A Validated RP‐HPLC Method for the Determination of Recombinant Human Insulin in Bulk and Pharmaceutical Dosage Form

Abstract

A modified RP-HPLC method was developed for the quantitative determination of recombinant human insulin in bulk and pharmaceutical dosage form with reduced retention time. Study of the effects of the column temperature, pH of the mobile phase and presence of vial additives (phenol andm-cresol), or impurities (A-21 Disamido) on the accuracy of the assay were assessed. Separation was achieved using a Hypersil BDS C-18 column and the mobile phase was composed of solution A (aqueous solution of 28.3 anhydrous Na2SO4g/L, pH 2.3) and solution B (28.5 g anhydrous Na2SO4g/L in 50:50 mixture of water and acetonitrile, pH 2.3) in a ratio 48:52 (v/v) at 45–50°C. The column temperature was 40°C, the flow rate was 1 mL/min and detection was performed at 216 nm. The procedures were validated according to international conference on harmonization (ICH) guidelines. Recovery study was done applying standard addition technique for further validation of the procedure. The retention time of recombinant human insulin was 19.7 min as compared to 29 min obtained by the reference method. Analytical conditions fluctuations or presence of vial additives or impurities did not show any significant effect on the accuracy of the method. The prepared standard insulin solution in 0.01 N HCl was found to be stable for 5 days. Statistical comparison showed no significant difference between the described method and reference method regarding the accuracy and precision. The modified method can be applied for routine quality control applications for determination of recombinant human insulin.

Reliability of Analytical Methods for Recombinant Human Insulin Quantification in the Bulk Crystals and in-Process Control

“…The internal standards SFP, RFP, and rHI-C and the USP reference standard (sHI) were characterized by chromatographic analyses (Moussa et al, 2010).…”

Development, Optimisation, Validation and Inter-Laboratory Verification of a Reversed Phase HPLC Method for Quantification of Human Recombinant Insulin

“…The HPLC method was validated according to ICH guidelines in terms of specificity, accuracy, precision, linearity, limits of detection/ quantification and stability of insulin solutions [5,10].…”

“…These assays, though specific, were limited by their ineffectiveness at detecting very low doses, and could not identify contaminant or decomposition products; hence, the increased use of HPLC [8]. However, the chemical nature and size of proteins makes their HPLC difficult because various factors, such as temperature and pH, need to be optimised [9,10]. The official monographs (British Pharmacopoeia, United States Pharmacopoeia, European Pharmacopoeia) contain methods for the detection and assay of insulin by HPLC, but these methods require extended runtimes, or the use of elevated temperature [9,11].…”

“…The official monographs (British Pharmacopoeia, United States Pharmacopoeia, European Pharmacopoeia) contain methods for the detection and assay of insulin by HPLC, but these methods require extended runtimes, or the use of elevated temperature [9,11]. Various researchers have also employed HPLC for quantification of insulin in literature using buffered [9,10,[12][13][14] and unbuffered mobile phases [5,15]. However, these methods require either the use of elevated temperatures, specialised detectors or resulted in long run times.…”

Development and validation of simple and rapid high performance liquid chromatographic method for routine analysis of human insulin in formulations

“…These methods employed complex (Chen et al, 2002;Vuppugalla et al, 2003;Sarmento et al, 2006) long run time (Asahara et al, 1991;Chen et al, 2002;Vuppugalla et al, 2003;Moussa et al, 2010) high flow rate (Vuppugalla et al, 2003) that could be impractical for routine analysis of multiple samples. Use of a special column or column temperature controllers, which are not popular in most of the analytical laboratories, is another limitation (Farid et al, 1989;Yomota et al, 1996).…”