Miniaturisation of solid phase extraction method for determination of retinol,alpha- and gamma-tocopherol in human serum using new technologies

The aim of this study was to develop rapid and simple solid phase extraction (SPE) and HPLC methods for simultaneous determination of retinol, gamma- and alpha-tocopherol in human serum using a special auto sampler with micro titration plates.

Separation of vitamins was performed at ambient temperature using monolithic column on a HPLC containing rack changer for micro titration plates. As the mobile phase methanol with flow rate 2.5 mL min^?1 was used. The injection volume was 20 µL. Retinol was detected at 325 nm, gamma- and alpha-tocopherol were carried out at 295 nm, respectively. The total time of analysis was 1.8 minutes. Extraction method was developed using Spe-ed 96 C18, 100 mg/2 mL micro titration plates and SPE vacuum manifold. The consumption of the sample was 50 µL. Time of the analysis for 96 samples on one micro titration plate was 1.5 hour. In order to validate the developed method, precision, accuracy, linearity, detection and quantitation limits were evaluated. This method is suitable for rapid automated large-batch analysis of retinol, alpha- and gamma-tocopherol in small sample volumes of human serum.