The role of matrix effects on the quantification of abscisic acid and its metabolites in the leaves of Bauhinia variegata L. using liquid chromatography combined with tandem mass spectrometry

Abstract

Phytohormone analysis using liquid chromatography combined with tandem mass spectrometry is a very important tool for studies involving plant metabolism; however, this analysis can be affected by matrix composition. Although it is necessary to understand this effect to produce reliable results, it has been widely neglected in analyses of plant hormones. Leaf extracts from Bauhinia variegata were obtained by solvent extraction followed by solid-phase cleanup. The extract was analyzed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in negative ionization mode, using the multiple reaction monitoring mode with two or three transitions to enhance analytical quality control. Although deuterated standards were used as surrogates, pronounced matrix effects were detected for phaseic acid (PA), abscisic acid (ABA) -glycosyl ester (ABA-GE) and dihydrophaseic acid (DPA), whereas ABA, neoPA and 7`-hydroxy-ABA (7′OHABA) showed negligible matrix effects. The method was validated using spiked samples and was applied to monitor ABA, PA, DPA, neoPA, 7´OHABA and ABA-GE on a daily basis. Analyte recoveries from spiked samples ranged from 67% to 87%. The highest leaf concentration of phytohormones was found at 2:00 pm and 5:00 pm, which represent typical day times related to the decrease of stomatal conductance. Despite the use of deuterated phytohormones as internal standards, we showed that the use of a calibration curve constructed with a matrix extract is mandatory for the reliable quantification of ABA and its metabolites, especially PA, ABA-GE and DPA. Daily variations in endogenous ABA leaf concentration were discussed.Keywords: ABA-β-D-glucosyl ester, ABA daily variations, dihydrophaseic acid, LC-ESI-MS/MS, phaseic acid.

Abscisic Acid Related Metabolites in Sweet Cherry Buds (Prunus avium L.)

“…Parameters for LC-ESI-MS/MS analysis were adapted from Zheng et al [1] and details are given in Supplementary Table S1. For PA, a 1 st transition m/z 279 → 139 and a 2 nd transition m/z 279 → 205 was found, where the 1 st transition is generally used to characterize the compound identity [1,36]. In case of Neo-PA, the same two transitions are also possible, where the transition m/z 279 → 205 prevails [36].…”

“…ABA is biologically inactivated in a stepwise manner during the course of catabolism. The mass spectrometric methods for targeting ABA catabolism are much better documented and can be utilized with more reliability [1,36,25]. Concerning ABA catabolism, three different hydroxylation pathways have been documented leading to oxidation of one of the methyl groups of the ring structure (C-7´, C-8´, and C-9´) [6].…”

“…The results for the relevant catabolites are presented in Figure 4B-D. Tandem analysis with mass spectrometry is widely used to identify ABA and its catabolites and can result in similar fragmentation profiles [36]. Both PA and Neo-PA have been demonstrated to have MRM transitions with identical fragmentation patterns due similar structural constellations [36]. Parameters for LC-ESI-MS/MS analysis were adapted from Zheng et al [1] and details are given in Supplementary Table S1.…”

“…For PA, a 1 st transition m/z 279 → 139 and a 2 nd transition m/z 279 → 205 was found, where the 1 st transition is generally used to characterize the compound identity [1,36]. In case of Neo-PA, the same two transitions are also possible, where the transition m/z 279 → 205 prevails [36]. The corresponding results for these two catabolites are given in Figure 4B and C. Both follow a similar pattern, and a 2.3 to 1.9 fold increase as compared to LF is noted respectively for PA and Neo-PA at the OC stage.…”

Identification and Characterization of Bauhinia Species by Spectroscopic and Spectrometric Fingerprints

“…The results were compared with those available in the scientific literature about the chemical composition of Bauhinia genus. 7,17 Ion fragmentations were analyzed and correlated with data reported. In addition, exact mass data observed by LC-MS/MS, the molecular formula was obtained, and the data was compared with compounds already reported for this genus.…”