Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS
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...
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MDPI AG
2021-01-01
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| Online Access: | https://www.mdpi.com/2218-1989/11/1/47 |
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| author | Kourosh Hooshmand Inge S. Fomsgaard |
| author_facet | Kourosh Hooshmand Inge S. Fomsgaard |
| author_sort | Kourosh Hooshmand |
| collection | DOAJ |
| description | 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 <i>Arabidopsis thaliana</i> (<i>Arabidopsis</i>) 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 <i>Arabidopsis</i> 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 <i>m</i>/<i>z</i> 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 <i>Arabidopsis</i> root crude extract. |
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| language | English |
| last_indexed | 2024-03-09T05:16:26Z |
| publishDate | 2021-01-01 |
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| series | Metabolites |
| spelling | doaj.art-dad094a74a7e4f6bba199ceaa9f890e72023-12-03T12:45:49ZengMDPI AGMetabolites2218-19892021-01-011114710.3390/metabo11010047Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MSKourosh Hooshmand0Inge S. Fomsgaard1Department of Agroecology, Aarhus University, DK-4200 Slagelse, DenmarkDepartment of Agroecology, Aarhus University, DK-4200 Slagelse, DenmarkGlucosinolates 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 <i>Arabidopsis thaliana</i> (<i>Arabidopsis</i>) 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 <i>Arabidopsis</i> 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 <i>m</i>/<i>z</i> 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 <i>Arabidopsis</i> root crude extract.https://www.mdpi.com/2218-1989/11/1/47intact glucosinolatesreversed-phase liquid chromatography (RPLC)triple quadrupole-linear ion trap mass spectrometrymultiple reaction monitoring (MRM)method validationglucosinolate profiling |
| spellingShingle | Kourosh Hooshmand Inge S. Fomsgaard Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS Metabolites intact glucosinolates reversed-phase liquid chromatography (RPLC) triple quadrupole-linear ion trap mass spectrometry multiple reaction monitoring (MRM) method validation glucosinolate profiling |
| title | Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS |
| title_full | Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS |
| title_fullStr | Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS |
| title_full_unstemmed | Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS |
| title_short | Analytical Methods for Quantification and Identification of Intact Glucosinolates in <i>Arabidopsis</i> Roots Using LC-QqQ(LIT)-MS/MS |
| title_sort | analytical methods for quantification and identification of intact glucosinolates in i arabidopsis i roots using lc qqq lit ms ms |
| topic | intact glucosinolates reversed-phase liquid chromatography (RPLC) triple quadrupole-linear ion trap mass spectrometry multiple reaction monitoring (MRM) method validation glucosinolate profiling |
| url | https://www.mdpi.com/2218-1989/11/1/47 |
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