Predictive kinetic method for quantitation of acetoacetate in body fluids and kinetic study of the reaction of acetoacetate with glycine and sodium nitroprusside
Abstract
This thesis describes a detailed study of the kinetic behavior of reactions used in the quantitation of acetoacetate in body fluids, and the development and evaluation of two mathematical models that offer quantitative prediction and a mechanistic interpretation of the system. Reactions studied are those between acetoacetate and glycine to form a transiently stable imine intermediate that reacts further to form enamine, reaction of the imine intermediate with sodium nitroprusside, and simultaneous reaction of acetoacetate, glycine, and nitroprusside. Variables studied include pH, temperature and concentrations of all reactants. The primary reaction between acetoacetate and glycine follows pseudo-first-order kinetics at high pH ($>$8.6) at which the enamine is transiently stable; the other reactions deviate from first-order behavior at longer times because the reaction product is unstable. A proposed mechanism for the total reaction sequence involves the reaction of the imine intermediate with nitroprusside to generate an unstable product. Two mathematical models were developed, based on the proposed mechanism, that adequately described the behavior of the system with time for a wide range of concentrations of all reagents involved. The mechanism is consistent with the data and it may be correct. Several options involving nonlinear curve-fitting methods applied to all reaction sequences above are evaluated in aqueous samples. Also, the two-step and combined reaction sequences are evaluated for quantitation of acetoacetate in urine samples. For acetoacetate in the concentration range 0.47-4.25, mmol L$\sp{-1}$, the methods yield relative standard deviations in the range 1.0-1.8% with absolute standard deviations of 3.5-35 $\mu$mol L$\sp{-1}$. Results of a previous study with an initial-rate method applied to the combined reactions in urine and serum samples are also reported.
Degree
Ph.D.
Advisors
Pardue, Purdue University.
Subject Area
Analytical chemistry
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