Thiols are a class of organic compounds, which play a crucial role in maintenance of the redox homeostasis in plants. Furthermore, they are involved in oxidative stress response and in regulation of cellular metabolism. These compounds are characterized by a nucleophilic ˗SH group and they represent the principal source of reduced sulfur in plants, in the forms of protein thiols and low molecular weight (LMW) thiols. The latter are involved in deactivation of xenobiotics, in enzymatic reactions and in the modification of redox state of sensitive molecules in plants. Several analytical methods were developed for the quantification of thiols and disulfides in different matrices. The most studied thiols in plants are glutathione and cysteine, whereas the knowledge related to other LMW thiols are limited. They are biological thiols, such as homocysteine, cysteamine and cysteinylglycine, or volatile thiols, studied in food and beverages (e.g. wine) for their flavouring properties. Recent studies show that a huge amount of LMW thiols exists, but several of them still need to be identified and, due to their low concentration in plants, their identification represent a major challenge. In order to deepen the study of this class of compounds, in this work two new analytical methods, for the identification and quantification of thiols, were carried out through the utilization of mass spectrometry techinques. After the derivatisation with 4,4’-dithiodipyridine (DTDP) of plant extracts, samples were analysed by high performance liquid chromatography (HPLC) coupled with triple quadrupole (QqQ) mass spectrometer. 6 The preventive analysis of authentic thiol standards led to the definition of instrumental parameters and to 4-TP-thiol-derivatives fragmentation pattern. Compounds as glutathione, cysteine and cysteinylglycine were used as references in order to define a fragmentation rule for the development of both quantification and identification methods. The quantification methods was developed through the analysis in SRM mode of authentic thiol standards. The fragmentation pattern for each compounds were defined after the direct infusion of standards in QqQ. This method allows to determine the concentration level of glutathione, glutathione disulfide, cysteine, cystine, cysteamine, cysteinylglycine and homocysteine in plant extracts. The method was validated for cauliflower leaves, rocket, garlic, onion and walnut, showing high sensitivity and selectivity. The identification method was developed through the distinctive fragmentation spectra produced by 4-TP-thiols-derivatives, characterised by the formation of m/z 144, m/z 112, m/z 111 or the neutral loss of 111 from the 4-TP part of the molecules. These signals were used as markers to confirm the presence of thiol-containing compounds in plant extracts. By the analysis of cauliflower leaves, rocket, garlic and onion extracts in precursor ion scan and neutral loss scan, a list of m/z values was obtained for each matrix. Unknown thiols were identified with the exact molecular mass and molecular formula determined using high-resolution mass spectrometer (HRMS) quadrupole-time of flight (QTOF). Identified compounds were confirmed by the analysis of authentic standards in SRM scan. Furthermore, any matrix was spiked with the authentic standard and then analysed after derivatisation. Identification method was applied to different Brassicaceae species from the Botanical Garden of the University of Padova, showing distinct thiol compositions, with several species-specific compounds.

Method development for thiols analysis: identification and quantitation in plant products through mass spectrometry techniques / Millan, Silvia. - (2020 Jul 27).

Method development for thiols analysis: identification and quantitation in plant products through mass spectrometry techniques

Millan, Silvia
2020-07-27

Abstract

Thiols are a class of organic compounds, which play a crucial role in maintenance of the redox homeostasis in plants. Furthermore, they are involved in oxidative stress response and in regulation of cellular metabolism. These compounds are characterized by a nucleophilic ˗SH group and they represent the principal source of reduced sulfur in plants, in the forms of protein thiols and low molecular weight (LMW) thiols. The latter are involved in deactivation of xenobiotics, in enzymatic reactions and in the modification of redox state of sensitive molecules in plants. Several analytical methods were developed for the quantification of thiols and disulfides in different matrices. The most studied thiols in plants are glutathione and cysteine, whereas the knowledge related to other LMW thiols are limited. They are biological thiols, such as homocysteine, cysteamine and cysteinylglycine, or volatile thiols, studied in food and beverages (e.g. wine) for their flavouring properties. Recent studies show that a huge amount of LMW thiols exists, but several of them still need to be identified and, due to their low concentration in plants, their identification represent a major challenge. In order to deepen the study of this class of compounds, in this work two new analytical methods, for the identification and quantification of thiols, were carried out through the utilization of mass spectrometry techinques. After the derivatisation with 4,4’-dithiodipyridine (DTDP) of plant extracts, samples were analysed by high performance liquid chromatography (HPLC) coupled with triple quadrupole (QqQ) mass spectrometer. 6 The preventive analysis of authentic thiol standards led to the definition of instrumental parameters and to 4-TP-thiol-derivatives fragmentation pattern. Compounds as glutathione, cysteine and cysteinylglycine were used as references in order to define a fragmentation rule for the development of both quantification and identification methods. The quantification methods was developed through the analysis in SRM mode of authentic thiol standards. The fragmentation pattern for each compounds were defined after the direct infusion of standards in QqQ. This method allows to determine the concentration level of glutathione, glutathione disulfide, cysteine, cystine, cysteamine, cysteinylglycine and homocysteine in plant extracts. The method was validated for cauliflower leaves, rocket, garlic, onion and walnut, showing high sensitivity and selectivity. The identification method was developed through the distinctive fragmentation spectra produced by 4-TP-thiols-derivatives, characterised by the formation of m/z 144, m/z 112, m/z 111 or the neutral loss of 111 from the 4-TP part of the molecules. These signals were used as markers to confirm the presence of thiol-containing compounds in plant extracts. By the analysis of cauliflower leaves, rocket, garlic and onion extracts in precursor ion scan and neutral loss scan, a list of m/z values was obtained for each matrix. Unknown thiols were identified with the exact molecular mass and molecular formula determined using high-resolution mass spectrometer (HRMS) quadrupole-time of flight (QTOF). Identified compounds were confirmed by the analysis of authentic standards in SRM scan. Furthermore, any matrix was spiked with the authentic standard and then analysed after derivatisation. Identification method was applied to different Brassicaceae species from the Botanical Garden of the University of Padova, showing distinct thiol compositions, with several species-specific compounds.
4,4’-dithiodipyridine, thiol derivatisation, vegetable, identification, quantification, mass spectrometry
Method development for thiols analysis: identification and quantitation in plant products through mass spectrometry techniques / Millan, Silvia. - (2020 Jul 27).
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11577/3425916
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