCG (Human Chorionic Gonadotropin) by RP-HPLC method by using human urine.
Methods: The chromatographic separation was performed using Phenom enex C18 (250 x 4.6 mm, 5Î¼, 300 Â°A) column. Mobile phase composed of sodium phosphate buffer (pH 7.0, 0.05M) and acetonitrile (87.5:12.5 % v/v) at a flow rate of 1 ml/min. Detection was carried out using UV detector at 215 nm. Bovine serum albumin (BSA) was used as an internal standard (ISTD) and extraction was carried out using protein precipitation method. The method was validated as per USFDA guidelines.
Results: The method was linear over the concentration range of 0.37 to 48.4 Âµg/ml. and correlation coefficient (R2) was found to be 0.9983 and the Lower limit of quantitation (LLOQ) was 0.37 Âµg/ml. Recovery was found more than 94.0% for HCG. The % CV for interday and intraday precision was found to be less than Ë‚1%.
Conclusion: A simple, rapid, specific, accurate and precise analytical method was developed and validated by using human urine.
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