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Published:
Journal of Analytical Toxicology,
ISSN 0146-4760,
Volume 25,
Number 1, January/February,
pp. 8-14
A
Mass Spectrometric Method for Quantitation of Intact Insulin in Blood Samples
Shauna
M. Darby[1], Mark L. Miller[2], Ralph O. Allen[1], and Marc LeBeau[3]
[1]University of Virginia, Department of Chemistry, Charlottesville, Virginia
22903; [2]FBI Laboratory, Forensic Science Research Unit, Quantico, Virginia
22135; and [3]FBI Laboratory, Chemistry Unit, Washington, D.C. 20535
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.
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