Assays photometric methods

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Gross, K.C. 1982. A rapid and sensitive spectro-photometric method for assaying polygalacturonase using 2-cyanoacetamide. Hort. Sci. 17 9.33-934. 

El-Kousy and Bebawy [31] described two stability-indicating spectro-photometric methods for the determination of omeprazole in the presence of its photodegradation products. In the first method, omeprazole from capsules or vials were dissolved in acetonitrile/water (1 1) and UV-VIS spectrophotometry used to determine the first-, second-, and third-derivative absorption curves between 200 and 400 nm. The level of omeprazole was assayed from the values of ordinates of the three curves at 290.4,... 

Since uric acid is a risk factor for gout and other diseases, diagnosis of hyperuricemia is increasingly important. The Glukometer has been equipped with a uricase membrane and employed for uric acid assay in serum [374], Satisfactory agreement with the uricase-catalase reference method was obtained the deviation of the mean value was as low as -1-2.4 pM. The reagent costs of the method amount to only one tenth of those required for the manual photometric method. 

Although unlike RIA, enzyme immunoassays can be carried out in homogeneous systems without a separation step (based on the change in enzyme activity during the immune reaction), in practice, heterogeneous (enzyme-linked immunosorbent assay) methods are much more frequently used. The antibody, or in the case of the double-antibody method the second antibody, is immobilized, either covalently or by coating enzyme multiplied immunoassay technique (EMIT). This can be done on the walls of microtiter plates. After the immunogenic reaction, the enzyme activity, which is the equivalent of radioactivity in RIA systems, can be measured by suitable photometric methods on the microtiter plates themselves. 

The reaction is the basis of various methods for the determination of amino acids since it is possible to measure (1) the CO2 produced, (2) the NH3 produced and (3) the colour intensity obtained when the liberated ammonia reacts with a further molecule of ninhydrin to produce a purple compound which can be assayed photometrically. This is the method by which amino acids are estimated using an amino acid analyser. The imino acids (proline and hydroxyproline) give yellow products instead of a purple one. Amino acids give a strong reaction with ninhydrin, but proteins and polypeptides, which contain far fewer free amino groups, give a much weaker reaction. 

Recently, Green and his co-workers have described a new assay for CoA which is based on a different principle from the previous assays. It has been reported that CoA is required for the oxidation of a-ketoglu-tarate. Since this reaction also requires the presence of DPN, a spectro-photometric method in which the reactants are so adjusted that the reduction of DPN is dependent upon CoA concentration is possible. This method is rapid and very sensitive. 

The electrode has not been used as much as the ultraviolet spectro-photometric method for routine catalase assays, and does not cover a sufficiently wide range of peroxide concentration to be suitable for studying the activity-substrate concentration relationship. The technique is suitable for simultaneous measurements of catalase activity and the catalase complex as described below. 

Active immobilized enzyme determination of heat changes in the proximity of immobilized enzymes with an enzyme thermistor, and the use of an enzyme thermistor in assays for metabolites Active immobilized enzyme Active immobilized enzyme spectro-photometric methods for determining bound protein... 

A microbiological assay is the method of choice (see p. 813) but spectro-photometric methods are also applicable. One spectrophotometric method is based on the formation of a yellow colour with an absorption maximum at 380 m//, when tetracycline is dissolved in 0-25N sodium hydroxide, another on the orange-brown colour (maximum absorption 490 m//) formed on mixing a dilute hydrochloric acid solution of the sample with ferric chloride solution. The former method which is described below is also applicable to oxytetracycline but not to chlortetracycline while the ferric chloride reaction, which is given in detail under oxytetracycline, applies to all three. 

The bathochromic shift which occurs in the spectra of many phenolic compounds with change of pH may be used as the basis of a simple spectro-photometric assay (AE method) when they are present in complex formulations. A pre-requisite of the method is the absence of spectral changes of the other constituents under the same conditions. The method has been adapted by Elvidge and Peutrell for the determination of hexachloro-phane and other phenols (phenol, resorcinol, cresol and methyl hydroxy-benzoate) in various pharmaceutical products. 

Liu, J. Zhou, X. Zhang, H. Zhang, T. A simple spectro-photometric method for hyaluronic acid assay. Wuxi Qinggongye Xueyuan Xuebao 1995, 14, 43-48 Chem. Abstr. 1995, 123, 159809. 

The microbial assay is based on the growth of l ctobacillus casei in the natural (72) or modified form. The lactic acid formed is titrated or, preferably, the turbidity measured photometrically. In a more sensitive assay, l euconostoc mesenteroides is employed as the assay organism (73). It is 50 times more sensitive than T. casei for assaying riboflavin and its analogues (0.1 ng/mL vs 20 ng/mL for T. casei). A very useful method for measuring total riboflavin in body fluids and tissues is based on the riboflavin requirement of the proto2oan cHate Tetrahjmenapyriformis which is sensitive and specific for riboflavin. 

Table 1 Hsts several of the chemical deterrninations and the corresponding reactions uti1i2ed, which are available on automated clinical analy2ers. With the exception of assays for various electrolytes, eg, Na", K", Cl , and CO2, deterrnination is normally done by photometric means at wavelengths in the ultraviolet and visible regions. Other means of assay include fluorescence, radioisotopic assay, electrochemistry, etc. However, such detection methods are normally required only for the more difficult assays, particularly those of semm or urine constituents at concentrations below )Tg/L. These latter assays are discussed more fully in the Hterature (3,4).

For kinetic investigations and for activity measurements, either photometric assays or - because of the higher complexity of the reactants converted by biocatalysts - HPEC methods can often be used. Here the ionic liquid itself or impurities may interfere with the analytical method. 

Subcase b2 This case, called the paired f-test , is often done when two test procedures, such as methods A and B, are applied to the same samples, for instance when validating a proposed procedure with respect to the accepted one. In practicular, an official content uniformity" 5 assay might prescribe a photometric measurement (extract the active principle from a tablet... 

The Sheldon group [87] prepared aquagels of different HNLs and compared them in the synthesis reaction of different cyanohydrins with the CLEAs and the free enzymes. The activity recovery for the aquagels and CLEAs measured by a photometric assay were quite low. Using the same loadings, the aquagels turned out to be much faster than the free enzyme. This confirms the underestimation of the recovery of activity by fast assays due to diffusion problems, as reported earlier [74, 75]. The stability and the catalytic performance of the immobilized HNLs are strongly influenced by the solvent, the immobilization method, and the enzyme source. 

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