IDENTIFICATION OF INSULIN VARIANTS USING RAMAN SPECTROSCOPY

Abstract Drop coating deposition Raman (DCDR) spectroscopy is used to obtain high-quality normal Raman spectra from small volumes (10 μl) of dilute insulin solutions (3–400 μM) for spectral identification and chromatographic detection. The results are used to demonstrate the spectroscopic classification (identification) of three natural insulin variants—human, bovine, and porcine—that differ by between one and three amino acid residues. DCDR measurements were performed on solutions obtained from reverse phase high-performance liquid chromatography (RP-HPLC) eluent fractions, either before or after lyophilization. Classification is demonstrated using replicate DCDR measurements, followed by normalized Savitsky–Golay second derivative preprocessing and partial least squares training with either leave-one-out or batch-to-batch testing.