A RAPID-DETERMINATION OF ALLANTOIN BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY USING TRIS(HYDROXYMETHYL)AMINOMETHANE-HCL BUFFER AS A MOBILE-PHASE

A rapid and simple method for the determination of allantoin in pharma ceuticals by reversed-phase ion-pair high-performance liquid chromatog raphy using an ODS column was presented. In general, it is difficult t o retain allantoin to the ODS column owing to its very low hydrophobic ity. We solved these problems by the use of a Tris-HCl buffer (pH 7.5) containing tetra-n-hexyl-ammonium bromide (THAB) as an ion-pair reage nt for the mobile phase. Comparatively low concentrations of Tris-HCl buffer (0.9 mM) and THAB (0.5 mM) gave a high capacity factor (k'). As a results of the er;amination of the chromatographic behavior, it is confirmed that the retention mechanism of allantoin to the ODS column on the present method was not the ion-pair mode, but the ion-exchange made. Calibration curves for allantoin showed a good linearity in the range of 10 to 400 mu g/ml (r = 0.9999). The reproducibility (R.S.D., n = 6) was invariably good (0.37%). The lowest concentration of allant oin for the determination was 200 nb per 20 mu l of injection. The pre sent method was successfully applied to the determination of allantoin in commercial eyedrops with good recovery (99.4%). It was found that allantoin in pharmaceuticals could be determined by the present method in short time and without any complicated derivatization.