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A novel and rapid sample pretreatment technique based on a combination of ultracentrifugation and solid-phase extraction for the determination of α-tocopherol in human erythrocyte membranes by high-performance liquid chromatography with ultraviolet detection is presented in this work. Red blood cell samples were ultracentrifuged (288 000 × g, 3 min, 4°C) in the presence of d-mannitol, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and calcium chloride. The α-tocopherol was then extracted from the erythrocyte membranes by solid-phase extraction with n-hexane in the presence of ascorbic acid. Tocopherol acetate was used as the internal standard. The extract was dissolved in methanol and separated on the monolithic column Chromolith Performance RP-18e (100 × 4.6 mm) using 100% methanol as the mobile phase. The absorbance of α-tocopherol was measured at a wavelength of 295 nm. The method was validated and showed sufficient accuracy and precision, ranging from 96.4 to 100.8% and from 4.5 to 6.3%, respectively. Moreover, the developed method was applied to the determination of erythrocyte α-tocopherol in real samples from patients. The combined ultracentrifugation and solid-phase extraction technique substantially decreased the time for the sample pretreatment step compared to liquid-liquid extraction and could be applicable for the quantitation of other analytes in erythrocyte membranes., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Biomarkers, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , are important indicators of the vitamin D general status and are monitored in several pathophysiological disorders, such as osteoporosis, diabetes, heart disease, etc. A novel ultra-HPLC with MS/MS methodology for the analysis of 25-hydroxyvitamin D derivatives coupled with a very simple and highly rapid sample preparation step was developed. Analytical parameters obtained showed linearity (R(2) ) above 0.999 for both vitamins with accuracies between 95.8 and 102%. The LODs were as low as 0.22 and 0.67 nmol/L for 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 , respectively. Intra-assay precision (%RSD) was lower than 4.5%, and inter-assay precision (%RSD) was lower than 6.5%. The feasibility of the developed methodology to be applied in clinical routine analysis has been proved by its application in blood samples from non-agenarian patients, patients with familial hypercholesterolemia and patients suffering from age-related macular degeneration., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The reduction of analysis time, cost, and improvement of separation efficiency are the main requirements in the development of high-throughput assay methods in bioanalysis. It can be achieved either by ultra-high-performance liquid chromatography (UHPLC) using stationary phases with small particles (<2 μm) at high back pressures or by using opposite direction--monolithic stationary phases with low back pressures. The application of new types of monolithic stationary phases for UHPLC is a novel idea combining these two different paths. The aim of this work was to test the recently introduced second-generation of monolithic column Chromolith® HighResolution for UHPLC analysis of liposoluble vitamins in comparison with core-shell and fully porous sub-2 μm columns with different particle sizes, column lengths, and shapes. The separation efficiency, peak shape, resolution, time of analysis, consumption of mobile phase, and lifetime of columns were calculated and compared. The main purpose of the study was to find a new, not only economical option of separation of liposoluble vitamins for routine practice., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)