Quantification of g-Oryzanol in crude cereal bran oils and commercial products by a rapid and reliable NP-HPLC method

A complex mixture of esters, between ferulic acid and triterpenols or sterols, is collectively termed Oryzanol. The main source of γ-oryzanol is the bran of some cereals, in particular brown and red rice, where its content is about 0.3-0.6% w/w. Rice bran is about 15-20 % oil (RBO) which solubilises oryzanol ( 0.2-1.7 % oil ) and vitamin E. Interest in RBO and oryzanol has been growing because of their technological importance and putative health benefits. RBO is used as functional food and oryzanol found application as additives in the alimentary industry [1]. The large number of commercial products which contain oryzanol as ingredient, demand for a fast and reliable procedure of detection and quantization. This is a hard task because of the complexity and variability of its composition and the necessity to achieve the analysis on samples very different each others. Several analytical methods have been proposed for the quantification of oryzanol in samples of bran or oil and for the identification of its constituents. Most efforts were devoted to reversed-phase chromatography (RP-HPLC) after multi-step procedures of chemical and chromatographic purifications of the sample. Our approach to the quantitative determination of γ oryzanol revealed some drawbacks in the RP-HPLC methods. In fact, apolar stationary phases are highly retentive of TGA, FFA and waxes. Efficient clean-up procedure in the preparation of sample is thus mandatory: it constitutes a solvent and time consuming step where loss of analyte may occur as well. Moreover, the quantization of many terpene ferulates in each sample may be redundant and computationally demanding. To our opinion, RP-HPLC methods are more suitable for qualitative studies of oryzanol components for a small number of samples. Besides, p-coumarate esters and minor cis isomers co-elute with abundant ferulates and trans isomers in RP-HPLC. On the other hand, normal-phase chromatography (NP-HPLC) allows the separation of p-coumarates from ferulates, cis from trans isomers and the differentiation of triterpene alcohol from sterol esters. The screening of a large number of samples requires a fast and reliable sample preparation and the possibility of automated and replicated HPLC injections. In NP-HPLC, the huge amount of apolar waxes, TAG and FFA are eluted before the analytes of interest thus eliminating the need of long column rinsing and tedious preliminary clean-up of samples. Our analysis was performed on cyanopropyl bonded column using a hexane/MTBE gradient system. The optimized method provided the best linear relation (r2 = 1), very good precision (RSD < 1) and good recovery (> 97%). The method was successfully applied to analysis of crude bran of some cereals and of commercial products.