Simultaneous and direct determination of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in serum by derivative synchronous fluorescence spectroscopy.

Simultaneous and direct assays of pyridoxal, pyridoxal-5'-phosphate, and pyridoxic acid in human serum are described. The method applied is based on the reaction of these compounds with beryllium in an ammoniacal medium to yield highly fluorescent derivatives. Overlapping of conventional fluorescence spectra is resolved by using second-derivative fluorescence spectroscopy, thus making the use of separation techniques unnecessary. The proposed method is simple (only beryllium and an ammoniacal buffer are needed to develop fluorescence), rapid (the derivative formation is instantaneous and serum treatment only requires deproteinization), and inexpensive (no sophisticated detection equipment is necessary, any conventional modern spectrofluorimeter being adequate for use). The analytical recovery achieved was of about 96% for pyridoxal, 97% for pyridoxal-5'-phosphate, and 100% for pyridoxic acid. Measurements were carried out in a single scan.