Electron ionization mass spectral studies of a series of N' -trimethylsilyl derivatives of N,N -dialkylacetimidamides

Abstract

A series of N'-trimethylsilyl (TMS) derivatives of N,N-dialkylacetimidamides (amidines) (1-10) was investigated using gas chromatography-mass spectrometry (GC-MS) under electron ionization (EI) conditions. These amidines are of particular interest as precursors and degradation products of highly toxic Novichok nerve agents and are classified as non-scheduled reportable chemicals under the Chemical Weapons Convention (CWC) by the Organisation for the Prohibition of Chemical Weapons (OPCW). Structural elucidation and identification of the N'-TMS derivatives were achieved through comprehensive analysis of their EI fragmentation patterns. Three sets of isomeric compounds (3–5, 6–7, and 8–10) were successfully distinguished based on characteristic fragmentation pathways and the relative abundances of diagnostic ions. To further support the mass spectral interpretation, retention indices were determined for all analytes in accordance with standardized OPCW protocols. The combined use of EI-MS fragmentation data and retention indices establishes a reliable and robust analytical approach, enabling sensitive and unambiguous identification of these amidines at trace levels in complex environmental matrices following derivatization.

Keywords

Silylated amidines GC-MS (EI)isomer differentiation chemical warfare agents

Introduction

Chemical warfare agents (CWAs) represent some of the most toxic substances ever created, designed specifically to kill, injure, or incapacitate through their chemical properties. The global effort to eliminate these weapons is led by the Chemical Weapons Convention (CWC) and its implementing body, the Organisation for the Prohibition of Chemical Weapons. It is a multilateral treaty that bans the development, production, stockpiling, and use of chemical weapons.^1,2 Due to these factors, analytical laboratories around the world have been consistently working to develop methods for their direct detection and that of their associated degradation products.^3,4 In terms of organophosphorus nerve agents, most of us are accustomed to the G- and V-series and are well informed with their use in a number of events; particularly during the Iran–Iraq war, the Aum Shinrikyo subway attack in Tokyo, Japan, and chemical attacks in Damascus, Syria, to name a few.^5,6 In 2018, the world witnessed first-hand the use of a new kind of nerve agents, designated as A-series or Novichoks, when these were used in the attempted assassination of Sergei and Julia Skripal in England. And then, in 2020, these were at the center of another assassination attempt, this time of Alexei Navalny, Russia.^7,8

Novichok agents are thought to operate as binary systems, where comparatively less dangerous precursor compounds are combined to generate a toxic active agent. Their mechanism of toxicity is based on acetylcholinesterase inhibition, leading to disruption of nervous system function, respiratory paralysis, and death.^9,10 Some members of this class have been described as more toxic than VX, making them a serious challenge to international chemical security and necessitating the advancement of analytical and forensic detection capabilities.¹¹

Novichoks are now recognized as a highly lethal fourth generation of CWAs.¹² Given their extreme toxicity, the comprehensive characterization and identification of these compounds, including both their starting materials and their environmental markers, is of critical importance for treaty verification and decontamination.¹³ Specifically, N,N-dialkylacetimidamides (amidines) have been identified as key chemical signatures because they serve a dual role as both the precursors used in the synthesis of these agents and the primary degradation products formed after their breakdown.¹⁴ These compounds are categorized under Schedule 1.A.13 and 1.A.14 and are listed among additional non-scheduled reportable chemicals in accordance with the CWC.¹⁵

Liquid chromatography-mass spectrometry is well-suited for the detection of polar and semipolar precursors and degradation products of CWAs. However, analysis of these compounds by gas chromatography-mass spectrometry (GC-MS) requires prior derivatization to enhance analyte volatility and thermal stability. The resulting derivatized mass spectral data can be incorporated into established reference libraries, such as the National Institute of Standards and Technology database and the OPCW Central Analytical Database (OCAD), thereby facilitating the identification of these analytes in aqueous, organic, and environmental matrices in future investigations.^16,17

Recent advancements in GC-MS and its tandem mass spectral analysis (MS/MS) have significantly improved trace-level detection of CWAs, providing the analytical robustness necessary for CWC verification and global security. This high-quality data is essential for successful performance in OPCW proficiency tests, which utilize spectral libraries like OCAD and Validation Group Working Database for definitive chemical identification.^18,19 In our earlier reports, we systematically examined amidines using ESI-HRMS, and studied amidines along with their methylated derivatives under EI conditions.^19,20 Recently, Ataş thoroughly studied mass spectral behavior of N’-trimethylsilyl (TMS) derivatives of a set of isomers: N-methyl-N-propylethanimidamide, N-methyl-N-isopropylethanimidamide, and N,N-diethylethanimidamide, under different mass spectral conditions.²¹ Building on these prior investigations and recent findings, we have extended the scope to examine the EI mass spectral characteristics of a series of silylated amidine derivatives.

Experimental procedure

Chemicals and reagents

Following established procedures, the N,N-dialkylacetimidamides investigated herein were prepared in-house.^22–24 Their structures were confirmed by ¹H NMR spectroscopy and mass spectrometry, and GC analysis indicated >90% purity. The amine precursors—dimethylamine, methylpropylamine, isopropylmethylamine, diethylamine, ethylmethylamine, ethylisopropylamine, ethylpropylamine, diisopropylamine, isopropylpropylamine, and dipropylamine were purchased from TCI Chemicals (Hyderabad, India). NaOH was obtained from M/s. S.D. Fine Chemicals (Mumbai, India). Deionized water (18 MΩ·cm) from a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout. N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was purchased from Sigma-Aldrich (Steinheim, Germany), and all solvents for GC-MS analysis were obtained from E. Merck (Mumbai, India).

Silylation by N,O-bis(trimethylsilyl)trifluoroacetamide

The TMS derivatives (1–10) were prepared by mixing 200 μL of each 100 ppm stock solution with 200 μL of the derivatizing agent in a 1:1 (v/v) ratio. This mixture was then vortexed and subjected to heating at 60 °C for 45 min to facilitate silylation. Following this derivatization process, the resulting silylated compounds were subsequently analyzed by GC-MS.

Gas chromatography-mass spectrometry

GC-MS (EI) analyses were performed using an Agilent 8890B gas chromatograph coupled to an Agilent 7000D triple-quadrupole mass selective detector. Separation was achieved on an HP-5MS capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness). EI at 70 eV was employed for detection. The oven temperature was initially set at 50 °C and held for 2 min, followed by a ramp of 10 °C/min to 280 °C, which was maintained for 10 min. The injector and GC-MS transfer line temperatures were maintained at 280 °C. Helium served as the carrier gas at a constant flow rate of 1.0 mL/min. A 1 µL aliquot of the sample was injected in a 1:2 split mode. Mass spectra were acquired over an m/z range of 29–600 at a scan rate of 1 s/scan. Data processing and analysis were conducted using Agilent MassHunter software.

Results and discussions

The N′-TMS derivatives of amidines yield characteristic fragmentation patterns under EI conditions, facilitating robust structural elucidation and identification. A homologous series of 10 TMS derivatives of amidines (1–10) was subjected to EI mass spectral analysis in the current study. The series comprises compounds bearing nitrogen-linked alkyl groups ranging from methyl to propyl and isopropyl substituents (Scheme 1). This series contains three sets of isomers (3–5; 6–7; 8–10). The EI mass spectra of all amidines showed a prominent molecular ion (M^•+) and a set of fragment ions attributable to (N–C) and (C–C) bond cleavages relative to the N-alkyl substituents. Moreover, alkene elimination was frequently observed when the substituent exceeded a methyl group. The resulting fragmentation products and their subsequent dissociation routes for compounds 1–10 are presented in Scheme 2.

Scheme 1.

Molecular structure of N’-TMS derivatives of N,N-dialkylacetimidamides (1–10). N’-TMS: N’-trimethylsilyl.

Scheme 2.

Proposed possible α-cleavages and further fragmentation pattern for compounds 1–10.

All the compounds showed a common characteristic fragment ion at m/z 73 corresponding to the TMS cation. In addition, compounds 1–10 exhibited notable fragment ions at m/z 114 (a) and b generated via N–C bond cleavage. Further alkene loss from b in compounds 3, 6–10 leads to the formation of their respective iminium cations j. A distinctive fragment ion at [M − 15]⁺ (c), arising from the loss of a methyl radical (^•CH₃, 15 Da) from the molecular ion, was observed across the entire series, albeit with low intensity (2–14%). Alkene elimination from ion c subsequently produced fragment ions d, d’, and e; this pathway was observed for compounds containing alkyl groups greater than a methyl substituent on the amine nitrogen. For compounds 2–10, α-cleavage-I resulted in alkyl radical loss (^•R¹) to form ion f, which underwent further alkene loss to generate ion g in compounds 3, 6–10, based on the N-alkyl substituent. Analogously, α-cleavage-II, entailing loss of the (^•R³) radical, afforded ion h, which then eliminated an alkene to yield ion i. A fragment ion is observed (k), which was formed by the loss of the CH₃CN unit from the molecular ion involving migration of the TMS group to the amine nitrogen. Most of the ions corresponding to this loss were found to be of lower abundance. Elimination of a methyl radical from k leads to the formation of l, which is found in high abundance in all the compounds (19–100%). α-Cleavage-III is observed from k due to the loss of alkyl radical (^•R¹), which gives the ion m followed by an alkene loss (n) for 3, 6–10 compounds. Similarly, α-cleavage-IV prompts the loss of the (^•R³) with a subsequent loss of the alkene group, giving rise to ions o and p, respectively. The EI mass spectral data of all the compounds (1–10) are listed in Table 1.

Table 1.

EI mass spectral data for compounds 1–10.

Ion, m/z (relative abundance, %)
Compound ID	1	2	3	4	5	6	7	8	9	10
M& ⁺	158 (54)	172 (58)	186 (61)	186 (61)	186 (53)	200 (63)	200 (49)	214 (18)	214 (50)	214 (45)
a	114 (94)	114 (98)	114 (100)	114 (100)	114 (100)	114 (100)	114 (100)	114 (90)	114 (100)	114 (100)
b	44 (2)	58 (10)	72 (9)	72 (20)	72 (5)	86 (13)	86 (8)	100 (30)	100 (29)	100 (21)
c	143 (9)	157 (8)	171 (5)	171 (5)	171 (9)	185 (4)	185 (6)	199 (2)	199 (7)	199 (3)
d	-	129 (1)	143 (2)	129 (8)	129 (14)	143 (4)	143 (10)	157 (7)	157 (14)	157 (7)
d'	-	-	*d	-	-	157 (17)	157 (3)	*d	171 (7)	*d
e	-	-	115 (16)	-	-	115 (14)	115 (11)	115 (14)	115 (20)	115 (15)
f	-	*c	*c	*c	157 (<1)	*c	171 (2)	*c	185 (<1)	*c
g	-	-	*d	-	-	*d'	*d	*d	143 (14)	*d
h	-	-	*c	-	-	*c	*c	*c	*f	185 (<1)
i	-	-	*d	-	-	*d	*d	*d	*g	143 (3)
j	-	-	44 (4)	-	-	58 (4)	58 (3)	58 (7)	58 (6)	58 (4)
k	117 (22)	131 (7)	145 (2)	145 (3)	145 (2)	159 (1)	159 (1)	173 (2)	173 (2)	173 (2)
l	102 (100)	116 (96)	130 (75)	130 (74)	130 (78)	144 (52)	144 (55)	158 (19)	158 (56)	158 (36)
m	-	*l	*l	*l	116 (12)	*l	130 (8)	*l	144 (12)	*l
n	-	-	102 (4)	-	-	116 (13)	102 (11)	116 (17)	102 (8)	116 (13)
o	-	-	*l	-	-	*l	*l	*l	*m	144 (2)
p	-	-	*n	-	-	102 (9)	*n	*n	*n	102
q	-	144 (<1)	158 (1)	144 (4)	144 (7)	158 (3)	158 (10)	172 (8)	172 (11)	172 (9)
q'	-	-	*q	-	-	172 (6)	172 (1)	*q	*q	*q
Other ions	86 (1), 73 (96), 64 (4), 59 (22), 45 (6)	100 (11), 86 (3), 73 (100), 59 (16), 45 (5)	157 (9), 116 (13), 100 (26), 86 (5), 73 (88), 59 (14), 45 (4)	157 (9), 100 (26), 88 (5), 73 (61), 59 (14), 45 (2)	158 (1), 100 (17), 88 (8), 73 (92), 59 (12), 45 (4)	171 (40), 130 (5), 100 (30), 73 (93), 59 (12), 45 (4)	116 (8), 100 (22), 73 (86), 59 (11), 45 (4)	171 (65), 142 (1), 141 (<1), 130 (8), 73 (100), 59 (11), 45 (5)	186 (<1), 130 (12), 116 (12), 86, 59 (10), 45 (4)	186 (<1), 171 (42), 130 (11), 73 (79), 59 (8), 45 (4)

Ion coincides *c with “c,” *d with “d,” *d’ with “d’,” *f with “f,” *g with “g,” *l with “l,” *m with “m,” *n with “n,” and *q with “q.”

Isomeric compounds 3–5

The EI mass spectrum of compound 3 exhibits a characteristic fragment ion at m/z 157, corresponding to the loss of an ethyl radical from the molecular ion. This diagnostic fragment is absent from the spectra of compounds 4 and 5 (Figure 1). A fragment at m/z 129 (d), attributed to the loss of C₃H₆ from c, appears exclusively in compounds 4 and 5. Due to the presence of isopropyl and propyl groups in compounds 4 and 5, a common ion at m/z 88, generated via C₃H₆ loss from l, is observed; this fragment is not detected in compound 3. Compound 5 exhibits a peak at m/z 144 (q), which is due to the elimination of the propene group from its molecular ion through McLafferty rearrangement, which is negligible or less abundant in compounds 3 and 4. The differentiation of compounds 3–5 was achieved unambiguously using the above-observed diagnostic ions.

Figure 1.

EI mass spectra of N’-TMS derivatives of: (a) N,N-diethylacetimidamide (3), (b) N-isopropyl-N-methylacetimidamide (4), and (c) N-methyl-N-propylacetimidamide (5). N’-TMS: N’-trimethylsilyl.

Isomeric compounds 6 and 7

Compounds 6 and 7 exhibit almost similar mass spectra, with differences in the relative intensities of certain fragment ions (Supplemental Figure S1). In compound 6, the ions at m/z 171 and 157, arising from the loss of ^•C₂H₅ and ^•C₃H₇ from the molecular ion, respectively, appear as relatively prominent peaks (40% and 17%). This pattern is not observed in compound 7, where the intensities of these ions are considerably lower (2% and 3%). In contrast, compound 7 displays an ion at m/z 158 corresponding to [M^•+ − C₃H₆], attributable to a McLafferty rearrangement; this fragment ion is relatively less abundant in the spectrum of compound 6. Compounds 6 and 7 can be reliably differentiated based on the above-observed variations in relative abundances of the fragment ions.

Isomeric compounds 8–10

The EI mass spectra of isomeric compounds 8–10 (Figure 2) display a series of common fragment ions; however, their relative intensities vary significantly among the three isomers. For compound 8, the base peak appears at m/z 73, while the ion at m/z 114 is the most intense signal in compounds 9 and 10. In the spectra of compounds 8 and 10, a prominent fragment ion at m/z 171 is observed, corresponding to the loss of a propyl radical (^•C₃H₇) from [M^•+], with relative abundances of 65% and 42%, respectively. In contrast, this fragment is relatively less abundant for compound 9 (∼7%). Additionally, loss of C₃H₆ from the molecular ions via McLafferty rearrangement occurs in all three cases, yielding ions with m/z 172; however, in the case of 8, that peak could be mostly due to ¹³C- and ²⁹Si-isotopologs of the abundant ions with m/z 171. In contrast, the molecular ions of 9 and 10 clearly undergo elimination of C₃H₆, giving rise to diagnostic peaks at m/z 172. This behavior is particularly pronounced for isomer 9, containing two N-propyl groups, whereas the “mixed” N-isopropyl-N-propyl isomer 10 shows a less intense m/z 172 ion because its molecular ion more readily loses a ^•C₃H₇ radical. The fragment ions at m/z 143 are observed in both 9 and 10 on the loss of ^•C₂H₅ radical, followed by C₃H₆ from the molecular ion, whereas this fragment is not observed in compound 8. Isomeric species (8–10) were readily differentiated based on their distinct EI-MS fragmentation profiles.

Figure 2.

EI mass spectra of N’-TMS derivatives of: (a) N,N-diisopropylacetimidamide (8), (b) N,N-dipropylacetimidamide (9), and (c) N-isopropyl-N-propylacetimidamide (10). N’-TMS: N’-trimethylsilyl.

Retention indices

In this study, the retention indices for compounds 1–10 were determined in accordance with OPCW Work Instructions.²⁵ Notably, the N’-TMS derivatives of N,N-dialkylacetimidamides exhibited higher Retention Index (RI) values relative to their intact and methylated counterparts,²⁰ reflecting an increase in the chromatographic retention of these specific derivatives. These RI data are summarized in Table 2.

Table 2.

Retention indices of compounds 1–10.

N’-TMS derivatives of N,N-dialkylacetimidamides (1–10)
Compound ID	Retention index
1	1017.18
2	1095.71
3	1136.13
4	1130.97
5	1159.35
6	1176.77
7	1198.06
8	1217.24
9	1267.59
10	1242.76

Conclusion

This study provides a systematic evaluation of the EI mass spectral profiles for a series of TMS derivatives of amidines (1–10). All investigated compounds exhibited diagnostic fragment ions characteristic of the amidine moiety. Furthermore, structural isomers were effectively differentiated by their unique fragmentation pathways and variations in relative ion abundances. Moreover, retention index values were determined for all analytes according to established OPCW protocols. This synergistic use of EI mass spectral data and RI values facilitates the reliable and unambiguous identification of amidines in complex mixtures, while contributing essential data to established spectral libraries.

Supplemental Material

sj-docx-1-ems-10.1177_14690667261460791 - Supplemental material for Electron ionization mass spectral studies of a series of N'-trimethylsilyl derivatives of N,N-dialkylacetimidamides

Supplemental material, sj-docx-1-ems-10.1177_14690667261460791 for Electron ionization mass spectral studies of a series of N'-trimethylsilyl derivatives of N,N-dialkylacetimidamides by Manasa Masna, S. Babu Dadinaboyina, Sandhya Bobbali, Ramunaidu Addipilli, Srinivas Kantevari and Jagadeshwar Reddy Thota in European Journal of Mass Spectrometry

Footnotes

Acknowledgements

The authors thank the Director of CSIR-Indian Institute of Chemical Technology for Facilities (IICT Communication Number: IICT/Pubs./2026/085). MM acknowledges ICMR, New Delhi, for the fellowship. SB acknowledges DST, New Delhi, for Senior Research Fellowship.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Jagadeshwar Reddy Thota

Supplemental material

Supplemental material for this article is available online.

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