Development of a human insulin certified reference material with SI-traceable purity.

A human insulin (hINS) certified reference material (CRM) was developed by the National Institute of Metrology (NIM). Three milligrams of purified solid hINS was packed into a brown sealed tube. The candidate material was identified by de novo sequence using mass spectrometry and Edman degradation methods. The content of insulin-related impurities, aggregation, moisture, volatile organic compounds (VOCs), anions, and ignition residues was also determined. Both mass balance (MB) and amino acid analysis-based isotope dilution mass spectrometry (AAA-IDMS) were used for the certified value assessment, which was determined to be (0.857 ± 0.024) g/g. The certified value was validated by liquid chromatography-circular dichroism spectroscopy (LC-CD) and quantitative nuclear magnetic resonance (qNMR) methods, which were in good agreement. No inhomogeneity was observed during a homogeneity examination. A stability examination showed that the CRM was stable for at least 12 months when stored at - 70 °C, and for 7 days when stored at 4, 25, or 40 °C. The CRM is expected to be used as a primary calibrator for matrix insulin CRM development and for quality control in biopharmaceutical production and clinical diagnostics.