Determination of seven biothiols in rice by high performance liquid chromatography-fluorescence detection with pre-column derivatization

Abstract

A high performance liquid chromatographic method with fluorescence detection and precolumn derivatization (HPLC-FLD) has been developed for the determination of seven biothiols including Cys, GSH, and phytochelatins (PCs: PC2, PC3, PC4, PC5 and PC6) in rice. The samples were ultrasonically extracted with 0.1% trifluoroacetic acid (TFA) containing 6. 3 mmol/L diethylenetriaminepentaacetic acid (DTPA), and then the seven biothiols were derivatized with monobromobimane (mBrB) as derivatization agent in 4-(2-hydroxyethyl)-1-piperazine propanesulfonic acid (HEPPS) buffer solution (pH 8.0). The separation was performed on an Agilent Eclipse Plus C18 column (50 mm x 4.6 mm, 5 microm) with gradient elution of 0.1% TFA solution (the pH value was adjusted to 2.5 with hydrochloric acid) and acetonitrile as mobile phases at a flow rate of 0.8 mL/min. The detection was performed at 380 nm for excitation and 470 nm for emission. The calibration curves of the seven biothiols showed good linearity in the concentration range of 0.7-100.0 mg/L with the correlation coefficients (r2) > or = 0.9991. The limits of detection were 0.03-0.20 mg/L. The recoveries of standard addition were in the range of 89.26%-99.42% with the relative standard deviations (RSDs, n = 6) of 2.05%-5.87%. The method is sensitive, accurate, reproducible and suitable for the simultaneous determination of Cys, GSH, PC2, PC3, PC4, PC5 and PC6 in rice.