Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

Peng Che; J. Tyler Davidson; Jeroen Kool; Isabelle Kohler

doi:10.1016/j.aca.2023.341962

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

Peng Che
, J. Tyler Davidson
, Jeroen Kool
, Isabelle Kohler^*

^*Corresponding author for this work

Research output: Contribution to Journal › Article › Academic › peer-review

Abstract

Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS²) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS². Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

Original language	English
Article number	341962
Pages (from-to)	1-12
Number of pages	12
Journal	Analytica Chimica Acta
Volume	1283
Early online date	26 Oct 2023
DOIs	https://doi.org/10.1016/j.aca.2023.341962
Publication status	Published - 1 Dec 2023

Bibliographical note

Funding Information:
M1 and M2: The protonated molecules of these two metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. In the CID MS2 mass spectrum of M1, the product ion at m/z 190.1244 (C12H16NO+, Δ = 1.2 mDa) was formed from an initial loss of water and led to the formation of product ions at m/z 172.1173 (C12H14N+, Δ = 4.6 mDa) and m/z 160.1204 (C11H14N+, Δ = 7.7 mDa) through the sequential loss of water or formaldehyde, respectively. Therefore, the hydroxyl was introduced to the tolyl functionality of 4-MEC, which was also reported by Helfer et al. [34]. Additional fragmentation leading to the formation of product ions at m/z 144.0840 (C10H10N+), m/z 132.0844 (C9H10N+), m/z 131.0757 (C9H9N+•), and m/z 117.0639 (C8H7N+•) provides further support for the location of the hydroxyl on the tolyl functionality of 4-MEC. Such fragmentation was also observed in the EAD product ion spectrum of M1, even if the product ion at m/z 190.1244 was less abundant (less than 2% of the total abundance). In addition, the EAD product ion spectrum was dominated by the product ions at m/z 164.0848 (C10H12O2+•, Δ = 1.0 mDa) and m/z 193.1112 (C11H15NO2+•, Δ = 0.9 mDa), which were not observed in the CID product ion spectrum (see Fig. 5a). The CID MS2 mass spectrum of M2 showed a highly abundant product ion at m/z 119.0499 (C8H7O+, Δ = 0.2 mDa), which indicated the hydroxyl group was neither introduced to the aromatic ring nor the tolyl functionality. However, the CID spectrum was not sufficient to elucidate the location of the other hydroxyl group on the molecule. Pedersen et al. also faced this issue when elucidating the structure of one hydroxylated metabolite of methylone (i.e., methylenedioxymethcathinone), whose CID product ion spectrum was dominated by only one abundant ion at m/z 149.0243 (C8H5O3+, Δ = 0.4 mDa). They therefore proposed two possible structures for that metabolite [35]. This is where EAD can play a role as a supplementary tool to provide additional structural information. Indeed, the EAD spectrum of M2 was exclusively dominated by a radical product ion at m/z 148.0896 (C10H12O+•, Δ = 0.7 mDa), supporting that the hydroxyl group was introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5b).M6: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M2, the CID product ion spectrum of this di-hydroxylated metabolite was dominated by the product ion at m/z 135.0452 (C8H7O2+, Δ = 0.6 mDa), which indicated the occurrence of aromatic hydroxylation but did not give sufficient information on the location of the other hydroxyl group. The presence of product ions in the CID MS2 mass spectrum at m/z 107.0499 (C7H7O+, Δ = 0.3 mDa) and m/z 89.0396 (C7H5+, Δ = 0.5 mDa) provides further support for aromatic hydroxylation through the loss of CO and water, respectively. The radical product ion at m/z 164.0842 (C10H12O2+•, Δ = 0.4 mDa) exclusively detected in the EAD MS2 mass spectrum indicated that the hydroxyl group was most likely introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5f).M13: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M6, the EAD MS2 product ion mass spectrum provided a complementary tool to the CID MS2 mass spectrum in support of hydroxylation on the amine and aromatic functionalities (Fig. S1g).M14-M16: The protonated molecules of these metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. The diagnostic ions in the CID fragmentation patterns of M14, such as the product ions at m/z 190.1278 (C12H16NO+, Δ = 4.6 mDa), 172.1134 (C12H14N+, Δ = 0.7 mDa) and 160.1133 (C11H14N+, Δ = 0.6 mDa), are observed, therefore the hydroxyl group was introduced to at the tolyl functionality of 2-MEC. Interestingly, the CID product ion spectrum of M14 displayed an abundant iminium ion at m/z 72.0822 (C4H10N+, Δ = 0.8 mDa) consistent with the fragmentation for other N-alkylated synthetic cathinones [31,37]. In comparison, the EAD product ion spectrum of this metabolite was dominated by additional product ions at m/z 190.1290 (C12H16NO+, Δ = 5.8 mDa), m/z 119.0513 (C8H7O+), and m/z 91.0589 (C7H7+), which were much more abundant than that in the EAD spectra of M1 (Fig. 5a). and M7 (Fig. S2a). The diagnostic product ions at m/z 161.0844 (C10H11NO+•, Δ = 0.3 mDa) and m/z 135.0813 (C9H11O+, Δ = 0.3 mDa) in the CID MS2 mass spectrum of M15 indicate the hydroxyl group was likely to be introduced to the aromatic ring. The diagnostic ion at m/z 135.0456 (C8H7O2+, Δ = 0.9 mDa) in the EAD MS2 mass spectrum of M15 provides further support for this elucidation (Fig. S2b). The CID MS2 mass spectrum of M16 is similar to M2 and M8, whose product ion spectra were dominated by the product ion at m/z 119.0501 (C8H7O+, Δ = 0.4 mDa). However, the EAD MS2 mass spectrum of this metabolite failed to provide helpful elucidation information in contrast to EAD MS2 mass spectra of M2 and M8. Based on the empirical results of M2 and M8, the hydroxyl group was introduced to the amine group of M16 (Fig. S2c).Peng Che was funded by a China Scholarship Council (CSC) fellowship (No.202006210045). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation.

Funding Information:
Peng Che was funded by a China Scholarship Council (CSC) fellowship (No. 202006210045 ). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation.

Publisher Copyright:
© 2023 The Authors

Funding

M1 and M2: The protonated molecules of these two metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. In the CID MS2 mass spectrum of M1, the product ion at m/z 190.1244 (C12H16NO+, Δ = 1.2 mDa) was formed from an initial loss of water and led to the formation of product ions at m/z 172.1173 (C12H14N+, Δ = 4.6 mDa) and m/z 160.1204 (C11H14N+, Δ = 7.7 mDa) through the sequential loss of water or formaldehyde, respectively. Therefore, the hydroxyl was introduced to the tolyl functionality of 4-MEC, which was also reported by Helfer et al. [34]. Additional fragmentation leading to the formation of product ions at m/z 144.0840 (C10H10N+), m/z 132.0844 (C9H10N+), m/z 131.0757 (C9H9N+•), and m/z 117.0639 (C8H7N+•) provides further support for the location of the hydroxyl on the tolyl functionality of 4-MEC. Such fragmentation was also observed in the EAD product ion spectrum of M1, even if the product ion at m/z 190.1244 was less abundant (less than 2% of the total abundance). In addition, the EAD product ion spectrum was dominated by the product ions at m/z 164.0848 (C10H12O2+•, Δ = 1.0 mDa) and m/z 193.1112 (C11H15NO2+•, Δ = 0.9 mDa), which were not observed in the CID product ion spectrum (see Fig. 5a). The CID MS2 mass spectrum of M2 showed a highly abundant product ion at m/z 119.0499 (C8H7O+, Δ = 0.2 mDa), which indicated the hydroxyl group was neither introduced to the aromatic ring nor the tolyl functionality. However, the CID spectrum was not sufficient to elucidate the location of the other hydroxyl group on the molecule. Pedersen et al. also faced this issue when elucidating the structure of one hydroxylated metabolite of methylone (i.e., methylenedioxymethcathinone), whose CID product ion spectrum was dominated by only one abundant ion at m/z 149.0243 (C8H5O3+, Δ = 0.4 mDa). They therefore proposed two possible structures for that metabolite [35]. This is where EAD can play a role as a supplementary tool to provide additional structural information. Indeed, the EAD spectrum of M2 was exclusively dominated by a radical product ion at m/z 148.0896 (C10H12O+•, Δ = 0.7 mDa), supporting that the hydroxyl group was introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5b).M6: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M2, the CID product ion spectrum of this di-hydroxylated metabolite was dominated by the product ion at m/z 135.0452 (C8H7O2+, Δ = 0.6 mDa), which indicated the occurrence of aromatic hydroxylation but did not give sufficient information on the location of the other hydroxyl group. The presence of product ions in the CID MS2 mass spectrum at m/z 107.0499 (C7H7O+, Δ = 0.3 mDa) and m/z 89.0396 (C7H5+, Δ = 0.5 mDa) provides further support for aromatic hydroxylation through the loss of CO and water, respectively. The radical product ion at m/z 164.0842 (C10H12O2+•, Δ = 0.4 mDa) exclusively detected in the EAD MS2 mass spectrum indicated that the hydroxyl group was most likely introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5f).M13: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M6, the EAD MS2 product ion mass spectrum provided a complementary tool to the CID MS2 mass spectrum in support of hydroxylation on the amine and aromatic functionalities (Fig. S1g).M14-M16: The protonated molecules of these metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. The diagnostic ions in the CID fragmentation patterns of M14, such as the product ions at m/z 190.1278 (C12H16NO+, Δ = 4.6 mDa), 172.1134 (C12H14N+, Δ = 0.7 mDa) and 160.1133 (C11H14N+, Δ = 0.6 mDa), are observed, therefore the hydroxyl group was introduced to at the tolyl functionality of 2-MEC. Interestingly, the CID product ion spectrum of M14 displayed an abundant iminium ion at m/z 72.0822 (C4H10N+, Δ = 0.8 mDa) consistent with the fragmentation for other N-alkylated synthetic cathinones [31,37]. In comparison, the EAD product ion spectrum of this metabolite was dominated by additional product ions at m/z 190.1290 (C12H16NO+, Δ = 5.8 mDa), m/z 119.0513 (C8H7O+), and m/z 91.0589 (C7H7+), which were much more abundant than that in the EAD spectra of M1 (Fig. 5a). and M7 (Fig. S2a). The diagnostic product ions at m/z 161.0844 (C10H11NO+•, Δ = 0.3 mDa) and m/z 135.0813 (C9H11O+, Δ = 0.3 mDa) in the CID MS2 mass spectrum of M15 indicate the hydroxyl group was likely to be introduced to the aromatic ring. The diagnostic ion at m/z 135.0456 (C8H7O2+, Δ = 0.9 mDa) in the EAD MS2 mass spectrum of M15 provides further support for this elucidation (Fig. S2b). The CID MS2 mass spectrum of M16 is similar to M2 and M8, whose product ion spectra were dominated by the product ion at m/z 119.0501 (C8H7O+, Δ = 0.4 mDa). However, the EAD MS2 mass spectrum of this metabolite failed to provide helpful elucidation information in contrast to EAD MS2 mass spectra of M2 and M8. Based on the empirical results of M2 and M8, the hydroxyl group was introduced to the amine group of M16 (Fig. S2c).Peng Che was funded by a China Scholarship Council (CSC) fellowship (No.202006210045). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation. Peng Che was funded by a China Scholarship Council (CSC) fellowship (No. 202006210045 ). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation.

Keywords

Collision-induced dissociation
Electron activated dissociation
Liquid chromatography
Mass spectrometry
Phase I metabolite identification
Positional isomers
Synthetic cathinones

Access to Document

10.1016/j.aca.2023.341962

Persistent URL (handle)

Persistent link

Cite this

@article{ae2d1e6d6091416d9c91a22dbd0831ae,

title = "Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers",

abstract = "Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.",

keywords = "Collision-induced dissociation, Electron activated dissociation, Liquid chromatography, Mass spectrometry, Phase I metabolite identification, Positional isomers, Synthetic cathinones",

author = "Peng Che and Davidson, \{J. Tyler\} and Jeroen Kool and Isabelle Kohler",

note = "Funding Information: M1 and M2: The protonated molecules of these two metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. In the CID MS2 mass spectrum of M1, the product ion at m/z 190.1244 (C12H16NO+, Δ = 1.2 mDa) was formed from an initial loss of water and led to the formation of product ions at m/z 172.1173 (C12H14N+, Δ = 4.6 mDa) and m/z 160.1204 (C11H14N+, Δ = 7.7 mDa) through the sequential loss of water or formaldehyde, respectively. Therefore, the hydroxyl was introduced to the tolyl functionality of 4-MEC, which was also reported by Helfer et al. [34]. Additional fragmentation leading to the formation of product ions at m/z 144.0840 (C10H10N+), m/z 132.0844 (C9H10N+), m/z 131.0757 (C9H9N+•), and m/z 117.0639 (C8H7N+•) provides further support for the location of the hydroxyl on the tolyl functionality of 4-MEC. Such fragmentation was also observed in the EAD product ion spectrum of M1, even if the product ion at m/z 190.1244 was less abundant (less than 2\% of the total abundance). In addition, the EAD product ion spectrum was dominated by the product ions at m/z 164.0848 (C10H12O2+•, Δ = 1.0 mDa) and m/z 193.1112 (C11H15NO2+•, Δ = 0.9 mDa), which were not observed in the CID product ion spectrum (see Fig. 5a). The CID MS2 mass spectrum of M2 showed a highly abundant product ion at m/z 119.0499 (C8H7O+, Δ = 0.2 mDa), which indicated the hydroxyl group was neither introduced to the aromatic ring nor the tolyl functionality. However, the CID spectrum was not sufficient to elucidate the location of the other hydroxyl group on the molecule. Pedersen et al. also faced this issue when elucidating the structure of one hydroxylated metabolite of methylone (i.e., methylenedioxymethcathinone), whose CID product ion spectrum was dominated by only one abundant ion at m/z 149.0243 (C8H5O3+, Δ = 0.4 mDa). They therefore proposed two possible structures for that metabolite [35]. This is where EAD can play a role as a supplementary tool to provide additional structural information. Indeed, the EAD spectrum of M2 was exclusively dominated by a radical product ion at m/z 148.0896 (C10H12O+•, Δ = 0.7 mDa), supporting that the hydroxyl group was introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5b).M6: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M2, the CID product ion spectrum of this di-hydroxylated metabolite was dominated by the product ion at m/z 135.0452 (C8H7O2+, Δ = 0.6 mDa), which indicated the occurrence of aromatic hydroxylation but did not give sufficient information on the location of the other hydroxyl group. The presence of product ions in the CID MS2 mass spectrum at m/z 107.0499 (C7H7O+, Δ = 0.3 mDa) and m/z 89.0396 (C7H5+, Δ = 0.5 mDa) provides further support for aromatic hydroxylation through the loss of CO and water, respectively. The radical product ion at m/z 164.0842 (C10H12O2+•, Δ = 0.4 mDa) exclusively detected in the EAD MS2 mass spectrum indicated that the hydroxyl group was most likely introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5f).M13: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M6, the EAD MS2 product ion mass spectrum provided a complementary tool to the CID MS2 mass spectrum in support of hydroxylation on the amine and aromatic functionalities (Fig. S1g).M14-M16: The protonated molecules of these metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. The diagnostic ions in the CID fragmentation patterns of M14, such as the product ions at m/z 190.1278 (C12H16NO+, Δ = 4.6 mDa), 172.1134 (C12H14N+, Δ = 0.7 mDa) and 160.1133 (C11H14N+, Δ = 0.6 mDa), are observed, therefore the hydroxyl group was introduced to at the tolyl functionality of 2-MEC. Interestingly, the CID product ion spectrum of M14 displayed an abundant iminium ion at m/z 72.0822 (C4H10N+, Δ = 0.8 mDa) consistent with the fragmentation for other N-alkylated synthetic cathinones [31,37]. In comparison, the EAD product ion spectrum of this metabolite was dominated by additional product ions at m/z 190.1290 (C12H16NO+, Δ = 5.8 mDa), m/z 119.0513 (C8H7O+), and m/z 91.0589 (C7H7+), which were much more abundant than that in the EAD spectra of M1 (Fig. 5a). and M7 (Fig. S2a). The diagnostic product ions at m/z 161.0844 (C10H11NO+•, Δ = 0.3 mDa) and m/z 135.0813 (C9H11O+, Δ = 0.3 mDa) in the CID MS2 mass spectrum of M15 indicate the hydroxyl group was likely to be introduced to the aromatic ring. The diagnostic ion at m/z 135.0456 (C8H7O2+, Δ = 0.9 mDa) in the EAD MS2 mass spectrum of M15 provides further support for this elucidation (Fig. S2b). The CID MS2 mass spectrum of M16 is similar to M2 and M8, whose product ion spectra were dominated by the product ion at m/z 119.0501 (C8H7O+, Δ = 0.4 mDa). However, the EAD MS2 mass spectrum of this metabolite failed to provide helpful elucidation information in contrast to EAD MS2 mass spectra of M2 and M8. Based on the empirical results of M2 and M8, the hydroxyl group was introduced to the amine group of M16 (Fig. S2c).Peng Che was funded by a China Scholarship Council (CSC) fellowship (No.202006210045). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation. Funding Information: Peng Che was funded by a China Scholarship Council (CSC) fellowship (No. 202006210045 ). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation. Publisher Copyright: {\textcopyright} 2023 The Authors",

year = "2023",

month = dec,

day = "1",

doi = "10.1016/j.aca.2023.341962",

language = "English",

volume = "1283",

pages = "1--12",

journal = "Analytica Chimica Acta",

issn = "0003-2670",

publisher = "Elsevier B.V.",

}

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers. / Che, Peng; Davidson, J. Tyler; Kool, Jeroen et al.
In: Analytica Chimica Acta, Vol. 1283, 341962, 01.12.2023, p. 1-12.

Research output: Contribution to Journal › Article › Academic › peer-review

TY - JOUR

T1 - Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

AU - Che, Peng

AU - Davidson, J. Tyler

AU - Kool, Jeroen

AU - Kohler, Isabelle

N1 - Funding Information: M1 and M2: The protonated molecules of these two metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. In the CID MS2 mass spectrum of M1, the product ion at m/z 190.1244 (C12H16NO+, Δ = 1.2 mDa) was formed from an initial loss of water and led to the formation of product ions at m/z 172.1173 (C12H14N+, Δ = 4.6 mDa) and m/z 160.1204 (C11H14N+, Δ = 7.7 mDa) through the sequential loss of water or formaldehyde, respectively. Therefore, the hydroxyl was introduced to the tolyl functionality of 4-MEC, which was also reported by Helfer et al. [34]. Additional fragmentation leading to the formation of product ions at m/z 144.0840 (C10H10N+), m/z 132.0844 (C9H10N+), m/z 131.0757 (C9H9N+•), and m/z 117.0639 (C8H7N+•) provides further support for the location of the hydroxyl on the tolyl functionality of 4-MEC. Such fragmentation was also observed in the EAD product ion spectrum of M1, even if the product ion at m/z 190.1244 was less abundant (less than 2% of the total abundance). In addition, the EAD product ion spectrum was dominated by the product ions at m/z 164.0848 (C10H12O2+•, Δ = 1.0 mDa) and m/z 193.1112 (C11H15NO2+•, Δ = 0.9 mDa), which were not observed in the CID product ion spectrum (see Fig. 5a). The CID MS2 mass spectrum of M2 showed a highly abundant product ion at m/z 119.0499 (C8H7O+, Δ = 0.2 mDa), which indicated the hydroxyl group was neither introduced to the aromatic ring nor the tolyl functionality. However, the CID spectrum was not sufficient to elucidate the location of the other hydroxyl group on the molecule. Pedersen et al. also faced this issue when elucidating the structure of one hydroxylated metabolite of methylone (i.e., methylenedioxymethcathinone), whose CID product ion spectrum was dominated by only one abundant ion at m/z 149.0243 (C8H5O3+, Δ = 0.4 mDa). They therefore proposed two possible structures for that metabolite [35]. This is where EAD can play a role as a supplementary tool to provide additional structural information. Indeed, the EAD spectrum of M2 was exclusively dominated by a radical product ion at m/z 148.0896 (C10H12O+•, Δ = 0.7 mDa), supporting that the hydroxyl group was introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5b).M6: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M2, the CID product ion spectrum of this di-hydroxylated metabolite was dominated by the product ion at m/z 135.0452 (C8H7O2+, Δ = 0.6 mDa), which indicated the occurrence of aromatic hydroxylation but did not give sufficient information on the location of the other hydroxyl group. The presence of product ions in the CID MS2 mass spectrum at m/z 107.0499 (C7H7O+, Δ = 0.3 mDa) and m/z 89.0396 (C7H5+, Δ = 0.5 mDa) provides further support for aromatic hydroxylation through the loss of CO and water, respectively. The radical product ion at m/z 164.0842 (C10H12O2+•, Δ = 0.4 mDa) exclusively detected in the EAD MS2 mass spectrum indicated that the hydroxyl group was most likely introduced to the amine through the loss of a 60 Da (•NOHCH2CH3) radical (Fig. 5f).M13: The protonated molecule of this metabolite was observed at nominal m/z 224, suggesting the occurrence of di-hydroxylation. Similar to the elucidation of M6, the EAD MS2 product ion mass spectrum provided a complementary tool to the CID MS2 mass spectrum in support of hydroxylation on the amine and aromatic functionalities (Fig. S1g).M14-M16: The protonated molecules of these metabolites were observed at nominal m/z 208, indicating that one hydroxyl group was introduced to the molecule. The diagnostic ions in the CID fragmentation patterns of M14, such as the product ions at m/z 190.1278 (C12H16NO+, Δ = 4.6 mDa), 172.1134 (C12H14N+, Δ = 0.7 mDa) and 160.1133 (C11H14N+, Δ = 0.6 mDa), are observed, therefore the hydroxyl group was introduced to at the tolyl functionality of 2-MEC. Interestingly, the CID product ion spectrum of M14 displayed an abundant iminium ion at m/z 72.0822 (C4H10N+, Δ = 0.8 mDa) consistent with the fragmentation for other N-alkylated synthetic cathinones [31,37]. In comparison, the EAD product ion spectrum of this metabolite was dominated by additional product ions at m/z 190.1290 (C12H16NO+, Δ = 5.8 mDa), m/z 119.0513 (C8H7O+), and m/z 91.0589 (C7H7+), which were much more abundant than that in the EAD spectra of M1 (Fig. 5a). and M7 (Fig. S2a). The diagnostic product ions at m/z 161.0844 (C10H11NO+•, Δ = 0.3 mDa) and m/z 135.0813 (C9H11O+, Δ = 0.3 mDa) in the CID MS2 mass spectrum of M15 indicate the hydroxyl group was likely to be introduced to the aromatic ring. The diagnostic ion at m/z 135.0456 (C8H7O2+, Δ = 0.9 mDa) in the EAD MS2 mass spectrum of M15 provides further support for this elucidation (Fig. S2b). The CID MS2 mass spectrum of M16 is similar to M2 and M8, whose product ion spectra were dominated by the product ion at m/z 119.0501 (C8H7O+, Δ = 0.4 mDa). However, the EAD MS2 mass spectrum of this metabolite failed to provide helpful elucidation information in contrast to EAD MS2 mass spectra of M2 and M8. Based on the empirical results of M2 and M8, the hydroxyl group was introduced to the amine group of M16 (Fig. S2c).Peng Che was funded by a China Scholarship Council (CSC) fellowship (No.202006210045). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation. Funding Information: Peng Che was funded by a China Scholarship Council (CSC) fellowship (No. 202006210045 ). Ruben Kranenburg from the Amsterdam Dutch Police is kindly acknowledged for providing the analytical standards of synthetic cathinones. Wilfried Niessen is warmly acknowledged for his assistance in data interpretation. Publisher Copyright: © 2023 The Authors

PY - 2023/12/1

Y1 - 2023/12/1

N2 - Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

AB - Over the last decade, a remarkable number of new psychoactive substances (NPS) have emerged onto the drug market, resulting in serious threats to both public health and society. Despite their abundance and potential toxicity, there is little information available on their metabolism, a crucial piece of information for clinical and forensic purposes. NPS metabolism can be studied using in vitro models, such as liver microsomes, cytosol, hepatocytes, etc. The tentative structural elucidation of metabolites of NPS formed using in vitro models is typically carried out using liquid chromatography combined with high-resolution tandem mass spectrometry (LC-HRMS2) with collision-induced dissociation (CID) as a fragmentation method. However, the thermally-excited ions produced with CID may not be sufficient for unambiguous identification of metabolites or their complete characterization. Electron-activated dissociation (EAD), a relatively new fragmentation approach that can be used to fragment singly-charged ions, may provide complementary structural information that can be used to further improve the confidence in metabolite identification. The aim of this study was to compare CID and EAD as fragmentation methods for the characterization and identification of synthetic cathinone positional isomers and their metabolites. The in vitro metabolism of 2-methylethcathinone (2-MEC), 3-methylethcathinone (3-MEC) and 4-methylethcathinone (4-MEC) was investigated with both CID and EAD methods using LC-HRMS2. Four, seven and six metabolites were tentatively identified for the metabolism of 2-MEC, 3-MEC and 4-MEC, respectively. Here, the metabolism of 3-MEC and 2-MEC is reported for the first time. The EAD product ion mass spectra showed different fragmentation patterns compared to CID, where unique and abundant product ions were observed in EAD but not in CID. More importantly, certain EAD exclusive product ions play a significant role in structural elucidation of some metabolites. These results highlight the important role that EAD fragmentation can play in metabolite identification workflows, by providing additional fragmentation data compared with CID and, thus, enhancing the confidence in structural elucidation of drug metabolites.

KW - Collision-induced dissociation

KW - Electron activated dissociation

KW - Liquid chromatography

KW - Mass spectrometry

KW - Phase I metabolite identification

KW - Positional isomers

KW - Synthetic cathinones

UR - https://www.scopus.com/pages/publications/85175329601

UR - https://www.scopus.com/pages/publications/85175329601#tab=citedBy

U2 - 10.1016/j.aca.2023.341962

DO - 10.1016/j.aca.2023.341962

M3 - Article

AN - SCOPUS:85175329601

SN - 0003-2670

VL - 1283

SP - 1

EP - 12

JO - Analytica Chimica Acta

JF - Analytica Chimica Acta

M1 - 341962

ER -

Electron activated dissociation - a complementary fragmentation technique to collision-induced dissociation for metabolite identification of synthetic cathinone positional isomers

Abstract

Bibliographical note

Funding

Keywords

Access to Document

Persistent URL (handle)

Other files and links

Fingerprint

Cite this