Analytical Method Development Report for the Determination of Assay & Related Substances of Ibutilide Fumarate in Ibutilide Fumarate Injection

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INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616 Analytical Method Development Report For The Determination Of Assay & Related Substances Of Ibutilide Fumarate In Ibutilide Fumarate Injection

Aakisetti Siva Sankar, Dr. G. Srinivasarao

Abstract: The aim of the study is to develop an effective assay to quantify ibutilide fumarate and its related substances in ibutilide fumarate injection. Initially, the API manufacturer’s method was followed which involves two different UV-HPLC assays for both ibutilide fumarate and its related impurities. Upon considering the heat degradation results of the API method, specificity and stability of the known impurities (related substances) was not clearly determined. So the API method was modifiedby using low-pressure gradient UV-HPLCfor accurate detection of ibutilide and its related impurities. The inference of the drug, under various physio-chemical conditions like acid stress, base stress, heat stress, dark and light exposed, peroxide treated, dry heat are also studied. Chromatogram results of the developed method observed to be highly stable and sensitivity as it is able to clearly differentiate the excipient, related impurities and degradation product. Therefore the developed method can be used for the determination of assay and related substances of Ibutilide fumarate in Ibutilide Fumarate injection.

Keywords: Ibutilide fumarate, HPLC, API manufactures’s method, Stress testing ————————————————————

Introduction: 4.6 with 0.1N hydrochloric acid and dilute to the volume Heart arrhythmia (cardiac dysrhythmia) is a cardiac disorder identified with water[19]. And for the related substances standard by irregular heartbeat. It is mainly due to the disturbance of electrical solution pipette out accurately 1.0ml of assay standard [1 ], [2] impulses which regulate the heart . As a result, the heart may beat solution (0.1mg/mL) into 100ml volumetric flask and dilute too slowly (bradycardia), or too quickly (tachycardia) or in an irregular to volume with water (0.001mg/mL)[20][21]. way. Anti-arrhythmic drugs can be classified according to their effects on the electrical behaviour of myocardial cells in to four which are Anti-arrhythmic drugs are classified into four types based on their Liquid chromatography conditions electrical behavior with myocardial cells namely membrane-stabilizers Chromatography conditions such as stationary and mobile (class I), beta-blockers (class II), potassium blockers (class III) and [3] phase composition, temperature of the column, flow rate, calcium influx blockers (class IV) . Ibutilide is one among the effective volume of the sample and wavelength for compound class III antiarrhythmic drug used intravenously to convert atrial flutter or fibrillation to normal rhythm[4], [5], [6]. Ibutilide contains both + and – detection were optimized. An effective and precise HPLC enantiomers [7] exist as a racemic mixture having one chiral centre. system consisting of a column Inertsil ODS-3V (250 X 4.6 IUPAC name and molecular formula of Ibutilide fumarate is mm; 5 µm) or equivalent with a column temperature: 30 °C Methanesulfonamide, N-{4-{4-(ethylheptylamino)-1 -hydroxybutyl} is utilized with a run time of 60 min and sample [8],[9] phenyl}, (+)(-), (E)-2-butenedioate (1:0.5) (hemifumarate salt) and concentration of 0.1mg/Ml[22]. The gradient programme for C22H38N2O5S respectively. Molecular weight of Ibutilide fumarate is 442.62 and its structural formula is revealed in the Figure 1. The the chromatography is given in Table1. Inject 100 µl each of injection solution is isotonic and colorless [10] having a concentration of blank, excipient solution, related substances standard 0.1 mg/ml of Ibutilide fumarate along with 0.189 mg of sodium acetate solution, assay standard solution, and test solution into the trihydrate, and 8.90 mg of sodium chloride, and the pH is adjusted to liquid chromatograph, record the chromatograms and [11],[12] 4.6 using HCl . Many commercial methods are available for the measure the response excluding the peaks due to blank assay of ibutilide fumarate injection, which utilize UV- Visible and excipients. Inject blank, excipient, standard and sample spectroscopy, HPLC, LCMS/MS, TLC,& GC as the principle detection method[13], [14]. Initially in this study we have used the API solutions as per Table2. manufacturer’s method which consist of two different methods for the determination of Assay & Related substances of Ibutilide fumarate, that Optimization of the quantification method under involves a HPLC method with a UV detection[15],[16]. Since Poor various physio-chemical conditions sensitivity and reproducibility of the reported methods led to the To ensure the specificity and stability of the new development of a novel method for determination of Ibutilide fumarate and its related substances in Ibutilide fumarate injection quantification method, the activity of the drug has been studied in various physio-chemical conditions. For the optimization of method both ibutilide fumarate formulation Materials And Methods and excipients were exposed in different conditions like acid stress, base stress, heat stress, dark and light exposed, Preparation of stock solution peroxide treated, dry heat and their effects were studied. Ibutilide fumarate Standard stock solution is prepared at a concentration of 1.0 mg/ml [17], [18] by dissolving 25 mg of Validation of ibutilide fumarate assay Ibutilide fumarate standard in 25 ml of double distilled The method for ibutilide fumarate was validated in terms of water. 1 ml of stock is diluted with water at a ratio of 1:10 reproduciblity, specificity, and robustness according to ICH and used for the development of the assay. harmonized tripartite guidelines.

Reagents and chemicals Specificity To prepare excipient solution take about 40ml of water into The specificity of method was evaluated to ensure that a 50ml volumetric flask; add accurately about 9.45 mg of there was no interference from the expients present in the Sodium acetate trihydrate and 445 mg of Sodium chloride formulations. The specificity was studied by injecting the then dissolve the contents. After that adjust the pH to about expients. 983 IJSTR©2019 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616

Robustness peak is due to excipient having different response at The reliability of the method to remain unaffected due to different conditions. Due to this reason, the method was deliberate variations is determined by measuring the revised stating that this peak at RRT 0.41 with respect to robustness of the analytical procedure. Ibutilide will be excluded during integration of chromatogram[24]. It was found that no other peaks due to Statistical analysis the excipients or the impurities were interfering with the The experiments were conducted in triplicates and the main peak ofIbutilide, and the two known process related results are expressed as mean with standard error. impurities are well separated in this method. The relative Statistical significance is evaluated using One way ANOVA retention times of these impurities with respect to Ibutilide is and 0.05 is used as threshold reference for P value. presented in Table 3.

Results and Discussion Specificity The specificity of method was monitored by analyzing the Optimization of HPLC condition ibutilide fumarate and standard solution. No peak was Chromatograms of blank, excipient, related substance detected in close to the retention time of ibutilide fumarate, standard, assay standard, ibutilide fumarate formulation are which proved to be high degree of specificity of the method depicted in Figure 2. The chromatograms of UV-HPLC (Figure 4). revealed that the compounds peaks are distinct and clear without any excipient interference thereby highlighting the Robustness efficiency of both sensitivity and selectivity of the optimized The robustness of the method was studied by deliberate protocol.The runtime of the procedure is found to be exactly changes in method like alteration in pH of mobile phase. It 60 minutes and the peaks were of good shape and was observed that there was no marked changes in the completely resolved. chromatogram, demonstrate that the HPLC method developed are robust (Figure 5). Optimization of the quantification method under various physio-chemical conditions Figures and tables The forced degradation samples, thermal and pH related stress samples were analyzed using this method (Figure 3). Table 1: Gradient Programme For The Chromatography During the heat stress, two potential degradation impurities Time Solution A Solution B observed at the relative retention time of 1.4 & 1.6 of about Elution (Minutes) (%) (%) 1.4% & 1.0% respectively. The acid degradation also 0.0 62 38 Equilibration showed one significant impurity at a different RRT of about 14.0 62 38 Isocratic 1.7. The base and light exposed does not showed any 18.0 60 40 Linear Gradient significant degradation pattern. But the peroxide stress 35.0 30 70 Linear Gradient resulted in complete degradation of ibutilide peak. Also in 45.0 30 70 Isocratic case of API degradation under similar conditions, the same 50.0 62 38 Equilibration 60.0 62 38 Equilibration pattern of impurity observed[23]. In case of thermal & pH stress, performed at different pH conditions, viz., at 3.0, 4.0, Table 2: Injection Sequence 4.6, 5.0 & 6.0. There observed a peak at about RRT 0.41 which is due to excipient appeared at variable response at Solution Injection different times during the analysis. During the lab batch analysis, it was also observed that the peak at RRT 0.41 Blank 1 was showing a variable response at aseptic fill and Excipient 1 terminally sterilized conditions. In aseptic fill at 40°C storage condition, this peak was quiet significant, whereas Related substances standard 5 in aseptic fill (50°C) & terminally sterilized (40°C, 50°C) it Assay standard 5 showed very insignificant response which proved that this Test solution 2

Table 3: Rrt Table Of Ibutilide Fumarate Process Related Impurity

Relative Chemical name In-house Remarks retention time N-ethyl-n-heptyl-gamma-oxo-4-[(methyl Impurity-A 2.5 Process related impurity sulfonyl)amino]-benzene butanamide. N-ethyl-n-heptyl-4-hydroxy-4-(4- Impurity-B 1.98 Process related impurity methanesulfonylamino-phenyl)-butyramide.

Fig. 1: structure of ibutilide fumarate

Fig. 2a: Chromatogram of blank

Fig. 2b: Chromatogram of excipient control

Fig.2c:Typical assay standard chromatogram

Fig.2d: Typical diluted standard chromatogram

Fig.2e: Typical Chromatogram of Ibutilide Fumarate formulation – Control

Fig.3a: Chromatogram of Ibutilide Fumarate formulation – Acid stress

Fig.3b: Chromatogram of Ibutilide Fumarate formulation – Base stress

Fig.3c: Chromatogram of Ibutilide Fumarate formulation – Heat stress

Fig.3d: Chromatogram of Ibutilide Fumarate formulation – Dark control 986 IJSTR©2019 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616

Fig.3e:Chromatogram of Ibutilide Fumarate formulation – Light exposed

Fig.3f:Chromatogram of Ibutilide Fumarate formulation – Peroxide treated

Fig.4a: HPLC chromatogram of mobilephase with 1mg/ml ibutilide fumarate

Fig.4b: HPLC chromatogram of mobilephase with .001mg/ml ibutilide fumarate in diluted mobile phase.

Fig.5a: HPLC chromatogram at different pH=3 condition to study the robustness of the method

Fig.5b: HPLC chromatogram at different pH=4 condition to study the robustness of the method

Fig.5c: HPLC chromatogram at different pH=5 condition to study the robustness of the method

Fig.5d: HPLC chromatogram at different pH=6 condition to study the robustness of the method

Figure 7: Chromatogram of Ibutilide API -Control

Figure 8: Chromatogram of Ibutilide fumarate API – Dry Heat Sample

Figure 9: Typical Chromatogram of Ibutilide fumarate process related impurity-A

Figure 10: Typical Chromatogram of Ibutilide fumarate process related impurity-B

Figure 11: Typical spiked Chromatogram of Ibutilide fumarate formulation with Impurities

Conclusion flutter. The American journal of cardiology, 78(8), The current assay developed for the detection of ibutilide 42-45 (1996). fumarate and its related substances in ibutilide fumarate [6] Naccarelli, G. V., Lee, K. S., Gibson, J. K., injection proven as a reliable and specific method. The &VanderLugt, J. Electrophysiology and developed assay provides rapid detection of ibutilide and pharmacology of ibutilide. The American journal of also highly sensitive to detect the presence of impurities cardiology, 78(8), 12-16 (1996). even in trace amount. Chromatogram infers the absence of [7] Antonicelli, R., Testarmata, P., &Recanatini, A. interference due to diluents, excipient and degradation Ibutilide in rapid conversion of atrial flutter in products. The inference of the drug, under various physio- octogenarians. Drugs & aging, 19(10), 787-791 chemical conditions like acid stress, base stress, heat (2002). stress, dark and light exposed, peroxide treated, dry heat [8] Franczyk, B., Gluba-Brzózka, A., Banach, M., are also studied. On comparison with earlier reported &Rysz, J. The Problem of Atrial Fibrillation in methods, the current developed assay is similar with the Patients with Chronic Kidney Disease. Current validation guidelines of ICH. On considering the integrity vascular pharmacology, 14(3), 260-265 (2016). and resolution of the observed peak, the present developed [9] Li, Y., Tian, L., Huang, Y., & Hua, L. assay for quantitative determination of ibutilide and related Pharmacokinetic and pharmacodynamic properties substances in ibutilide fumarate injection is precise, of a single intravenous dose of ibutilide fumarate: a accurate and made to use for human studies. phase I, randomized, open-label, increasing-dose study in healthy Chinese men. Clinical References therapeutics, 29(9), 1957-1966 (2007). [1] Cimini, M. G., Brunden, M. N., & Gibson, J. K. [10] Gowda, R. M., Punukollu, G., Khan, I. A., Wilbur, Effects of ibutilide fumarate, a novel antiarrhythmic S. L., Vasavada, B. C., &Sacchi, T. J.Ibutilide for agent, and its enantiomers on isolated rabbit pharmacological cardioversion of atrial fibrillation myocardium. European journal of and flutter: impact of race on efficacy and pharmacology, 222(1), 93-98 (1992). safety. American journal of therapeutics, 10(4), 259-263 (2003). [2] Labhasetwar, V., Underwood, T., Heil, J. R., Gallagher, M., Langberg, J., & Levy, R. J.Epicardial [11] Steinwender, C., Hönig, S., Kypta, A., Kammler, J., administration of ibutilide from polyurethane Schmitt, B., Leisch, F., & Hofmann, R. Pre-injection matrices: effects on defibrillation threshold and of magnesium sulfate enhances the efficacy of electrophysiologic parameters. Journal of ibutilide for the conversion of typical but not of cardiovascular pharmacology, 24(5), 826-840 atypical persistent atrial flutter. International journal (1994). of cardiology, 141(3), 260-265 (2010). [3] Soucier, R., Silverman, D., Abordo, M., Jaagosild, [12] Hohnloser, S. H., Dorian, P., Straub, M., P., Abiose, A., Madhusoodanan, K. P.,&Berns, E.. Beckmann, K., &Kowey, P. Safety and efficacy of Propafenone versus ibutilide for post operative intravenously administered tedisamil for rapid atrial fibrillation following cardiac surgery: neither conversion of recent-onset atrial fibrillation or atrial strategy improves outcomes compared to rate flutter. Journal of the American College of control alone (the PIPAF study). Medical Science Cardiology, 44(1), 99-104 (2004). Monitor, 9(3), PI19-PI23 (2003). [13] Tian, L., Li, Y., Huang, Y., Kuang, F., Li, F., & Hua, [4] Bernard, E. O., Schmid, E. R., Schmidlin, D., L. Sensitive method for the determination of Scharf, C., Candinas, R., &Germann, R. Ibutilide ibutilide in human plasma by liquid versus amiodarone in atrial fibrillation: a double- chromatography–tandem mass blinded, randomized study. Critical care spectrometry. Journal of Chromatography medicine, 31(4), 1031-1034 (2003). B, 816(1-2), 81-85 (2005). [5] Ellenbogen, K. A., Clemo, H. F., Stambler, B. S., [14] LIU, X. H., LI, H., & WANG, M. L. HPLC Wood, M. A., &VanderLugt, J. T. Efficacy of determination of the content and the related ibutilide for termination of atrial fibrillation and substances of ibutilide fumarate for injection [J]. Chinese Journal of Pharmaceutical Analysis, 8, 035 (2009). 990 IJSTR©2019 www.ijstr.org INTERNATIONAL JOURNAL OF SCIENTIFIC & TECHNOLOGY RESEARCH VOLUME 8, ISSUE 10, OCTOBER 2019 ISSN 2277-8616

[15] Hsu, C. L., & Walters, R. R. Chiral separation of ibutilide enantiomers by derivatization with 1- naphthyl isocyanate and high-performance liquid chromatography on a Pirkle column. Journal of Chromatography A, 550, 621-628 (1991). [16] Chang-yuan, L. H., & Walters, R. R. Assay of the enantiomers of ibutilide and artilide using solid- phase extraction, derivatization, and achiral-chiral column-switching high-performance liquid chromatography. Journal of Chromatography B: Biomedical Sciences and Applications, 667(1), 115-128 (1995). [17] Labhasetwar, V., Strickberger, S. A., Underwood, T., Davis, J., & Levy, R. J. Prevention of acute inducible atrial flutter in dogs by using an ibutilide- polymer-coated pacing electrode. Journal of cardiovascular pharmacology, 31(3), 449-455 (1998). [18] LI, G. L., & LI, M. Preparation and quality control of ibutilide fumarate for injection [J]. Chinese Journal of New Drugs, 3, 016 (2007). [19] Buist, S. C., Hsu, C. Y. L., & Walters, R. R. Sensitive determination of a new antiarrhythmic agent, trecetilide, in plasma by high-performance liquid chromatography with fluorescence detection1. Journal of Chromatography A, 828(1- 2), 259-265 (1998). [20] Perricone, S. C., Chidester, C. G., & Hester, J. B. Synthesis of (R)-N-[4-[4-(dibutylamino)-1- hydroxybutyl] phenyl] methanesulfonamide,(E)-2- butenedioate (2: 1) salt (artilide fumarate) and the enantiomers of N-[4-[4-(ethylheptylamino)-1- hydroxybutyl] phenyl] methanesulfonamide,(E)-2- butenedioate (2: 1) salt (ibutilide fumarate). Tetrahedron: Asymmetry, 7(3), 677-690 (1996). [21] Buchanan, L. V., Thompson, D. D., Hsu, C. Y. L., Walters, R. R., & Gibson, J. K. Antiarrhythmic and electrophysiologic effects of sublingual ibutilide fumarate. Drug development research, 34(4), 322- 328 (1995). [22] Mansouri, Z. U.S. Patent No. 9,707,177. Washington, DC: U.S. Patent and Trademark Office (2017). [23] Dorsey, J. G., Cooper, W. T., Siles, B. A., Foley, J. P., & Barth, H. G. Liquid chromatography: Theory and methodology. Analytical Chemistry, 68(12), 515-568 (1996). [24] Sengupta, S., Zhao, G., Capila, I., Eavarone, D., &Sasisekharan, R. U.S. Patent Application No. 11/495,947 (2007).