Fragmentation Considerations Using Amidoamine Oxide Homologs

Abstract

This study investigates the mass spectrometric analysis of 10 novel amidoamine oxide compounds, which are innovative hydrogelators for polar solvents. This research aims to identify characteristic fragment patterns for these amide compounds using high-resolution mass spectrometry. Methanol solutions of the compounds were analyzed in positive and negative ion modes, and MS1 and MS2 spectra at 6 collision energy levels were obtained via electrospray ionization and hybrid tandem mass spectrometry. The importance of low-intensity peaks in structure elucidation was emphasized because low-intensity fragments could provide crucial structural information, especially for compounds with similar structures. Chain-length-dependent fragmentation patterns were observed, which could aid in predicting the structures of related compounds. This research highlights the challenges of balancing informative low-intensity peaks with accurate spectral matching in databases. Based on our results, combining mass spectrometry with separation techniques, such as liquid chromatography, could enhance structural elucidation for unknown compounds. This study contributes to the broader field of mass spectrometry and structural chemistry, particularly in the analysis of amide compounds, and future directions are proposed for developing robust algorithms for selecting and interpreting low-intensity peaks to improve compound identification in complex mixtures.

INTRODUCTION

Amidoamine oxide (AAO) is a novel hydrogel capable of gelling polar solvents and has recently attracted attention as an innovative material in the field of soft materials.^1–3) These amide compounds require a stable structure during synthesis, necessitating the accurate acquisition of various mass spectra to comprehensively determine the unknown molecular structures formed during the synthetic process.

Generally, structural analysis of these amide compounds often yields characteristic fragment ions cleaved at the amide bond, similar to peptide analysis in proteins. However, while scattered examples of spectral analyses for protein structural studies and individual amide compound investigations exist, comprehensive information on the trends of characteristic fragment ions and spectra obtained from the simultaneous analysis of similar structures is lacking.

In our recent work,⁴⁾ we synthesized 10 types of AAOs with varying chain lengths. This study aimed to simultaneously acquire and analyze the mass spectra of these novel amide compounds to verify whether characteristic fragment patterns for amide compounds could be identified. Specifically, we obtained MS1 and MS2 spectra for 10 amide compounds using high-resolution mass spectrometry. The acquired data were subsequently analyzed using SCIEX OS software.

By disseminating the information on characteristic fragment ions of amide compound structures obtained in this study, we aim to elucidate the aspects of the fragmentation mechanism of molecules containing amide bonds. This knowledge is expected to facilitate the structural analysis of similar amide compounds, contributing to the broader field of mass spectrometry and structural chemistry.

EXPERIMENTAL PROCEDURES

Chemicals

All compounds used in this study are shown in Figs. 1 and 2. These compounds were synthesized in a previous report.⁴⁾ Each AAO was dissolved in methanol, diluted to a concentration of 0.1 μg/mL, and infused at 10 μL/min using a YSP-201 syringe pump (YMC, Kyoto, Japan). The structures and purities of all compounds were confirmed via nuclear magnetic resonance (NMR). The identifiers of all compounds are described in the Supplementary MassBank⁵⁾ record files.

Fig. 1. Chemical structure of alkyl amidoamine oxide. k, l, m, and n represent the length of the methylene chain in the hydrophobic part, between the nitrogen atoms of the amide group, between the carbonyl carbons of the amide groups, and between the amide and the amine oxide, respectively. Amidoamine oxide is denoted as k-l-m-n according to the length of the methylene of the methylene chain in four places.

Fig. 2. Chemical structures of the 10 amidoamine oxides.

Analytical conditions

The analytical conditions employed in this study are shown in Table 1.

Table 1. Analytical conditions employed in this study.

Mass spectrometry
Instrument	X500R QTOF (AB Sciex LLC, Framingham, MA, USA)
Instrument_Type	ESI-QTOF
Chromatography
Inlet_Type	Infusion
Solvent	Methanol
Mass_Spectrometry
MS_Type	MS2
Ion_Mode	Positive and negative ion mode
Capillary_Voltage	5500 V (positive), 4500 V (negative)
Collision_Energy	10, 20, 30, 40, 50, and 60 V
Collision_Gas	N2
Fragmentation_Mode	CID
Ionization	ESI

RESULTS AND DISCUSSION

Methanol solutions of the 10 compounds were analyzed by direct infusion into the mass spectrometer in both positive and negative ion modes. Initially, MS1 spectra were obtained, followed by MS2 spectra from precursor ions [M+H]⁺ and [M−H]⁻ at 6 collision energy levels. In total, 133 spectra were acquired. The MS1 and MS2 spectra showed that the effective product ions were converted into MassBank Records (see the Supplementary Tables and Files: m/z were automatically exported from the software).

Figure 3 shows an example of MS2 spectra of 13-2-2-6 in the positive-ion ESI. Although a lot of important information to annotate the molecular structure includes the noise peaks of the MS2 spectrum for the research fields of informatics, we have to select a threshold to show the spectrum. Therefore, MS2 obtained was up to 1% of the base peak at the present analyses. The molecular formula of each product was estimated, as shown in Table 2; these results indicated an estimation with a high mass accuracy of less than 5 ppm error. The AAO structure was readily interpretable because of the presence of fragment ions. These ions, resulting from cleavage between amide nitrogen and carbonyl carbon atoms, were analogous to b or y ions commonly observed in collision-induced dissociation of peptides.⁶⁾ Subsequently, the structural fragment assignment was performed, resulting in reasonable structural estimates (Fig. 4).

Fig. 3. MS2 of 13-2-2-6 amidoamine oxide acquired in positive ion mode.

Table 2. Annotation of ions observed in the product ion spectrum of 13-2-2-6 [M+H]⁺ (ESI+) with calculated errors.

Name	Accurate m/z	Formula	Exact m/z	Delta (Δ) ppm
13-2-2-6	513.4362	C28H57N4O4+	513.4374	−2.4
13-2-2-6’	495.4260	C28H55N4O3+	495.4269	−1.7
13-2-2-6†	452.3839	C26H50N3O3+	452.3847	−1.7
13-2-2-6†’	434.3734	C26H48N3O2+	434.3741	−1.6
b3	353.2793	C20H37N2O3+	353.2799	−1.6
y2	271.2739	C16H35N2O+	271.2744	−1.8
z2	254.2474	C16H32NO+	254.2478	−1.7
b2	243.1700	C12H23N2O3+	243.1703	−1.3
y1†	242.1859	C12H24N3O2+	242.1863	−1.7
y1†’	224.1753	C12H22N3O+	224.1757	−1.9
c2†	199.1437	C10H19N2O2+	199.1441	−2.0
b2†–2H	182.1172	C10H16NO2+	182.1176	−1.9
y3	161.1645	C8H21N2O+	161.1648	−2.1
y1b3	143.0812	C6H11N2O2+	143.0815	−2.1
z1b3–2H	126.0546	C6H8NO2+	126.0550	−2.8
	112.0390	C5H6NO2+	112.0393	−2.7
y3†	100.1117	C6H14N+	100.1121	−2.7
c2b3	100.0390	C4H6NO2+	100.0393	−3.7
	98.0961	C6H12N+	98.0964	−3.3
	58.0658	C3H8N+	58.0657	−12

Fig. 4. Fragment ion notation: (A) genetic notation, (B) prime means loss of H₂O, and (C) dagger symbol (†) indicates the loss of the N,N-dimethylamine oxide group.

The ions y₁† and z₁b₃ appeared to deviate from Nakata’s mass shift rule.⁷⁾ According to this rule, when bonds between a heteroatom and a carbon atom are broken in two places, with the charge on the carbon atom side, the mass shift in positive-ion mode should be 1. A reasonable mass shift was found for z₁† (m/z 227.1751); this fragment was produced by a similar cleavage, whereas y₁† and z₁b₃ showed no such ions and had two fewer hydrogen atoms. These results indicated that the cleavage was not simple but a rearrangement reaction, such as cyclization.

Figure 5 shows a pronounced increase in the b₂† signal at n = 3. A ring structure is known to be formed during the fragmentation of the ions containing multiple amide bonds, such as peptides.^8,9) The formation of ring structures during fragmentation has also been reported in phospholipids.¹⁰⁾

Fig. 5. MS2 spectra of the amidoamine oxides with different chain lengths at CE = 40 V. (A) k, l dependence and (B) m, n dependence.

In this study, the intensity of b₂† was highest when n = 3; these results indicated the presence of cyclic structures that were more likely to be formed at this chain length than other structures in Fig. 6. While some structures could be identified using only major peaks, fragment ions with an intensity of less than 1% could also provide meaningful information when verifying the fragmentation of congeners with different chain lengths. The b₃ ions were predominant in the AAO fragments with large l values, such as 13-5-2-6, whereas with shorter ions, such as 13-2-2-3, the b₃ ions showed only low-intensity peaks, and their m/z values were very accurate. One possible factor for the ease of cleavage at b₃ is its relationship to neutral loss formation. The reason may be that n = 3 is insufficient length to form a neutral loss with a stable ring structure. Thus, weak ions were not necessarily meaningless, and in many cases, they could be useful in reading a structure from mass spectra.

Fig. 6. Estimated fragment ion structures. (A) z2 and (B) b2†–2H (one of the predicted structures).

In some preceding studies, tropylium ions indicate the presence of benzyl and tolyl groups, and other ions are useful for analysis, even if their intensity is lower. When fragments derived from HF elimination were found, the interval of fragment ions with Δm/z = 20.006 was easily obtained from neutral loss. Therefore, when these intervals exist, it could be used to include fluorine as a candidate element in compositional formula estimation. Thus, a common concept is that more peaks contained in a spectrum need to be included for analysis. However, in general, if all fragments, even those with low intensity, are included in mass spectra libraries, misidentification can also occur during brute force library searches; thus, fragments with low intensity are usually removed. Some consensus is expected to be established on the required quality of the mass spectra provided in the public mass spectral repository.

In this study, MS1 and MS2 mass spectra were utilized and obtained by infusion for structural identification of each compound. The selected compounds featured varying chain lengths. This diversity enabled the determination of the structure-specific fragmentation patterns and efficiently determined the AAOs. On the other hand, the characteristic fragment ions could not potentially be detected in samples that contain unknown structures. In these cases, combining other separation methods could be useful for further structural identification. For example, when phospholipids are analyzed via liquid chromatography (LC), retention times often follow predictable trends. These trends correlate with the acyl chain length and the number of double bonds. In this study, the addition of LC separation could help quickly determine the unknown AAO structures.

CONCLUSION

The present study provides valuable insights into the mass spectrometric analysis of novel AAOs. We observed that the intensity of certain fragment ions, such as b₂†, varied systematically with the chain length of the compounds. This relationship could be particularly useful for predicting the structures of related compounds. Although high-intensity peaks often suffice for structural identification, our analysis revealed that low-intensity fragments could provide crucial structural information. For example, b₃ ions in shorter-chain AAOs had low and highly accurate m/z values, demonstrating their utility in structural elucidation. The intensity patterns of certain ions, particularly b₂†, indicated the formation of cyclic structures during fragmentation. These patterns were most prominent when n = 3; these results indicated a chain-length dependence on this type of fragmentation behavior. By analyzing the MS1 and MS2 spectra of the 10 structurally related compounds simultaneously, we were able to identify characteristic fragmentation patterns specific to AAOs. These findings highlight the complex balance between including informative low-intensity peaks and maintaining accurate mass spectral matching in databases. Future research needs to focus on developing robust algorithms for selecting and interpreting low-intensity peaks to increase the accuracy and efficiency of compound identification in complex mixtures.

However, considering the trade-off between comprehensive spectral information and database efficiency is crucial. As we incorporate more data on low-intensity ions, the database retrieval time may increase, potentially impacting the speed of spectral matching processes. Therefore, future research needs to address optimizing the database structures and search algorithms to manage this increased data volume without compromising analysis speed. Additionally, integrating our MS approach with separation techniques such as LC could further improve the structural elucidation of the unknown AAO compounds.

Our work contributes to the broader field of mass spectrometry and structural chemistry, particularly in the analysis of amide compounds. Our study highlights the need for comprehensive spectral analysis and the potential of systematic structural variations in enhancing our understanding of the fragmentation mechanisms.

ACKNOWLEDGMENTS

Each AAO mass spectrum was converted to MassBank records, courtesy of Dr. Takaaki Nishioka. This study is financially supported by Grant-in-Aid for Scientific Research A of Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number 23H00539).

Notes

Mass Spectrom (Tokyo) 2024; 13(1): A0158

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