Absorbance Assays

FRAP assay Absorbance of Fe + complex at 593 nm produced by antioxidant reduction of corresponding tripyridyltriazine Fe + complex... 

Carrier-free Na l (40mCi/mL) was produced at the Radiopharmaceuticals Division of the Bhabha Atomic Research Centre (BARC) in Mumbai, India. Yellow tetrazolium MTT (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetra-zolium bromide) from Sigma (USA) was used for the cytotoxicity assay. Absorbance was measured at 550 nm in an ELISA reader (Bio-Tek, USA). Culture media and supplements were obtained from Sigma, and aU other chemicals and solvents were of analytical reagent grade, procured from reputed manufacturers in India. 

This experiment describes the adaptation of the bicinchoninic acid (BCA) protein assay to a flow injection analysis. The assay is based on the reduction of Cu + to Cu+ by the protein, followed by the reaction of Cu+ with bicinchoninic acid to form a purple complex that absorbs at 562 nm. Directions are provided for the analysis of bovine serum albumin and rabbit immunoglobulin G, and suggestions are provided for additional analyses. 

Fibers and Fiber Sources. Fibers are present ia varyiag amounts ia food iagredients and are also added separately (see Dietary fiber). Some fibers, including beet pulp, apple pomace, citms pulp, wheat bran, com bran, and celluloses are added to improve droppiags (feces) form by providing a matrix that absorbs water. Some calorie-controUed foods iaclude fibers, such as peanut hulls, to provide gastroiatestinal bulk and reduce food iatake. Peanut hulls normally have a high level of aflatoxias. They must be assayed for aflatoxia and levels restricted to prevent food rejection and undesirable effects of mycotoxias. 

Medroxyprog esteroneAcetate. Accurate pharmacokinetic and metaboHsm studies on MPA have been difficult because the radioimmunoassays employed caimot differentiate between MPA and its metaboHtes (346). Comparison of MPA plasma levels assayed by hplc and radioimmunoassay show that radioimmunoassay may overestimate intact MPA concentrations by about fivefold (347). However, values of the mean elimination half-life of MPA were similar, being 33.8 and 39.7 h when measured by hplc and radioimmunoassay, respectively (347). Approximately 94% of MPA in the blood is bound to albumin (348). When taken orally, MPA is rapidly absorbed with Htde or no first-pass metaboHsm (13). Peak semm levels ate reached after 3 h. Steady state occurs after three days of daily adininistration (349). The pharmacokinetics of MPA when adininistered in a depot formulation have been described (350). 

Assays using equiUbrium (end point) methods are easy to do but the time requited to reach the end point must be considered. Substrate(s) to be measured reacts with co-enzyme or co-reactant (C) to produce products (P and Q) in an enzyme-catalyzed reaction. The greater the consumption of S, the more accurate the results. The consumption of S depends on the initial concentration of C relative to S and the equiUbrium constant of the reaction. A change in absorbance is usually monitored. Changes in pH and temperature may alter the equiUbrium constant but no serious errors are introduced unless the equihbrium constant is small. In order to complete an assay in a reasonable time, for example several minutes, the amount and therefore the cost of the enzyme and co-factor maybe relatively high. Sophisticated equipment is not requited, however. 

Assay of Enzymes In body fluids, enzyme levels aie measured to help in diagnosis and for monitoiing treatment of disease. Some enzymes or isoenzymes are predominant only in a particular tissue. When such tissues are damaged because of a disease, these enzymes or isoenzymes are Hberated and there is an increase in the level of the enzyme in the semm. Enzyme levels are deterrnined by the kinetic methods described, ie, the assays are set up so that the enzyme concentration is rate-limiting. The continuous flow analyzers, introduced in the early 1960s, solved the problem of the high workload of clinical laboratories. In this method, reaction velocity is measured rapidly the change in absorbance may be very small, but within the capabiUty of advanced kinetic analyzers. 

Most areas of research and appHcations involving the use of radioisotopes require a knowledge of what radiations come from each isotope. The particular apphcation determines what type of information is needed. If the quantity of a radionuchde in a particular sample or at a particular location is to be deterrnined and this value is to be deterrnined from the y-ray spectmm, the half-life of the nucHde and the energies and intensities or emission probabiUties of the y-rays of interest must be known. Usually it is preferable to use the y-rays for an assay measurement because the d- and P-rays ate much more readily absorbed by the source material, and may not reach the sample surface having their original energies. Once these energies are altered they caimot be used to identify the parent radionuchde. 

The fire assay, the antecedents of which date to ancient Egypt, remains the most rehable method for the accurate quantitative determination of precious metals ia any mixture for concentrations from 5 ppm to 100%. A sample is folded iato silver-free lead foil cones, which are placed ia bone-ash cupels (cups) and heated to between 1000 and 1200°C to oxidize the noimoble metals. The oxides are then absorbed iato a bone-ash cupel (ca 99%) and a shiny, uniformly metaUic-colored bead remains. The bead is bmshed clean, roUed fiat, and treated with CP grade nitric acid to dissolve the silver. The presence of trace metals ia that solution is then determined by iastmmental techniques and the purity of the silver determined by difference. 

Analytical Techniques. Sorbic acid and potassium sorbate are assayed titrimetricaHy (51). The quantitative analysis of sorbic acid in food or beverages, which may require solvent extraction or steam distillation (52,53), employs various techniques. The two classical methods are both spectrophotometric (54—56). In the ultraviolet method, the prepared sample is acidified and the sorbic acid is measured at 250 260 nm. In the colorimetric method, the sorbic acid in the prepared sample is oxidized and then reacts with thiobarbituric acid the complex is measured at - 530 nm. Chromatographic techniques are also used for the analysis of sorbic acid. High pressure Hquid chromatography with ultraviolet detection is used to separate and quantify sorbic acid from other ultraviolet-absorbing species (57—59). Sorbic acid in food extracts is deterrnined by gas chromatography with flame ionization detection (60—62). 

Since the avermectins exhibit unprecedented potency, they are used at unusually low doses of 6 —300 )-lg/kg, which makes the detection and isolation of residues and metaboUtes from animal tissue a new challenge. For this reason a sensitive analytical assay requires a derivative suitable for detection at concentrations down to 1/10 or 1/100 of one ppm. Ivermectin and avermectin B are therefore converted into an aromatic derivative which allows detection by fluorescence absorbance. To achieve this derivatization, avermectin B, ivermectin, or their derivatives are heated with acetic anhydride in pyridine at 100°C for 24 h (30). The reaction time can be reduced to 1 h by using /V-methylimidazole as a catalyst (31). The resultant... 

Separate sample blanking requires an additional analytical channel, and is therefore wasteflil of both reagents and hardware. An alternative approach that is used on several automated systems, eg, Du Pont ACA, BM-Hitachi 704, Technicon RA-1000, is that of bichromatic analysis (5) where absorbance measurements are taken at two, rather than one, wavelength. When the spectral curves for the interference material and the chromogen of the species measured differ sufficiently, this can be an effective technique for reducing blank contributions to assay error. Bichromatic analysis is effective for blanks of both the first and second type. 

A German process produces a high (99%) sodium cyanide assay by absorbing the gases from a BMA-type hydrogen cyanide reactor direcdy in sodium hydroxide solution (56). The resulting sodium cyanide solution is heated in a crystallizer to remove water, and form sodium cyanide crystals. 

Spectrophotometry, a simple and rehable technique, is often used in rate assays. This method can be used when the substrate or the product of the reaction absorbs in the uv or the visible region. In other cases, a nonabsorbing system can be coupled to a system in which the substrate or product absorbs in the uv or visible region. 

An enzymatic assay can also be used for detecting anatoxin-a(s). " This toxin inhibits acetylcholinesterase, which can be measured by a colorimetric reaction, i.e. reaction of the acetyl group, liberated enzymatically from acetylcholine, with dithiobisnitrobenzoic acid. The assay is performed in microtitre plates, and the presence of toxin detected by a reduction in absorbance at 410 nm when read in a plate reader in kinetic mode over a 5 minute period. The assay is not specific for anatoxin-a(s) since it responds to other acetylcholinesterase inhibitors, e.g. organophosphoriis pesticides, and would need to be followed by confirmatory tests for the cyanobacterial toxin. 

In the presence of cytochrome C, changes in transmittance at 550 nm reflect oxidant production as superoxide reduces the cytochrome C. This is usually done as an absorbance assay in a spectrophotometer, but it can be performed as a transmittance assay on the SLM fluorometer (20). 

Example 32 (see Section 2.2.1) assume that the measurement of a test article yields an absorbance of 0.445 what is the probable assay value Even for m = 10 repeat determinations, the true value of X(y ) is only loosely... 

Absorbance Reading [AU] Tablet Weight [mg] Assay [mg] Calibration Factor Type of Result... 

The sensitivity of enzyme assays can also be exploited to detect proteins that lack catalytic activity. Enzyme-linked immunoassays (ELlSAs) use antibodies covalently finked to a reporter enzyme such as alkafine phosphatase or horseradish peroxidase, enzymes whose products are readily detected. When serum or other samples to be tested are placed in a plastic microtiter plate, the proteins adhere to the plastic surface and are immobilized. Any remaining absorbing areas of the well are then blocked by adding a nonantigenic protein such as bovine serum albumin. A solution of antibody covalently linked to a reporter enzyme is then added. The antibodies adhere to the immobilized antigen and these are themselves immobilized. Excess free antibody molecules are then removed by washing. The presence and quantity of bound antibody are then determined by adding the substrate for the reporter enzyme. 

The physicochemical properties of the reactants in an eiKyme-catalyzed reaction dictate the options for the assay of enzyme activity. Spectrophotometric assays exploit the abihty of a substrate or product to absorb hght. The reduced coenzymes NADH and NADPH, written as NAD(P)H, absorb hght at a wavelength of 340 run, whereas their oxidized forms NAD(P) do not (Figure 7—9). When NAD(P)+ is reduced, the absorbance at 340 run therefore increases in proportion to—and at a rate determined by—the quantity of NAD(P)H produced. Conversely, for a dehydrogenase that catalyzes the oxidation of NAD(P)H, a decrease in absorbance at 340 run will be observed. In each case, the rate of change in optical density at 340 nm will be proportionate to the quantity of enzyme present. 

The assay of enzymes whose reactions are not accompanied by a change in absorbance or fluorescence is generally more difficult. In some instances, the product or remaining substrate can be transformed into a more readily detected compound. In other instances, the reaction product may have to be separated from unreacted substrate prior to measurement—a process facili-... 

Whole cell OPH activity was measured by following the increase in absorbancy of p-nitrophenol from the hydrolysis of substrate (0.1 mM Paraoxon) at 400 nm (sm = 17,000 M cm ). Samples of culture (1 ml) were centrifuged at 10,000 g and 4 C for 5 min. The cells were washed, resuspended with distilled water, and 100 pi was added to an assay mixture containing 400 pi 250 mM CHES [2-(N-cyclohexylamino)ethane-sulfonic acid] buffer, pH 9.0, 100 pi 1 mM Paraoxon, and 400 pi distilled water. One unit of OPH activity was defined as pmoles Paraoxon hydrolyzed per min. Each value and error bar represents the mean of two independent experiments and its standard deviation. 

CAO G, ALESSio H M and CUTLER R G (1993) Oxygen-radical absorbance capacity assay for antioxidants, Free Rad Biol Med, 14, 303-11. 

Comparative studies of the widely employed spectrophotometric readings at the Soret and Q bands (405 and 630 nm, respectively) and the elemental analysis of copper and nitrogen showed that the spectrophotometric assay based only on the Soret band can overestimate the purity of a preparation. Erroneous data were attributed to an increase in absorptivity at the Soret band when other colored compounds like metal-free analogs and carotenoids are present. Indeed, copper-free chlorin e6 exhibits a specific absorbance 3.6 times greater than that of its coppered counterpart. Therefore, measurements at the Q band (630 mn) and the establishment of the S Q ratio are preferred. 

Sodium copper chlorophyllin, approved by the FDA as a color additive in citrus-based dry beverage mixes, should have a ratio of absorbance (SoretQ band) not less than 3.4 and not more than 3.9. In Europe, purity criteria of the food additives E141[i] and E141[ii], which are copper complexes of chlorophyll and chlorophyllin, respectively, are set out in the EC color specifications that include identification and spectrophotometric assay tests. ... 

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