A new method for simultaneous estimation of unsaponifliable constituents of rice bran oil using HPTLC

Rice bran oil (RBO) is rich in a variety of bioactive phytochemicals otherwise known as unsaponifiable constituents (USC). Oryzanols, phytosterols, tocols, etc. are the major USC in RBO; the methods presently used for their estimation involve different techniques and require pretreatment of the sample. In this paper standardization of a simple method for simultaneous estimation of USC directly from RBO using HPTLC is presented. The method involves a two-stage separation of USC on a pre-coated silica gel 60 F254TLC plate viz.: TLC-1 to separate sterols, oryzanols and tocols; TLC-2 to separate steryl esters, wax, and squalene. Calibration plots using the respective standards were made to determine LOD, LOQ and linear regression equations. Recovery studies were also conducted and the values ranged from 93.45 to 101.97%. The LOD and LOQ values showed the sensitivity of the method. The instrumental precision was found to be in the range of 0.30 to 1.18 CV%. Quantitative estimation of USC in crude RBO and refined RBO using this method gave a concentration of 52.80 mg/g of USC in the crude and 33.48 mg/g in the refined oil. The present method for estimation of USC using HPTLC is fast, simple, accurate, precise, and sensitive, as demonstrated here.