Analytical Biochemistry
Analytical Biochemistry
3 years ago

Isotope-dilution liquid chromatography-tandem mass spectrometry for sensitive quantification of human insulin in serum using derivatization-technique

An isotope-dilution mass spectrometry (IDMS) method for measuring insulin levels in human serum was developed using C-terminal-derivatization method coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The carboxyl groups of Glu-C-cleavage products were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity and IDMS quantification, resulting in increases in LC-MS/MS peak areas of derivatized Glu-C-cleavage products of human insulin by ∼23-(A5–17 peptide) to 49-fold(B14–21 peptide), respectively, as compared with results observed in the absence of derivatization. Separation was achieved on a C18 column by gradient elution at 0.3 mL/min, with a mobile phase composed of 0.1% formic acid in acetonitrile and water. Validation studies of target peptides (B1–13 peptide and B14–21 peptide) revealed a linear response in the range of 0.05 ng/mL to 10 ng/mL (regression coefficient, r 2 = 0.9987 and 0.9988, respectively), a relative standard deviation within and between days of <8.6%, and spike and recovery test results indicating mean recoveries ranging from 100.2% to 106.6%. Comparison with an established commercial immunoassay showed high correlation (r 2 = 0.9943 and 0.9944, B1–13 peptide and B14–21 peptide, respectively) at serum concentrations of between 0.20 ng/mL and 1.51 ng/mL. These findings suggested that this IDMS-based approach was able to quantify human serum insulin with high sensitivity and precision in the reference interval and indicated a potential for determining serum-insulin reference-measurement procedures to allow traceable measurement.

Publisher URL: www.sciencedirect.com/science

DOI: S0003269717303615

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