Rapid determination of lipophilic vitamins in human serum by ultra‐high performance liquid chromatography using a fluorinated column and high‐throughput miniaturized liquid–liquid extraction

Abstract

A high-throughput miniaturized liquid-liquid extraction procedure followed by a simple ultra-high performance liquid chromatography method coupled with fluorescence detection for bioanalytical analysis of all tocopherol isomers and retinol in human serum has been developed and validated. In the extraction procedure, a synthetic internal standard tocol was used, which does not occur in the human body. The separation of structurally related vitamins was achieved using a new generation of pentafluorophenyl propyl core-shell stationary phase with elution using methanol and an aqueous solution of ammonium acetate. The fluorescence of retinol and tocopherol isomers was detected at λ = 325, 295 nm and λ = 480, 325 nm, respectively. The rapid baseline separation of all analytes was accomplished within 4.0 min. The sensitivity of method was demonstrated with lower limits of quantification: retinol 0.01 μM, α-tocopherol 0.38 μM, β-tocopherol 0.18 μM, γ-tocopherol 0.14 μM, and δ-tocopherol 0.01 μM. Possible application of this method in clinical practice was confirmed by the analysis of human serum samples from healthy volunteers. Finally, the simultaneous determination of retinol and all tocopherol isomers in human serum can enable the clarification of their role in metabolism and in diseases such as cancer.

Rapid Separation of All Four Tocopherol Homologues in Selected Fruit Seeds via Supercritical Fluid Chromatography Using a Solid-Core C18 Column

“…e RP-LC is favored over the NP-LC because of some advantages such as column stability and/or reproducibility of retention times. Nowadays, by using both NP-LC and RP-LC and an appropriate column such as silica (Si), diol (Diol), and amino (NH 3 ) for NP-LC [8] and pentafluorophenyl (PFP or F5), C30, naphthalene (πNAP), and planar pyrene (5PYE) for RP-LC [9][10][11][12][13], which allows for isomers β and c separation, all tocopherols can be determined. Tocopherol homologues are often determined by RP-LC with a C18 column to obtain rapid separation.…”

Determination of vitamin A in blood serum based on solid‐phase extraction using cetyltrimethyl ammonium bromide‐modified attapulgite

“…However, it is challenging to determine vitamin A in serum/plasma, due to the complex biological matrix, the limited sample volume, and the low concentration of vitamin A. Sample pretreatment has become an indispensable step, and LLE is most commonly used . By LLE, vitamin A is extracted from complex matrices by some organic solvent, then dried by a nitrogen stream, and finally re‐dissolved in mobile phase or other organic solvents for subsequent HPLC or HPLC‐MS analysis.…”

Analysis of retinol, α‐tocopherol, 25‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3 in plasma of patients with cardiovascular disease by HPLC–MS/MS method

“…The methods require multiple extraction procedures (Quesada et al, ), large volumes of plasma samples (Alvarez & De Mazancourt, ; Granado‐Lorencio et al, ; Quesada et al, ) and relatively long chromatographic run times (Alvarez & De Mazancourt, ; Chatzimichalakis et al, ; Granado‐Lorencio et al, ; Quesada et al, ). A validated HPLC method with fluorescence detection has been also published (Cervinkova, Krcmova, Klabackova, Solichova, & Solich, ), enabling determination of retinol and tocopherol isomers in human serum, which are present at higher concentrations than the 25‐hydroxy derivatives of vitamin D. HPLC‐MS/MS methods have also been reported for the determination of fat‐soluble vitamins in human plasma/serum, allowing improved sensitivity and simplified sample preparation (Albahrani, Rotarou, Roche, & Greaves, ; Hrvolová et al, ; Midttun et al, ; Priego Capote, Jiménez, Granados, & de Castro, ). The method described by Priego Capote et al () enabled determination of vitamins A, E, K and D and their hydroxyl derivatives but a plasma volume of 1 mL was required for the analysis.…”