A mass spectrometric method for quantitation of intact insulin in blood samples.
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 degrees 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.
Darby
S M
SM
University of Virginia, Department of Chemistry, Charlottesville, Virginia 22903, USA.
Miller
M L
ML
Allen
R O
RO
LeBeau
M
M
D000818:Animals; D002096:C-Peptide / Q000032:analysis; D004355:Drug Stability; D006461:Hemolysis; D006801:Humans; D007328:Insulin / Q000097:blood* / Q000737:chemistry; D013058:Mass Spectrometry; D011863:Radioimmunoassay; D013552:Swine
D002096:C-Peptide; D007328:Insulin