Bratton-Marshall method

For quantitative work, it is necessary to estimate the concentration of 5-amino-l-(P-D-ribofuranosyl)imidazole in aqueous solution. It seems that the only available method is the Bratton-Marshall assay, which was originally developed for the estimation of arylamines in biological fluids. The principle of the method is the spectrometric estimation of a salmon-pink colored dyestuff obtained by diazotation in situ, followed by coupling with /V-( 1 -naphthyl)ethyl-enediamine.65 The only remaining problem then is to know the molar extinction of this dye because pure samples of AIRs are not available. A value of 16800 at 520 nM was obtained for the dyes prepared from a model compound, 5-amino-l-cyclohexylimidazole-4-carboxylic acid (54), which is crystalline. A comparable molar extinction can be expected for the dye prepared from imidazole 55, if the carboxyl group does not exert too much influence on the chromophore. Actually, its influence is perceptible even with the naked eye, the dyestuff prepared from 53 having a somewhat different, wine-red color, with max>520 nM. The molar extinction for 55 is 17400 at 500 nM. When the decarboxylation of 54 was conducted under mild acidic conditions (pH 4.8, 50°C, 1 hour), estimation of 5-aminoimidazole 55 by the Bratton-Marshall method led to the conclusion that the reaction was almost quantitative.66 Similar conditions for the final decarboxylation were adopted in the preparation of samples of AIRs labeled with stable isotopes.58... 

Modifications of the Bratton-Marshall method mentioned in Section IV.D.3.g can be used for sensitive spectrophotometric detection and determination of primary aromatic and heterocyclic amines. Thus, a simple spectrophotometric determination of the cardioprotective agent acadesine (142) was developed, to measure concentrations of the drug in plasma during intravenous infusion to patients undergoing coronary artery bypass graft surgery336. 

SPE methods for organic environmental pollutants, including PAA (Table 1) and HA A (Table 2.B), coupled with electrophoresis end analysis were reviewed126. Analysis of PAA in environmental waters requires preconcentration and cleanup to remove interfering substances such as humic acids from the samples. This may be accomplished by several methods, for example (a) derivatization of the PAA by the Bratton-Marshall method followed by HPLC-UVD measuring at 460 nm (Section III.B.2) (b) flocculation of the water sample with Al3+ ions to selectively remove humic compounds before SPE (c) a... 

Methods based on HX (X = Br or I)-catalyzed denitrosation (g) HX denitrosation-TEA in water 5-80% Acetone in NOX (NO ) Griess or Bratton-Marshall reagent) Chemiluminescence to nitrosamides fully automated Applicable to most NOC 72... 

From the topically applied silver sulfadiazine only sulfadiazine is absorbed to some extent (about 10%). The analysis of sulfadiazine in biological fluids can be based on the methods described by Stober and De Witte (12). Delaveau and Friedrich-Noue (33) employed the Bratton-Marshall reaction for the measurement of sulfadiazine levels in plasma, serum and urine after topical application of silver sulfadiazine cream. 

A colorimetric method for microdetermination of sulfonamides based on diazotization of the drug with sodium nitrite and hydrochloric acid has been reported (59). The diazonium salt is then coupled with 8-hydroxyquinoline in alkaline medium and the absorbance of the developed color measured at its maximum wavelength. A similar method involves diazotization and coupling of the sulfonamide with indole in alkaline solution to form an intense yellow azo dye which exhibits maximum absorption at 449 nm. Beer s law is obeyed over the concentration range 1-32 /ig/ml with a relative standard deviation of less than 2% (60). The reaction of sulfonamides with chloramine-T in sulfuric acid gives a yellow product which is suitable for the determination of sulfonamides in different formulations. It has an accuracy similar to that of the Bratton and Marshall method (61). 

A modified method for the determination of sulfonamides based on the Bratton-Marshall reaction with derivative spectrophotometry has been proposed (64). Various sulfonamides give excellent precision by using first- to fourth-order derivative spectra. The method has been applied to the determination of sulfonamides in pharmaceutical formulations and urine without pretreatment of the sample. 

Examples of the chemical reaction method are the assay of sulfonamide by the Bratton-Marshall procedure. The drug in solution is reacted with sodium nitrite to form an unstable diazonium intermediate. This unstable salt couples with a reagent yielding a reaction product with an intense color. The dye-salt extraction method may also be exemplified by the assay of atropine. Atropine in solution reacts with bromthymol blue at an appropriate pH. After shaking, the color complex formed is extracted from the aqueous layer into the organic layer and measured quantitatively. 

W G Resetarits DE and Bates TR, Errors in chlorothiazide bioavailability estimates based on a Bratton-Marshall colorimetric method for chlorothiazide in urine, /. Pharm. Sci., 68, 126-127 (1979). 

R. Whelpton, G. Watkins and S.H. Curry, Bratton-Marshall and liquid-chromatographic methods compared for determination of sulfamethazine acetylator status, Clin. Chem., 1981, 27, 1911-1914. 

Averell and Norris (3) have developed an analytical method adapted to the determination of parathion in spray or dust residues, which is sensitive to about 20 micrograms. It is based upon the reduction of parathion with zinc to the amino compound, diazotiza-tion, and coupling with Bratton and Marshall s amine, which gives an intense magenta color with an absorption peak at 555 millimicrons. Bowen and Edwards (6) have used the polarograph to assay technical grades of parathion and its formulations. 

Pyrrolidone carboxylyl peptidase activity has been followed using pyrrolidone carboxylyl L-alanine (or other pyrrolidone carboxylyl-amino acids) as substrate by following the amount of amino acid released as determined by the ninhydrin method (137). In another assay procedure the release of -naphthyl amine from L-pyrrolidone carboxylic-/ -naphthylamide (135) is determined by the diazotization procedure of Bratton and Marshall (138) as modified by Goldberg and Rutenberg (139). 

The method of Bratton and Marshall was developed for the estimation of sulphonamides in body fluids for mixtures in which the concentration of sulphonamides is sufficiently large (this also generally applies to urine) the determination is done as for the filtrate in the method described below. The absence of substances such as local anaesthetics, which would give the same colour reaction as the sulphonamides, must be ascertained. To determine the total drug if conjugated sulphonamides are present a portion of the prepared test solution must be hydrolysed by heating on a water-bath for one hour in 0-2N hydrochloric acid and subsequent adjustment to volume. The following method is that applied to blood ... 

Chemical Methods. Assay of these peptides by chemical methods is based upon the following reactions. Pteroylglutamic acid is reduced by metallic zinc in acid solution, and the liberated arylamine is diazotized and coupled with Bratton and Marshall reactant (90). The coupled compound is colored and therefore determined by photometry (305). p-Aminobenzoylpolyglutamic acid is also determined by the same method. 

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