Analytical Methods for Quantification and Identification of Intact Glucosinolates in Arabidopsis Roots Using LC-QqQ(LIT)-MS/MS.

Kourosh Hooshmand,Inge S Fomsgaard

doi:10.3390/metabo11010047

Abstract

Glucosinolates are biologically active secondary metabolites in Brassicaceae plants that play a critical role in positive and negative interactions between plants and root-associated microbial communities. The aim of this study was to develop a reversed-phase liquid chromatography method to quantify and identify intact glucosinolates in the root of Arabidopsis thaliana (Arabidopsis) grown in non-sterile natural soil by using liquid chromatography-hybrid triple quadruple-linear ion trap (LC-QqQ(LIT)) mass spectrometry. The Synergi Fusion C18-based column was found to be effective for sufficient retention and separation of nine intact glucosinolates without the need for time-consuming desulfation or ion-pairing steps. Method validation results showed satisfactory inter-day and intra-day precision for all glucosinolates except for 4-hydroxyglucobrassicin. Good inter-day and intra-day accuracy and recovery results were observed for glucoiberin, gluconapin, glucobrassicin, 4-methoxyglucobrassicin and neoglucobrassicin. However, for 4-hydroxyglucobrassicin, glucoraphanin and glucoerucin corrections to quantification results might be necessary since the recovery and accuracy results were not optimal. Matrix effects were shown to have a negligible effect on the ionization of all target analytes. The established liquid chromatography–tandem mass spectrometry (LC-MS/MS) method was applied to quantify target intact glucosinolates in Arabidopsis root crude extract of four different wild-type accessions where differences in terms of concentration and composition of intact glucosinolates were observed. Employment of sensitive and selective precursor ion survey scan of m/z 97 in combination with the information-dependent acquisition (IDA) of the enhanced product ion (EPI) dependent scan (Prec97-IDA-EPI) using LC-QqQ(LIT) provided high confidence in structural characterization of diverse intact glucosinolate profiles in complex Arabidopsis root crude extract.

Highlights

Glucosinolates are anionic sulfur- and nitrogen-containing plant secondary metabolites that are largely limited to species within the family Crucifereae, including the model plant Arabidopsis thaliana [1]
Peak tailing for some of the glucosinolates (4msb (2), pOHB (3), 3but (4) and 4OHI3M (5)) was observed (Supplementary Figure S2), probably due to additional unwanted secondary interactions acid in both mobile phases A and B led to strong ion signal suppression in negative electrospray ionization (ESI) mode for the majority of the target compounds (Supplementary Figure S3B)
Our results are4 conof 18 sistent with the results of two different studies showing that the addition of acetic acid in mobile phases was more effective than formic acid in enhancing the signal-to-noise ratio phenolic acids andthe androgen receptor modulators in negative ion modeearly

Summary

Introduction

Glucosinolates are anionic sulfur- and nitrogen-containing plant secondary metabolites that are largely limited to species within the family Crucifereae, including the model plant Arabidopsis thaliana (hereafter Arabidopsis) [1]. Glucosinolates are characterized by having a common core structure containing a β-D-glucopyranose residue linked to a sulfated thiohydroximate (Figure 1). The biosynthesis of glucosinolates occurs in three independent steps; side-chain elongation (for methionine and phenylalanine-derived glucosinolates), core structure formation and secondary modification (Figure 1) [3]. Aliphatic glucosinolates undergo a wide range of secondary transformations, including oxidation, hydroxylation, alkenylations and benzoylations, Metabolites 2021, 11, 47 and secondary modification (Figure 1) [3]. Aliphatic glucosinolates undergo a wide range of secondary transformations, including oxidation, hydroxylation, alkenylations and benwhile for indole glucosinolates, secondary transformations include only hydroxylations zoylations, while for indole glucosinolates, secondary transformations include only hyand methoxylations (gray background in Figure 1)in[3]

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Conclusion