Abstract
A new fluorimetric method for the determination of monosubstituted guanidino compounds with benzoin is described. This is based on that fluorescence develops when the guanidino compound is heated in aqueous potassium hydroxide solution with benzoin in the presence of dimethylformamide, and the flu°rescence produced is stabilized by β-mercaptoethanol. The reaction conditions of the method were established by employing methylguanidine and arginine as model compounds. The prescribed method requires the heating time of 45min at 100°, and the fluorescence produced shows excitation and emission maxima around 325 and 435nm, respectively. The method is simple, selective for monosubstituted guanidino compounds including polypeptides with one or two arginyl residues, and sensitive (thelower limits of determination, 0.08-0.22nmol/ml).
Then, the method was revised for a rapid and a more sensitive method, based on the findings that the reaction mixture fluoresced most intensely when made weakly alkaline (pH, around 9) after heating for only several min. A mixture of sodium dihydrogen phosphate and Tris for the pH adjustment and 7min of the heating were employed in the revised method. The fluorescence from the guanidino compounds shows excitation maximum around 322 and emission maximum around 440nm. The rivised method is sensitive (the lower limits of determination, 0.09-0.17 and 0.04-0.06 nmol/ml for the compounds with one and two arginyl residues, respectively), precise (CV, 1.8% for 10nmol/m/ arginine, n=30), and selective (no interfering compounds of biological importance). The method should be applicable to the asssy of the guanidino compounds in biological samples, which are implicated as uremic toxins, after the compounds are separated (from the sample) by liquid chromatography.
