Retinol and alpha-tocopherol are biologically active compounds often monitored in blood samples because of their evident importance in human metabolism. In this study a novel ultra-performance liquid chromatographic (UPLC) method used for determination of both vitamins in human serum has been compared with conventional HPLC with particulate and monolithic C(18) columns. In UPLC a sub-two-micron particle-hybrid C(18) stationary phase was used for separation, in contrast with a five-micron-particle packed column and a monolithic column with a highly porous structure. Methanol, at flow rates of 0.48, 1.5, and 2.5 mL min(-1), respectively, was used as mobile phase for isocratic elution of the compounds in the three methods. Detection was performed at 325 nm and 290 nm, the absorption maxima of retinol and alpha-tocopherol, respectively. Analysis time, sensitivity, mobile-phase consumption, validation data, and cost were critically compared for these different chromatographic systems. Although cost and mobile-phase consumption seem to make UPLC the method of choice, use of the monolithic column resulted in almost the same separation and performance with a slightly shorter analysis time. These methods are alternatives and, in routine laboratory practice, more economical means of analysis of large numbers of biological samples than use of a traditional particulate column.
A novel, fast, and simple capillary electrophoresis method has been developed for the analysis of gentisin, isogentisin, and amarogentin in roots of Gentiana lutea (yellow gentian), an herb traditionally used as gastric stimulant. Gentisin and isogentisin are xanthones showing potent inhibition of monoamine oxidase type A and B, amarogentin represents one of the bitter principles of Gentiana, responsible for its gastric-roborant effects. Optimal CE-separation conditions comprise a 100 mM sodium tetraborate buffer of pH 9.3, containing 10 mM beta-cyclodextrin as additive; optimum temperature and applied voltage were found to be 30 degrees C and 25 kV, respectively. Direct diode array detection at 260 nm (gentisin, isogentisin) and 242 nm (amarogentin) was performed, and the required analysis time was only 11 min. The developed method was validated for linearity, sensitivity, precision, and accuracy, and utilized to assay several commercially available G. lutea samples. Quantitative data obtained with the developed CE method are compared with HPLC results, and the advantages of each approach are discussed.
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