Method Validation of Recombinant Human Growth Hormone by HPLC with UV and Fluorescence Detectors

Abstract

Recombinant growth hormone is a biotechnological product. As there may be variations in the purity or yield of an individual batch, quality control of the products is important for identification, efficacy, safety, and standardization. The pharmacopeial assay of growth hormone is done by high-performance liquid chromatography with a UV detector (HPLC-UV) using the single point method. This method requires a high amount of substance for analysis and can be costly. Therefore, the purpose of our study was to develop a sensitive, more efficient, and low cost assay for determination of growth hormone. Methods: The analytical principle was based on HPLC-Size Exclusion Chromatography by using TSKgel G2000SW_XL column and 0.063 M phosphate buffer solution: 2-propanol (97:3) as the mobile phase. The flow rate was 0.6 ml/ml. UV detection at 214 nm and fluorescence detection with the excitation at 275  nm and the emission at 337  nm were used in the experiments. Results and Discussion: For HPLC-UV, the retention time of growth hormone was 14.5 min. The limit of detection (LOD) was found to be 0.001 mg/ml. The correlation coefficient of the calibration curve was 0.9987. For HPLC-Fluorescence, the retention time of growth hormone was achieved at 14.8 min and the LOD was 0.9 µg/ml. The calibration curve was linear with the correlation coefficient of 0.9998. For accuracy, the recovery of both methods was found to be within 98.10-102.49%. Intra-day and inter-day precisions were less than 3%. Conclusions: The developed methods were linear, accurate, precise, and sensitive. The HPLC-Fluorescence exhibited an advantage to improve sensitivity which required lower concentration of growth hormone.