Stable isotope labeling assisted liquid chromatography-electrospray tandem mass spectrometry for quantitative...

In the current study, we developed a stable isotope labeling strategy for the absolute quantification of gibberellins (GAs) by high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS/MS). N,N-dimethyl ethylenediamine (DMED) and its deuterated counterpart d(4)-DMED were used to derivatize GAs extracted from plant tissue samples and GA standards respectively. The both derivatives of GAs were mixed and then subjected to HPLC-ESI-MS/MS analysis. The absolute quantification of GAs in plant tissues could be achieved by calculating the peak area ratios of DMED labeled GAs/d(4)-DMED labeled GAs. In the proposed strategy, the derivatization reaction of the labeling reagents with GAs could be completed rapidly (within 5 min) with high efficiency (>99%) under mild conditions. The resulting derivatives could produce specific fragments in collision induced dissociation (CID), leading to high selectivity in multiple-reaction monitoring (MRM) mode, thus enhanced the reliability of the LC-MS/MS method. Furthermore, the limits of quantitation (LOQs) of GAs were considerably decreased (2-32 folds) due to incorporating easily ionized moieties into GAs, and the quantification of GAs in plant tissue could be achieved without isotopically labeled GA standards. Good linearity was obtained with correlation coefficients R(2) values of >0.99. The limits of detection (LODs) and quantitation (LOQs) ranged from 0.02 to 0.74 pg and 0.07 to 2.45 pg, respectively. Eleven GAs could be successfully determined in spiked sample with 72-128% recoveries and the relative standard deviations (RSDs) were between 1.0% and 13.9%. Finally, the developed method was successfully applied for the detection of GAs in 50mg (fresh weight) Oryza sativa leaves.