Abstract
A simple and rapid HPLC method requiring small volumes (250 μL) of human serum after C18 SPE sample preparation was developed using monolithic technology for simultaneous determination of all‐__trans‐__retinoic acid, 13‐cis‐retinoic acid, retinol, gamma‐ and alpha‐tocopherol. The monolithic column, Chromolith Performance RP‐18e (100×4.6 mm), was operated at ambient temperature. The mobile phase consisted of a mixture of acetonitrile (ACN) and 1% ammonium acetate in water (AMC) at pH 7.0. The mobile phase started at 98:2 (v/v) ACN/AMC (column pre‐treatment) at a flow rate of 2 mL/min, then changed to 95:5 (v/v) ACN/AMC for 4 min at a flow rate of 1.5 mL/min and a further 3 min at a flow rate of 3.2 mL/min. Detection and identification were performed using a photodiode array detector. Retinol, 13‐cis‐ and all‐__trans‐__retinoic acid were monitored at 325 nm. Both alpha‐ and gamma‐tocopherol were detected at 295 nm. The total analysis time was 7.2 min. Tocol (synthesized tocopherol, not occurring in humans) was used as internal standard. The method was linear in the range of 0.125–10.00 μmol/L for all‐__trans‐__retinoic acid, 0.125–5.00 μmol/L for 13‐cis‐retinoic acid, 0.25–10.00 μmol/L for retinol, 0.5–50.00 μmol/L for gamma‐tocopherol, and 0.5–50.00 μmol/L for alpha‐tocopherol. The present method may be useful for monitoring of retinoids and tocopherols in clinical studies.