INTRODUCTION:

Ivabradine (INN) is a novel medication used for the symptomatic management of stable angina pectoris¹. Ivabradine is chemically 3-(3-((((7S)-3, 4-dimethoxybicyclo [4.2.0] octa-1, 3, 5-trien-7-yl) methyl) (methyl) amino) propyl)-7, 8-dimethoxy-1, 3, 4, 5-tetrahydro-2H-3-benzazepi n-2-one. Ivabradine acts by selectively inhibiting the "funny" channel pacemaker current in the senatorial node in a dose-dependent manner, ensuing in a lesser heart rate and thus more blood to flow to the myocardium. Metoprolol is a cardio selective β-adrenergic receptor antagonist used to treat pregnant women with cardiovascular diseases together with hypertension, cardiomyopathy, ischemic heart disease, and arrhythmias². Metoprolol is chemically 1-[4-(2-methoxyethyl) phenoxy]-3-(propan-2-ylamino) propan-2-ol. It may also be used for supraventricular and tachyarrhythmias and prophylaxis for migraine headaches. The β1-selectivity of these agents is thought to be due in part to the large substituents in the Para position.

At low doses, metoprolol selectively blocks cardiac β1-adrenergic receptors with little activity against β2-adrenergic receptors of the lungs and vascular smooth muscle. Receptor selectivity decreases with higher doses. Unlike propranolol and pindolol, metoprolol does not exhibit membrane-stabilizing or intrinsic sympathomimetic activity. Ivabradine in combination with metoprolol is used in the treatment of inappropriate sinus tachycardia³.

Fig 1a. Ivabradine structure

Fig 1b: Metoprolol structure

A wide literature survey is carried out and found that pharmacokinetics studies⁴, microbiology⁵, LC–MS/MS^6-7 spectroscopy^8,9 and HPLC^10-14were developed for Ivabradine and no techniques were accounted for simultaneous estimation of Ivabradine and Metoprolol by RP-HPLC and RP-UPLC. Hence an aim was made to develop easy and sensitive RP-UPLC method for the estimation of Ivabradine and Metoprolol in pure and drug sample.

MATERIALS AND METHODS:

Samples of Ivabradine and Metoprolol are acquired from Spectrum Pharma labs, Hyderabad, India. HPLC grade water, Acetonitrile, methanol, Potassium dihydrogen ortho phosphate buffer, ortho-phosphoric acid, and tri ethyl amine was procured from Merck, Mumbai, India. Waters UPLC Acquity system equipped with TUV detector with Empower 2 Software. Isocratic analytical method was run through SB C8 100 x 3.0mm, 1.8mm column. Mobile phase consist 0.01N Potassium dihydrogen ortho phosphate Buffer: Acetonitrile taken in the ratio 50:50v/v was pumped through column at a flow rate of 0.3ml/min. Temperature was keep at 30°C. Optimized wavelength fix at 260 nm and run time was set for 3 min. The volume of injection is 0.50µl.

Preparation of 0.01N Potassium dihydrogen ortho phosphate buffer:

Perfectly weighed 1.36gm of Potassium dihydrogen ortho phosphate in a 1000ml of volumetric flask added about 900ml of HPLC grade water added and degassed to sonicate and finally made up the volume with water, then PH adjusted to 4.0 with dill ortho phosphoric acid solution.

Preparation of diluents:

Acetonitrile and water taken in the ratio of 50:50v/v

Preparation of Standard stock solutions:

Accurately weighed 5mg of Ivabradine and 25mg of Metoprolol and transferred to 25ml volumetric flask and 10ml of diluents was added and sonicated for 10 minutes. Flask was filling up to the mark with diluents and mark as standard stock solution. 1ml from each stock solution was pipette out and taken into a 10ml volumetric flask and fill up to the mark with diluents.

Preparation of sample stock solutions:

20 tablets of IVA Met XL were weighed and powdered. The weight equivalent to one tablet was transferred into a 50ml volumetric flask, 25ml of diluents was added and sonicated for 25 min, further the volume was made up with diluents and filtered by 0.45µ PVDF filters. 2ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluents to get final concentrations of 20µg/ml Ivabradine and 100µg/ml Metoprolol.

RESULTS AND DISCUSSION:

Method development:

At first, reverse phase liquid chromatography separation was tried to develop using various ratios of Methanol and Water, Acetonitrile and Water as mobile phases, in which drugs did not respond correctly, and the resolution was also poor. The organic content of the mobile phase was also investigated to optimize the separation of both drugs. To get better the tailing factor, the pH of the mobile phase becomes an important factor. SB C8 100 x 3.0mm, 1.8mm with an isocratic mobile phase composed of 0.01N Phosphate buffer and Acetonitrile mixed in the ratio of 50:50v/v at a flow rate of 0.3ml/min. The column temperature was maintained at 30°C and the detection was carried out using a TUV detector at 260 nm was selected as the stationary phase to improve resolution and the tailing of both peaks was reduced considerably and brought close to 1. The retention times were found to about 0.810min and 1.156min for Metoprolol and Ivabradine. The method was validated in terms of accuracy, precision, LOD, LOQ, robustness and linearity as per ICH guideline.

Method Validation:

System Suitability:

The system suitability parameters were determined by preparing standard solutions of Ivabradine 20μg/ml and Metoprolol 100μg/ml. The solutions were injected six times and the variables like peak tailing, resolution and USP plate count were determined. The % RSD for the area of six standard injections results were not >2.0%.

Specificity:

Specificity was determined by standard solutions of 20µg/ml Ivabradine and 100µg/ml Metoprolol, blank and placebo solutions. There should not find interfering peaks in the blank and placebo at retention times of these drugs in this method. So this method was said to be specific.

Linearity:

By appropriate aliquots of the standard Ivabradine and Metoprolol prepared six working solutions ranging between 5-30µg/ml and 25-150µg/ml. Each experiment linearity point was performed in triplicate according to optimized chromatographic conditions. Calibration curves were plotted with observed peak areas against concentration followed by the determination of regression equations and calculation of the correlation coefficient on curves for Ivabradine and Metoprolol.

Accuracy:

Accuracy was carried out by % recovery studies of Ivabradine and Metoprolol at three altered concentration levels (50%, 100%, and 150%). Percentage recovery was calculated from the amount added and the amount recovered. The percentage recovery was within the acceptance criteria, this indicates the accuracy of the method. (Acceptance criteria: percentage of recovery between 98 to 102)

Precision:

The repeatability of the method was verified by calculating the % RSD of six replicate injections of 100% concentration (20μg/ml of Ivabradine and 100μg/ml of Metoprolol) on the same day and for intermediate precision % RSD was calculated from repeated studies on different days.

Limit of Detection (LOD) and Limit of Quantitation (LOQ):

The LOD and LOQ were calculated from the slope (s) of the calibration plot and the standard deviation (SD) of the peak areas using the formulae LOD = 3.3 σ/s and LOQ = 10 σ/s.

Robustness:

Robustness of the method were verified by altering the chromatographic conditions like flow rate, mobile phase ratio and temperature are made, but there were no recognized change in the result and all are within range as per ICH guidelines. Robustness conditions like flow minus (0.27ml/min), flow plus (0.33 ml/min), 45:55 mobile phase minus, 55:45 mobile phase plus, temperature minus (25°C) and temperature plus (35°C) were maintained and samples were injected in duplicate manner. System suitability parameter was passed. % RSD was within the limit.

Degradation Studies:

Acid degradation:

To 1 ml of stock solution of Ivabradine and Metoprolol, 1ml of 2N Hydrochloric acid was added and refluxed for 30 mins at 60°C. For UPLC study, the resultant solution was diluted to obtain 20μg/ml and 100μg/ml solutions and 0.5μl solutions were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Oxidative Degradation:

To 1ml of stock solution of Ivabradine and Metoprolol, 1 ml of 20% hydrogen peroxide (H₂O₂) was added individually. The solutions were kept for 30 min at 60°C. For UPLC study, the resultant solution was diluted to obtain 20μg/ml and 100μg/ml solution and 0.5μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Alkali Degradation:

To 1 ml of stock solution Ivabradine and Metoprolol, 1 ml of 2N sodium hydroxide was added and refluxed for 30 mins at 60°C. The resultant solution was diluted to obtain 20μg/ml and 100μg/ml solution and 0.5μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Thermal Degradation:

The standard drug solution was placed in oven at 105°C for 6Hrs to study dry heat degradation. For UPLC study, the resultant solution was diluted to 20μg/ml and 100μg/ml solution and 0.5μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Photo Degradation:

The photochemical stability of the drug was also studied by exposing the 200μg/ml and 1000μg/ml solution to UV light by keeping the beaker in UV chamber for 7 days. For UPLC study, the resultant solution was diluted to obtain 20μg/ml and 100μg/ml solutions and 0.5μl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

Neutral Degradation Studies:

Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60ºc. For UPLC study, the resultant solution was diluted to (20µg/mL and100µg/mL) solution and 0.50µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

After a number of trials with mobile phases of different composition, and mobile phase containing 0.01N Potassium dihyrogen Ortho phosphate buffer and Acetonitrile taken in the ratio of 50:50v/v was selected as mobile phase because of better resolution more no. of Theoretical plates and symmetric peaks. Ivabradine and Metoprolol were found to show appreciable absorbance at 260nm when determined spectro-photometrically and hence it was selected as the detection wavelength. An optimized chromatogram showing the separation of Ivabradine and Metoprolol.

System Suitability:

According to ICH guidelines plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable variables were passed and were within the limits.

Table 1: System suitability variables

Parameter	Metoprolol	Ivabradine
Retention time (min)	0.810	1.156
Theoretical plates (N)	5970	2355
Tailing factor (T)	1.45	1.30
Resolution	4.4	4.2
%RSD	0.9	0.8

Linearity:

Concentration range of 5-30μg/ml for Ivabradine and 25-150μg/ml of Metoprolol were found to be linear with correlation coefficients 0.999 were within limits. The result was shown in Fig. 3

Table 2: Results for linearity

Parameter	Ivabradine	Metoprolol
Y intercept	13738	19132
Correlation coefficient r²	0.999	0.999
Regression Equation	y =13738x +4034	y=19132x +26914
Linearity range	5-30μg/ml	25-150 μg/ml
LOD	0.03	0.12
LOQ	0.08	0.35

Accuracy:

The Percentage accuracy was a relative standard deviation for accuracy at each level is well within the limit. Over all the percentage recovery found to be 100.03% and 100.03% Ivabradine and Metoprolol respectively and over all the percentage relative standard deviation was found to be 0.44% and0.37 % Ivabradine and Metoprolol respectively for all the levels were within the limit.

Fig. 3: Calibration curve of Metoprolol and Ivabradine

Table 3: Results for Accuracy

Ivabradine				Metoprolol
Recovery level	Amount added (μg/ml)	Amount found	% of Recovery	Amount Added (μg/ml)	Amount Found (μg/ml)	% of Recovery
50%	10	10.02	100.26	50	50.11	100.24
100%	20	19.99	99.95	100	99.96	99.96
150%	30	29.96	99.87	150	149.82	99.98
Mean recovery			100.00	Mean recovery		100.03
%RSD			0.44	%RSD		0.37

Precision:

Percentage relative standard deviation of six results was within the limit. The areas of all the injections were taken and standard deviation, % relative standard deviation (RSD) was calculated.

Limit of Detection:

Limit of detection of target assay concentration of Ivabradine and Metoprolol by using formula method 0.03μg/ml and 0.12μg/ml.

Limit of Quantification:

Limit of quantification of the target assay concentration of Ivabradine and Metoprolol by using formula method 0.08μg/ml and 0.35μg/ml were within the limits.

Robustness:

Table 4: Opted Method conditions

S. No	Condition	Plus	Minus
1	Flow rate	0.27ml/min	0.33ml/min
2	Mobile phase	55B:45A	45B:55A
3	Temperature	35°C	25°C

In the above conditions the variables like % relative standard deviation (RSD) of peak area, tailing factor and theoretical plates showed were within the limit.

Forced Degradation Study:

Degradation studies demonstrated the specificity of the developed method in the presence of degradation products. Degradation was carried out in combination of two drugs and purity of drug peaks was confirmed by purity angles. Their combination drug products were exposed to acid, alkali, oxidative, thermal stress, and photo stress and water conditions. Then found to be no degradable substances presence and proved that the proposed method was stable towards acid, alkali, peroxide, thermal, photo and water conditions within the limits.

Fig. 4: Acid degradation

Fig. 5: Alkaline degradation

CONCLUSION:

The developed RP-UPLC method provides a convenient and efficient method for the estimation of Ivabradine and Metoprolol in dosage form. There was no interference from the Excipients used in the tablet formulation and hence the method is suitable for analysis of tablets. The results of validation showed that the proposed method is simple, linear, precise, accurate and selective and is employed in routine assay of Ivabradine and Metoprolol in bulk and tablets. The resulting LOD, LOQ of Ivabradine and Metoprolol were 0.03 and 0.12 μg/ml, 0.08 and 0.35 μg/ml respectively.

ACKNOWLEDGEMENT:

The Authors are thankful to theBharath Institute of Higher Education and Research for given us this opportunity to explore the research. The authors are very thankful to the Spectrum labs, Hyderabad for providing the gift samples of Ivabradine and Metoprolol.

CONFLICT OF INTEREST:

Nil.

REFERENCE:

1. Chatterjee, S. A short review of the I-f Channel Antagonist, Ivabradine. The Internet Journal of Cardiology, 2008, 6(2), 1-4.

2. _{Rachel
J. Ryu}; _Sara
Eyal.; _{Thomas R. Easterling}.; _{Steve N. Caritis}; _{Raman
Venkataraman. Pharmacokinetics of Metoprolol during Pregnancy and Lactation. J
Clin Pharmacol., 2016, 56(5), 581–589.}

3. Ptaszynski, P.; Kaczmarek, K.; Ruta, J.; Klingenheben T.; Cygankiewicz, I.; Wranicz JK. Ivabradine in combination with metoprolol succinate in the treatment of inappropriate sinus tachycardia. J.Cardiovasc Pharmacol Ther., 2013, 18(4), 338-344.

4. Wei Sun.; Ruimin Chen. Wanshu li.; Chengkehauang. Simultaneous determination of Ivabradine, metoprolol and their metabolites in rat plasma by ultra-performance liquid chromatography tandem mass spectrometry and its application in a pharmacokinetic study. Analytical Methods, 2015, 7 (19), 8459-8465.

5. François Mathiaux.; Sylvain Dulaurent.; Frédéric Julia.; Jean-Michel Gaulier. Case Report of Ivabradine Intoxication, Analytical Toxicology, 2014, 38(4), 231–232.

6. Zoerner, A.A.; Schroeder C.; Kayacelebi.; Suchy, A. A. Validated rapid UPLC-MS/MS method for simultaneous Ivabradine, reboxetine, and metoprolol analysis in human plasma and its application to clinical trial samples. J Chromatogr B Analyt Technol Biomed Life Sci., 2013, 15, 105-111.

7. Chengtao, Lu.; Yanyan, Jia.; JingYang.; XinJin.; YingSong.; Wenxing, Liu.; YiDing.; Xiaoli, Sun.; Aidong, Wena. Simultaneous determination of Ivabradine and N-desmethylivabradine in human plasma and urine using a LC-MS/MS method application to a pharmacokinetic study. Acta Pharmaceutica Sinic. 2012, 2(2), 205-212.

8. Sagarika, Panda. Srikanta, Patra. Rapid and Selective UV Spectrophotometric and RP-HPLC Methods for Dissolution Studies of Ivabradine controlled Release Formulations. Pharma Tutor, 2014, 2(8), 201-213.

9. Bhosale Swati, D.; Vanjari Suvarna, S.; Jag tap Nikhil, S. Development and validation of stability indicating spectrophotometric method for the estimation of Ivabradine hydrochloride in bulk and in tablet formulation. Wjpps. 2016, 5(7), 1919-1927.

10. Sunitha, Seerapu. Srinivasan, B. P. Development and Validation of RP-HPLC Method for the Estimation of Ivabradine Hydrochloride in Tablets. Indian J Pharm Sci, 2010, 72(5), 667–671.

11. Muzaffar-ur- Rehman, Md.; Nagamallika, G.Validated RP-HPLC Method for the Determination of Ivabradine Hydrochloride in Pharmaceutical Formulation. Int. J. Pharm. Sci. Drug Res., 2017, 9(5), 228-233.

12. Mahaparale, S.I, Gonjari, D.; Jaya veera, K.N. Stability indicating hplc method for simultaneous estimation of metoprolol succinate and Telmisartan. Journal of Liquid Chromatography and Related Technologies. 2013, 36(18), 2601-2611.

13. Selvakumar, Pandiyan. Rajagopal, Development and validation of stability indicating rapid HPLC method for estimation of Ivabradine hydrochloride in solid oral dosage form. IJPPS. 2014, 6(4), 378-382.

14. Naveen Kumar C.H.; Saraswathi Mannuri.; Vijaya Kuchana. Kannappan, N. Development and validation of a stability indicating RP-HPLC method for determination of metoprolol succinate in pharmaceutical dosage forms. Der Pharmacia Sinica., 2014, 5(6), 69-78.

Received on 20.06.2019 Modified on 23.07.2019

Research J. Pharm. and Tech. 2020; 13(1):250-254.

DOI: 10.5958/0974-360X.2020.00050.5