Spectrophotometric assay, calculation

Assays for Antioxidative Activity Spectrophotometric and polarographic (9) assays were utilized to monitor levels of antioxidative activity. In the spectrophotometric assay, a 2-ml portion of linoleic acid emulsion (9) was placed in a test tube along with 10 pi of a sample to be assayed, and from this, 200 pi were withdrawn and mixed with 2 ml of 100% methanol and 6 ml of 60% methanol-water. The remainder of the 2 ml of emulsion and sample mixture was then incubated at 37°C for 15 to 20 h. Meanwhile, the absorbance of the methanol solution to which the 200 pi of sample had been added was measured at 234 nm. After incubation, the absorbance of 200 pi of the emulsion and sample mixture was also measured at 234 nm in the same manner. Antioxidative activity (A.O.A.) was calculated using the equation ... 

A spectrophotometric assay was introduced by Liener.101 It is based on the observation that rabbit erythrocytes sediment at a rate proportional to the concentration of the hemagglutinin. Hemagglutinating activity is calculated from absorbance measurements at 620 nm of the unsedimented cell-suspension after a given time.97,101... 

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... 

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 ultraviolet assay of tablets which contain only Isopropamlde as the active ingredient Is the most direct method employed (lif). Twenty tablets (5 mg/ tablet) are placed In a 250 ml. volumetric flask, disintegrated In about 150 ml. of water, and diluted to volume with water. The solution is filtered through Whatman No. 1 filter paper (l5),50.0 ml. of the filtrate percolated through an anion exchange column (16), and the column rinsed thoroughly with water. All eluates are collected in a 100 ml. volumetric flask and diluted to volume with water. The ultraviolet absorption spectrum of this solution Is compared with that of a known standard of Isopropamlde under the same spectrophotometric conditions and the assay value per tablet calculated. 

The USP assay (10) of amodiaquine hydrochloride in pure form and in tablets involves ultraviolet spectrophotometric determination. A quantity of the drug equivalent to about 300 mg is dissolved in dilute hydrochloric acid (1 100) to obtain a concentration of about IS The absorbance of this solution, along with a solution of undried USP Amodiaquine Hydrochloride RS in the same medium having a known concentration of about IS Ag/nil, is determined at 342 run using dilute hydrochloric add (1 100) as the blank. The quantity, in mg, of C20H22CIN3O, 2HQ in the portion of amodiaquine hydrochloride taken is calculated the formula 20c (Au/As), in which C is the concentration, in /Amodiaquine Hydrochloride RS in the standard solution and Au and As are the absorbances of the solution of amodiaquine hydrochloride and the standard solution respectively. The same method is applied to the assay of amodiaquine hydrochloride in tablets after extraction of the base into chloroform and then re-extraction with dilute hydrochloric acid (1 100). 

In the MTT assay, after a 24 or 48 h incubation of the cultured cells, the culture medium was removed and 20 pi of the MTT solution prepared at 5 mg/ml (Sigma Chemical Company, MO, USA) was added to each well for 4 h. The resulting crystals were dissolved in dimethyl sulfoxide (DMSO). The controls included native cells and the medium itself. The spectrophotometric absorbance of each well was measured with the use of a microplate reader (ELx 800, Bio-Tek Instruments, Winooski, VT, USA) at 550 mn. The cytotoxicity percentage was calculated by the formula percent cytotoxicity (cell death) = (1 - [absorbance of experimental wells/ absorbance of control wells]) x 100%. 

All assays were performed under heavy-metal free conditions after extraction of heavy-metals and the acid treatment of glassware to remove contaminating heavy-metal ions. Except where stated otherwise, assays were carried out spectrophotometrically at 340 nm and SO C in a 1 ml reaction mixture containing 50 mM Tris, pH 7.8, 0.14 mM NADH, 0.1 p,g PGK and 40 p.g glyceraldehyde-phosphate dehydrogenase. Concentrations of free Mg2+, MgATp2-, and PGA were calculated as described previously (11). 

Details of the TIBC assay differ slightly from assay to assay. Typically, 1 mL of serum is mixed with 0.1 mL ferric ammonium citrate (1.25 mmol Fe /liter) for 10 min after which 2 mL barbital-NaCl buffer and 0.4-0.5 g of magnesium carbonate (light powder) are added. Mix for 15 min on a rotary mixer after capping with parafilm. Centrifuge for 10 min at ISOOxg remove 2 mL of the supernatant solution, and spectrophotometrically analyze for iron as described above. Correction for the serum dilution (3 1) is included in calculating the TIBC. 

The FI methods developed for automation of ABTS +-based assay are listed in Table 31.2. All the methods have been successfully employed for the rapid evaluation of the anti-oxidative abilities of pure compounds, beverages, and food extracts. The TEAC values were calculated according to formula (31.9) or (31.10) in case of pure compounds and unknown samples, respectively. As can be seen from the data presented in Table 31.3, the Tr equivalents obtained by the FI methods are in quite good agreement with each other and with the TEAC values determined by the classic spectrophotometric ABTS assay. 

It should be noted that Ellman s assay does not require the use of external standards, or the generation of a standard curve, which is very convenient. However, it is limited in its sensitivity. These assay limitations are directly because of the inherent limitations in spectrophotometric detection. For instance, if an Ellman s assay sample shows an absorbance of 0.05 at 412 nm (approaching the lower limit of detection), the thiol concentration is calculated to be 3.7 /zM. This may be suitable when assaying proteins in the mg/mL concentration range (1 mg/ mL bovine serum albumin =15 uM), but not for lower concentrations. In some cases, the presence of unmodified cysteine thiols is aberrant or undesirable a more sensitive assay to detect low levels of such species would be useful. 

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