Validation of a major modification of the mobile phase in the European Pharmacopoeia LC-UV human insulin assay

To prevent the buildup of salt crystals inside the chromatograph tubing and pump that occurs with the high mobile phase sodium sulfate concentration (148 mM) used in the European Pharmacopoeia method for the quantification of human insulin, the use of a lower concentration was proposed. A minimal concentration of 37 mM sodium sulfate maintained the chromatographic performance while avoiding instrumental damage. This buffer concentration modification is considered as a major change by the European Pharmacopoeia and this improved method therefore had to be validated. Using a single-point calibration (600 µg.mL−1) yielded adequate precision (the highest relative standard deviations for repeatability and intermediate precision were <5% and <8%, respectively) and accuracy (bias values between −8% and +7%) for the quantification of human insulin in solutions of known concentration and in commercial insulin preparations. The limits of detection and quantification (1.6 and 4.5 µg.mL−1, respectively) were ...