Identification of Auxin Metabolites in Brassicaceae by Ultra-Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry

Auxins are signaling molecules involved in multiple stages of plant growth and development. The levels of the most important auxin, indole-3-acetic acid (IAA), are regulated by the formation of amide and ester conjugates with amino acids and sugars. In this work, IAA and IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, along with some selected IAA metabolites, were studied in positive and negative electrospray ionization (ESI) modes, utilizing high-resolution mass spectrometry (HRMS) as a tool for their structural analysis. HRMS/MS spectra revealed the fragmentation patterns that enable us to identify IAA metabolites in plant extracts from eight vegetables of the Brassicaceae family using a fast and reliable ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QToF-MS) method. The accurate m/z (mass to charge) ratio and abundance of the molecular and fragment ions of the studied compounds in plant extracts matched those obtained from commercially available or synthesized compounds and confirmed the presence of IAA metabolites.

The metabolism of IAA occurs through two processes-the decarboxylative and the non-decarboxylative catabolism (Figure 1b). Decarboxylation of IAA is a minor catabolic pathway, catalyzed by plant peroxidases. Major degradation products that generate from IAA catabolism are indole-3-aldehyde (IAld), 2-oxindole-3-acetic acid (oxIAA), indole-3-carbinol (I3C), and indole-3carboxylic acid (ICOOH) [5]. Catabolism of IAA in Arabidopsis is usually performed through a non-decarboxylative process, involving oxidation steps of the indole ring of the free IAA or the IAA conjugates with glucose or aspartic acid (Asp) and glutamic acid (Glu) [5,7]. Plants produce various IAA-conjugated forms either with sugars (ester conjugates), or with amino acids, peptides, and proteins (amide conjugates), which contribute to many crucial processes within the plants, such as storage and transport of IAA [7,8], IAA catabolism [9], regulation of IAA levels [10], prevention against enzymatic degradation of IAA [11], and part of abiotic and biotic stress of plants [12,13].
Several amide conjugates with the amino acids leucine (Leu), alanine (Ala), Asp, and Glu have been identified in Arabidopsis [14,15], while amide conjugates with isoleucine (Ile), valine (Val), phenylalanine (Phe), Asp, Glu, and Ala have been quantified in rice [16]. Auxin conjugates with Asp, Glu, and tryptophan (Trp) have been identified in young tomato leaves, Ginkgo male flower, and coconut milk [17]. Pěnčík et al. [18] isolated IAA amino acid conjugates with Ala, Asp, Glu, Val, Leu, Phe, and glycine (Gly) from Helleborus niger by immunoaffinity extraction and quantified them using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample required pretreatment with diazomethane, prior to analysis, for the transformation of IAA conjugates into their corresponding methyl esters. This analytical protocol, although laborious, does not examine the possibility of the pre-existence of IAA amino acid methyl ester conjugates in the sample. Van Meulebroek et al. [19] identified IAA conjugates with aspartic acid dimethyl ester and glutamic acid dimethyl ester in tomato fruit, along with IAA conjugates of Ala, Asp, Trp, and Val using ultra-high performance liquid chromatography high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap-MS). In the same study, IAA conjugates with alanine methyl ester, glutamic acid dimethyl ester, isoleucine, Ala, and Asp were identified in tomato leaf [19]. As indicated in the literature, the MS study of IAA conjugates was performed in positive electrospray ionization (ESI) mode [16][17][18][19].
Considering that almost 90% of the total IAA in Arabidopsis are amide-linked conjugates with amino acids and peptides [5,20], a need has emerged for more extensive studies focused on the identification of auxin amide conjugates, which will contribute substantially to our knowledge and understanding of the overall mechanism of IAA. Despite the research on IAA conjugates in Arabidopsis, no reports yet exist in the literature for studies about other members of the Brassicaceae family. Plants produce various IAA-conjugated forms either with sugars (ester conjugates), or with amino acids, peptides, and proteins (amide conjugates), which contribute to many crucial processes within the plants, such as storage and transport of IAA [7,8], IAA catabolism [9], regulation of IAA levels [10], prevention against enzymatic degradation of IAA [11], and part of abiotic and biotic stress of plants [12,13].
Several amide conjugates with the amino acids leucine (Leu), alanine (Ala), Asp, and Glu have been identified in Arabidopsis [14,15], while amide conjugates with isoleucine (Ile), valine (Val), phenylalanine (Phe), Asp, Glu, and Ala have been quantified in rice [16]. Auxin conjugates with Asp, Glu, and tryptophan (Trp) have been identified in young tomato leaves, Ginkgo male flower, and coconut milk [17]. Pěnčík et al. [18] isolated IAA amino acid conjugates with Ala, Asp, Glu, Val, Leu, Phe, and glycine (Gly) from Helleborus niger by immunoaffinity extraction and quantified them using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). The sample required pretreatment with diazomethane, prior to analysis, for the transformation of IAA conjugates into their corresponding methyl esters. This analytical protocol, although laborious, does not examine the possibility of the pre-existence of IAA amino acid methyl ester conjugates in the sample. Van Meulebroek et al. [19] identified IAA conjugates with aspartic acid dimethyl ester and glutamic acid dimethyl ester in tomato fruit, along with IAA conjugates of Ala, Asp, Trp, and Val using ultra-high performance liquid chromatography high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap-MS). In the same study, IAA conjugates with alanine methyl ester, glutamic acid dimethyl ester, isoleucine, Ala, and Asp were identified in tomato leaf [19]. As indicated in the literature, the MS study of IAA conjugates was performed in positive electrospray ionization (ESI) mode [16][17][18][19].
Considering that almost 90% of the total IAA in Arabidopsis are amide-linked conjugates with amino acids and peptides [5,20], a need has emerged for more extensive studies focused on the identification of auxin amide conjugates, which will contribute substantially to our knowledge and understanding of the overall mechanism of IAA. Despite the research on IAA conjugates in Arabidopsis, no reports yet exist in the literature for studies about other members of the Brassicaceae family.
Identification and quantification of auxins and IAA conjugates is challenging, due to their low concentration (nmol g −1 to pmol g −1 fresh weight), interferences from other co-extracted substances, and differences in concentration levels between IAA and auxin conjugates [5]. High-resolution mass spectrometric (HRMS) techniques, such as quadrupole time-of-flight (QToF), provide high selectivity against matrix interferences, as well as accurate mass and structural information, allowing the screening, identification, and target analysis of compounds [21]. These techniques have also been previously applied in the elemental composition analysis of fragmentation ions [22][23][24][25] and in the identification of bioactive substances in plant extracts [26][27][28][29][30]. Ultra-high-performance liquid chromatography (UHPLC) allows physical separation of complex elutes. The speed, resolution, and sensitivity of UHPLC make it ideally suited for use with HRMS. The combination of HRMS with UHPLC provides a powerful technique with very high sensitivity [31].
Herein, we report the mass spectrometry study and the fragmentation pathways for 20 IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, as well as IAA and selected IAA metabolites (IAld, IAN, IAM, and 4-Cl-IAA), using high-resolution mass spectrometry in positive and negative ESI modes. The HRMS study and accurate masses enabled us to elucidate the major fragmentation pathways through the determination of elemental composition analyses of the fragment ions, and to develop a fast and reliable UPLC-QToF-MS method for the identification of the target class of compounds in plant extracts. The application of the method in methanolic extracts of the Brassicaceae family vegetables is also presented and a wide range of indoles is identified, which may permit further elucidation of Brassicaceae signaling pathways.

High-Resolution Mass Spectrometry Study
MS spectra for all compounds were recorded both in ESI positive and negative ion modes and are given in the Supplementary Materials file along with the extracted ion chromatograms. Characteristic ions that can be used for the identification of IAA metabolites are summarized in Table 1. For the proposed chemical formulae, Agilent MassHunter software was used, taking into account the nitrogen rule, the mass error being lower than 5 ppm, and the isotopic abundance distribution match. Identification and quantification of auxins and IAA conjugates is challenging, due to their low concentration (nmol g −1 to pmol g −1 fresh weight), interferences from other co-extracted substances, and differences in concentration levels between IAA and auxin conjugates [5]. High-resolution mass spectrometric (HRMS) techniques, such as quadrupole time-of-flight (QToF), provide high selectivity against matrix interferences, as well as accurate mass and structural information, allowing the screening, identification, and target analysis of compounds [21]. These techniques have also been previously applied in the elemental composition analysis of fragmentation ions [22][23][24][25] and in the identification of bioactive substances in plant extracts [26][27][28][29][30]. Ultra-high-performance liquid chromatography (UHPLC) allows physical separation of complex elutes. The speed, resolution, and sensitivity of UHPLC make it ideally suited for use with HRMS. The combination of HRMS with UHPLC provides a powerful technique with very high sensitivity [31].
Herein, we report the mass spectrometry study and the fragmentation pathways for 20 IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, as well as IAA and selected IAA metabolites (IAld, IAN, IAM, and 4-Cl-IAA), using high-resolution mass spectrometry in positive and negative ESI modes. The HRMS study and accurate masses enabled us to elucidate the major fragmentation pathways through the determination of elemental composition analyses of the fragment ions, and to develop a fast and reliable UPLC-QToF-MS method for the identification of the target class of compounds in plant extracts. The application of the method in methanolic extracts of the Brassicaceae family vegetables is also presented and a wide range of indoles is identified, which may permit further elucidation of Brassicaceae signaling pathways.

High-Resolution Mass Spectrometry Study
MS spectra for all compounds were recorded both in ESI positive and negative ion modes and are given in the Supplementary Materials file along with the extracted ion chromatograms. Characteristic ions that can be used for the identification of IAA metabolites are summarized in Table  1. For the proposed chemical formulae, Agilent MassHunter software was used, taking into account the nitrogen rule, the mass error being lower than 5 ppm, and the isotopic abundance distribution match. In positive ionization mode, indole compounds bearing an alkyl chain as the substituent in the pyrrole ring undergo a cleavage of the β-bond with respect to the aromatic system and lead to 3methylene-3H-indol-1-ium, 1 ( Figure 2). This ion can be rearranged to the more stable quinolinium cation, 2, with a calculated m/z 130.0651 [32,33]. MS 2 spectra of IAA and all studied IAA metabolites, generated from the protonated ions [M + H] + , showed the adoption of the same fragmentation pathway. 4-Chloro-indole acetic acid exhibited two chlorinated quinolinium cations with m/z 164.0252 and m/z 166.0231, respectively, in a ratio of 3:1, due to the presence of the two chlorine isotopes. Indole-3-carbaldehyde, lacking a β-bond, generated 1H-indol-1-ium, 3, with m/z 118.0651, due to the cleavage of the α-bond with respect to the pyrrole ring. Prinsen et al. [34] studied IAA and related metabolites in positive ESI mode and suggested the quinolinium cation as a diagnostic ion In positive ionization mode, indole compounds bearing an alkyl chain as the substituent in the pyrrole ring undergo a cleavage of the β-bond with respect to the aromatic system and lead to 3-methylene-3H-indol-1-ium, 1 ( Figure 2). This ion can be rearranged to the more stable quinolinium cation, 2, with a calculated m/z 130.0651 [32,33]. MS 2 spectra of IAA and all studied IAA metabolites, generated from the protonated ions [M + H] + , showed the adoption of the same fragmentation pathway. 4-Chloro-indole acetic acid exhibited two chlorinated quinolinium cations with m/z 164.0252 and m/z 166.0231, respectively, in a ratio of 3:1, due to the presence of the two chlorine isotopes. Indole-3-carbaldehyde, lacking a β-bond, generated 1H-indol-1-ium, 3, with m/z 118.0651, due to the cleavage of the α-bond with respect to the pyrrole ring. Prinsen et al. [34] studied IAA and related metabolites in positive ESI mode and suggested the quinolinium cation as a diagnostic ion for IAN, IAM, and IAld determination. The same cation, confirmed by the findings of the current HRMS study, for IAN, IAM, and IAld determination. The same cation, confirmed by the findings of the current HRMS study, has also been reported as the main fragment of amino acid conjugates IATrp [17], IAAla, IALeu, IAAsp, and IAGlu, [14], generated by the complete breakage of the molecules under the ESI conditions used.   The full scan spectra of all studied indole amide conjugates which have the amino acid either as a free carboxylic group or as a methyl ester presented the ions [M + H] + and [M + Na] + . Their MS 2 spectra generated from the protonated ions [M + H] + exhibited, apart from the quinolinium cation (exact mass: 130.0651), a second characteristic fragment assigned to the amino acid residue cation, in low to moderate abundances, due to the amide bond cleavage. The quinolinium cation alone is not a strong confirmation of the presence of IAA amino acid conjugates, because it can be generated from a variety of indole compounds that can undergo β-cleavage [32][33][34]. Nevertheless, since every amino acid has a unique nominal molecular mass, the fragment assigned to the amino acid along with the ions [M + H] + , [M + Na] + , and quinolinium may allow the identification of every IAA amino acid conjugate. As an example, the full scan and MS/MS spectra of indole-3-acetyl-l-serine and indole-3-acetyl-l-serine methyl ester in positive ESI mode are given in In the negative ion mode, IAA and all IAA metabolites exhibited the deprotonated molecular ions [M − H] − , which were selected as the precursor ions for the MS 2 spectra. IAA and IAM gave a fragment with a proposed chemical formula of C 9 H 8 N − (exact mass: 130.0662) corresponding to 3-methyleneindolin-1-ide, 4, formed after a β-cleavage of the aliphatic chain at C-3 position of the indole ( Figure 4). 4-Chloro-indole acetic acid due to the presence of the two chlorine isotopes exhibited two chlorinated fragments with m/z 164.0271 (exact mass: 164.0273, ∆ 1.2 ppm) and m/z 166.0246 (exact mass: 166.0244, ∆ 1.2 ppm), respectively, in a ratio of 3:1. It has been noticed that the quinolinium ion 2 with exact mass m/z 130.0651 or the 4-chlorinated one with m/z 164.0252 obtained in positive ion mode and 3-methyleneindolin-1-ide 4 with m/z 130.0662 or 4-chloromethyleneindolin-1-ide with m/z 164.0271 obtained in negative ion mode seem to be identical. However, they are different due to their opposite charge and their accurate masses are separated by two electrons. This was also observed in fragmentation pathways of nonsteroidal anti-inflammatory drugs [35]. The full scan spectra of all studied indole amide conjugates which have the amino acid either as a free carboxylic group or as a methyl ester presented the ions [M + H] + and [M + Na] + . Their MS 2 spectra generated from the protonated ions [M + H] + exhibited, apart from the quinolinium cation (exact mass: 130.0651), a second characteristic fragment assigned to the amino acid residue cation, in low to moderate abundances, due to the amide bond cleavage. The quinolinium cation alone is not a strong confirmation of the presence of IAA amino acid conjugates, because it can be generated from a variety of indole compounds that can undergo β-cleavage [32][33][34]. Nevertheless, since every amino acid has a unique nominal molecular mass, the fragment assigned to the amino acid along with the ions [M + H] + , [M + Na] + , and quinolinium may allow the identification of every IAA amino acid conjugate. As an example, the full scan and MS/MS spectra of indole-3-acetyl-L-serine and indole-3acetyl-L-serine methyl ester in positive ESI mode are given in  To the best of our knowledge, there are no existing literature reports on the negative ESI mass spectrometry study of auxin amino acid conjugates. In the MS 2 spectra of the amide conjugates carrying the amino acid with a free carboxylic group, a prominent fragment ion with a chemical formula depending on the amino acid was observed. These specific fragments were found to correspond to the anion form of the corresponding amino acid, formed as a result of the amide bond cleavage. Both the deprotonated and the fragment ions can be used for the identification and confirmation of the amino acid conjugates with IAA, since each amino acid has a different nominal molecular mass. As an example, in negative ESI mode, indole-3-acetyl-L-serine can be identified by  To the best of our knowledge, there are no existing literature reports on the negative ESI mass spectrometry study of auxin amino acid conjugates. In the MS 2 spectra of the amide conjugates carrying the amino acid with a free carboxylic group, a prominent fragment ion with a chemical formula depending on the amino acid was observed. These specific fragments were found to correspond to the anion form of the corresponding amino acid, formed as a result of the amide bond cleavage. Both the deprotonated and the fragment ions can be used for the identification and confirmation of the amino acid conjugates with IAA, since each amino acid has a different nominal molecular mass. From the present mass spectrometry study, it can be concluded that auxin IAA amide conjugates generated diagnostic amino acid-specific fragment ions, both in positive and negative ion modes, which permit their identification. However, especially for IAA conjugates, positive ion mode seems to be more helpful, although both modes showed almost the same level of reliability and accuracy. In positive ion mode, all IAA conjugates studied, apart from the protonated molecular ion and adduct with sodium, additional diagnostic fragments, fragment 2 related to the presence of the indole ring, and an amino acid-specific fragment, were recorded. Thus, both the indole ring and the amino acid are detected. In negative ion mode, apart from the deprotonated molecular ions, conjugates with an amino acid bearing a free carboxylic group generated the amino acid-specific fragment ion, while conjugates with an amino acid bearing a methyl ester group generated the diagnostic for the indole ring fragment 5. From the present mass spectrometry study, it can be concluded that auxin IAA amide conjugates generated diagnostic amino acid-specific fragment ions, both in positive and negative ion modes, which permit their identification. However, especially for IAA conjugates, positive ion mode seems to be more helpful, although both modes showed almost the same level of reliability and accuracy. In positive ion mode, all IAA conjugates studied, apart from the protonated molecular ion and adduct with sodium, additional diagnostic fragments, fragment 2 related to the presence of the indole ring, and an amino acid-specific fragment, were recorded. Thus, both the indole ring and the amino acid are detected. In negative ion mode, apart from the deprotonated molecular ions, conjugates with an amino acid bearing a free carboxylic group generated the amino acid-specific fragment ion, while conjugates with an amino acid bearing a methyl ester group generated the diagnostic for the indole ring fragment 5.

Analysis of Brassicaceae Vegetables
The Brassicaceae family utilizes the indole ring to produce a diverse set of compounds such as glucosinolates (glucobrassicin), phytoalexins (camalexin), and the phytohormone IAA. The active amount of IAA is tightly regulated by metabolic processes and IAA amino acid conjugates are classified, up to now, as storage and catabolism molecules.
Since the study of IAA amide conjugates in the Brassicaceae family is limited in Arabidopsis species and within our project dedicated to the analysis of phytonutrients in cruciferous vegetables [32,[36][37][38], we decided to apply our high-resolution mass spectrometry study of the IAA metabolites to explore the presence of IAA amide conjugates in other members of the Brassicaceae family. We studied extracts from eight vegetables, namely, Brassica oleracea L. var.  Table 1.
Extracted ion chromatograms for a standard solution of IASer and for a purple broccoli extract are presented in Figure 6  The compounds identified in the examined cruciferous vegetable extracts applying our methodology are summarized in Table 2. IAA, IAld, and IAAla were detected in all cruciferous vegetables. 4-Cl-IAA was found only in B. oleracea var. rubra and R. raphanistrum subsp. sativus. The intermediate in IAA biosynthesis, IAN, was absent only in E. sativa and IAM was present in B. oleracea var. italica cv. Viοlleto and R. raphanistrum subsp. sativus. IATrp, IAVal, IAPhe, IAVal-Me, and IASer-Me were found in almost all Brassicaceae species studied, whereas IATrp-Me and IAMet-Me were absent in all cases. IAMet was detected in B. oleracea var. rubra and B. oleracea var. capitata. In addition, conjugates with amino acids tyrosine (R. raphanistrum subsp. sativus), glycine (in B. oleracea var. capitata, R. raphanistrum subsp. Sativus, and E. sativa), aspartic acid (B. rapa subsp. rapifera), and glutamic acid (in B. rapa subsp. rapifera, B. oleracea var. italica cv. Calabrese, and R. raphanistrum subsp. sativus), as well as their corresponding methyl esters, were detected in some cases. It must be noted that indole-3-acetyl-aspartic acid and indole-3-acetyl-glutamic acid dimethyl esters were detected only in B. oleracea var. capitata. IASer was detected in purple broccoli and radish, whereas IAAla-Me was detected in B. rapa subsp. rapifera, B. oleracea var. italica cv. Calabrese, and R. raphanistrum subsp. sativus. The compounds identified in the examined cruciferous vegetable extracts applying our methodology are summarized in Table 2. IAA, IAld, and IAAla were detected in all cruciferous vegetables. 4-Cl-IAA was found only in B. oleracea var. rubra and R. raphanistrum subsp. sativus. The intermediate in IAA biosynthesis, IAN, was absent only in E. sativa and IAM was present in B. oleracea var. italica cv. Violleto and R. raphanistrum subsp. sativus. IATrp, IAVal, IAPhe, IAVal-Me, and IASer-Me were found in almost all Brassicaceae species studied, whereas IATrp-Me and IAMet-Me were absent in all cases. IAMet was detected in B. oleracea var. rubra and B. oleracea var. capitata. In addition, conjugates with amino acids tyrosine (R. raphanistrum subsp. sativus), glycine (in B. oleracea var. capitata, R. raphanistrum subsp. Sativus, and E. sativa), aspartic acid (B. rapa subsp. rapifera), and glutamic acid (in B. rapa subsp. rapifera, B. oleracea var. italica cv. Calabrese, and R. raphanistrum subsp. sativus), as well as their corresponding methyl esters, were detected in some cases. It must be noted that indole-3-acetyl-aspartic acid and indole-3-acetyl-glutamic acid dimethyl esters were detected only in B. oleracea var. capitata. IASer was detected in purple broccoli and radish, whereas IAAla-Me was detected in B. rapa subsp. rapifera, B. oleracea var. italica cv. Calabrese, and R. raphanistrum subsp. sativus.
Up to now, little has been known regarding the spectrum of IAA conjugates present in a given plant tissue, mainly due to the difficulties of their individual analysis. Most of the knowledge is based on the total IAA obtained after hydrolysis of its conjugates. Studies in the dicot Arabidopsis have shown a remarkable difference between the total IAA and free IAA, indicating the presence of a considerable amount of IAA conjugates [20]. Since only IAA conjugates with aspartic acid and glutamic acid were determined in that work by gas chromatography-mass spectrometry (GC/MS) at low concentrations, it was concluded that the major part (98%) of amide conjugates remain to be identified [20]. Kowalczyk and Sandberg, using also an indirect GC/MS method, found that the content of IAA conjugates with Leu, Asp, Glu, and Ala did not significantly contribute to the total IAA in Arabidopsis [14]. Pěnčík et al. quantified IAA amino acid conjugates with Ala, Asp, Glu, Val, Leu, Phe, and Gly from a higher plant, Helleborus niger, using HPLC-MS/MS in positive ion mode and they clearly demonstrated the vast difference (of about four orders of magnitude) between the contents of free IAA and the IAA conjugate with Ala, the least abundant of the quantified conjugates [18]. Thus, the development of methodologies employing techniques such as HRMS will help in the exploration of IAA conjugates. Developing such an approach, we can identify a series of IAA conjugates in cruciferous vegetables. The very low concentrations of IAA conjugates in the plant tissues could be a limitation of our methodology, one that can possibly be overcome if a larger quantity of the lyophilized vegetable in the extraction step is used.

General Experimental Procedures
The HRMS spectra were recorded on an Agilent 6530 Quadrupole Time of Flight LC-MS system (UPLC-QToF-MS), with an ESI source, coupled with Agilent 1290 Infinity UPLC system and an autosampler (Agilent Technologies, Santa Clara, CA, USA). Nitrogen was used as the collision gas and electrospray ionization (ESI)-negative and positive mode-was used for the MS experiments. The data acquisition was carried out with Agilent MassHunter software (version B.06.00). The following QToF conditions were used: drying gas, 12 L/min; gas temperature, 300 • C; fragmentor, 170V; skimmer, 65 V; capillary voltage, 4000 V; nebulizer gas, 45 psi; acquisition rate, 1 spectra/s (threshold 200 Abs, 0.01% rel.); MS scan range, 50-1500. For the MS/MS experiments, an auto-MS/MS method was developed with the following parameters: MS/MS acquisition rate, 1 spectra/s (threshold 5 Abs, 0.01% rel.); MS/MS scan range, 50-1500; collision energy slope, 5 V; offset, 2.5 V; preferred charge state, 2, 1, unknown. The mass accuracy of the QToF-MS was calibrated before each analysis using a reference solution for scanning up to m/z 1500. Mass calibration of the QToF-MS was controlled by constant infusion of a reference mass solution (obtained from Agilent Technologies) into the source of the QToF-MS during the analysis. The ions selected were the reference ions 121.0509 and 922.0098 for positive ESI mode, whereas in negative ESI mode, the selected reference ions were 112.9856 and 1033.9881. The raw data files were processed with Agilent MassHunter Qualitative Analysis software (version B.07.00).
The extraction from cruciferous vegetables was performed using a simple extraction method with 80% MeOH, followed by classical filtration. The simultaneous identification of the studied compounds using UPLC-QToF-MS was conducted in negative and positive ESI mode. Identification was based on retention time relative to that of the standard compound, the accurate mass of ions, and their MS/MS spectra.

Reagents and Materials
Indole-3-acetic acid was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). 4-Chloroindole-3-acetic acid was purchased from Fluorochem Ltd. (Derbyshire, UK). MeOH and formic acid (LC-MS grade) were obtained from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All indole amide conjugates were synthesized by a coupling reaction between IAA and the corresponding amino acid methyl esters [36]. IAld was synthesized by Vilsmeier-Haack reaction, from indole and oxalyl chloride in dimethylformamide [39]. IAN was synthesized from IAld by treatment with sodium borohydride and potassium cyanide [40]. IAM was synthesized from hydrolysis of IAN with sodium hydroxide [41]. Ultra-pure water was provided by MilliQ purification system (Millipore Direct-Q, Bedford, MA, USA). A Heidolph 2 rotary evaporator (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany) and Whatman filter paper grade 1 (Whatman Ltd., Maidstone, UK) were used. were used for the preparation of extracts. Samples were lyophilized and ground to a fine homogenous powder using mortar and pestle.

Preparation of Extracts
The preparation of extracts was performed according to the literature [16] with the following modifications: 1 g of dry tissue was extracted at 4 • C for 24 h with 20 mL of 80% MeOH. The solvent was collected, and the residue re-extracted with 20 mL of 80% MeOH for 30 min at 4 • C. The organic layers were combined, dried with 1 g of anhydrous sodium sulfate, and filtered using Whatman filter paper grade 1. After filtration, the extract was evaporated to dryness at 35 • C under reduced vacuum using a rotary evaporator. The residue was dissolved in 1 mL MeOH. A 50 µL amount of the extract was diluted with 50 µL of MeOH and then injected into the LC-MS system.

Conclusions
We employed high-resolution mass spectrometry as an instrumental technique for the structural elucidation of indole acetic acid major metabolites found in plants. The molecular and fragment ions of IAA and IAA metabolites, particularly of IAA amide conjugates with amino acids bearing a free carboxylic group or a methyl ester group, were described and 25 compounds were identified in Brassicaceae using an easy, rapid, and accurate UPLC-QToF-MS analytical protocol in a single run. To the best of our knowledge, this is the first report employing high-resolution mass spectrometry especially for the study of IAA amide conjugates, in cruciferous vegetables. This work will provide a helpful methodology for further study of previously undescribed plant hormone conjugates in plant tissues and could open new pathways to generate a whole-plant organ distribution map of IAA and IAA metabolites depending on the developmental stage of the plant. Funding: This research received no external funding.

Conflicts of Interest:
The authors declare no conflict of interest.