The aim of this project was to develop and validate an analytical testing method for qualitative and quantitative analysis of Human Insulin, USP, by tandem High Performance Liquid Chromatography and Mass Spectroscopy Detection (LC/MS).

The experimental work was carried out by using a High Performance Liquid Chromatography unit coupled with a Mass Spectroscopic Detection system controlled by Chemstation, which was qualified to be in compliance of CFR part 11. Method conditions were established based on the physical characteristics of the Humana Insulin.

Once the chromatographic conditions were established, a single point standard calibration was developed. All of the responses of the Insulin in each replicate standard injection were used in the calculation of the mean standard response. Upon completion of the analytical testing, the overall relative standard deviation was included in the suitability requirements for reproducibility. A check standard at the nominal concentration was used as a part of the system suitability by comparing the concentration to area counts to that of the standard. Standard samples were injected in replicates at the beginning of the run and also at the end of the run to demonstrate the integrity of the system during function.

Validation parameters of Human Insulin, USP, with a molecular mass of 5807.58 m/z showed the calibration curve was linear in the range of 0.75 to 2.25 mg/mL and accurate using the following conditions. The chromatographic analysis for Human Insulin was performed on a reversed-phase C18 column. The flow rate was 0.3 mL/minute and the LC/MS positive detection scan range was 200 to 3000 m/z. Mobile phase A for consisted of 950 ml distilled water/50 ml acetonitrile/5 ml acetic acid (pH=3.04) and 950 ml distilled water/50 ml acetonitrile/5 ml acetic acid (pH=4.46). The retention time of Insulin was typically between 6.0 to 7.0 minutes.

This study shows that High Performance Liquid Chromatography unit coupled with a Mass Spectroscopic Detection system can be a useful way for analysis of Insulin, USP.