Determination of B2 and B6 vitamers in serum by capillary electrophoresis-molecular fluorescence-charge coupled...

A method for the separation by capillary zone electrophoresis and determination of vitamers of two important water-soluble vitamins, i.e., B2 and B6, is proposed here. The working conditions for optimal separation were obtained by a multivariate methodology in order to succeed in the best resolution in the shortest analysis time. The optimization of the buffer composition together with other variables, such as analysis and injection voltage, temperature of the capillary cassette, and injection time, resulted in a solution of 30 mM KH2PO4 adjusted to pH 8.5 with formic acid. Concerning the detection step, the target analytes were quantified by molecular fluorescence, for which two different detectors, a photomultiplier tube (PMT) and a charge-coupled detector (CCD), were compared in terms of resolution, sensitivity, and precision. A Xe-Hg lamp was used as an irradiation source in the two cases. The best option was the CCD, which provides three-dimensional electropherograms and enables to solve the overlapped peaks. Besides, the sensitivity of the CCD was similar to that of the PMT, due to the treatment data, obtaining limits of detection and quantification from 1.16 to 27.1 ng/g and from 3.83 to 89.4 ng/g, respectively. The method was applied to the serum samples for which a prior liquid-liquid extraction using ethanol in an acid medium was mandatory for eliminating the interferences and concentrating the analytes by a factor of 5. The rapidity of the analysis (13 min for the electrophoretic separation) and the excellent precision (repeatability and within-laboratory reproducibility between 2.86 and 4.11% and 7.03 and 8.45%, respectively, both expressed as relative standard deviation) demonstrated the capability of the proposed method for a clinical routine analysis.