HPLC method for simultaneous determination of retinoids and tocopherols in human serum for monitoring of anticancer therapy

Abstract

A simple and rapid HPLC method requiring small volumes (250 microL) 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 (100x4.6 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 micromol/L for all-trans-retinoic acid, 0.125-5.00 micromol/L for 13-cis-retinoic acid, 0.25-10.00 micromol/L for retinol, 0.5-50.00 micromol/L for gamma-tocopherol, and 0.5-50.00 micromol/L for alpha-tocopherol. The present method may be useful for monitoring of retinoids and tocopherols in clinical studies.

An optimized and validated RP-HPLC/UV detection method for simultaneous determination of all-trans-Retinol (Vitamin A) and α-Tocopherol (Vitamin E) in human serum: Comparison of different particulate reversed-phase HPLC columns

“…The suggested method is simple, accurate, rapid, and novel (comparison of different particulate columns, simultaneous determination of all-trans retinol and ␣-tocopherol at the same wavelength of UV detector, mobile phase composition, temperature effect, stability study, and optimization of other chromatographic parameters) as compared with the previous reported methods [4,50,51,57,58]. The method was validated for simultaneous determination of all-trans-retinol and ␣-tocopherol in human serum and various chromatographic conditions and other experimental parameters such as mobile phase composition, stationary phase selection, detector wavelength, flow rate, column oven temperature, internal standard selection and sample preparation were optimized.…”

“…However, the columns used vary in length, particle size, and internal diameter. Although the use of various normal phase columns [30,46,47] and reversed-phase monolithic [48][49][50][51] and octyl-modified silica (OS) [46] columns has been reported, reversed-phase conventional particulate octadecylsilane (ODS) columns seem to be the method of choice for the analysis of all-trans-retinol and ␣-tocopherol in serum/plasma as monolithic columns are expensive in comparison to particulate columns [22,32,[52][53][54][55][56]. In a study separation of both the analytes in serum has been achieved on 3 m column with DAD and fluorescence detector within 13 min; however, comparison of various particulate columns has not been performed [57].…”

Comparison of a new high-resolution monolithic column with core-shell and fully porous columns for the analysis of retinol and α-tocopherol in human serum and breast milk by ultra-high-performance liquid chromatography†

“…The higher consumption of mobile phase is compensated for by the longer lifetime and lower price of the column in comparison with the other tested columns. Based on our practical experience, the monolithic column allows >1000 repetitions of analysis of retinol and α‐tocopherol in biological material without any loss of performance. This fact is because of the monolithic silica structure and the absence of column frits.…”

Applications of monolithic columns in liquid chromatography‐based clinical chemistry assays

“…The CV for all analytes is r8.1%. The third method was published in 2009 and measures all-trans-retinoic acid, 13-cis-retinoic acid, retinol, g-and a-tocopherol within a run time of 7.2 min using HPLC-diode array detection monitored at 325 and 295 nm [52]. The CV of the assay is 1.4-2.4% and the LOQ is 0.06-2.00 mmol/L for all analytes.…”