Comparison of a novel ultra-performance liquid chromatographic method for determination of retinol and α-tocopherol in human serum with conventional HPLC using monolithic and particulate columns

Abstract

Retinol and α-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 C₁₈ columns. In UPLC a sub-two-micron particle-hybrid C₁₈ 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 α-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.