Assays diphenylamine

This is a spectrophotometric assay based on the reaction of diphenylamine with the deoxyribose moiety of DNA to produce a complex that absorbs at 600 nm. The reaction is specific for deoxyribose and RNA does not interfere. It can be used on relatively crude extracts where direct spectrophotometric determinations of DNA concentration are not possible. 

To determine recovery, amounts of amphetamine and diphenyl-amine in the range 0-20 yg are added to normal urine and then extracted and assayed according to the procedure. Quantification is done and the mean percent recovery for amphetamine was 82% and for diphenylamine it was 83%. 

Deveaux et al. [21] reported that mefenamic acid, being an N-aryl derivative of anthranilic acid, can be characterized by two color reactions. The color reactions, negative with N-aryl derivatives of aminonicotinic acid, are associated with the diphenylamine structure. For the first color reaction, add to a test tube approximately 0.5 g of oxalic acid dihydrate and at least 1 mg of the test material. Place the tube into an oil bath at 180-200°C for 4-5 min. After cooling, dissolve the residue in 10 mL of 95% ethanol to obtain a stable, intense blue solution (absorption maximum at 586-590 nm). To use the reaction for capsule formulations, extract the active ingredient with acetone and filter prior to the assay. For the second color reaction, add mefenamic acid (100-800 pg) in 1 mL of H0Ac-H2S04 (98 2, v/v), 5 mL of HOAc-HCl (50 50, v/v) and 1 mL of 0.10% (w/w) aqueous levulose. Heat the mixture for 25 min at 100°C, and after cooling, measure the absorbance at 597 nm. 

Assay Transfer about 170 mg of sample, previously ground to a fine powder and accurately weighed, into a 250-mL wide-mouth Erlenmeyer flask, and dissolve in 10 mL of methanol. Add 150 mL of water, 1 mL of 1 N sulfuric acid, and 4 drops of diphenylamine indicator (3 mg of / -di phenyl ami ncsu I Ionic acid sodium salt per milliliter of 0.1 N sulfuric acid), and titrate with 0.1 N ceric sulfate to the first complete color change from yellow to red-violet. Record the volume, in milliliters, of 0.1 N ceric sulfate required as V. Calculate the percentage of C10H14O2 in the sample, uncorrected for hydroquinone and 2,5-di-tm-butylhydroquinone, by the formula... 

The DPA method is based on the colorimetric measurement of products formed by reaction of co-hydroxylevulinylaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 1 M perchloric acid.14-16 The reaction yields a mixture of products, such as that shown in Eq. 1.4,17 which together have an absorbance maximum at 595 nm. This method has a similar dynamic range to the DABA assay with colorimetric measurement. Interferences include proteins, lipids, saccharides, and RNA. 

Dumas et al. also developed a micro-method for the same determination in urine and blood. Samples of 3 ml blood were treated with ammonium oxalate and filtered. 1 ml plasma was made alkaline and extracted with chloroform three times, the chloroform extract was evaporated and the residue solved in 10 yl of a solution of 5 ng diphenylamine/ul (internal standard) in ethyl acetate. The solution was used for the gas chromatographic assay. A specific N-detector was used and the sensitivity was 0.2 ng for nicotine and 1 ng for cotinine. The recovery is given in Table 5.9. 

TLC and HPLC. To determine the best solvents for HPLC, 5pl of the C18 concentrated PVP fraction were separated on analytical TLC plates coated with either C18 reversed phase adsorbant or with silica. One cm sections of the plates were removed, placed in buffer, hydrolyzed, extracted and the p-damascenone charm determined. Standards containing 5 mg each of 1 -O-n-octyl- p-D-glucopyranoside and a-methyl-D-mannoside were run simultaneously on the same plate and visualized by spraying with aniline-diphenylamine-acetone-phosphoric acid 80% (4 mL 4 g 200 mL 30 mL) (10). HPLC separations were performed on a 0.46 mm x 25 cm column packed with 5 im C18, eluted with 60/40 methanol water at a flow of 0.8 mL/min. The refractive index of samples was recorded with a Knauer differential refractometer. Fractions were collected every minute and assayed for p-damascenone charm. 

Step 4. Treatment with hot acid releases all the adenine and guanine (99) and some desoxyribose and inorganic phosphate from DNA, the remainder appearing as acid-soluble pyrimidine-nucleotide combinations (36). Diphenylamine reacts only with the free desoxyribose or purine de-soxyribosides. Orcinol reacts slightly with DNA hence its use for RNA assay in the presence of large amounts of DNA requires a correction (93). 

Detailed colorimetric assay methods for nucleic acid derivatives will be confined to descriptions of slight modifications of three procedures diich have withstood well the test of time (f) the Mejbaum (76) modification of the Bial orcinol reaction for pentose (2) the Dische (42) diphenylamine method for desoxypentose and (S) the method of Fiske and Subbarow (45) for inorganic orthophosphate. Many other modifications of the original techniques have been advanced but, in our opinion, none offer a complete solution of such difficulties as may be encountered in nudmc acid studies. Discussions of the colorimetric techniques will be foimd in Schlenk s review (89), in the manual edited by Umbreit cf oZ. (61), and in the review by Dische (41). 

The initial attempts to develop a quantitative assay for sialic acids were colorimetric. The majority of these early methods were unsuitable because of their lack of specificity and/or sensitivity required for sialic acid analysis in biological materials or for enzymic assays. Some of these methods included diphenylamine (Ayala et al. 1951), tryptophane/perchloric acid (Seibert et al. 1948), sulphuric acid/acetic acid (Hess et al. 1957), hydrochloric acid (Folch et al. 1951) and the Ehrlich reagent, dimethyl-aminobenzaldehyde (Werner and Odin 1952). 

The earliest methods for detection and assay of sialic acids were colorimetric procedures that capitalized on the more or less specific chromogens derived from these carbohydrates. Over the years a variety of colorimetric agents have been employed orcinol (Klenk and Langer-beins, 1941), diphenylamine (Ayala et aL, 1951), hydrochloric acid... 

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