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GC–MS analysis of regioisomeric substituted N-benzyl-4-bromo-2,5-dimethoxyphenethylamines
Forensic Chemistry 14 (2019) 100164
Contents lists available at ScienceDirect
Forensic Chemistry journal homepage: www.elsevier.com/locate/forc
GC–MS analysis of regioisomeric substituted N-benzyl-4-bromo-2,5dimethoxyphenethylamines
T
Ahmad J. Almalkia,b, C. Randall Clarka, , Jack DeRuitera ⁎
a b
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
HIGHLIGHTS
analytical properties of six dimethoxy-NBOMe derivatives are described. • The yields a number of equivalent regioisomeric major fragments. • EI-MS of the TFA derivatives allowed complete regioisomer differentiation. • EI-MS • Capillary GC yields excellent resolution of these six compounds. ABSTRACT
The six N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine regioisomers are potential designer compounds related to the common NBOMe drug N-(2methoxy)benzyl-4-bromo-2,5-dimethoxyphenethylamine (25B-NBOMe). These six compounds represent the incorporation of one additional methoxy-group into the common NBOMe molecular framework. The compounds were prepared from commercially available precursor materials and their electron ionization mass spectra (EI-MS) are quite similar yielding nearly identical fragment ions. The 2′,3′-dimethoxybenzyl regioisomer gave a unique fragment ion of significant abundance in the EI-MS at m/z 136. Baseline gas chromatographic resolution of the six regioisomers was achieved using a midpolarity phase of 50% phenyl and 50% dimethyl polysiloxane and the more crowded dimethoxy substitution patterns eluted first under temperature programming conditions. The EI mass spectra for the TFA-derivatives of these six regioisomers gave a molecular ion of significant abundance at m/z 505/507, unlike the parent compounds. Differentiation and specific identification of all six of the regioisomers was possible based on a combination of different base peak ions (m/z 151 or 242/244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91.
1. Introduction The halogenated 25-NBOMe compounds such as N-(2-methoxy) benzyl-4-bromo-2,5-dimethoxyphenethylamine (25B-NBOMe) represent a new class of hallucinogenic or psychedelic drugs. Compounds of this structural class were first reported in the scientific literature in 2003 [1] as derivatives of the psychedelic phenethylamine, 4-chloro2,5-dimethoxylphenethylamine. Numerous phenethylamines containing multiple aromatic ring substituents produce psychoactive/hallucinogenic effects [2] and these unbranched primary amines have been described as 2C phenethylamines referring to the unbranched primary amine two carbon side-chain [3]. The pharmacological actions of 25I-NBOMe and its derivatives appear to result from their potent agonist action at the serotonin 2A (5HT2A) receptors often ten-times more potent than the corresponding primary phenethylamine [4,5]. The addition of the 2-methoxybenzyl group significantly enhances the affinity and potency of these ⁎
secondary amines [4] compared to the primary amines. Furthermore, the radiolabelled form of some of the more potent members of this class of NBOMe derivatives have been used for mapping the distribution of 5HT2A receptors in the brain [6]. Serotonin 2A receptors are implicated in the pathophysiology of depression and schizophrenia and their stimulation is responsible for the hallucinogenic effects of many recreational drugs including lysergic acid diethylamide (LSD) and representatives of the 2C-series as demonstrated in animal models [7–10]. Compounds of the NBOMe structural class have virtually no history of human use prior to 2010 when they first became available online [11]. Reports from human users suggest that the 25-NBOMe compounds are active hallucinogens at doses as little as 200–1000 μg when administered intranasally or sublingually; and from 50 to 500 μg when smoked (as freebase), making these substances only slightly less potent than LSD. Casale and Hays [12] reported the preparation and characterization of a limited number of 25-NBOMe variants, where the iodine atom of
Corresponding author. E-mail address: [email protected] (C.R. Clark).
https://doi.org/10.1016/j.forc.2019.100164 Received 8 January 2019; Received in revised form 27 March 2019; Accepted 7 April 2019 Available online 08 April 2019 2468-1709/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Structures of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
the 2C ring was replaced with another halogen or other organic functional groups, and where the methoxy group at position 2- of the Nbenzyl moiety was moved to positions 3- or 4- of this ring. In this study the authors noted the significant similarities in the mass spectra and infrared spectra within each series of regioisomers (i.e. the 2-, 3- and 4methoxy-N-benzyl isomers of 4-I 2C). These spectral similarities highlight the forensic challenge of identification of any one of these isomers to the exclusion of the other regioisomers. In recent years a number of designer derivatives of the N-BOMe series have been synthesized which contain additional substituents in the N-benzyl ring and many of these new analogues display very high serotonin receptor binding profiles [13,14]. Adding substituents to this portion of the NBOMe structure further complicates specific compound identification because it significantly increases the number of possible regioisomers. For example, there are six possible regioisomeric dimethoxy-benzyl derivatives of 25B-NBOMe as shown in Fig. 1, and each of these isomers would be expected to display very similar spectral properties, presenting a significant challenge for analytical differentiation. A number of these isomers were shown [15] to be potent ligands for serotonin 5-HT2A and 5-HT2C receptor subtypes with pKi values greater than 7. In this report we describe GC–MS methods to differentiate and specifically identify all six members of this dimethoxy-benzyl 25B-NBOMe series. Furthermore, these analytical approaches could be applied to the specific identification of other regioisomeric derivatives with varying substitution patterns in the N-benzyl substituent of the core NBOMe structure.
2.2. Synthetic methods Precursor materials including the six dimethoxybenzaldehydes were purchased from Aldrich Chemical Company (Milwaukee, Wisconsin) or VWR Chemical Company (Radnor, Pennsylvania). The synthesis of 2,5dimethoxyphenylnitroethene was accomplished using 2,5-dimethoxybenzaldehyde, nitromethane and anhydrous ammonium acetate and the structure of the product was confirmed by GC–MS and NMR spectroscopy. The 2,5-dimethoxyphenylnitroethene was reduced via a suspension of LiAlH4 in dry tetrahydrofuran. The excess LiAlH4 reagent was neutralized by successive dropwise addition of tetrahydrofuran in water, 10% sodium hydroxide, and finally water. The product oil was extracted into dichloromethane and evaporated under reduced pressure. The dried product oil 2,5-dimethoxyphenethylamine was converted to the hydrochloride salt via gaseous HCl. Bromination of this compound was accomplished using bromine dissolved in glacial acetic acid. The yellow precipitate was isolated by filtration and converted to the HCl salt. The six N-(dimethoxy)benzyl-4-bromo-2,5-dimethoxyphenethyl amine final products were prepared by treating a solution of 4-bromo2,5-dimethoxyphenethylamine HCl with triethylamine and the appropriately substituted dimethoxybenzaldehyde and NaBH4. The free base form of the products were isolated by extraction and all six compounds in this series were also converted to HCl salts. 3. Results and discussion
2. Experimental
The compounds in this study are analogues of N-(2-methoxy)benzyl4-bromo-2,5-dimethoxy-phenethylamine (25B-NBOMe), modified to contain the N-dimethoxybenzyl aromatic ring substituents and substitution patterns (Fig. 1). Small alkyl ethers such as methoxy groups are common structural modifications used in designer drug development. The synthetic reaction scheme for the preparation of the compounds is shown in Fig. 2. The nitroethene intermediate was prepared by reaction of commercially available 2,5-dimethoxybenzaldehyde with nitromethane followed by reduction with lithium aluminum hydride to yield the desired 2,5-dimethoxyphenethylamine. Bromination of this phenethylamine hydrochloride with bromine in acetic acid gave 4-bromo-2,5dimethoxyphenethylamine in excellent yield. The position of bromination was confirmed by proton NMR and the spectra for the aromatic region of the precursor and the brominated dimethoxyphenethylamine are shown in Fig. 3. The two remaining protons in the aromatic region of the spectrum appear as two singlets and these protons are clearly not coupled by ortho or longer range meta-coupling. The only way to obtain such a pattern in the proton NMR would be if the bromine atom was added to the 4-position of the aromatic ring. Using general reductive alkylation methods, 4-bromo-2,5-dimethoxyphenethylamine
2.1. Instrumentation The GC–MS system consisted of an Agilent Technologies (Santa Clara, CA) 7890A gas chromatograph and an Agilent 7683B auto injector coupled with a 5975C VL Agilent mass selective detector. The GC was operated in splitless injection mode with a helium (ultra-high purity, grade 5, 99.999%) flow rate of 0.48 mL/min and the injection volume was 1 µL. The MS was operated in the electron ionization (EI) mode with an ionization voltage of 70 eV, a scan rate of 2.86 scans/s and a source temperature of 230 °C. The GC injector was maintained at 230 °C and the transfer line at 230 °C. The GC–MS chromatographic separations were carried out on a column (30 m × 0.25 mm i.d.) coated with 0.25 μm film of midpolarity Crossbond® silarylene phase similar to 50% phenyl, 50% dimethylpolysiloxane (Rxi®-17Sil MS) purchased from Restek Corporation (Bellefonte, PA). The temperature program consisted of an initial hold at 70 °C for 1.0 min, ramped up to 250 °C at a rate of 30 °C/min followed by a hold at 250 °C for 25 min then increased to 340 °C at 15 °C/min.
2
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Fig. 2. Synthesis of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
hydrochloride was used to prepare the six N-(dimethoxybenzyl)-4bromo-2,5-dimethoxyphenethylamine isomers. This reductive alkylation method consisted of a two-step approach involving imine formation first, followed by reduction with sodium borohydride (NaBH4) to yield the desired secondary amines shown in Fig. 1. The electron ionization mass spectra (EI-MS) for the compounds in this study are quite similar yielding nearly identical fragment ions as shown in Fig. 4. The most abundant ions in the EI-MS spectra for this series of dimethoxybenzyl isomers occurs at m/z 121, 151 and 180. The predominant ion at m/z 151 is the dimethoxybenzyl cation and the ion at m/z 180 is the iminium cation formed by the dissociation of bond between α- and β-carbon atoms of the ethylene linker group eliminating the 4-bromo-2,5-dimethoxybenzyl radical. Finally, the ion at m/ z 121 likely forms from loss of CH2O from the dimethoxybenzylcation. The molecular weight for each of the regioisomeric compounds in this study was confirmed by the (M+H)+ ion at 410/412 using chemical ionization (CI) techniques since no molecular ion was observed in the EI-MS. Only one isomer in this dimethoxybenzyl series (the 2′,3′-dimethoxybenzyl regioisomer) gave a unique fragment ion of significant abundance in the EI-MS and this ion occurred at m/z 136. This characteristic ion is absent from the spectrum of the other five dimethoxybenzyl regioisomers and corresponds to the loss of methyl (CH3) from
the dimethoxybenzyl group. A similar fragmentation pathway was observed in previous studies when the corresponding m/z 136 fragment was generated from 2,3-dimethoxybenzyl-piperazine [16]. The gas chromatographic separation of the six N-(dimethoxy) benzyl-4-bromo-2,5-dimethoxyphenethylamine regioisomers in Fig. 5 was achieved using a midpolarity Crossbond® silarylene phase containing a 50% phenyl and 50% dimethyl polysiloxane polymer (Rxi®17Sil MS). Under temperature programming conditions, the dimethoxybenzyl regioisomers eluted over a 2.0 min window (approx.) in the 37 min range and this chromatographic system allowed for baseline resolution for all six compounds. The isomers with a 2′-methoxy group (2′,3′-, 2′,4′-, 2′,5′- and 2′,6′-dimethoxy) eluted before the two regioisomers that did not contain a 2′-methoxy group. Furthermore, the two derivatives with the greatest degree of steric crowding relative to the benzyl side chain (2,3- and 2,6-dimethoxy) eluted prior to all other members of the series. The 3,5-isomer having the maximum distance between aromatic ring substituents eluted last. The EI mass spectra for the TFA-derivatives of these six regioisomeric N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl amines are shown in Fig. 6. Each of the TFA derivatives gave a molecular ion of significant abundance at m/z 505/507, unlike the parent compounds. Two major fragment ions common in the spectra of all six regioisomeric derivatives occur at m/z 242/244 and m/z 151. The m/z
Fig. 3. Proton NMR of the aromatic region for 2,5-dimethoxyphenethylamine and 4-bromo-2,5-dimethoxyphenethylamine. 3
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Fig. 4. Mass spectra of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines. 4
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Fig. 5. Gas chromatographic separation of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
the relative abundance of significant ions at m/z 121 and m/z 91. In the case of the 2′,4′-isomer, the m/z 121 ion is the third most abundant ion in the spectrum and is present in greater abundance than the m/z 91 ion. This is reversed in the 2′,6′-isomer and the m/z 91 ion is the third most abundant ion and is present in greater intensity than the m/z 121 ion. Thus, the combination of different base peak ions (m/z 151 or 242/ 244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91, allows for differentiation and specific identification of all six of the regioisomers of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxy phenethylamine series in their TFA-derivatized form. The presence of a m/z 263 fragment ion of high relative abundance in the EI-MS of the TFA derivative of the 2′,5′-dimethoxy isomer of this series prompted further studies to elucidate its structure. This ion does not contain bromine and may form following initial H migration to the oxygen atom of the carbonyl group followed by cleavage of the N–C bond from the phenylethyl side of the compound to yield the m/z 263 radical cation as shown in Fig. 8. The elemental composition of the m/z 263 ion was obtained by high resolution accurate mass analysis (EITOF) yielding an experimental value of 263.0765 consistent with C11H12NO3F3 (263.0769 theoretical, −1.5 PPM). The high abundance of the m/z/ 263 ion in the TFA derivatives of the 2′,5′- and 3′,4′-dimethoxy derivatives may result from methoxy group conjugation in these isomers which would enhance stabilization of the radical cation species. The absence of this ion in the spectrum of the 2′,3′-isomer where the methoxy groups are also conjugated may result from this isomer preferentially fragmenting to yield the unique m/z 136 fragment described previously. To investigate the origin of the m/z 263 fragment ion in the mass spectra of the N-(2′,5′-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine TFA derivative some additional derivatives and analogues of this compound were prepared and subjected to EI-MS analysis. Fig. 9 shows the EI mass spectrum for the pentafluoropropionylamide (PFPA) derivative of N-(2′,5′-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine. This derivative contains an additional CF2 unit (+50 mass units) relative to the TFA derivative, and thus yields an analogous m/z 313 ion by the fragment pathway shown in Fig. 8. The expected m/z 313 ion is present in the spectrum of this PFPA derivative while other fragment ions are similar to those observed in the TFA derivative in terms of mass and intensities (m/z 151 > 313 > 242/244 = 121). Thus confirming the perfluoroacyl group (TFA and PFPA) as a component of this unique radical cation fragment. If the m/z 263 ion is characteristic for derivatives of this particular benzyl substitution pattern, then other 2′,5′-dimethoxy-NBOMe
242/244 ion contains bromine and is likely the phenylethyl radical cation formed by hydrogen migration followed by the dissociation of benzylic N–C bond from the phenylethyl side of the compound (Fig. 7). The m/z 151 ion forms by the cleavage of the N-C bond yielding the dimethoxybenzyl cation as observed with the parent underivatized compounds. While the m/z 242/244 and m/z 151 fragment ions are present in all six regioisomeric N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine TFA derivatives, a number of other ions varying in mass or relative abundance are present in these spectra and allow for complete differentiation among these six regioisomeric compounds. For example, only the 2′,3′- and 3′,5′-dimethoxy compounds have the base peak at m/z 242/244, setting them apart from the other four regioisomers (Fig. 6). But only the 2′,3,-dimethoxy isomer contains a m/z 136 ion of high abundance in its EI mass spectrum, differentiating this compound from the 3′,5′-dimethoxy isomer and the other four regioisomers of this series. This m/z 136 ion is also observed in the underivatized form of the 2,3-dimethoxy isomer and distinguishes it from the other five regioisomers of this series. The 2′4′-, 2′5′-, 2′,6′- and 3′,4′-dimethoxy isomers all have a base peak at 151 m/z in their EI-MS (Fig. 6). However, only the 2′5′-dimethoxy compound has an intense m/z 263 fragment ion second in abundance only to the base peak and in greater relative abundance than the m/z 242/244 ion. The 3′,4′-isomer also contains a m/z 263 fragment ion in its spectrum, but its abundance is significantly lower than the base peak (m/z 151) and the m/z 242/244 ion. In addition to the m/z 263 ion, the 2′5′-dimethoxy compound has a m/z 121 ion of nearly equal intensity to the m/z 242/244 ion and an ion of this mass is not present in any significant abundance in the spectrum of the 3′,4′isomer. Therefore, the presence and relative abundance of the m/z 263 and m/z 121 fragment ions differentiates the 2′,5′-isomer from all other regioisomers of this series. It should be noted that the mass spectrum of the 3′,5′-isomer also contains a m/z 263 fragment ion of relatively low abundance (Fig. 6). However this isomer is differentiated based on its base peak of m/z 242/244 as noted above, and a more intense m/z 229/ 231 ion (third most abundant) than the other five isomers. The proposed structures for these various fragment ions are shown in Fig. 7. The remaining 2′,4′- and 2′,6′-dimethoxy isomers have a base peak at m/z 151 and a m/z 242/244 fragment ion of secondary abundance. The EI-MS of these two isomers lack significant fragment ions at m/z 136 and 263, distinguishing them from the other four members of this series. Their spectra also differ from the other four regioisomers in the relative abundance of the m/z 229/231 fragment ion. This ion is present in significantly greater abundance in the 2′,3′-, 2′,5′-, 3′,4′- and 3′,5′isomers than in the 2′,4′- and 2′,6′-dimethoxy isomers. The mass spectra of the 2′,4′- and 2′,6′-dimethoxy regioisomers differ from each other in 5
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Fig. 6. Mass spectra of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-trifloroacetamide regiosiomers.
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Fig. 7. Proposed EI-MS fragmentation pathway for the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-trifloroacetamides. *The m/z 136 ion only occurs in the N-(2,3-dimethoxybenzyl)-isomer.
Fig. 8. Structure of the N-(2,5-dimethoxybenzyl)trifloroacetamide m/z 263 radical cation.
Fig. 9. EI-MS of N-(2,5-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-pentafloropropionamide.
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Fig. 10. EI-MS of N-(2,5-dimethoxybenzyl)-2,5-dimethoxyphenethyl-trifloroacetamide.
analogues with varied substituents in the phenethyl ring or phenethyl side chain would also be expected to yield a m/z 263 ion in their mass spectra. The TFA derivative of the non-halogenated derivative (N-(2′,5′dimethoxybenzyl)-2,5-dimethoxyphenethylamine) was prepared and subjected to EI-MS analysis. As shown in Fig. 10 the non-halogenated derivative also yielded the m/z 263 fragment ion. The m/z 164 in the mass spectrum of this hydrogen derivative corresponds to the 242/244 ion in the spectrum of the bromine containing derivative. Also, the EIMS of the TFA derivative of 4-iodo-(2′,5′-dimethoxybenzyl)-2,5-dimethoxyphenethylamine (25I-NBOMe) gave a base peak in the MS at m/z 151 with a significant m/z 263 ion.
Conflict of interest There are no known conflicts of interest. Acknowledgements This project was supported by Award No. 2016-DN-BX-K0175, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author (s) and do not necessarily reflect those of the Department of Justice. Appendix A. Supplementary data
4. Conclusions
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.forc.2019.100164.
The N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine series of regioisomeric compounds represents the incorporation of one additional methoxy-group into the common N-(2-methoxybenzyl)-molecular framework for the NBOMe drugs. All six of these regioisomeric secondary amines show essentially equivalent EI-MS fragment ions however the extensive fragmentation yields very low molecular ion current. Acylation of the amines to yield perfluoroacyl derivatives such as the trifluoroacetamines allow for the individual identification of each isomer as well as significant peaks in the molecular ion region of the EI spectrum. The combination of different base peak ions (m/z 151 or 242/244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91, allows for differentiation and specific identification of all six of the regioisomeric TFA derivatives. The m/z 242/244 ion contains bromine and is likely the phenylethyl radical cation formed by hydrogen migration followed by the dissociation of benzylic N–C bond from the phenylethyl side of the compound. This rearrangement process accounts for the base peak in the EI-MS for the 2′,3′- and 3′,5′-dimethoxy isomers. Thus setting them apart from the other four regioisomers which show the m/z 151 as the base peak. This m/z 151 ion forms by the cleavage of the N–C bond yielding the dimethoxybenzyl cation as observed with the parent underivatized compounds. The sequential loss of 30 Da (CH2O) from the m/z 151 cation accounts for the fragment ions at m/z 121 and m/z 91.
References [1] Heim, Synthesis and pharmacology of potent 5-HT2A receptor agonists with N-2methoxybenzyl partial structure (Ph.D. Thesis), Free University, Berlin, 2003. [2] G. Appendino, A. Minassi, O. Taglialatela-Scafati, Recreational drug discovery: natural products as lead structures for the synthesis of smart drugs, Nat. Prod. Rep. 31 (2014) 880–904. [3] A. Shulgin, A. Shulgin, PiHKAL, A Chemical Love Story, Transform Press, 1991. [4] M.R. Braden, J.C. Parrish, J.C. Naylor, D.E. Nichols, Molecular interaction of serotonin 5-HT2A receptor residues Phe339(6.51) and Phe340(6.52) with superpotent N-benzyl phenethylamine agonists, Mol. Pharmacol. 2006 (2006) 70 pp. 1956–1964. [5] A.L. Halberstadt, M.A. Geyer, Effects of the hallucinogen 2,5-dimethoxy-4-iodophenethylamine (2C-I) and superpotent N-benzyl derivatives on the head twitch response, Neuropharmacology 2013 (77C) (2013) 200–207. [6] A. Ettrup, M. Hansen, M.A. Santoni, J. Paine, N. Gillings, M. Palner, S. Lehel, M.M. Herth, J. Madsen, J. Kristensen, M. Begtrup, G.M. Knudsen, Radiosynthesis and in vivo evaluation of a series of substituted 11C-phenethylamines as 5-HT2A agonist PET tracers, Eur. J. Nucl. Med. Mol. Imaging 38 (2011) 681–693. [7] J. Gonzalez-Maeso, N.V. Weisstaub, M. Zhou, Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior, Neuron 53 (2007) 439–452. [8] S.L. Hill, T. Doris, S. Gurung, S. Katebe, A. Lomas, M. Dunn, P. Blain, S.H. Thomas, Severe clinical toxicity associated with analytically confirmed recreational use of 25I-NBOMe: case series, Clin. Toxicol. 51 (2013) 487–492. [9] D.E. Nichols, Hallucinogens, Pharmacol. Ther. 101 (2004) 131–181. [10] J.L. Poklis, S.K. Demposey, K. Liu, J.K. Ritter, C. Wolf, S. Zhang, A. Pioklis, Identification of metabolite biomarkers of the designer hallucinogen 25I-NBOMe in mouse hepatic microsomal preparations and human urine samples associated with clinical intoxication, J. Anal. Toxicol. 39 (2015) 607–616.
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A.J. Almalki, et al. [11] http://www.erowid.org/chemicals/nbome/ [10.07.12]. [12] J.F. Casale, P.A. Hays, Characterization of eleven 2,5-dimethoxybenzyl-N-(2methoxybenzyl)phenethylamine (NBOMe) derivatives and differentiation from their 3- and 4-methoxybenzyl analogues – Part I, Microgram 9 (2012) 84–109. [13] J.L. Poklis, S.A. Raso, K.N. Alford, A. Pioklis, M.R. Peace, Analysis of 25I-NBOMe, 25C-NBOMe and other dimethoxyphenyl-N-[(2-methoxy) methyl]ethanamine derivatives on blotter paper, J. Anal. Toxicol. 39 (2015) 617–623. [14] D. Zuba, K. Sekuła, Analytical characterization of three hallucinogenic N-(2methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs, Drug Test.
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Contents lists available at ScienceDirect
Forensic Chemistry journal homepage: www.elsevier.com/locate/forc
GC–MS analysis of regioisomeric substituted N-benzyl-4-bromo-2,5dimethoxyphenethylamines
T
Ahmad J. Almalkia,b, C. Randall Clarka, , Jack DeRuitera ⁎
a b
Department of Drug Discovery and Development, Harrison School of Pharmacy, Auburn University, Auburn, AL 36849, USA Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia
HIGHLIGHTS
analytical properties of six dimethoxy-NBOMe derivatives are described. • The yields a number of equivalent regioisomeric major fragments. • EI-MS of the TFA derivatives allowed complete regioisomer differentiation. • EI-MS • Capillary GC yields excellent resolution of these six compounds. ABSTRACT
The six N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine regioisomers are potential designer compounds related to the common NBOMe drug N-(2methoxy)benzyl-4-bromo-2,5-dimethoxyphenethylamine (25B-NBOMe). These six compounds represent the incorporation of one additional methoxy-group into the common NBOMe molecular framework. The compounds were prepared from commercially available precursor materials and their electron ionization mass spectra (EI-MS) are quite similar yielding nearly identical fragment ions. The 2′,3′-dimethoxybenzyl regioisomer gave a unique fragment ion of significant abundance in the EI-MS at m/z 136. Baseline gas chromatographic resolution of the six regioisomers was achieved using a midpolarity phase of 50% phenyl and 50% dimethyl polysiloxane and the more crowded dimethoxy substitution patterns eluted first under temperature programming conditions. The EI mass spectra for the TFA-derivatives of these six regioisomers gave a molecular ion of significant abundance at m/z 505/507, unlike the parent compounds. Differentiation and specific identification of all six of the regioisomers was possible based on a combination of different base peak ions (m/z 151 or 242/244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91.
1. Introduction The halogenated 25-NBOMe compounds such as N-(2-methoxy) benzyl-4-bromo-2,5-dimethoxyphenethylamine (25B-NBOMe) represent a new class of hallucinogenic or psychedelic drugs. Compounds of this structural class were first reported in the scientific literature in 2003 [1] as derivatives of the psychedelic phenethylamine, 4-chloro2,5-dimethoxylphenethylamine. Numerous phenethylamines containing multiple aromatic ring substituents produce psychoactive/hallucinogenic effects [2] and these unbranched primary amines have been described as 2C phenethylamines referring to the unbranched primary amine two carbon side-chain [3]. The pharmacological actions of 25I-NBOMe and its derivatives appear to result from their potent agonist action at the serotonin 2A (5HT2A) receptors often ten-times more potent than the corresponding primary phenethylamine [4,5]. The addition of the 2-methoxybenzyl group significantly enhances the affinity and potency of these ⁎
secondary amines [4] compared to the primary amines. Furthermore, the radiolabelled form of some of the more potent members of this class of NBOMe derivatives have been used for mapping the distribution of 5HT2A receptors in the brain [6]. Serotonin 2A receptors are implicated in the pathophysiology of depression and schizophrenia and their stimulation is responsible for the hallucinogenic effects of many recreational drugs including lysergic acid diethylamide (LSD) and representatives of the 2C-series as demonstrated in animal models [7–10]. Compounds of the NBOMe structural class have virtually no history of human use prior to 2010 when they first became available online [11]. Reports from human users suggest that the 25-NBOMe compounds are active hallucinogens at doses as little as 200–1000 μg when administered intranasally or sublingually; and from 50 to 500 μg when smoked (as freebase), making these substances only slightly less potent than LSD. Casale and Hays [12] reported the preparation and characterization of a limited number of 25-NBOMe variants, where the iodine atom of
Corresponding author. E-mail address: [email protected] (C.R. Clark).
https://doi.org/10.1016/j.forc.2019.100164 Received 8 January 2019; Received in revised form 27 March 2019; Accepted 7 April 2019 Available online 08 April 2019 2468-1709/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Structures of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
the 2C ring was replaced with another halogen or other organic functional groups, and where the methoxy group at position 2- of the Nbenzyl moiety was moved to positions 3- or 4- of this ring. In this study the authors noted the significant similarities in the mass spectra and infrared spectra within each series of regioisomers (i.e. the 2-, 3- and 4methoxy-N-benzyl isomers of 4-I 2C). These spectral similarities highlight the forensic challenge of identification of any one of these isomers to the exclusion of the other regioisomers. In recent years a number of designer derivatives of the N-BOMe series have been synthesized which contain additional substituents in the N-benzyl ring and many of these new analogues display very high serotonin receptor binding profiles [13,14]. Adding substituents to this portion of the NBOMe structure further complicates specific compound identification because it significantly increases the number of possible regioisomers. For example, there are six possible regioisomeric dimethoxy-benzyl derivatives of 25B-NBOMe as shown in Fig. 1, and each of these isomers would be expected to display very similar spectral properties, presenting a significant challenge for analytical differentiation. A number of these isomers were shown [15] to be potent ligands for serotonin 5-HT2A and 5-HT2C receptor subtypes with pKi values greater than 7. In this report we describe GC–MS methods to differentiate and specifically identify all six members of this dimethoxy-benzyl 25B-NBOMe series. Furthermore, these analytical approaches could be applied to the specific identification of other regioisomeric derivatives with varying substitution patterns in the N-benzyl substituent of the core NBOMe structure.
2.2. Synthetic methods Precursor materials including the six dimethoxybenzaldehydes were purchased from Aldrich Chemical Company (Milwaukee, Wisconsin) or VWR Chemical Company (Radnor, Pennsylvania). The synthesis of 2,5dimethoxyphenylnitroethene was accomplished using 2,5-dimethoxybenzaldehyde, nitromethane and anhydrous ammonium acetate and the structure of the product was confirmed by GC–MS and NMR spectroscopy. The 2,5-dimethoxyphenylnitroethene was reduced via a suspension of LiAlH4 in dry tetrahydrofuran. The excess LiAlH4 reagent was neutralized by successive dropwise addition of tetrahydrofuran in water, 10% sodium hydroxide, and finally water. The product oil was extracted into dichloromethane and evaporated under reduced pressure. The dried product oil 2,5-dimethoxyphenethylamine was converted to the hydrochloride salt via gaseous HCl. Bromination of this compound was accomplished using bromine dissolved in glacial acetic acid. The yellow precipitate was isolated by filtration and converted to the HCl salt. The six N-(dimethoxy)benzyl-4-bromo-2,5-dimethoxyphenethyl amine final products were prepared by treating a solution of 4-bromo2,5-dimethoxyphenethylamine HCl with triethylamine and the appropriately substituted dimethoxybenzaldehyde and NaBH4. The free base form of the products were isolated by extraction and all six compounds in this series were also converted to HCl salts. 3. Results and discussion
2. Experimental
The compounds in this study are analogues of N-(2-methoxy)benzyl4-bromo-2,5-dimethoxy-phenethylamine (25B-NBOMe), modified to contain the N-dimethoxybenzyl aromatic ring substituents and substitution patterns (Fig. 1). Small alkyl ethers such as methoxy groups are common structural modifications used in designer drug development. The synthetic reaction scheme for the preparation of the compounds is shown in Fig. 2. The nitroethene intermediate was prepared by reaction of commercially available 2,5-dimethoxybenzaldehyde with nitromethane followed by reduction with lithium aluminum hydride to yield the desired 2,5-dimethoxyphenethylamine. Bromination of this phenethylamine hydrochloride with bromine in acetic acid gave 4-bromo-2,5dimethoxyphenethylamine in excellent yield. The position of bromination was confirmed by proton NMR and the spectra for the aromatic region of the precursor and the brominated dimethoxyphenethylamine are shown in Fig. 3. The two remaining protons in the aromatic region of the spectrum appear as two singlets and these protons are clearly not coupled by ortho or longer range meta-coupling. The only way to obtain such a pattern in the proton NMR would be if the bromine atom was added to the 4-position of the aromatic ring. Using general reductive alkylation methods, 4-bromo-2,5-dimethoxyphenethylamine
2.1. Instrumentation The GC–MS system consisted of an Agilent Technologies (Santa Clara, CA) 7890A gas chromatograph and an Agilent 7683B auto injector coupled with a 5975C VL Agilent mass selective detector. The GC was operated in splitless injection mode with a helium (ultra-high purity, grade 5, 99.999%) flow rate of 0.48 mL/min and the injection volume was 1 µL. The MS was operated in the electron ionization (EI) mode with an ionization voltage of 70 eV, a scan rate of 2.86 scans/s and a source temperature of 230 °C. The GC injector was maintained at 230 °C and the transfer line at 230 °C. The GC–MS chromatographic separations were carried out on a column (30 m × 0.25 mm i.d.) coated with 0.25 μm film of midpolarity Crossbond® silarylene phase similar to 50% phenyl, 50% dimethylpolysiloxane (Rxi®-17Sil MS) purchased from Restek Corporation (Bellefonte, PA). The temperature program consisted of an initial hold at 70 °C for 1.0 min, ramped up to 250 °C at a rate of 30 °C/min followed by a hold at 250 °C for 25 min then increased to 340 °C at 15 °C/min.
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Fig. 2. Synthesis of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
hydrochloride was used to prepare the six N-(dimethoxybenzyl)-4bromo-2,5-dimethoxyphenethylamine isomers. This reductive alkylation method consisted of a two-step approach involving imine formation first, followed by reduction with sodium borohydride (NaBH4) to yield the desired secondary amines shown in Fig. 1. The electron ionization mass spectra (EI-MS) for the compounds in this study are quite similar yielding nearly identical fragment ions as shown in Fig. 4. The most abundant ions in the EI-MS spectra for this series of dimethoxybenzyl isomers occurs at m/z 121, 151 and 180. The predominant ion at m/z 151 is the dimethoxybenzyl cation and the ion at m/z 180 is the iminium cation formed by the dissociation of bond between α- and β-carbon atoms of the ethylene linker group eliminating the 4-bromo-2,5-dimethoxybenzyl radical. Finally, the ion at m/ z 121 likely forms from loss of CH2O from the dimethoxybenzylcation. The molecular weight for each of the regioisomeric compounds in this study was confirmed by the (M+H)+ ion at 410/412 using chemical ionization (CI) techniques since no molecular ion was observed in the EI-MS. Only one isomer in this dimethoxybenzyl series (the 2′,3′-dimethoxybenzyl regioisomer) gave a unique fragment ion of significant abundance in the EI-MS and this ion occurred at m/z 136. This characteristic ion is absent from the spectrum of the other five dimethoxybenzyl regioisomers and corresponds to the loss of methyl (CH3) from
the dimethoxybenzyl group. A similar fragmentation pathway was observed in previous studies when the corresponding m/z 136 fragment was generated from 2,3-dimethoxybenzyl-piperazine [16]. The gas chromatographic separation of the six N-(dimethoxy) benzyl-4-bromo-2,5-dimethoxyphenethylamine regioisomers in Fig. 5 was achieved using a midpolarity Crossbond® silarylene phase containing a 50% phenyl and 50% dimethyl polysiloxane polymer (Rxi®17Sil MS). Under temperature programming conditions, the dimethoxybenzyl regioisomers eluted over a 2.0 min window (approx.) in the 37 min range and this chromatographic system allowed for baseline resolution for all six compounds. The isomers with a 2′-methoxy group (2′,3′-, 2′,4′-, 2′,5′- and 2′,6′-dimethoxy) eluted before the two regioisomers that did not contain a 2′-methoxy group. Furthermore, the two derivatives with the greatest degree of steric crowding relative to the benzyl side chain (2,3- and 2,6-dimethoxy) eluted prior to all other members of the series. The 3,5-isomer having the maximum distance between aromatic ring substituents eluted last. The EI mass spectra for the TFA-derivatives of these six regioisomeric N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl amines are shown in Fig. 6. Each of the TFA derivatives gave a molecular ion of significant abundance at m/z 505/507, unlike the parent compounds. Two major fragment ions common in the spectra of all six regioisomeric derivatives occur at m/z 242/244 and m/z 151. The m/z
Fig. 3. Proton NMR of the aromatic region for 2,5-dimethoxyphenethylamine and 4-bromo-2,5-dimethoxyphenethylamine. 3
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Fig. 4. Mass spectra of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines. 4
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Fig. 5. Gas chromatographic separation of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamines.
the relative abundance of significant ions at m/z 121 and m/z 91. In the case of the 2′,4′-isomer, the m/z 121 ion is the third most abundant ion in the spectrum and is present in greater abundance than the m/z 91 ion. This is reversed in the 2′,6′-isomer and the m/z 91 ion is the third most abundant ion and is present in greater intensity than the m/z 121 ion. Thus, the combination of different base peak ions (m/z 151 or 242/ 244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91, allows for differentiation and specific identification of all six of the regioisomers of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxy phenethylamine series in their TFA-derivatized form. The presence of a m/z 263 fragment ion of high relative abundance in the EI-MS of the TFA derivative of the 2′,5′-dimethoxy isomer of this series prompted further studies to elucidate its structure. This ion does not contain bromine and may form following initial H migration to the oxygen atom of the carbonyl group followed by cleavage of the N–C bond from the phenylethyl side of the compound to yield the m/z 263 radical cation as shown in Fig. 8. The elemental composition of the m/z 263 ion was obtained by high resolution accurate mass analysis (EITOF) yielding an experimental value of 263.0765 consistent with C11H12NO3F3 (263.0769 theoretical, −1.5 PPM). The high abundance of the m/z/ 263 ion in the TFA derivatives of the 2′,5′- and 3′,4′-dimethoxy derivatives may result from methoxy group conjugation in these isomers which would enhance stabilization of the radical cation species. The absence of this ion in the spectrum of the 2′,3′-isomer where the methoxy groups are also conjugated may result from this isomer preferentially fragmenting to yield the unique m/z 136 fragment described previously. To investigate the origin of the m/z 263 fragment ion in the mass spectra of the N-(2′,5′-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine TFA derivative some additional derivatives and analogues of this compound were prepared and subjected to EI-MS analysis. Fig. 9 shows the EI mass spectrum for the pentafluoropropionylamide (PFPA) derivative of N-(2′,5′-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine. This derivative contains an additional CF2 unit (+50 mass units) relative to the TFA derivative, and thus yields an analogous m/z 313 ion by the fragment pathway shown in Fig. 8. The expected m/z 313 ion is present in the spectrum of this PFPA derivative while other fragment ions are similar to those observed in the TFA derivative in terms of mass and intensities (m/z 151 > 313 > 242/244 = 121). Thus confirming the perfluoroacyl group (TFA and PFPA) as a component of this unique radical cation fragment. If the m/z 263 ion is characteristic for derivatives of this particular benzyl substitution pattern, then other 2′,5′-dimethoxy-NBOMe
242/244 ion contains bromine and is likely the phenylethyl radical cation formed by hydrogen migration followed by the dissociation of benzylic N–C bond from the phenylethyl side of the compound (Fig. 7). The m/z 151 ion forms by the cleavage of the N-C bond yielding the dimethoxybenzyl cation as observed with the parent underivatized compounds. While the m/z 242/244 and m/z 151 fragment ions are present in all six regioisomeric N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine TFA derivatives, a number of other ions varying in mass or relative abundance are present in these spectra and allow for complete differentiation among these six regioisomeric compounds. For example, only the 2′,3′- and 3′,5′-dimethoxy compounds have the base peak at m/z 242/244, setting them apart from the other four regioisomers (Fig. 6). But only the 2′,3,-dimethoxy isomer contains a m/z 136 ion of high abundance in its EI mass spectrum, differentiating this compound from the 3′,5′-dimethoxy isomer and the other four regioisomers of this series. This m/z 136 ion is also observed in the underivatized form of the 2,3-dimethoxy isomer and distinguishes it from the other five regioisomers of this series. The 2′4′-, 2′5′-, 2′,6′- and 3′,4′-dimethoxy isomers all have a base peak at 151 m/z in their EI-MS (Fig. 6). However, only the 2′5′-dimethoxy compound has an intense m/z 263 fragment ion second in abundance only to the base peak and in greater relative abundance than the m/z 242/244 ion. The 3′,4′-isomer also contains a m/z 263 fragment ion in its spectrum, but its abundance is significantly lower than the base peak (m/z 151) and the m/z 242/244 ion. In addition to the m/z 263 ion, the 2′5′-dimethoxy compound has a m/z 121 ion of nearly equal intensity to the m/z 242/244 ion and an ion of this mass is not present in any significant abundance in the spectrum of the 3′,4′isomer. Therefore, the presence and relative abundance of the m/z 263 and m/z 121 fragment ions differentiates the 2′,5′-isomer from all other regioisomers of this series. It should be noted that the mass spectrum of the 3′,5′-isomer also contains a m/z 263 fragment ion of relatively low abundance (Fig. 6). However this isomer is differentiated based on its base peak of m/z 242/244 as noted above, and a more intense m/z 229/ 231 ion (third most abundant) than the other five isomers. The proposed structures for these various fragment ions are shown in Fig. 7. The remaining 2′,4′- and 2′,6′-dimethoxy isomers have a base peak at m/z 151 and a m/z 242/244 fragment ion of secondary abundance. The EI-MS of these two isomers lack significant fragment ions at m/z 136 and 263, distinguishing them from the other four members of this series. Their spectra also differ from the other four regioisomers in the relative abundance of the m/z 229/231 fragment ion. This ion is present in significantly greater abundance in the 2′,3′-, 2′,5′-, 3′,4′- and 3′,5′isomers than in the 2′,4′- and 2′,6′-dimethoxy isomers. The mass spectra of the 2′,4′- and 2′,6′-dimethoxy regioisomers differ from each other in 5
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Fig. 6. Mass spectra of the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-trifloroacetamide regiosiomers.
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Fig. 7. Proposed EI-MS fragmentation pathway for the N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-trifloroacetamides. *The m/z 136 ion only occurs in the N-(2,3-dimethoxybenzyl)-isomer.
Fig. 8. Structure of the N-(2,5-dimethoxybenzyl)trifloroacetamide m/z 263 radical cation.
Fig. 9. EI-MS of N-(2,5-dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethyl-pentafloropropionamide.
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Fig. 10. EI-MS of N-(2,5-dimethoxybenzyl)-2,5-dimethoxyphenethyl-trifloroacetamide.
analogues with varied substituents in the phenethyl ring or phenethyl side chain would also be expected to yield a m/z 263 ion in their mass spectra. The TFA derivative of the non-halogenated derivative (N-(2′,5′dimethoxybenzyl)-2,5-dimethoxyphenethylamine) was prepared and subjected to EI-MS analysis. As shown in Fig. 10 the non-halogenated derivative also yielded the m/z 263 fragment ion. The m/z 164 in the mass spectrum of this hydrogen derivative corresponds to the 242/244 ion in the spectrum of the bromine containing derivative. Also, the EIMS of the TFA derivative of 4-iodo-(2′,5′-dimethoxybenzyl)-2,5-dimethoxyphenethylamine (25I-NBOMe) gave a base peak in the MS at m/z 151 with a significant m/z 263 ion.
Conflict of interest There are no known conflicts of interest. Acknowledgements This project was supported by Award No. 2016-DN-BX-K0175, awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author (s) and do not necessarily reflect those of the Department of Justice. Appendix A. Supplementary data
4. Conclusions
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.forc.2019.100164.
The N-(dimethoxybenzyl)-4-bromo-2,5-dimethoxyphenethylamine series of regioisomeric compounds represents the incorporation of one additional methoxy-group into the common N-(2-methoxybenzyl)-molecular framework for the NBOMe drugs. All six of these regioisomeric secondary amines show essentially equivalent EI-MS fragment ions however the extensive fragmentation yields very low molecular ion current. Acylation of the amines to yield perfluoroacyl derivatives such as the trifluoroacetamines allow for the individual identification of each isomer as well as significant peaks in the molecular ion region of the EI spectrum. The combination of different base peak ions (m/z 151 or 242/244), unique fragment ions (m/z 136 and m/z 263), along with differences in the relative abundance of ions at m/z 121 and m/z 91, allows for differentiation and specific identification of all six of the regioisomeric TFA derivatives. The m/z 242/244 ion contains bromine and is likely the phenylethyl radical cation formed by hydrogen migration followed by the dissociation of benzylic N–C bond from the phenylethyl side of the compound. This rearrangement process accounts for the base peak in the EI-MS for the 2′,3′- and 3′,5′-dimethoxy isomers. Thus setting them apart from the other four regioisomers which show the m/z 151 as the base peak. This m/z 151 ion forms by the cleavage of the N–C bond yielding the dimethoxybenzyl cation as observed with the parent underivatized compounds. The sequential loss of 30 Da (CH2O) from the m/z 151 cation accounts for the fragment ions at m/z 121 and m/z 91.
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