Biological analysis physiological methods

Coulometry is an electrolytic method of analysis. In general, electrolytic methods have limited applications in analytical biochemistry but they are useful in the analysis of substances which, while not strictly biochemical, are often important in biological and physiological chemistry. 

Trichloroacetic acid (TCA) and perchloric acid (PCA) has traditionally been used for the precipitation of proteinaceous materials from biological fluids. These methods of sample preparation have been used extensively for the analysis of nucleotides and nucleosides in physiological fluids (B1, C3, C7, D6, E5, HIO, K9, K12, K27, K28, M3, M4, N3, R3-R5, S9, Tl, V3, W5), and also for the analysis of tissue nucleotides (L9, R3). 

A comparative study was made of the RP-HPLC analysis of free amino acids in physiological concentrations in biological fluids, with pre-column derivatization by one of the four major reagents o-phthalaldehyde (73) in the presence of 2-mercaptoethanol, 9-fluorenylmethyl chloroformate (90), dansyl chloride (92) and phenyl isothiocyanate (97, R = Ph) (these reagents are discussed separately below). Duration of the analysis was 13-40 min. Sensitivity with the latter reagent was inferior to the other three however, its use is convenient in clinical analysis, where sample availability is rarely a problem. The derivatives of 73 were unstable and required automatized derivatization lines. Only 92 allowed reliable quantation of cystine. All four HPLC methods compared favorably with the conventional ion-exchange amino acid analysis188. 

Potential interfering substances in a biological matrix include endogenous matrix components, metabolites, decomposition products, and in the actual study, concomitant medication. Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes. For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted. Method selectivity should be evaluated during method development and method validation and can continue during the analysis of actual study samples. 

The ease of oxidation of reduced ascorbic acid is the basis for a simple method of analysis by dye titration (58j. Ascorbic acid as it occurs in citrus juice is in the reduced form. When subjected to oxidation, ascorbic acid changes to the dehydro form. Dehydroascorbic acid has nearly the same physiological activity as the reduced form and is easily converted to the latter. Further oxidation of the dehydroascorbic acid converts it to 2,3-diketo-gulonic acid. This reaction is irreversible, and the oxidized product is devoid of biological activity. These reactions are shown in Figure 2. Nearly 90 percent or more of the vitamin C found in citrus juice and citrus products is in the reduced form (Table X) (59). 

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