Improving the Specificity of the Nitrosonaphthol Procedure for Urinary 5-Hydroxyindoleacetic Acid

The nitrosonaphthol procedure for urinary 5-HIAA has been modified by using an excessof NaCI and adjusting the urine to a constant pH during the first chloroform purification step. Thus, the variable salt concentration of different urine samples, which has been claimed to be responsiblefor the reported inconsistencyof the meth-od, has been eliminated. IN RECENT STUDIES (1) in which the urinary levels of 5-hydroxiiidoie-acetic acid (5-HIAA) were measured by the nitrosonaphthol procedure (2), a chromophore was formed with the endogenous metabolite ill about 10 % of the specimens from hospitalized patients which was not identical with that of the authentic reference compound. Although this method is widely used and is generally reiiable, the question of spe-cificitv and consisteiicy has occasionally been raised (3-5). This prob-leni apparently has been resolved ill the present procedure by using an excess of sodium chloride during tile purification step with chloroform. Also, adjusting tile urine samples to tile same pH, rather than adding the same amount of acid to each, appears to have improved time Con-sistency and the specificity of the method. Furthermore, with the de-scribed extraction procedure, treatment of the urine with the dinitro-phenylhydrazine reagent was not necessary. Method Collect urine in a glass or plastic jar containing 10 ml. of 5 N HC1 per 24-hr. (1000-2000 ml.) sample; use Baker’s analytic grade reagents unless otherwise specified. Pipet up to 10 nIl, of uriiie (make up with water if less is used) int