Develop a Bioanalytical Method for Insulin in Different Dosage Forms
The primary technological objective of this project was to develop a more sensitive and reproducible analytical method for detection and quantification of insulin and its metabolites in animal blood serum. The secondary technological objective was to develop a procedure to monitor drug dose uniformity as well as the in-vitro release rate.
Insulin is the most important regulatory hormone in the control of glucose homeostasis, consisting of 51 amino acids shared between two intramolecular chains and with a molecular weight of 5800. Insulin, like other proteins, is not a stable entity but is liable to modification by chemical reactions with molecules in its vicinity. Thus during storage and use of pharmaceutical preparations, insulin is degraded by hydrolytic reactions or is transformed by formation of intermolecular covalent bonds with other insulin molecules, leading to higher molecular weight transformation products.
The currently accepted method of determination of the concentration of insulin in a given preparation is based on biological assays. Currently, High Performance Liquid Chromatography (HPLC) methodologies have been applied to determine the concentration of insulin in various preparations. Although HPLC technology has been utilized in these studies, there have been several problems associated with the published assays. Retention time of 16 minutes and 20 minutes for the compound of interest may be considered excessive when it is necessary to analyze multiple samples. No single analytical method can detect all possible chemical, physical and immunological changes in the protein structure, thus several analytical techniques such as electrophoresis, spectroscopy, chromatography, thermal analysis, immunoassays and bioassays may be required to completely characterise a protein and examine its degradation profiles. Most of these HPLC determinations of insulin controlled the degree of ionization of insulin by using a mobile phase with low pH and high salinity.
An alternative approach to increase sensitivity and reproducibility with better resolution is to use an ion-pairing reagent. However, some of these methods suffer from inadequately resolving insulin contained in different dosage forms including new alternatives such as liquid suspension or solid powder form, due to inadequate selectivity or efficiency. Therefore, any improvement in the sensitivity and enhanced reproducibility would be considered a major advancement.
Diteba was able to achieve this breakthrough advancement. The method developed by Diteba is based on the separation of insulin from other excipients for quantifying the insulin in finished product. Several parameters such as composition of the mobile phase, flow rate and detection wavelength were evaluated for their effect on location and shape of the peak of insulin while developing the method. Human inhaled insulin, like other multiple dose injection formulations, contains preservatives. Many of these preservatives exhibit high ultra-violet absorption, making it necessary to have analytical techniques capable of separating the protein from the preservative. Ideally, it would be desirable to have quantitative separation methods capable of resolving the preservatives and the degradation product with no interfering interaction with the analyte peak.
All this considered, the final optimized method utilized, a mobile phase consisting of 60 volume of 1 mmol sodium sulphate and 0.2% triethylamine in water, pH 3.2 adjusted by phosphoric acid, and 40 vol of Acetonitrile. The solution was filtered through a 0.2 µm membrane. The eluent was monitored with a UV detector at 214 nm with flow rate of 1 ml/min. The retention time for human insulin was 4.67 minutes. It should be noted that the retention time varied considerably unless the relative content of acetonitrile in the mobile phase was carefully kept constant, but a slight change in the pH did not affect the retention time of insulin.
The peak area for human insulin varied linearly with the amount 10-100 µg/ml. Since higher concentrations cause overlapping of insulin and preservative-related peaks, it is recommended to dilute the administered test samples to the desired concentration. The mean absolute recovery values of the method were 97± 0.31.
The results for variation of the method were determined over a concentration range of 10-100 µg/ml. This data indicated a considerable degree of precision and reproducibility for the method, both between and within analytical runs. The limits of detection and quantitation of the method were 0.25 and 0.75 µg/ml respectively. The % CV values of the peak heights of six successive injections in each case were 7.58 and 5.69 respectively. The stability of different insulin concentrations at room temperature was also determined. Approximately a 10% decrease in concentration of samples after 24 hours of storage at room temperature was observed, but it was negligible after 12 hours. Therefore, it was recommended to use freshly prepared samples. As the results indicated, the method is remarkably accurate and this ensures the attainment of reliable results.
The advantages of this method are the analysis of human insulin from formulation, the wide range of linearity with considerable accuracy and precision of analysis, greater resolution and reproducibility and non-dependency on requirements such as a column temperature controller.