DEVELOPMENT OF CLINICAL METHODOLOGY USING LIQUID CHROMATOGRAPHY WITH AMPEROMETRIC DETECTION
Abstract
The goal of any bioanalytical method is to selectively extract information from a complex sample matrix which contains a great deal of chemical "noise." Liquid chromatography with electrochemical detection (LCEC) is an analytical technique that provides both the selectivity and the sensitivity needed for many of these measurements. A number of potentially useful clinical assays have been developed based on this new technique for determination of small amounts of certain key metabolites. Dopamine-(beta)-hydroxylase (D(beta)H) activity was measured in human serum. Dopamine was used as the substrate and the enzymatic product, norepinephrine, was isolated by selective adsorption onto aluminum oxide at high pH. The norepinephrine was eluted from the alumina by dilute acid and measured by LCEC. A unique column switching scheme was employed to flush the excess substrate to waste and thereby increased the sample throughput. The overall precision of the method was 5.5% relative standard deviation. Catechol-O-methyltransferase (COMT) activity was determined in various tissues. Rat liver, brain, heart, and human erythrocyte COMT were studied after the development of this method. Dopamine was used as the substrate and the O-methyl metabolites were isolated on short cation exchange columns prior to the LC. The ability to determine multiple product information (meta/para product preference ratios) was a significant improvement over previous methods. The measurement of serum and plasma catecholamines was accomplished by the modification of an earlier LCEC procedure for urinary catecholamines. The combination of cation exchange isolation columns with alumina liquid/solid adsorption resulted in a significant sample clean-up. Norepinephrine was measured in human and rat serum and sheep plasma. Using the sheep as a model, a stress study was conducted. The fourfold increase in plasma norepinephrine above basal levels confirmed the utility of circulating norepinephrine determinations as a biological indicator of the sympathetic nervous system. The coenzyme, nicotinamide adenine dinucleotide (NAD('+)), is an important component in many oxidoreductase enzyme systems. The reduced form, NADH, is electroactive at carbon electrodes. This opens the way to measuring many non-electroactive compounds that are substrates of redox enzymes. Using alcohol dehydrogenase as a model system, ethanol was determined in blood and serum by measuring the NADH produced from the enzymatic oxidation. The method was linear over the range of ethanol found in blood after drinking. The precision was 2.5% relative standard deviation. The determination of serum lactate was approached in a similar manner, except that column switching was employed to eliminate weakly retained components that overload the column, necessitating longer retention times for NADH. The method was applied to the determination of lactate in diabetic serum. The overall precision of the lactate pre-column sample enrichment method was 2.6% relative standard deviation.
Degree
Ph.D.
Subject Area
Analytical chemistry
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