Development and validation of an HPLC method for the simultaneous determination of tocopherols, tocotrienols and carotenoids in cereals after solid‐phase extraction

Abstract

The increasing interest in antioxidant properties of cereal and cereal-based products has prompted the development of a simple and reliable HPLC method for the simultaneous determination of important phytochemicals like tocopherols (T), tocotrienols (T3) and carotenoids. Separation was carried out on a Nucleosil 100 C(18) column, 5 μm (250 mm × 4.6 mm) thermostated at 25 °C, using a linear gradient elution system starting with methanol and ending with a mixture of methanol-isopropanol-acetonitrile. All separated compounds including the internal standard (α-tocopherol acetate) were eluted within 16 min and detected by dual detection: fluorescence for tocopherols and tocotrienols at 290 nm excitation and 320 nm emission and UV-vis photodiode array detection for lutein and β-carotene at 450 nm. Detection limits ranged from 0.2 μg/g (β-carotene) to 1.60 μg/g (α-tocopherol). The intra- and inter-assay coefficients of variation were calculated by using cereals with different levels of lipophilic antioxidants. The extraction method involved sample saponification and clean-up by solid-phase extraction (SPE). The extraction recoveries obtained using OASIS HLB SPE cartridges and dichloromethane as eluent were in the range of 90.2-110.1%, with RSD lower than 10%. The method was successfully applied to cereals: durum wheat, bread wheat, rice, barley, oat, rye, corn and triticale.

Simultaneous analysis of carotenoids and tocopherols in botanical species using one step solid–liquid extraction followed by high performance liquid chromatography

“…The LOD and LOQ values for atocopherol acetate (1.02 and 2.04 lg g À1 DM) were more than 14 times higher than for non-esterified tocopherols (Table 2). In this respect, other authors who used a-tocopherol acetate as internal standard and analysed it with fluorescence detector reported very weak signal but did not provide numerical values (Darnet, Serra, Rodrigues, & da Silva, 2011;Irakli et al, 2011). LOD and LOQ for b-carotene detected by ultraviolet-visible absorption were 0.629 and 1.88 lg g À1 DM, respectively.…”

“…For HPLC analysis, dry extracts were dissolved in 1.5 mL of n-hexane/2-propanol (99/1, v/v), and filtered through a 0.22 lm syringe filter (hydrophilic Durapore PVDF, Millipore, Darmstadt, Germany). All the redissolved extracts were kept at À20°C in amber-coloured vials, and analysed before a week to ensure reproducible results (Irakli, Samanidou, & Papadoyannis, 2011;Rodrigo, Alegría, Barberá, & Farré, 2002).…”

Phytochemical profiles and antioxidant capacity of pigmented and non-pigmented genotypes of rice (Oryza sativaL.)

“…The combined extracts were applied to the Oasis cartridges; the retained constituents were eluted with 2 mL dichloromethane, and subsequently evaporated to dryness. Finally, the residue was redissolved in methanol and was analyzed as described by Irakli et al (2011).…”

Fluorometric Determination of Vitamin Constituents in Human Plasma Using Ultra Performance Liquid Chromatography

“…Although normal phase methods have been employed for the separation of some compounds [9][10][11][12][13], the vast majority of high performance liquid chromatography based fat soluble vitamin assay methods have employed reversed phase chromatography to facilitate analyte separation . Ultraviolet detection of these compounds has been most common in the literature [42][43][44][45][46][47], but fluorescence detection has also been used in the detection of tocopherol and retinol [32][33][34][35][36][37][38][39][40][41][42]. The use of fluorescence detection has allowed for various compounds to be analyzed at low limits of quantification and detection in plasma-human [32][33][34][35][36][37]40] or otherwise [39] as an alternative to ultraviolet detection methods.…”