EVALUATION OF LIQUID CHROMATOGRAPHY WITH THIN-LAYER AMPEROMETRIC DETECTION FOR THE DETERMINATION OF TYROSINE METABOLITES OF CLINICAL AND BIOCHEMICAL SIGNIFICANCE
Abstract
Electrochemical detection for liquid chromatography (LCEC) has been shown to be a very versatile tool for biochemical analysis. The principles and operational aspects of the thin-layer amperometric detector are discussed. The electrochemical response depends on the electrode material and cell geometry, the mobile phase composition, and the electrochemical characteristics of the analyte. Carbon paste, glassy carbon, or amalgamated gold are the most useful electrode materials for LCEC. The linear range can be extended by two orders of magnitude (on the high end) by using a modified cell geometry. In addition, highly resistive mobile phases can be used with the improved design. The selectivity is determined by the ease at which an electrophore can undergo an oxidation or reduction at the electrode surface. The response of the detector follows hydrodynamic theory. Current output is directly proportional to the cube root of the flow velocity, and is inversely proportional to the cube root of the channel thickness. The maximum signal to noise ratio is obtained when the area of the electrode and the thickness of the gasket are as small as possible. At present, reverse-phase liquid chromatography offers the greatest compatability with electrochemical detection. Selectivity can be altered by proper choice of mobile or stationary modifiers. Analysis of complex biological mixtures usually require pre-LC isolation procedures. Techniques commonly employed are liquid-liquid extractions, liquid-solid adsorptions, column chromatography and/or thin-layer chromatography. All of these techniques are discussed with respect to their application for the determination of tyrosine metabolites. Sample analysis time can be reduced by using mobile and stationary phase programming. Step changes to a mobile phase having greater eluting power can improve resolution as well as reduce the time per chromatogram. Column switching or stationary phase programming accomplishes the same goals, but does not disrupt selective equilibria between components of the mobile and stationary phases. The LCEC technique was used to determine important tyrosine metabolites from urine, serum, and brain tissue samples. A method is described for determining homogentisic acid (HGA) in urine and serum. The minimum detectable quantity was 0.1 (mu)g/mL. Acidic and neutral metabolites of the tyrosine-catecholamine biochemical pathway were determined in urine and brain tissue samples. 3,4-Dihydroxyphenylacetic acid (DOPAC) was determined by a procedure taking advantage of the selective adsorption of cis-diol compounds on alumina at high pH. DOPAC was desorbed from the alumina and quantitated using LCEC. A chromatogram requiring less than 15 minutes was obtained using this procedure. Homovanillic acid (HVA), vanilmandelic acid (VMA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were determined from urine samples using a combination TLC-LCEC technique. Concentrations determined from normal urine pools fell within the range given by other investigators. (HVA = 4.3 (mu)g/mL, VMA = 2.1 (mu)g/mL, and MHPG = 0.65 (mu)g/mL). The limit of detection was 0.1 ng at a signal to noise level of 2. Procedures are described for the simultaneous determination of VMA and HVA using the column-switching technique. HVA and VMA were determined in neuroblastoma patients to illustrate the utility of the technique. MHPG was determined in psychiatric patients using the procedure described. These compounds were also determined in whole rat and mouse brain tissue samples. Isolation procedures are described for the individual or simultaneous determinations.
Degree
Ph.D.
Subject Area
Analytical chemistry
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