Studies on the absorption and excretion of medicaments. II. Separatory determination of isoniazid and its metabolites in urine

Abstract

Separatory determination of isonicotinic acid hydrazide (INAH) (I) and its metabolites, 1-acetyl-1-isonicotinoylhydrazine (II), isonicotinic acid (III), glucose isonicotinoylhydrazone (IV), and pyruvic acid isonicotinoylhydrazone (V) was established by the use of ion exchange resins, Dowex 1-X8 (Cl type) and Amberlite CG-50 (H type) and Amberlite CG-50 (Hform) (100-200 mesh). Separation of (I), (III), and (V) is effected by passing the sample urine through Dowex-1 column and its effluent is continuously passed through the Amberlite CG-50 column, by which (III) and (V) are adsrobed by the Cl-type resin and (I) by H-type resin. The Dowex-1 column is eluted with sodium chloride solution to desorb (III) alone and then eluted with 0.5N hydrochloric acid to desorb (V). Amberlite CG-50 column is eluted with 0.5N hydrochloric acid to desorb (I). For the separation of (II) and (IV), two Dowex-1 columns are prepared. The sample urine is passed through the first Dowex-1 column to remove (III) and (V), the effluent is acidified with acetic acid, and oxidized with potassium dichromate to convert (I) and (IV) into isonicotinic acid. This solution is neutralized and passed through the second Dowex-1 column to desorb isonicotinic acid and (II) alone is flown out. Isonicotinic acid is eluted with 0.5N hydrochloric acid. From the sum of these (I) and (IV), the amount of (I) determined separately is substracted and the difference is the quantity of (IV).Each of these metabolites was determined colorimetrically using cyanogen bromide. The separated sample solution, neutralized if necessary, was oxidized and decomposed with the bromine reagent to convert all to isonicotinic acid, colored by 7.5% cyanogen bromide in sodium carbonate-sodium hydrogen carbonate buffer, and determined colorimetrically (E425). This method makes it possible to determine INAH and its metabolites in the urine when INAH was used with streptomycin, PAS, thioacetazone, or pyrazinamide.