Assay chromophoric procedure

Halcinonide has been quantitated in various formulations or as bulk powder by a differential ultraviolet, borohydride reduction assay. 2 This differential assay involves measuring the ultraviolet absorbance of an aliquot of methanolic steroid solution containing sodium borohydride decomposed prior to the addition of steroid. Its absorbance is determined against a methanolic reference solution of steroid reduced by sodium borohydride to destroy the 3-one-4-ene chromophore. The utility of this procedure is that many interferences from excipients and other, unconjugated, steroids can be eliminated in the assay of a formulation. 

Colorimetric assays are commonly used in molecular biology and biotechnology laboratories for determining protein concentrations because the procedures and their instrumentation requirements are simple. Two forms of assays are used. The first involves reactions between the protein and a suitable chemical to yield a colored, fluorescent, or chemiluminescence product. Second, a colored dye is bound to the protein and the absorbance shift is observed. Disadvantages of both these methods include limited sensitivity at below 1 pg/mL, interferences from buffers, and unstable chromophores (Jain et al. 1992). 

Apart from the application of dyed proteins as substrates of proteolytic activity assay so far mentioned (see also 7.2), proteins are labeled with chromophores for studying membrane surfaces, for detecting proteins after several different [ ation procedures (i.e. chromatographic and electrophoretic techniques), for the determination of proteins in the mixture with many other substances, etc. 

DHA can be separated from AA and most ionic compounds by ion exchange columns. Such columns do not separate DHA from other AA metabolites and neutral carbohydrates. DHA can be separated from these compounds by reverse phase HPLC using water or water-acetonitrile eluants. Good separations of DHA from biological samples can be expected to be achieved by HPLC. Detection is a problem since UV absorption is inadequate. The red chromophore with amino acids is not very sensitive. Perhaps DHA could be reduced to AA after separation and detected by the strong 263-nm absorption of AA or by an electrochemical detector. DHA levels and DHA/AA ratios are probably quite important in biology and medicine, and good procedures for these assays are of considerable interest. 

The reactivity of 5-acetamido-3,5-dideoxy-L-flra6/7io-2-heptulopyranosonic acid (a seven-carbon analogue of iV-acetylneuraminic acid) in the resorcinol and thiobarbituric acid procedures - which are commonly used for the determination of sialic acids - has been evaluated. The chemistry involved in the formation of the characteristic chromophores in these assays was also discussed. 

A modification of the periodic acid/thiobarbituric acid method has been described by excitation of the usual chromophore at 550 nm and measurement of the emission at 570 nm (Hammond and Papermaster 1976). The assay is 500-fold more sensitive than the conventional spectrophotometric procedures and can detect lOng sialic acid. Deoxyribose remains a problem with this assay and the precautions detailed for the periodic acid/thiobarbituric acid assay are required if accurate quantitation is to be made (Hammond and Papermaster 1976). 

Quantitative estimation of the O-acyl content of sialic acid preparations can be made using the hydroxamic acid reaction as described by Ludowieg and Dorfman (1960), modified from the method of Hestrin (1949). Calibration with ethyl acetate or other ester standards provides a simple quantitative assay, measuring the chromophore at 520 nm. Volume reductions in the assay procedure allow quantitation of 0.05(i,mole of sialic acid acyl ester (Schauer 1978). Qualitative identification of different acylhydroxamates can be made on thin-layer chromatography (see section III.3.b)). 

The other widely employed colorimetric assay is the thiobarbituric acid procedure which is useful for free but not bound sialic acid. Proposed independently by Warren (1959) and Aminoff (1959), the method is based on periodate oxidation of sialic acid to )8-formylpyruvic acid (Figure 11) which then reacts with two moles of 2-thiobarbituric acid to form a chromophore (XLIX) with absorption maximum at 549 nm. The identity of this chromophore was established through synthesis... 

A scaled-down version of the Warren method was developed that can be used to assay as little as 25 ng of NANA (Hahn et aL, 1974). A number of automated procedures based on periodate-thiobarbituric acid have been described (Kendal, 1968 Delmotte, 1968 Fidgen, 1973 Gerbant et aL, 1973). Additional spectrophotometric techniques have employed the sulfo-phospho-vanillin reaction (Saifer and Feldman, 1971) and 1,10-phenanthroline (Dimitrov, 1973). The direct Ehrlich reaction, utilizing dimethylaminobenzaldehyde, played a key role in the early studies of sialic acid chemistry and analysis (Gottschalk, 1960) and still finds occasional use although its sensitivity is considerably below that of resorcinol and thiobarbituric acid (Werner and Odin, 1952 Onodera et aL, 1965). In comparing this method with others, Onodera et al. (1965) concluded that the possibility of false chromophores requires at least two colorimetric methods for reliable estimate of sialic acid in biological materials. 

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