MS: Mechanistic explanations of hydrolytic, oxidative and photocatalytic rearrangement of degradation products

Journal Pre-proof Study on forced degradation behaviour of dofetilide by LC-PDA and Q-TOF/MS/MS: Mechanistic explanations of hydrolytic, oxidative and photocatalytic rearrangement of degradation products Bhargavi Thalluri, Vivek Dhiman, Shristy Tiwari, Shandaliya Mahamuni Baira, M.V.N. Kumar Talluri

PII:

S0731-7085(19)32033-3

DOI:

https://doi.org/10.1016/j.jpba.2019.112985

Reference:

PBA 112985

To appear in:

Journal of Pharmaceutical and Biomedical Analysis

Received Date:

20 August 2019

Revised Date:

6 November 2019

Accepted Date:

10 November 2019

Please cite this article as: Thalluri B, Dhiman V, Tiwari S, Baira SM, Kumar Talluri MVN, Study on forced degradation behaviour of dofetilide by LC-PDA and Q-TOF/MS/MS: Mechanistic explanations of hydrolytic, oxidative and photocatalytic rearrangement of degradation products, Journal of Pharmaceutical and Biomedical Analysis (2019), doi: https://doi.org/10.1016/j.jpba.2019.112985

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Study on forced degradation behaviour of dofetilide by LC-PDA and QTOF/MS/MS: Mechanistic explanations of hydrolytic, oxidative and photocatalytic rearrangement of degradation products

Bhargavi Thalluri, Vivek Dhiman, Shristy Tiwari, Shandaliya Mahamuni Baira, M. V. N. Kumar Talluri* [email protected], [email protected]

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Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education &

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Research, IDPL R&D Campus, Balanagar, Hyderabad- 500 037. India.

*Corresponding author: Dr. M.V.N. Kumar Talluri, Department of Pharmaceutical Analysis, National

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Institute of Pharmaceutical Education & Research (NIPER), Hyderabad, India;

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Tel: +91-40-23074750, Ext: 2012, Fax: +91-40-23073751.

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Graphical abstract

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Highlights

Forced degradation studies was carried on dofetilide according to ICH Q1A (R2)

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guidelines.

Eight DPs were identified and separated on RP-HPLC.

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Characterization of degradation products using LC-Q-TOF/MS/MS in ESI and APCI

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positive mode.

Mechanistic explanation and mass fragmentation pathway was established for

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dofetilide and its DPs.

Abstract

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A solution and solid state forced decomposition study was carried on dofetilide under diverse stress conditions of hydrolysis, oxidation, photolysis and thermal as per International Council for Harmonisation guidelines (ICH) Q1A(R2) to understand its degradation behaviour. A total of eight degradation products (DPs) were identified and separated on reversed phase kromasil 100 C8 column (4.6 mm x 250 mm x 5 µm) using gradient elution with ammonium acetate (10 mM, pH 6.2) and acetonitrile as mobile phase. The detection wavelength was selected as 230 nm. The high performance liquid chromatography (HPLC) study found that

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the drug was susceptible to hydrolytic stress condition, but it was highly unstable to photolytic and oxidative conditions. The solid drug was stable in thermal and photolytic conditions. Initially comprehensive mass fragmentation pattern of the drug was accomplished

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with the LC/ESI/QTOF/MS/MS studies in positive ionization mode. The same was followed for all the eight degradation products to characterise their structure. The DP4 was N-oxide

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and the structure was confirmed by LC/APCI/QTOF/MS/MS in positive ionization mode.

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The complete mass fragmentation pattern of the drug and its DPs were established which in turn helped the characterisation of their structures. The mechanistic pathway for the

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formation of all the DPs was explained.

Keywords: Dofetilide, Forced degradation study, HPLC-PDA, Degradation products,

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Characterization, LC-MS.

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1. Introduction

Arrhythmia is a condition characterise by irregular single heartbeat (arrhythmic beat), or as an irregular group of heartbeats (arrhythmic episode) [1]. Clinically arrhythmia is classified according to the site of origin of the abnormality (atrial, junctional, or ventricular) and whether

the

heart

rate

is

increased

(trachycardia)

or

decreased

(bradycardia).

Antidysrhythmic drugs are classified into different classes (class I, II, III, IV) based up on 3

their electrophysiological effect, among them Dofetilide (TIKOSYN) comes under Class III [2]. Chemically dofetilide is N-[4-(2-{[2-(4-methane sulfonamidophenoxy) ethyl] (methyl) amino} ethyl) phenyl]methanesulfonamide [3]. The presence of a tertiary amine and methanesulfonamide linkage in the chemical structure of the drugs may induce degradation during the storage conditions and various stages of formulation development [4-5]. The drug stability can risk patient safety by formation of toxic DPs or influence the bioavailability of

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the drug [6-7]. The allergic response to sulfonamides is due to arylamino at position N4 present in sulfamethoxazole, sulfasalazine, and sulfadiazine antibiotics [7]. As dofetilide contains methanesulfonamide group, it is important to perform a comprehensive degradation

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study (hydrolytic, oxidative, thermal and photolytic) on dofetilide to identify its likely DPs and arylamino formation. Therefore, today’s regulatory necessity is identification and

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characterisation of very low quantities of toxic or genotoxic degradation products [8].

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Different hyphenated analytical techniques are available for identification, separation and characterisation of DPs or impurities (IMPs) which further helps in establishment of degradation pathways of the drugs [9-14].

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Dofetlide is official in United States Pharmacopeia (USP), an impurity also reported in USP monograph [15]. There are no report on the degradation behaviour of dofetilide according to

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ICH Q1A(R2) [16] and WHO [17] regulatory guidelines. Few of stability indicating methods

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were reported in the literature for its stability study. According to ICH Q1A (R2) stress testing or forced degradation study of the drug substance can help identify the likely degradation products, which can in turn help establish intrinsic stability of the molecule [16]. Dofetilide was subjected to different stress conditions for identifying its degradation behaviours. HPLC method was developed to identify dofetilide and its degradation products.

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The liquid chromatography mass spectrometry (LC-MS) study was carried out for characterisation and establishment of degradation pathway of dofetilide. 2. Experimental 2.1. Chemicals and reagents Pure drug was obtained as a gratis sample from MSN Laboratories Pvt. Ltd. (Hyderabad, India). All chemicals and reagents were of analytical grade were used in this study. Sodium hydroxide (NaOH) and ammonium acetate (CH3COONH4) was procured from SRL

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Chemicals (Hyderabad, India), Hydrochloric acid (HCl) (35%) from Merck (Mumbai, India), Hydrogen peroxide (H2O2) (6%) from S D Fine-Chem Ltd. (Mumbai, India), HPLC grade acetonitrile (ACN) was procured from Duksan pure chemicals (South Korea). HPLC grade

2.2. Apparatus and equipments

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Chennai India) were used to prepare all solutions.

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water (H2O) was obtained from Ultra Clear TWF system, (Evoqua Water Technologies,

Dofetilide stressed samples were analyzed using Alliance HPLC system-e2695 (Waters

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Corp., Milford, MA, USA) equipped with photodiode array (PDA) detector (model 2998). The separation was achieved using Kromasil 100 C8 column (4.6 mm x 250 mm x 5 µm).

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The HPLC chromatogram was acquired using Empower-3 software (Waters corp.). Accurate mass spectra was attained by using an Agilent1200 infinity series LC

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instrument (Agilent Technologies, Santa Clara, California, USA) coupled with quadrupole time-of flight mass spectrometer (Q-TOF, LC/MS G6540B, Agilent Technologies).

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Instrument control and accurate mass data was processed using Mass Hunter Workstation software. The ionization was carried out in electrospray ionization (ESI positive mode) and atmospheric pressure chemical ionization (APCI positive mode). In

ESI positive mode

different parameters were optimised as follows: the fragmentor voltage was set at 130 V, the capillary at 4000 V, the skimmer at 65 V, and nitrogen was used as the drying (325 C,

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10L/min) and nebulizing (275.79 kPa) gas. For collision-induced dissociation experiments, ultra high pure nitrogen gas was used as collision gas (30 eV). The formation of N-oxide was confirmed by APCI +ve mode using 400 C vaporizer temperature and source current was set as 4 µA. The photostability studies were carried out in Newtronic photostability chamber (Newtronic lifecare equipment Pvt. Ltd. Umbergaon, Maharashtra) equipped with ultraviolet (UV) light

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and fluorescent lamp, according to ICH Q1B [18]. The solid state thermal degradation studies were carried out in the Osworld laboratory oven (Osworld Scientific Pvt. Ltd., Mumbai, India). An ultra-sonicator from PCI Analytes and pH meter (Eutech Instruments, Singapore) were used to dissolve the sample and to measure the pH of the mobile phase, respectively.

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Heating plate (IKA RCT Basic, Bengaluru India) was used for forced degradation studies.

different chemicals used in this study.

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2.3. Stress studies

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Weighing balance Sartorius CPA225D (Mumbai, India) was used for weighing the drug and

Dofetilide was subjected to different stress conditions like hydrolytic (at different pH

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conditions), oxidative, thermal and photolytic condition (solid and solutions at different pH conditions) as per ICH Q1A (R2) [16] and ICH Q1B [18] and followed the protocol

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published by Singh and Bakshi,s [19]. All the stress studies were carried at a final drug concentration of 500 ppm (w/v). Acidic and alkaline hydrolytic study was initiated with

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lower stressor concentration and increased to higher concentrations for that, 5mg of the drug was mixed in 10 ml of 5N HCl as well as 2N NaOH solutions in separate volumetric flasks to the final concentration of the drug was 500 ppm and subjected to heating at 80 °C. For the neutral hydrolytic conditions, drug was added in diluent (ACN:H2O, 20:80%) was heated at 80 °C. Oxidative degradation was carried out by using 0.1% H2O2, solution was kept at room temperature for 6 h in dark place. Photolytic studies were carried in solution (0.5 N HCl, 0.2 6

N NaOH) and solid form of drug, by exposing them to ultraviolet (UV) and fluorescent lamp for 12 days of time. Thermal decomposition studies were performed on solid sample of the drug in amber color vial to dry heat at 100 °C for 7 days by keeping it in hot air oven. The samples were withdrawn at their respective time points and refrigerated till analysis. 2.4. HPLC method development and optimisation The process of method development for stressed samples were initiated using Chromocil C8 (4.6mm x 250mm x 5µm) column using ACN and CH3COONH4 (10 mM, 6.2 pH) as mobile

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phase. Various chromatography conditions like pH of the buffer, gradient conditions, flow rate, column, and column temperature were varied systematically to achieve optimised HPLC method. The detection wavelength was set at 230 nm which was the maximum of the UV

2.5. LC-MS/MS studies on the drug and DPs

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absorption spectrum of the drug.

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Mass fragmentation pattern of the drug and DPs were established with the help of LC-MS in

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ESI and APCI positive mode. About 5 ppm concentration of drug solution and stressed samples were injected into LC/ESI/QTOF/MS/MS and mass parameters were optimized in

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order to get clear information regarding the molecular ion peak of the drug and its DPs. 3. Results and discussion

3.1. HPLC chromatogram and stress degradation behaviour of dofetilide

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The HPLC method for stressed samples was developed and optimised using reversed phase

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Kromasil 100 C8 (4.6 mm×250 mm×5 µm) column, column temperature was set as 35 C. Acetonitrile (A) and 10 mM aamonium acetate (B) with pH 6.2 was used as mobile phase with gradient method (A: T0.01/10, T2.0/10, T4.0/13, T5.0/24, T15.0/90, T16.0/90, T18/10, T20/10 and B: T0.01/90, T2.0/90, T4.0/87, T5.0/76, T15.0/10, T16.0/10, T18/90, T20/90). The wavelength was set as 230 nm. The drug was shown significant degradation behaviour in 5N HCl (80°C, 48h), 2N NaOH (80°C, 48h) respectively. It was highly prone to degradation in the presence 7

of mild oxidative 0.1 % and photolytic conditions. A total eight DPs were formed in different stress conditions as shown the figure [Fig.1] and table [Tab.1]. 3.2. Mass fragmentation behaviour of the drug Dofetilide is encompassing of two prominent rings, 4-aminophenol (A), p-toluidine (B) and tertiary amine as shown in figure [Fig.S1]. The mass fragmentation pattern of the drug was established using LC/ESI/QTOF/MS/MS in positive ionisation mode. The mass spectrum of the drug was shown in figure [Fig.S2] and labelled as a to t and the molecular ion peak as

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[M+H] +. The most possible molecular formulae of the fragments were determined using an elemental composition calculator. Accurate mass, exact mass, molecular formula, along with error in mmu of the drug and its fragments were shown in table 2. This information was taken

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into consideration for establishing the fragmentation pathway of the drug as shown in the figure [Fig.2]

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The MS/MS spectra of the drug revealed that m/z 442 [M+H]+ was protonated parent ion

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shown in the figure [Fig.S2]. The elimination of hydrosulfomethane (m/z 78) from m/z 442, resulted in two fragment ions m/z 363 based on the protonation to 4-aminophenol (A) and ptoluidine (B). The fragment ion m/z 363 formed from 4-aminophenol (A) gives m/z 348, m/z

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284 and m/z 271 on loss of CH3, CH3O2S and C4H6NO2S respectively. The m/z 179 and m/z 163 were formed from m/z 348 on the loss of C7H7NO2S and C7H7NO3S respectively. After

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that fragment ions m/z 149 and m/z 71 were formed from m/z 163 on the elimination of CH2

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and C6H6N respectively. The fragment ion m/z 119 was formed from m/z 179 and m/z 149 on the loss of C2H5NO and CH3N respectively. The fragment ion m/z 58 was generated from m/z 71 on the loss of CH2. The m/z 271 yielded a fragment ion m/z 245 on the loss of C2H2. Further m/z 245 generated an m/z 214 on the loss of CH3NH2. The fragment ion m/z 135 was formed from m/z 214 on

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the loss of CH3O2S. The protonation on p-toludine (B) as shown in figure [Fig.2] was generated a fragment ion m/z 363 on the loss of CH3O2S. Further m/z 363 was generated a fragment ion m/z 255 on the loss of C6H6NO. After that on the loss of CH3 m/z 228 was formed from m/z 255. The fragment ion m/z 198 was formed from m/z 228 on the loss CH4N. After that fragment ion m/z 120 was formed from 198 on the loss of CH4O2S. Two fragment ions m/z 107 and m/z 103 formed from m/z 120 on the loss of CH radical and NH2 respectively. Further m/z 91 was formed from m/z 107 on the loss of

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NH2. The fragment ions m/z 198, 120, 107, 103 were found to be common in drug and DPs fragmentation pattern.

3.3 Characterization of the degradation products by LC/ESI/QTOF/MS/MS

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The stressed samples were also subjected to LC/ESI/QTOF/MS/MS positive mode to characterise their structure. The mass line spectra of DPs are shown figures Fig. (S3-S4).

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Most plausible structures could be proposed for all the DPs with the help of

the table [Tab.3]. 3.3.1. DP1 (364.1690)

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LC/ESI/QTOF/MS/MS. Accurate mass, exact mass, error in mmu, RDB of DPs is shown in

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The accurate mass of DP 1 was found to be m/z 364.1690 [M + H]+ [Fig.S3a] formed by hydrolysis of methane-sulphonamide groups. The two fragment ions m/z 255 and m/z 167

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were formed from m/z 364 on the loss of C6H7NO and C9H12NO2S respectively. The m/z 255

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generated two fragments ions m/z 229 and 198 on the loss of C2H4 and C3H7N respectively. Further m/z 58 was formed from m/z 229 on the loss of C7H9NO2S. The fragment ion m/z 120 was formed from m/z 198 on the loss of CH3O2S. After that fragment ion m/z 103 was formed from m/z 198 and m/z 120 on the loss of CH5NO2S and NH3 respectively. The m/z 77 was formed from m/z 103 on the loss of C2H2. The fragment ion m/z 136 was formed from m/z 167 on the loss of CH5N [Fig.3]. DP 1 was confirmed as N-(4-(2-((2-(4-aminophenoxy)

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ethyl (methyl)amino)ethylphenyl) methanesulfonamide based upon the m/z 255 a characteristic fragment and followed the drug fragmentation pattern shown in figure [Fig.2].

DP 2 (286.1915) The accurate m/z value of DP 2 was found to be 286.1915 [M + H]+ [Fig. S3b]. The DP 2 was formed by hydrolysis of two methane-sulphonamide. Formation of DP 2 was confirmed by the fragmentation pattern as shown in figure [Fig. 3]. The three fragment ions m/z 177, m/z 163 and m/z 136 was formed from m/z 286 on the loss of C6H6NO, C7H9NO and

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C9H14N2. The m/z 198 and m/z 245 were not present in mass spectra of DP 2 confirmed that loss of two methane sulphonamide molecules. The fragment ion m/z 120 was base peak formed form m/z 163 on the loss of C2H5N. Further m/z 103 was formed from m/z 120 on the

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loss of NH3. After that fragment ion m/z 77 was generated from m/z 103 on the loss of C2H2. The DP 2 was confirmed as 4-(2-((2-(4-aminophenoxy)ethyl)(methyl)amino) ethyl)

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benzenaminium.

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DP 3 (428.1303)

The accurate m/z value of DP 3 was found to be 428.1303 [M + H]+ [Fig. S3c]. The fragment

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ion m/z 349 was formed as a characteristic fragment on the loss of CH3O2S from m/z 428 confirmed that demethylation observed at tertiary amine [Fig. 4]. The m/z 241 was formed

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from m/z 349 on the loss of C6H6NO. Further two fragment ions m/z 198 and m/z 163 were formed from m/z 241 on the loss of C2H5N and CH3O2S respectively. The m/z 119 was

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formed from m/z 198 on the loss of CH3O2S. The fragment ion m/z 107 was formed from m/z 163 on the loss of C3H6N. The m/z 91 was formed from the m/z 119 on the loss of C2H4. DP 3 was confirmed as N-(4-(2-((4-(methylsulfonamide) phenethyl) amino) ethoxy) phenyl) methanesulfonamide with molecular formula. DP 4 (458.1418)

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The accurate m/z value of DP 4 was found to be 458.1418[M + H]+ with an increased mass of 16 D to the drug m/z 442.1465 [M + H]+. The MS/MS spectra of DP 4 in ESI positive ionization mode is shown in figure [Fig. S3d]. The fragment ion m/z 440 was observed on the loss of m/z 18(H2O) from m/z 458. To confirm the DP4 was N-oxide or hydroxide degradation product, DP4 sample was subjected to LC/APCI/MS positive mode. The m/z 261 was formed as a molecular ion peak instead of m/z 458 [Fig. S3f]. Further loss of oxygen from m/z 261 generated the fragment ion m/z 245 confirmed that DP 4 was N-oxide product

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as shown in figure (Fig. 5). In ESI mode two fragment ions m/z 257 and m/z 198 were formed from 440 on the loss of C8H9NO2S and C10H14N2O3S respectively. Further m/z 214 was formed from m/z 257 on the loss of C2H6N. The m/z 198, m/z 119, was common

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fragments between drug (Fig. 2) and DP 4 (Fig. 5). The chemical name of DP 4 was found to

ethan-1-N-oxide with molecular formula. DP 5 (245.0951)

phenoxy)

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be N-methyl-N-(4-(methylsulfonamido) phenethyl)-2-(4-(methylsulfonamido)

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The accurate mass of DP 5 was 245.0951 [M + H]+. In MS/MS spectra [Fig. S3e] of DP 5 the m/z 214 and m/z 166 were formed on the loss of CH5N and CH3O2S from m/z 245

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respectively. The two fragment ions m/z 186 and m/z 135 were observed on the loss of C2H4 and CH3SO2 from m/z 214 respectively. The m/z 58 was formed on the loss of C6H6NO from

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m/z 166 [Fig. 6]. Chemically DP 5 was found to be M-(4-(2-(methylamino)ethoxy) phenyl)

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methanesulfonamide with molecular formula. DP 6 (273.1270)

DP 6 was formed by ether hydrolysis, accurate mass found to be m/z 273.1270 [M + H]+ [Fig. S3g]. Two fragments ion m/z 255 and m/z 76 were formed from m/z 237 on the loss of H2O and C9H11NO2S respectively. Formation of m/z 76 fragment was confirmed that ether hydrolysis lead to formation of DP 6, further loss of water, m/z 58 was formed. The m/z 229

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and m/z 176 was formed form m/z 255 on the loss of C2H3 and CO2SH respectively. Further fragmentation yielded m/z 198 on the loss of CH5N from m/z 229. The fragment ion m/z 120 was formed from m/z 198 on the loss of CH3O2SH. The m/z 103 was formed from m/z 198 on the loss of CH5NO2S, further m/z 120 yields a fragment ion m/z 91 on the loss of C2H5. The fragment ion m/z 163 was formed from m/z 176 on the loss of CH2 [Fig 7]. The chemical structure of DP 6 was confirmed as N-(4-2-((2-hydroxyethyl)(methyl)amino)ethyl)phenyl) methane sulfonamide.

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DP 7 (229.1005)

The DP 7 was a dealkylation degradation product with accurate mass m/z 229.1005 [M+H]+ [Fig. S3h]. Two fragment ions m/z 198 and m/z 150 were formed from m/z 299 on the loss of

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CH5N and CH3O2S respectively. Fragment ion m/z 120 was formed on the loss of CH3O2S from m/z 198. Further fragmentation of m/z 120 yielded fragment ion of m/z 107 and 103 on

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the loss of CH and NH2. The m/z 91 was formed from the m/z 120 on the loss of C2H5, after

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that m/z 77 was formed from m/z 103 on the loss of C2H2. The m/z 119 was formed from m/z 150 on the loss of CH5N shown in [Fig. 7]. DP 7 was confirmed as N-(4-(2-( methylamino)

DP 8 (362.1550)

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ethyl)phenyl) methanesulfonamide.

Accurate mass of DP 8 is found to be 362.1550 [M+H]+ [Fig. S3i]. Fragment ions of DP 8

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was different to that fragment ions of drug representing the structural rearrangement in drug

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molecule. Two fragment ions m/z 348, m/z 283 were formed from m/z 362 on the loss of CH3 and CH3O2S respectively. Further m/z 331 and m/z 305 fragment ions were formed from 348 on the loss of NH2 and C2H5N respectively. Two fragment ions m/z 226 and m/z 212 on the loss of CH3O2S and CH4NO2S respectively. Further m/z 105 was formed from m/z 212 on the loss of C6H7NO. The DP 8 was confirmed as 4-((2-methyl-6-(methylsulfonamido)1,2,3,4-tetrahydroisoquinolin-1-yl)methoxy)benzenaminium shown in figure [Fig. 8].

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3. Proposed mechanistic explanation for the formation of degradation products The outline of DPs formation was shown in figure [Fig. 9]. DP 1 and DP 2 was formed under acidic and photoacidic conditions by the elimination of methane sulfonic acid, leaving arylamino group which helps in retaining the aromaticity of the benzene ring [20]. The drug underwent dealkylation in photo neutral conditions mediated by a free radical mechanism leading to the formation of DP3 [21]. Under alkaline and neutral hydrolytic conditions, DP3 was possibly formed by the hydrolysis of amine [22]. DP 4 was the N-oxide degradation

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product of dofetilide formed under oxidative, neutral and photo neutral conditions and is the characteristic degradation product of tertiary amines containing molecules. In photoneutral condition the N-oxide was formed by photolytic transition of the ground state oxygen to

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singlet state oxygen by a transient ‘’flip’’ of one of the spins. This singlet oxygen act a photooxidative mediator and results in series of one-electron reduction steps generating

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superoxide anion radical and finally hydrogen peroxide. The tertiary amine nitrogen acts as a nucleophile in this reaction by attacking an oxygen atom of the peroxide resulting in the

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formation of hydroxy ammonium ion and hydroxide anion. Subsequently, the latter reacts with the hydrogen atom of the hydroxyl group at the nitrogen releasing the amine oxide [23].

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DP 5 was formed by base catalysed dealkylation of tertiary amine in alkaline and photobase conditions [24]. In photoacidic and photoneutral conditions DP 6 was formed by ether

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hydrolysis. Formation of DP 7 was similar to that of DP 5. DP 8 was formed in photo neutral conditions by undergoing rearrangement reaction. The amine radical cation was formed by

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loss of one electron. The one electron oxidation process was initiated by using UV-visible light-mediated photochemistry. After that amine radical cation was formed and α-amino radical was formed by deprotonation leads to formation of new C-C bond [25]. 4. Conclusion Dofetilide was subjected to diverse stress conditions to find out intrinsic stability of the drug. The drug was found to be unstable under different solution state stress conditions. HPLC13

PDA method was developed for identification and separation of stressed samples. A total of eight DPs were separated on HPLC, which were formed under different stress conditions. The HPLC study found that the drug was susceptible to acidic and alkaline stress degradation, but it was highly unstable to photolytic and oxidative conditions. However the solid drug was stable in thermal and photolytic conditions. The degradation products were characterized by LC/ESI/QTOF/MS/MS in positive ionization mode. In oxidative stress

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condition the formation of N-oxide was confirmed by LC/APCI/QTOF/MS/MS.

Conflict of Interest

The authors declared that there is no conflict of interest in publishing the manuscript. We wish to be

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considered the revised manuscript for publication in Journal of Pharmaceutical and Biomedical

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Analysis

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Acknowledgements

The authors thank Dr. Shashi Bala Singh, Director, NIPER-Hyderabad, for facilities and

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encouragement. The authors gratefully acknowledge MSN Laboratories Pvt. Ltd (Hyderabad) India for the gift sample of the dofetilide. The authors are also thankful to the Department of

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Pharmaceuticals, Ministry of Chemicals and Fertilizer, New Delhi, India, for providing a

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Research Fellowship.

5. References 1. M. G. Tsipourasa, D. I. Fotiadisa, D. Sideris, An arrhythmia classification system based on the RR-interval signal, Artif. Intell. Med. 33 (2005), 237-250.

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2. H. P. Rang, M. M. Dale, J. M. Ritter, R. J. Flower, Rang and dales Pharmacology. Sixth ed; Livingstone, London, 2019. 3. https://pubchem.ncbi.nlm.nih.gov/compound/dofetilide, (Accessed on 16 May 2019). 4. G. Bansal, M. Singh, K.C. Jindal, S. Singh, LC-UV-PDA and LC-MS studies to characterize degradation products of glimepiride, J. Pharm. Biomed. Anal., 48 (2008) 788–795. 5. S. W. Baertschi, Pharmaceutical stress testing predicting drug degradation second ed; Taylor & Francis Group, New York, 2005.

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6. H. Bhutani, S. Singh, K. C. Jindal, A. K. Chakraborti, Mechanistic explanation to the catalysis by pyrazinamide and ethambutol of reaction between rifampicin and isoniazid in anti-TB FDCs, J. Pharm. Biomed. Anal., 39 (2005) 892-899. 7. R. Sankar, N. Sharda, S. Singh, A critical review of the probable reasons for the poor variable bioavailability of rifampicin from anti-tubercular fixed-dose combination (FDC) products, and the likely solutions to the problem, Int. J. Pharm., 228 (2001) 5-17.

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8. J. K. Aronson, Side effects of drug annual 26., first ed; United kingdom 2003.

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9. S. Mehta, R. P. Shah and S. Singh, Strategy for identification and characterization of small quantities of drug degradation products using LC and LC-MS: Application to valsartan, a model drug, Drug Test. Anal., 2 (2010) 82-90

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10. M. Narayanam, T. Handa, P. Sharma, S. Jhajra, P. K. Muthe, P. K. Dappili, Ravi P. Shah, S. Singh, Critical practical aspects in the application of liquid chromatography-mass spectrometric studies for the characterization of impurities and degradation products, J. Pharm. Biomed. Anal., 87 (2014) 191– 217.

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11. P.D. Kalariya, P.N. Patel, R. Srinivas, M.V.N.K. Talluri, Quality by design based development of a selective stability-indicating UPLC method of dolutegravir and characterization of its degradation products by UPLC-QTOF-MS/MS, New J. Chem., 39 (2015) 6303-6314.

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12. M.V.N.K. Talluri, S. Dharavath, P.D. Kalariya, B. Prasanth, R. Srinivas, Structural characterization of alkaline and oxidative stressed degradation products of lurasidone using LC/ESI/QTOF/MS/MS, J. Pharm. Biomed. Anal., 105 (2015) 1-9. 13. R.N. Rao, M.V.N.K. Talluri, D.D. Shinde, Simultaneous separation and determination of coenzyme Q10 and its process related impurities by NARP-HPLC and atmospheric pressure chemical ionization-mass spectrometry (APCI-MS), J. Pharm. Biomed. Anal., 47 (2008) 230-237. 14. R.N. Rao, D. D. Shinde, M.V.N.K. Talluri, Enantioselective HPLC resolution of synthetic intermediates of armodafinil and related substances, J. Sep. Sci., 31 (2008) 981-989.

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15. S. M. Baira, P. D. Kalariya, R. Nimbalkar, P. Garg, R. Srinivas, M.V.N.K. Talluri, Characterization of forced degradation products of canagliflozine by LC/QTOF/MS/MS and in silico toxicity predictions, Rapid Commun. Mass Spectrom., 3 (2018) 212-220. 16. ICH, Impurities in new drug substances Q3A(R2)., International conference on Harmonisation, IFPMA, Geneva (Switzerland)., (2006). 17. WHO, Expert committee on specifications for pharmaceutical preparations, World Health Organization, Geneva, (Switzerland)., (2005). 18. ICH, Photostability testing of new drug substances and products Q1B, International Conference on Harmonisation, IFPMA, Geneva (Switzerland), 2005.

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19. S. Singh, M. Bakshi, Stress tests to determine inherent stability of drugs, Pharm. Technol., 4 (2000) 1-14. 20. X. Xu, Determination of degradation products of sumatriptan succinate using LC-MS and LC-MS-MS, J. Pharm. Biomed. Anal., 26 (2001) 367-377.

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21. S. B. Landge, S. A. Jadhav, K. P. Nimbalkara, A. C. Mali, V. T. Mathad, Stability indicating RP-HPLC method for the determination of dronedarone hydrochloride and its potential process-related impurities in bulk drug and pharmaceutical dosage form, Am. J. Anal. Chem., 4 (2013) 323.

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22. Z. Ling, L. Yun, L. Liu, B. Wu, X. Fu, Aerobic oxidative N-dealkylation of tertiary amines in aqueous solution catalyzed by rhodium porphyrins, Chem Commum., 49 (2013) 4214-4216.

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23. I. Ahmad, S. Ahmed, Z. Anwar, M.A. Sheraz, M. Sikorski, Photostability and photostabilization of drugs and drug products, Int. J. PHOTOENERGY., 16 (2016) 530549.

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24. O. Okazaki, F. P. Guengerichs, Evidence for Specific Base Catalysis in N-Dealkylation reaction Catalyzed by Cytochrome P450 and Chloroperoxidase, J. Biol. Chem. 3 (1993) 1546-1552

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25. J. Hu, J. Wang, T. H. Nguyen and N. Zheng, The chemistry of amine radical cations produced by Visible light photoredox catalysis, Beilstein J. Org. Chem. 9 (2013) 19772001.

Figure captions

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Figure 1. HPLC chromatogram showing degradation products of dofetilide under different

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solution state stress conditions.

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Figure 2. Proposed mass fragmentation pathway of dofetilide.

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Figure 3. Proposed mass fragmentation pathway of DP1 and DP2.

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Figure 4. Proposed mass fragmentation pathway of DP3.

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Figure 5. Proposed mass fragmentation pathway of DP4.

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Figure 6. Proposed mass fragmentation pathway of DP5.

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Figure 7. Proposed mass fragmentation pathway of DP6 and DP7.

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Figure 8. Proposed mass fragmentation pathway of DP8.

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Figure 9. Proposed degradation pathway and mechanism of hydrolysis, oxidation and photocatalytic rearrangement of dofetilide.

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Table 1. Stress conditions for dofetilide degradation products formation. DP 1

DP 2

DP 3

DP 4

DP 5

A, PA

A

B, N, PB, PN

O, PN

N, PB

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DPs Stress conditions

DP 6

DP 7

DP 8

PA, PN

PB

PN

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*Acid (A), Photoacid (PA), Base (B), Photobase (PB), Neutral (N), Photoneutral (PN), Oxidative (O)

Table 2. LC/ESI/QTOF/MS/MS data of dofetilide.

Peak no.

Experimental mass

Best possible molecular formula

Exact mass of most probable structure

Error in mmu

RDB

Possible parent fragment

Difference from parent ion

Possible molecular formulae for losses

[M+H]+

442.1485

C19H28N3O5S2

442.1465

2.0

7.5

-

-

-

24

363.1630

C18H25N3O3S

363.1611

1.8

8.0

[M+H]+

79

CH3O2S

b c d e f g h i j k l m n o p q r s t

348.1393 284.1777 271.1116 255.1172 245.0950 228.0937 214.0542 198.0594 179.1188 163.1232 149.1077 135.0681 120.0808 119.0740 107.0739 103.0548 91.0551 71.0738 58.0660

C17H22N3O3S C17H22N3O3 C12H19N2O3S C12H19N2O2S C10H17N2O3S C10H16N2O2S C9H12NO3S C9H12NO2S C10H15N2O C10H15N2 C9H13N2 C8H9N O C8H10N C8H9N C7H9N C8H7 C7H7 C4H9N C3H8N

348.1376 284.1757 271.1111 255.1162 245.0954 228.0927 214.0532 198.0583 179.1179 163.1230 149.2165 135.0679 120.0808 119.0730 107.0730 103.0542 91.0542 71.0730 58.0651

1.6 1.9 0.5 0.0 -0.4 1.0 0.9 1.0 0.9 0.2 0.3 0.2 0.0 1.0 0.9 0.6 13.4 0.8 0.8

8.5 8.5 4.5 4.5 3.5 4.0 4.5 4.5 4.5 4.5 4.5 5.0 5.0 5.0 4.0 5.5 4.5 1.0 0.5

a a a a d e f g b b k h i j j i n k k

15 78 92 108 26 15 45 30 169 185 30 79 78 60 72 95 29 92 105

CH3 CH3O2S C6H6N6 C6H6NO C2H2 CH3 CH5N2 CH4N C7H7NO2S C7H7NO3S CH2O CH3O2S CH4O2S C2H6NO C3H6NO CH5NO2S C2H5 C6H6N C7H7N

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a

Experimental mass

Exact mass of most probable structure

Error in mmu

C18H26N3O5S2

RDB

364.1689

-1.2

7.5

286.1914

0.0

7.5

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DP 1 364.1677

Best possible molecular formula

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DPs

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Table 3. LC/ESI/QTOF/MS/MS data of dofetilide degradation products (DP1 to DP8).

DP 2 286.1914

C17H24N3O

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Major fragments (chemical formulae, error in mmu, RDB)

255.1162 (C12H19N2O2S,-1.4,4.5), 229.1005 (C10H17N2O2S,-0.8,3.5), 198.0583 (C9H12NO2S,0.5,4.5), 167.1179 (C9H15N2O,0.3,3.5), 136.0757(C8H10NO,0.1,4.5), 120.0809 (C8H10N,0.1,4.5), 103.0543 (C8H7,0.5,5.5), 77.0386 (C6H5,0.6,5.5), 58.0651(C3H8N,0.7,0.5) 177.1386 (C11H17N2,-0.3,4.5), 136.0757 (C8H10NO,-0.3,4.5), 163.1230 (C10H15N2,-0.4,4.5), 120.0808 (C8H10N,0.1,4.5), 103.0542 (C8H7, 0.2,5.5), 77.0386 (C6H5,0.2,4.5)

428.1308

-1.5

7.5

DP 4 458.1410

C19H28N3O6S2

458.1414

0.4

7.5

DP 5 245.0938

C10H17N2O3S

245.0954

-1.6

3.5

DP 6 273.1293

C12H21N2O3S

273.1267

2.6

3.5

DP 7 229.0996

C10H17N2O2S

DP 8 362.1547

C18H24N3O3S

349.1455 (C17H23N3O3S,-0.5,8), 241.1005 (C11H17N2O2S,-0.6,4.5), 198.0583 (C9H12NO2S,-0.6,4.5), 163.1230 (C10H15N2,-0.8,4.5), 119.0733(C8H9N,-0.6,5), 107.0730 (C7H9N,0.0,4) , 91.0542 (C7H7,13.4,4.5 ) 440.1308 (C19H26N3O5S2,0.0,8.5), 257.0954 (C11H17N3O3S,0.8,4.5), 214.0532(C9H12NO3S,-1.1,4.5), 198.0578 (C9H12NO2S,-1.7,4.5), 119.0730 (C8H9N,-0.7,4.5), 214.0532 (C9H12NO3S,-2.4,4.5), 186.0219(C7H8NO3S,-0.5,4.5), 166.1101 (C9H14N2O,-14,4), 135.0679 (C8H9NO,-2,5), 58.0656 (C3H8N,0.2,0.5)

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C18H26N3O5S2

255.1178 (C12H19N2O2S,1.6,4.5), 229.1005 (C10H17N2O2S,-1.4,3.5), 198.0583 (C9H12NO2S,1.2,4.5), 176.1308 (C11H16N2,-0.3,5), 163.1230 (C19H27N2O3S,0.1,4.5), 120.0808 (C8H10N,0.9,4.5), 103.0542 (C8H7,-0.0.5.5), 91.0542 (C7H7,13.4,4.5), 76.0757 (C3H10NO, 0.9) 58.0654(C3H8N, 0.2,0.3) 198.0584 (C9H12NO2S, -0.1,4.5), 150.1151 (C9H14N2, -0.6,4.0) 120.0808 (C8H10N, -0.1,4.5), 119.0730 (C8H9N, ,-0.0,5.0) 107.0730 (C7H9N,0.0,4) 103.0542(C8H7,0.0,5.5), 91.0546 (C6H7, 13.4 ,4.5), 77.0386 (C6H5.1.7,4.5) 348.1376(C17H22N3O,0.7,8.5), 331.1111(C17H19N2O3S,0.0.9.5), 305.0960(C15H17N2O3S,0.5,8.5), 283.1679(C17H21N3O,1.1,9.0), 226.1106(C14H14N2O,0.4,9.0), 212.1070(C14H14NO,-11,8.5)

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DP 3 428.1313

362.1533

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229.1005

26

-0.9

3.5

1.4

8.5