Introduction: A fragmentation pathway of valsartan protonated ion proposed based on rational interpretation workflows. Method: The mass spectral data (MS, MS/MS, and MS3) of valsartan protonated ion, were attained by electrospray positive ionization with flow injection analysis; using liquid chromatography systems hyphenated with hybrid tandem mass spectrometer (Q-trap). Results: The pathway of fragm entation was established using product ions of the protonated ion; elemental composition, molecular structure and mechanism of formation for significant product ions were proposed. A pathway was proposed based on the MS/MS and MS3 spectral data in combination with basic interpretation rules and rational workflows. Conclusion: Workflow used for data interpretation can be useful for writing the fragmentation pathway, a mechanism for the formation of fragments, and can be applied for mass spectral data interpretation of similar small organic molecules.
Nowadays the use of Mass spectrometry (MS) is amplified in the analytical research laboratories of pharmaceutical research and development. Mass spectrometry plays an essential role for the characterization and identification of small and large drug molecules. Understanding mass spectrometry data interpretation became necessary for the research scientist working in the field of pharmaceutical research. The application of mass spectrometry is kept on increasing day by day. Therefore in this study spectral data generated using advanced mass spectrometric systems, along with reasonable data interpretation is beneficial for comprehensive structural analysis study, i.e., to study the fragments and to propose a pathway [
The focus of this study solely towards the mass spectral data interpretation, during this study unit-resolution tandem mass analyzer (Q-trap) mass spectrometry systems with trap functionality, was used. Q-trap analyzer was very helpful to generate MS3 spectral data by using the third quadrupole as a trap. MS3 information directed to write a fragmentation pathway for parent and product ions. A small nitrogenous organic molecule with a carboxylic acid functional group; valsartan was selected for this study and spectral data were generated using positive ion mode. Followed by prediction of fragmentation pathway of protonated ion, using similar methodology or workflow as described or applied in [
Mass spectrometry spectral data interpretation is the most time taking and complicated process. So, here use of a step by step, rational workflow to interpret MS (protonated peak) and MS/MS (collision-induced dissociation or fragmentation), and MS3 spectra of valsartan (small drug molecule) depicted.
Valsartan [
During this study mass, spectral data of valsartan was generated using electrospray ionization, and collision-induced dissociation; followed by interpretation, workflow [
Valsartan was extracted from the generic dosage form. A final concentration was about ten µg/ml in a mixture of water, methanol, and acetonitrile.
The ultrapure water (18.2 MΩ) was obtained using MilliQ apparatus from Millipore (Milford, USA), acetonitrile HPLC grade and the HPLC grade methanol was purchased from J. T. Baker.
Shimadzu Prominence 20 AD HPLC (Kyoto Japan) and Waters Aquity HClass were coupled with Q-trap 5500 (AB SCIEX), equipped with electrospray ionization source (ESI) were used for this analysis.
The experimental data (MS and MS/MS spectral data) of valsartan was generated using high-performance liquid chromatography (HPLC) coupled with Q-trap mass analyzer via Flow Injection Analysis (FIA) mode and Electro-spray Ionization (ESI+) ion source. Collision-induced fragmentation (CID) technique in collision cell (Q2) of MS/MS spectrometer was used for the fragmentation of molecular ion into further fragments. The fragmentation pathways are preferably based on even-electron (EE) and least favorable to the formation of odd-electron (OE) ions in spectra, EE ions will generally result from the charge migration or transfer across the cleavage sites, rearrangement and multiple bond cleavage are common in CID spectra [
The mass spectrometry technique is a modern method for the identification of small molecules and their impurities. The full scan MS and product ion spectra of valsartan were obtained from experiments.
The MS/MS spectrum of valsartan was acquired at collision energy (CE) 10, 20, and 30; to understand and differentiate between the low and high energy fragments and MS/MS spectra of m/z 436.2 at different collision energy were presented in Figures 2-4 and summarized in
MS/MS spectra in Figures 2-4 showing the shift of low energy fragments to high energy fragments, aforesaid MS/MS information along with MS3 was helpful for writing the fragmentation pathway. During CID fragmentation of parent ion 436.2 (M + H)+ ion fragmented into 11 fragments (9 major and 2 trace); m/z 418.2 (PI-1), 408.3 (PI-2), 390.3 (PI-3), 362.4 (PI-4), 352.2 (PI-5), 306.2 (PI-6), 291.1 (PI-7), 235.1 (PI-8), 207.0 (PI-9), 190.0 and 180.0.
Major productions of MS/MS m/z 418.2 (PI-1), 362.4 (PI-4), 352.2 (PI-5), 306.4 (PI-6) and 291.1 (PI-7) are subjected of MS3 analysis, and MS3 spectra are presented in Figures 5-10. The fragmentation outcome of MS3 analysis summarized in
Parent ion (m/z) | CE | Product ions (m/z) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
PI-1 | PI-2 | PI-3 | PI-4 | PI-5 | PI-6 | PI-7 | PI-8 | PI-9 | |||
436.2 C24H30N5 O 3 + | 10 | 418.2 | 408.3 C22H26N5 O 3 + | - | 362.4 | 352.2 | 306.2 | 291.1 | 235.1 | - | |
20 | 418.2 | 408.3 | 390.2 | 362.4 | 352.2 | 306.2 | 291.1 | 235.1 | 207.0 | ||
30 | - | - | - | 362.4 | 352.2 | 306.2 | 291.1 | 235.1 | 207.0 |
EE = even electron; EN = even nitrogen; ON = odd nitrogen; CE = Collision energy; PI = product ion.
Measured Mass (m/z) (MS/MS) | ID | Measured Mass (m/z) | Electron Paring | Nitrogen Rule | No. of Nitrogen (s) | Proposed Formula | Calculated Mass |
---|---|---|---|---|---|---|---|
418 (PI-1) C24H28N5 O 2 + Calculated: 418.2238 | PI-3 | 390.0 | EE | ON | 5 | C22H24N5 O 2 + | 390.2 |
PI-4 | 362.2 | EE | ON | 5 | C20H20N5 O 2 + | 362.2 | |
PI-10 | 345.1 | OE | ON | 5 | C21H23N5•+ | 345.2 | |
PI-6 | 306.1 | EE | ON | 5 | C18H20 N 5 + | 306.2 | |
PI-7 | 291.2 | OE | ON | 5 | C17H17N5•+ | 291.1 | |
PI-8 | 235.0 | EE | EN | 4 | C14H11 N 4 + | 235.1 | |
PI-9 | 207.2 | EE | EN | 2 | C14H11 N 2 + | 207.1 | |
362 (PI-4) C20H20N5 O 2 + Calculated: 362.1612 | PI-4 | 362.2 | EE | ON | 5 | C20H20N5 O 2 + | 362.2 |
PI-10 | 345.1 | OE | ON | 5 | C21H23N5•+ | 345.2 | |
PI-7 | 291.1 | OE | ON | 5 | C17H17N5•+ | 291.1 | |
352 (PI-5) C19H22N5 O 2 + Calculated: 352.1768 | PI-6 | 306.2 | EE | ON | 5 | C18H20 N 5 + | 306.2 |
PI-8 | 235.0 | EE | EN | 4 | C14H11 N 4 + | 235.1 | |
PI-9 | 207.1 | EE | EN | 2 | C14H11 N 2 + | 207.1 | |
345.0 (PI-11) C21H23N5•+ (MS3) | PI-6 | 306.2 | EE | ON | 5 | C18H20 N 5 + | 306.2 |
PI-8 | 235.1 | EE | EN | 4 | C14H11 N 4 + | 235.1 | |
PI-9 | 207.2 | EE | EN | 2 | C14H11 N 2 + | 207.1 | |
306 (PI-6) C18H20 N 5 + Calculated: 306.1713 | PI-8 | 235.1 | EE | EN | 4 | C14H11 N 4 + | 235.1 |
PI-9 | 207.1 | EE | EN | 2 | C14H11 N 2 + | 207.1 | |
291(PI-7) C17H17N5•+ Calculated: 291.1478 | PI-8 | 235.1 | EE | EN | 4 | C14H11 N 4 + | 235.1 |
PI-11 | 194.1 | EE | ON | 1 | C14H12N+ | 194.1 | |
PI-12 | 192.1 | EE | ON | 1 | C14H10N+ | 192.1 | |
PI-13 | 167.1 | EE | EN | 0 | C13H11+ | 167.1 |
EE: even electron; EN: even nitrogen; ON: odd nitrogen; PI: Product ion.
During this study, fragment pathway was proposed for the valsartan protonated ion and mechanism for the formation of the signification fragments ions also suggested. The protonated ion peak as [M + H]+ of valsartan appeared at m/z 436. The CID fragmentation of protonated [M + H] + ion, basic massspectromety interpreation rules and MS3 fragment ions are supporting to the proposed fragmentation pathway. The suggested reaction mechanism will help to designate the mechanism for the similar small molecules, and a fragment ion with similar functional groups, workflow applied in this study was found efficient and can be applied for structure verification studies.
This paper is part of the Ph.D. thesis of Dev Kant Shandilya. The author expresses his gratitude to the Dean, Department of Research, Bhagwant University, Ajmer, Rajasthan, India for extending his constant support.
The authors declare no conflicts of interest regarding the publication of this paper.
Shandilya, D.K., Israni, R. and Joseph, P.E. (2018) Prediction of the Fragmentation Pathway of Valsartan Protonated Ion. Open Access Library Journal, 5: e4764. https://doi.org/10.4236/oalib.1104764
LC: Liquid chromatography; HPLC: High performance liquid chromatography; MS: Mass spectrometry; MS/MS: Tandem mass spectrometer; MS3: Tandem mass spectrometer with trap functionality; m/z: mass-to-charge ratio; API: Atmospheric pressure ionization; APCI: Atmospheric pressure chemical ionization; ESI: Electrospray ionization; CID: Collision-induced dissociation; FIA: Flow Injection Analysis.