Abstract

A rapid, accurate and sensitive HPLC method has been developed and validated for the quantitative determination of lispro and glargine insulin analogues in pharmaceutical preparations. Pharmacopeial method for the determination of insulin lispro employed mobile phase constituted by phosphate buffer solution 0.1mol/L (pH 2.3) and acetonitrile (74 + 26, v/v) and flow rate of 0.8mL/min. Retention time was estimated to be 25 minutes and the temperature of the column maintained to 40ºC. No pharmacopeial method was found to the determination of insulin glargine. For this reason, the aim of this study was to develop and validate a new chromatographic method for the determination of lispro and glargine insulin analogues in pharmaceutical preparations. Solutions were prepared using the mobile phase (methanol-water, 70:30) as solvent and filtered through a 0.2 µm membrane. Aliquots of 20µL were injected into the HPLC. The method validation parameters yielded good results and included range, linearity, precision, accuracy, specificity, and recovery. The calibration curves for lispro and glargine insulin analogues were linear from 0.1 to 3.5UI/mL, with correlation coefficients of 0.9990 and 0.9995, respectively. The interday and intraday precisions (relative standard deviation) were less than 1%. The accuracy was studied and the recovery test indicated mean absolute of 100.99% and 98.76% for lispro and glargine insulin analogues, respectively. The results obtained by HPLC method were calculated by analysis of variance (ANOVA). We concluded that the HPLC method proposed is satisfactory for the quantification of lispro and glargine insulin analogues in pharmaceutical preparations.

Keywords

HPLC, High-Performance Liquid Chromatography, ANOVA, Analysis of Variance, UV, Ultraviolet, LOD, Limit of Detection, LOQ, Limit of Quantification, CV, Coefficient of Variation, UI, International Unit, Insulin Analogues