Prediction of the Fragmentation Pathway of Valsartan Protonated Ion

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.

• Valsartan
• Small Organic Molecules
• Fragmentation Pathway
• Protonated Ion
• Fragmentation
• Interpretation

1. Introduction

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 [1] - [13] .

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 [14] [15] [16] .

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 [17] [18] [19] is an angiotensin-receptor blocker (ARB) that may be used to treat a variety of cardiac conditions including hypertension, diabetic nephropathy, and heart failure. Valsartan lowers blood pressure by antagonizing the renin-angiotensin-aldosterone system (RAAS); it competes with angiotensin II for binding to the type-1 angiotensin II receptor (AT1) subtype and prevents the blood pressure increasing effects of angiotensin II. Unlike angiotensin-converting enzyme (ACE) inhibitors, ARBs do not have the adverse effect of a dry cough. Valsartan may be used to treat hypertension, isolated systolic hypertension, left ventricular hypertrophy and diabetic nephropathy. It may also be used as an alternative agent for the treatment of heart failure, systolic dysfunction, myocardial infarction and coronary artery disease (drug bank and rxlist). Valsartan is chemically described as N-(1-oxopentyl)-N-[[2'-(1H-tetrazol-5-yl) [1,1'-biphenyl]-4-yl]methyl]-L-valine. Its empirical formula is C₂₄H₂₉N₅O₃, its molecular weight is 435.5, and its structural formula presented in Figure 1.

During this study mass, spectral data of valsartan was generated using electrospray ionization, and collision-induced dissociation; followed by interpretation, workflow [15] and basic rules were used for the interpretation of the full scan atmospheric pressure ionization mass spectra (MS), collision-induced dissociation fragmentation spectra (MS/MS) and MS³ data.

2. Experimental

2.1. Drug Sample

Valsartan was extracted from the generic dosage form. A final concentration was about ten µg/ml in a mixture of water, methanol, and acetonitrile.

2.2. Chemicals and Reagents

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.

2.3. Instrumentation

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.

2.4. Chromatographic and Mass Spectrometric Conditions

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 [8] - [13] . Interpretation workflow of [15] was applied for data interpretation. The same work flow can also be applied for de-protonated ions or negative ionization mode [20] .

3. Results and Discussion

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. Figure 1 shows the ESI+ mass spectrum (MS) of valsartan acquired using ESI (+ve). The protonated ion peak appeared at m/z 436.2 Da as [M + H]⁺ (calculated formula C₂₄H₃₀N_{5 O 3 +} ). The protonated ion peak was further confirmed by calculating the mass difference with an adduct ion at m/z 458.1 as [M + Na]⁺ and 474.1 [M + K]⁺ as exhibited in MS spectrum.

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 Table 1.

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 Table 2. During MS³ analysis four new fragments 345.1 (PI-10), 194.1 (PI-11), 192.1 (PI-12) and 167.1 (PI-13). Fragmentation of PI-10 fragment ion (m/z 345) obtained and presented in Figure 8. Based on MS/MS and MS³ spectral data, fragmentation pathway was predicted or depicted and presented in Figure 11 and interpretation of fragment ions, presented in Table 1 and Table 2. Fragmentation pathway presented in Figure 11, for medium and low molecular weight fragments there are multiple source for formation as presented in Table 2 (MS³ information). Proposed mechanism for the formation of all significant fragment ions PI-1 to PI-13 presented in Figures 12-24.

Table 1

MS/MS spectra of atorvastatin based on rational work flow

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.

Table 2

Fragment ions (MS3) interpretation

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.

4. Conclusion

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.

Acknowledgements

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.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

Abbreviations Used

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.

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17. https://en.wikipedia.org/wiki/Valsartan

18. https://www.drugs.com/cdi/valsartan-capsules.html

19. https://www.rxlist.com/diovan-drug.htm

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