A Dual Functional-Group Derivatization Liquid Chromatography–Tandem Mass Spectrometry Method: Application for Quantification of Human Insulin

Recently, we developed a sensitive and accurate quantification method for short-chain peptides using dual functional-group derivatization. Sensitive and accurate quantification was achieved in our study by separating the sensitivity-enhancement and isotope-labeling derivatization reactions. In this study, we applied the abovementioned method to quantify human insulin in serum using enzymatic digestion. The amino groups in human insulin were reductively alkylated with acetaldehyde or acetaldehyde- d 4 to afford N -alkylated products with different masses. This process is simple, quick, produces high yields, and helps in the accurate comparative analysis of mass-differentiated human insulin. After mixing the N -alkylated products with different masses, the obtained mixtures were digested using Glu-C. The carboxyl groups were then derivatized with 1-(2-pyrimidinyl)piperazine to increase sensitivity and analyzed using liquid chromatography–tandem mass spectrometry. Excellent accuracy (97.2–107.5%) and precision (RSD, 2.2–10.6%) were achieved using this method. Furthermore, the quantification values obtained agreed with the quantification values obtained using isotope dilution mass spectrometry in a previous study.