Assay spectrophotometric methods

In addition to the agar plate disk-diffusion assay, spectrophotometric methods have frequently been used to measure microbial growth inhibition. Spectrophotometric methods generally require that the test microorganism be grown in liquid... 

Identification of dyes on dyed textiles is traditionally carried out by destructive techniques [493], TLC is an outstanding technique for identification of extracted dyestuffs and examination of inks. Figure 4.9 shows HPTLC/SERRS analysis of acridine orange [492], Wright et al. [494] have described a simple and rapid TLC-videodensitometric method for in situ quantification of lower halogenated subsidiary colours (LHSC) in multiple dye samples. The results obtained by this method were compared with those obtained by an indirect TLC-spectrophotometric method and those from HPLC. The total time for the TLC-videodensitometric assay of five standards and four samples applied to each plate was less than 45 min. The method is applicable for use in routine batch-certification analysis. Loger et al. [495,496] have chromatographed 19 basic dyes for PAN fibres on alumina on thin-layer with ethanol-water (5 2) and another 11 dyes on silica gel G with pyridine-water... 

A multiwavelength approach might have been considered as an alternative to chemical derivatisation. Ruddle and Wilson [62] reported UV characterisation of PE extracts of three antioxidants (Topanol OC, Ionox 330 and Binox M), all with identical UV spectra and 7max = 277 nm, after reaction with nickel peroxide in alkaline ethanolic solutions, to induce marked differentiation in different solvents and allow positive identification. Nonionic surfactants of the type R0(CH2CH20) H were determined by UV spectrophotometry after derivatisation with tetrabromophenolphthalein ethyl ester potassium salt [34]. Magill and Becker [63] have described a rapid and sensitive spectrophotometric method to quantitate the peroxides present in the surfactants sorbitan monooleate and monostearate. The method, which relies on the peroxide conversion of iodide to iodine, works also for Polysorbate 60 and other surfactants and is more accurate than a titrimetric assay. 

Selective differential UV spectrophotometric method was presented for the determination of niclosamide in bulk and in its pharmaceuticals [43]. The method was based on measuring niclosamide in alkaline solutions against their neutral ethanolic solutions as blanks. The proposed method was sensitive, highly specific, and advantageous over the conventional UV assays, since the interference of the excipients, impurities, degradation products, or other accompanying drugs was nullified. 

Fernandez-Gonzales et al. [16] described a method for determination of OTC in medicated premixes and feeds by second-derivative synchronous spectrofluorome-try. The assay based on the reaction of oxytetracycline with divalent metal ion (Ca2+) at pH 6-10 to form a yellow complex that can be analyzed by synchronous spectrofluorometry (AX = 115 nm). Rao et al. [17] described a spectrophotometric method for the determination of OTC in pharmaceutical formulations based on the color reaction with uranium, which was detected at 413 nm. 

Buyuktimkin and Buyuktimkin [20] described a spectrophotometric method of assay of penicillamine and its tablets. An aqueous solution of 100 mg/mL of penicillamine was added to ethanolic 5 mM Sanger reagent (l-fluoro-2,4-dinitrobenzene), solid NaHC03, and water. The solution was diluted and heated at 70 °C for 45 min. After cooling, the solution was diluted with 3% ethanolic HC1. The absorbance of the resulting yellow complex was measured at 355 nm (molar absorptivity = 19,721). Recovery of the drug from commercial tablets was 99.1 0.7%. 

Fakhr Eldin et al. [22] described a simple sequential spectrophotometric method for the assay of penicillamine. The method is based on the complex formed when the drug is reacted with Fe(III) solution in hydrochloric acid media. The deep blue colored drug Fe(III) complex is monitored at a maximum wavelength of 600 nm. 

A number of spectrophotometric methods for the quantification of phenolic compounds in plant materials have been developed. Based on different principles, these assays are used to determine various structural groups present in phenolic compounds. Spectrophotometric methods may quantify all extractable phenolics as a group (Marshall and others 2008), or they may determine a specific phenolic substance such as sinapine (Ismail and Eskin 1979) or a given class of phenolics such as phenolic acids (Brune and others 1989). 

Niewola et al. [183, 185] have described a rapid, convenient and accurate method, based upon an enzyme-based immunosorbent assay (ELISA) for the determination of Paraquat residues in soil. Polystyrene plates, coated with paraquat-keyhole limpet haemocyanin (KLH) conjugate, are incubated with the test samples and a known amount of monoclonal antibody. Residual antibody that has not reacted with free Paraquat in the sample combines with paraquat-KLH on the plate. The determination of the fixed antibody is achieved by the addition of peroxidase labelled rabbit antimouse immunoglobulin G followed by reaction with a chromogenic substrate. The enzyme activity of the solid phase is determined from the absorbance measurements, which are inversely proportional to the concentration of Paraquat. The method shows high specificity and correlates well with the traditional ion exchange-spectrophotometric method for the determination of Paraquat [178]. 

A few typical examples for the assay of pharmaceutical substances by UV-spectrophotometric method are described below ... 

Table 8.3 Examples of kinetic spectrophotometric methods of enzyme assay... 

The suggested derivative spectrophotometric method and PLS-1 were applied to determirration of caffeine in energy drirrks (Table 31.2). The assay results obtained by both methods were statistically compared at the 5% level. As shown in Table 31.2 there was no sigrrifrcant differences between the mean values and precisiorrs of two methods. 

Dimethylaniline is both a manufacturing impurity in bupivacaine and since it is formulated in injections a possible breakdown product, although hydrolysis of amides is much slower than hydrolysis of esters. The BP uses a spectrophotometric method to assay for this impurity but GC provides a more sensitive and specific method for this determination. 

The United States Pharmacopoeia 23 [11] and Indonesian Pharmacopoeia IV [9] describe the assay of benzoic acid and salicylic acid in ointments. Two chromatographic columns (20 x 2.5 cm) are used to effect the separation. One transfers a mixture of 1 g siliceous earth and 0.5 mL diluted phosphoric acid (3 in 10) to the first column (A), then packs above this a mixture of 4 g siliceous earth and ferric chloride-urea reagent. A mixture of 4 g siliceous earth and 2mL of sodium bicarbonate solution (1 in 12) is packed into the second column (B). For analysis, column A is mounted directly above column B. The sample solution is inserted onto column A, allowed to pass into the column, and then washed with 2-40 mL of chloroform. Benzoic acid can be eluted from column B by using a 3 in 100 solution of glacial acetic acid in chloroform. The benzoic acid content then can be determined by a spectrophotometric method such as that described earlier (section 4.5). 

Sethi et al. reported the assay by two methods of diloxanide furoate and tinidazole in combined dosage forms, [16]. One of these was a dualwavelength spectrophotometric method, and the other a difference spectrophotometric method. In the first method, the absorbance of sample solution was measured at 259 and 311 nm. The concentration of tinidazole was calculated from absorbance at 311 nm, and the concentration of diloxanide furoate was calculated with the use of a given equation. In the second method, the absorbance of an aqueous solution of... 

Reznick A, Packer L (1994) Oxidative damage to proteins spectrophotometric method for carbonyl assay. Methods Enzymol 233 357-363... 

Blood samples were taken at 0.25, 0.5, 1.5, 2.5, 4.5, 6.5, 8.5, 10.5 and 12.5 h and the concentration of theophylline in serum was assayed by a spectrophotometric method [7]. The samples were analysed in duplicate. Bioavailability was calculated from the area under the concentration curve following the trapezoidal rule. 

The first problem is deciding on which of these two common models to use. It has been argued that for spectrophotometric methods where the Beer-Lambert Law is known to hold, Y = bX + e, the force through zero model is the correct model to choose if the absorbance values are corrected for the blank." The correct way to carry out the calibration regression is to include the blank response at assumed zero concentration and use the model Y = bX + a + instead. This may be a nicety from a practical standpoint for many assays but there are instances where a force through zero model could produce erroneous results. Note that the e denotes the random error term. Table 15 contains a set of absorbance concentration data from a UV assay. 

The greatest advantage of the spectrophotometric method is that it is direct and rapid, requires no sample workup, and allows for continuous assays of lipase activity compared to the multiple fixed-time-point analyses incumbent within Basic Protocols 1 and 2. The spectrophotometric method can also be done using very small volumes (as small as 1 ml) and is suitable for following the course of purification (such as in chromatographic fractions) or adaptable to 96-well plates (and subject to automation, if available). Thus, it is the method of choice for screening several samples or preparations for lipase (esterase) activity. 

In addition to assay features already mentioned, other factors may influence the choice of assay by the user. In terms of sensitivity of the assay, the threshold of detection of lipase activity, using the procedures as described in this unit, is on the order of 10 2 U for titrimetry, 10H U for colorimetry, and 10 4 U for spectrophotometry (where U is the amount of enzyme required to yield 1 imol product per minute). The smallest amounts (volumes) of materials, including enzyme, are required for the spectrophotometric method, and progressively more material is required for the colorimetric and titrimetric methods. Unless a flow cell adapter is available, the spectrophotometric method is not suitable for analysis of particulate (immobilized) enzyme preparations, whereas the other assay procedures are. 

Aside from the time required to prepare reagents, the least amount of time is required per lipase assay by the spectrophotometric method, and the greatest amount of time is required per assay for the titrimetric method. Although all assays are described as requiring up to 30 min for the reaction mixture to be subsampled, time savings can be realized by subsampling more frequently over a shorter period of time, as long as one obtains valid initial rate data. Thus, for all assays, the time involved to run the lipase reaction can be normalized to be the same at -10 to 15 min. The difference in time requirements for the protocols becomes embedded in sample workup procedures. 

For the spectrophotometric method, there is no sample workup, allowing one to run -4 assays/hr. This can be increased to -16 to 100 or more samples/hr depending on equipment features and automation, such as multiple cuvette holders/changers and 96-well microplate readers. For the colorimetric procedure, sample workup requires -10 min/subsample, but several samples can be batch processed simulta-... 

Describes a simple and sensitive spectrophotometric method to estimate the content of total carbonyl compounds in rancid fats and foods by trapping them with 2,4-DNPH the technique determines total carbonyls, including those that are nonvolatile, decreasing the ability of the assay to correlate well with sensory data. Although gas chromatographic techniques are better suited for determining volatile carbonyl compounds from lipid oxidation, this is still the classical colorimetric assay. 

Extraction using diethyl ether has also been used in another traditional method to determine the level of preservatives in a sample. In this case, benzoic acid can be extracted from a product at low pH using diethyl ether. By adjusting the pH of the product, and hence the ionisation of the acids themselves, it is possible to quantify benzoic acid in a soft drink in the presence of saccharin. After extraction the benzoic acid can be assayed spectrophotometrically or by titration (Egan et al., 1990c). 

Methanol can be determined colorimetrically but must usually be distilled from the reaction mixture before being analyzed. Wood and Siddiqui (27) described a simple and precise spectrophotometric method for measuring methanol in an assay for PE. The method involved permanganate oxidation of methanol to formaldehyde and reaction of the formaldehyde with pentane-2,4-dione. The color reaction was developed directly in the PE reaction mixture without interference from the pectin. However, Termote et al. (28)... 

Plasma CHE was determined by a radiometric assay (9) using acetylcholine and Btf 284c51. Plasma creatine kinase fCK) was determined by a spectrophotometric method (10). 

Sastry et al. [27] described four simple and sensitive spectrophotometric methods for the assay of omeprazole in pure and in dosage forms based on the formation of chloroform soluble ion-associated under specified experimental conditions. Four acidic dyes Suprachen Violet 3B (SV 3B, method A), Tropaeolin 000 (TP 000, method B), Boromocresol Green (BCG, method C), and Azocarmine G (AG, method D) are utilized. 

Wahbi et al. [32] used a spectrophotometric method for the determination of omeprazole in pharmaceutical formulations. The compensation method and other chemometric methods (derivative, orthogonal function, and difference spectrophotometry) have been applied to the direct determination of omeprazole in its pharmaceutical preparations. The method has been validated the limits of detection was 3.3 x 10 2 /ig/ml. The repeatability of the method was found to be 0.3-0.5%. The linearity range is 0.5-3.5 /ig/ml. The method has been applied to the determination of omeprazole in its gastro-resistant formulation. The difference spectrophotometric (AA) method is unaffected by the presence of acid induced degradation products, and can be used as a stability-indicating assay method. 

Another IR spectrophotometric method for the determination of salicylamide among other medicinally used amides was reported (1 5). The amide carbonyl absorption band at 1732 cm l was used as the basis of quantitative assay. 

Quantitative spectrophotometric methods for pectin utilize carbazole (diphenyleneimine Bitter and Muir, 1962) and m-hydroxydiphenyl (Kintner and Van Buren, 1982). The intense red-to-brown color with carbazole in sulfuric acid, relatively specific for uronans (pectin and alginate), is much less intense with ketohexoses, aldohexoses, and pentoses (Snell and Snell, 1953). The m-hydroxydiphenyl assay is subject to less interference than the carbazole assay. 

Assay Dissolve about 500 mg of sample, accurately weighed, in 25 mL of 50% alcohol previously neutralized with 0.1 IV sodium hydroxide, add phenolphthalein TS, and titrate with 0.1 IV sodium hydroxide. Each milliliter of 0.1 A sodium hydroxide is equivalent to 12.21 mg of C7H602. Lead Determine as directed in the Flame Atomic Absorption Spectrophotometric Method under Lead Limit Test, Appendix IIIB, using a 10-g sample. 

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