Determination of Phenytoin and Its Intermediates by RP-HPLC with UV-Detection by Timing Wavelength Switching

Benzoin was oxidized by air in the presence of iron salt as catalyst and using dimethyl sulfoxide (acidified with HCl) as solvent to give benzil, which was in turn condensed with urea to give the object product phenytoin. In the course of this synthetic process, it was required to determine phenytoin, the intermediate benzil, and also the starting material benzoin. A method for determination of the 3 compounds mentioned above by RP-HPLC in combination with the UV-detection by timing wavelength switching was tested and proposed. The sample containing the analytes was dissolved with acetonitrile and solution obtained was filtered through 0.45 μm filtering membrane. The filtrate was used for LC analysis. The kromasil 100-5C₁₈ chromatographic column was used as stationary phase, and acetonitrile-H₂O solution with the volume ratio of 80:20 was used as mobile phase in isogradient elution. Absorbances (expressed as their peak areas) of the eluates were measured by the programme of timing wavelength switchings as follows:at 0-3.0 min and wavelength of 218 nm (for phenytoin); at 3.0-4.0 min and wavelength of 247 nm (for benzoin); and at 4.0-6.0 min and wavelength of 260 nm (for benzil). Linearity ranges of the standard curves of the 3 compounds were in the same range of 0.1-100 mg·L^-1. Detection limits (3S/N) found were 0.010 mg·L^-1, 0.003 0 mg·L^-1 and 0.007 0 mg·L^-1 respectively. Test for recovery was made by addition of mixed standard solutions at 3 concentration levels to matrixes of substantial sample, results of recovery found for the 3 analytes were in the range of 95.4%-105%. Values of RSDs (n=6) were found in the range of 1.8%-4.2%.