Determination of Phytochelatins in Rice by Stable Isotope Labeling Coupled with Liquid Chromatography–Mass Spectrometry

Abstract

A highly sensitive method was developed for the detection of phytochelatins (PCs) in rice by stable isotope labeling coupled with liquid chromatography–electrospray ionization–tandem mass spectrometry (IL–LC–ESI–MS/MS) analysis. A pair of isotope-labeling reagents [ω-bromoacetonylquinolinium bromide (BQB) and BQB-<i>d</i>⁷] were used to label PCs in plant sample and standard PCs, respectively, and then combined prior to LC/MS analysis. The heavy labeled standards were used as the internal standards for quantitation to minimize the matrix and ion suppression effects in MS analysis. In addition, the ionization efficiency of PCs was greatly enhanced through the introduction of a permanent charged moiety of quaternary ammonium of BQB into PCs. The detection sensitivities of PCs upon BQB labeling improved by 14–750-fold, and therefore, PCs can be quantitated using only 5 mg of plant tissue. Furthermore, under cadmium (Cd) stress, we found that the contents of PCs in rice dramatically increased with the increased concentrations and treatment time of Cd. It was worth noting that PC₅ was first identified and quantitated in rice tissues under Cd stress in the current study. Taken together, this IL–LC–ESI–MS/MS method demonstrated to be a promising strategy in detection of PCs in plants with high sensitivity and reliability