Electrophoretically mediated microanalysis with small molecules: the Jaffé method for creatinine carried out in a capillary tube.

Abstract

An eletrophoretically mediated microanalysis (EMMA) approach, used to perform online chemistry between two small molecules, has been characterized and optimized. The "plug-plug" type EMMA method involved electrophoretic mixing and subsequent reaction of nanoliter plugs of creatinine-containing samples and alkaline picrate (Jaffe reaction) within the confines of the capillary column, which acts as a microreactor. Analyses were performed by pressure injecting a plug of picrate followed by a plug of the creatinine-containing sample. A potential was then applied to electrophoretically mix the two reactants, and an incubation time of up to 6 min allowed the reaction to proceed prior to the application of a 27 kV separation potential with absorbance detection at 485 nm. The use of a 50 microm inner diameter(ID) extended light path capillary (150 microm pathlength) was found to be adequate for determining elevated levels of creatinine in human blood sera, but could not be used to quantify normal levels. Quantification of both normal and elevated levels of creatinine in sera was possible with a 75 microm ID high-sensitivity cell (1200 microm pathlength). Calibration plots using the latter for creatinine in human blood sera spanned the expected clinical range and were linear between 40 microM and 1.2 mM (r2 = 0.996) with an estimated limit of detection of 17 microM (signal-to-noise ratio S/N = 3). A quantitative comparison of results obtained with the reported EMMA method and accepted clinical methodology correlated very well (slope = 1.001).