Basic Absorption Assays

Although direct densitometry is becoming increasingly important, the spot elution method is still very widely used throughout the world. Many selective and sensitive analyses can be performed in a simple and relatively inexpensive fashion by using TLC for separation and a basic absorption or fluorescence spectrometer for quantification. For example, assay of some drugs by TLC with elution is specified in the United States Pharmacopeia (USP). 

The derivatization process (5) is accomplished in aqueous media at basic pH (pH 7-10) in a matter of approximately 15 min to yield a 2-cyanobenz[f]isoindole (CBI), which is stable for 10 to 12 hr in solution. As shown in Figure 1, the absorption characteristics of the CBI adducts are also readily accessible for assay by standard fluorescence or ultraviolet detection. In addition to the absorption between 200 and 300 nm, there are two maxima in the visible spectrum at approximately 420 and 440 nm accessible for fluorescence or ultraviolet detection. A probable mechanism (5,11) for the CBI formation is illustrated in Scheme 1. 

For quantitative analysis of protein concentration the colorimetric Bradford-assay [147] is most commonly used. Here another Coomassie dye, Brilliant Blue G-250, binds in acidic solutions to basic and aromatic side chains of proteins. Binding is detected via a shift in the absorption maximum of the dye from 465 nm to 595 nm. Mostly calibration is performed with standard proteins like bovine serum albumin (BSA). Due to the varying contents of basic and aromatic side chains in proteins, systematic errors in the quantification of proteins may occur. 

As reviewed in detail by Rosenberg and Godwin (R18), folate absorption has been measured by three basically different methods (1) measurement of rises in blood folate after an oral dose, (2) measurement of folate compounds in urine after an oral dose, and (3) administration of isotopically labeled folate by mouth followed by measurement of isotope appearing in plasma and excreted in urine and feees. Folate in plasma and urine is assayed with bacteria, usually strains of Lactobacillus casei or Streptococcus faecalis, which require folate for growth. They differ somewhat in the forms of folate they can utilize, but in general these microbiological assays measure unconjugated folate in either their reduced or unreduced forms. 

MEASUREMENT/ASSAY. The assay of vitamin A is accomplished by two basic methods biological, or chemical. The bioassay procedure is based on a biological response such as growth of rats or chicks deficient in vitamin A. It measures the total vitamin A, including provitamin A, present. But, because of the difficulties and time factor in bioassays, chemical assays are usually used. Until recently, dietary allowances of vitamin A were stated in terms of either International Units (lU) or United States Pharmacopeia (USP) units, which are equal. An International Unit (lU) of vitamin A is defined on the basis of rat studies as equal to 0.344 meg of crystalline retinylacetate (which is equivalent to 0.300 meg of retinol, or to 0.60 meg of beta-carotene). These standards were based on experiments that showed that in rats only about 50% of the beta-carotene is converted to vitamin A. In man, however, beta-carotene is not as available as in the rat, due to poorer absorption in the intestines and other factors, with the result that various factors have been used to compensate for this when vitamin A activity of foods and diets have been expressed in lU. 

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