Abstract
This article describes the development of a new paradigm in lipid testing wherein seven major human plasma lipids are determined simultaneously without the need for analytical separations. Included are cholesterol and the esters of linoleic, conjugated linoleic (CLA), arachidonic, linolenic, eicosapentaenoic, (EPA), and docosohexaenoic acids, (DHA). The simple quantitative colorimetric assay is rapid, rugged, inexpensive and specific to the โCH=CHโCH~2~โ functional group in both cyclic and acyclic structures. The critical moiety in the functional group is the ฮฑโmethylene (allylic) group that triggers the reaction. Without it, a double or triple bond is not reactive. The visible spectrum for a typical plasma sample turned out to be the linear sum of the weighted contributions from all seven analytes that โ given the heterogeneity of blood samples โ leads to a broad diversity in the spectral patterns. To resolve the spectral data, a number of chemometric techniques were investigated. Initially, calculations were made on spectral data that originated from prepared synthetic mixtures whose molar concentrations were known. The most encouraging algorithms employed are: coupling ridge regression (RR) with Kโmatrix, partial least squares regression (PLS), and generalized standard addition method (GSAM). An attempt to apply GSAM to serum samples is validated using GCโMS. The same serum samples are analyzed using GCโMS and spectrophotometry and the ratios of omegaโ6 to omegaโ3 obtained from the analyses are compared. Results using both methods correspond very well. (The reaction between acetyl chloride and olefinic systems is described as a FriedelโCrafts reaction but the mechanism of this process, the structure of the products, and the nature of the resulting chromophore remains to be fully identified.)