A Mass Spectrometric Method for Quantitation of Intact Insulin in Blood Samples

Abstract

An analytical method was developed for the quantitation of intact insulin in blood samples. Solid-phase extraction (SPE) was used to purify and concentrate the protein after the plasma is separated. Analysis is performed by electrospray liquid chromatography-mass spectrometry (LC-MS) using a trifluoroacetic acid mobile phase. The limit of quantitation of the SPE LC-MS method has been determined to be 1.0 ng/mL for endogenous levels of insulin. Base levels of human insulin in plasma have been quantitated, and values ranging from 1.0 to 1.4 ng/mL were observed. In a single analysis, the method can determine human, porcine, and bovine insulin. Reproducibility was tested for both blood samples and aqueous standards and produced relative standard deviations of approximately 10% and lower. Calibration curves were constructed corresponding to plasma levels of 0.4 to 80 ng/mL and found to be linear with R² values greater than 0.99. Stability studies of human and porcine insulin were performed over a period of 21 days for whole human blood samples stored at both room temperature and 4°C. Hemolyzed blood samples were also analyzed using the developed method and were found to produce quantitatable levels of insulin. The advantage of the application of SPE and LC-MS for the quantitation of insulin is the high specificity compared to other techniques such as radioimmunoassay (RIA). In addition, the developed LC-MS method is not subject to interferences that cause problems with RIA, such as hemolysis. The method is efficient and rapid and produces results more specific than those obtained with RIA.