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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.

Liquid chromatography/mass spectrometry (LC/MS) and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS methods were developed for the presumptive identification of ricin toxin and the alkaloid marker ricinine from crude plant materials. Ricin is an extremely potent poison, which is of forensic interest due to its appearance in terrorism literature and its potential for use as a homicide agent. Difficulties arise in attempting to analyze ricin because it is a large heterogeneous protein with glycosylation. The general protein identification scheme developed uses LC/MS or MALDI-TOF for size classification followed by the use of the same instrumentation for the analysis of the tryptic digest. Fragments of the digest can be searched in an online database for tentative identification of the unknown protein and then followed by comparison to authentic reference materials. LC fractionation or molecular weight cutoff filtration was used for preparation of the intact toxin before analysis. Extracts from two types of castor beans were prepared using a terrorist handbook procedure and determined to contain 1% ricin. Additionally, a forensic sample suspected to contain ricin was analyzed using the presented identification scheme (data not shown). The identification of the alkaloid ricinine by GC/MS and LC/MS was shown to be a complementary technique for the determination of castor bean extracts.

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A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.