INTRODUCTION:

The chemically ivabradine hydrochloride is 3-(3-{[((7S)-3,4-Dimethoxybicyclo[4,2,0]octa-1,2,3-trien-7-yl)methyl]methy amino} propyl)- 1,3,4,5-tetrahydro-7,8-dimethoxy-2H-3-b enzazepin-2-one,hydrochloride (figure A) corresponding to the molecular formula C₂₇H₃₆N₂O₅.HCL. Ivabradine HCL has a relative molecular mass of 505.05 g/mole.¹

Recently, ivabradine was approved by the U. S. Food and Drug Administration (USFDA) as aheart-rate-lowering agent in 2015. Ivabradine is the first drug with a novel mechanism of action acting by specifically and selectively inhibiting the cardiac pacemaker current (If), a mixed sodium-potassium inward current that controls the spontaneous diastolic depolarization in the sinoatrial (SA) node and hence regulates the heart rate.²The molecular channel of ivabradineHcl belongs to the HCN family. If ion current flow is disrupts by the inhibition of this channel, thereby prolonging diastolic depolarization, slowing firing in the SA node, and eventually reducing the heart rate.³ The Cardiac effects of ivabradine are specific to the SA node, and the drug has no effect on blood pressure, intracardiac conduction, ventricular repolarization, myocardial contractility.⁴

The sinoatrial node is unique in that its cells have an innate ability to generate a cyclical change in their resting membrane potential, which drives it toward the threshold needed for spontaneous depolarization. This depolarization, in turn, generates repetitive, spontaneous action potentials accounting for itsautomaticity. This depolarization is initiated by theopening of specific ion channels that conduct a slow, inward-depolarizing mixed sodium-potassium current, referred to as the pacemaker or “funny” current (If). If is generated via a nonselective, hyperpolarization-activated cyclic nucleotide–gatedtransmembrane channel. Ivabradineblocks the intracellular aspect of this transmembrane channel by inhibiting cation movementwith a high degree of selectivity, leading to a reduction in the slope of the diastolic depolarization of the pacemaker action potential, thereby slowing the heart rate. Ivabradine blocks the channel in its open state, creating a particularly favorable attribute, use dependence (i.e., it becomes more potent at faster heart rates). Ivabradine causes a dose-dependent reduction in heart rate and, as a consequence of its specific mechanism of action; it is able to do so without affecting cardiac inotropy or systemic vascular resistance.^5,6

The literature survey reveals that numerous methods for determinations of Ivabradine HCL in singlein pharmaceutical dosage forms^7,8spectrophotometric methods, HPTLC⁹stability indicating spectrophotometric methods^10,11,12and methods in combination with other drugs including RP-HPLC.¹³

In this paper we described a very simple, sensitiveRP-HPLC method. This method show very simple and accurate approach for the analysis of ivabradine HCL without need of sophisticated instruments, expensive solvents or a large number of samples.

MATERIALS AND METHODS:

Materials:

Pure sample of ivabradine HCL was kindly supplied as a gift sample by Lupin Pharmaceuticals (Aurangabad, Maharashtra) India. All solvents and chemicals were of analytical grade. Marketed Tablet dosage form used in this research work was IVABRAD 5mg (LupinLaboratories) acquired from local market.

Instruments:

Spectrophotometric measurements were carried out on Shimadzu UV 1800 double beam spectrophotometer. Infrared spectroscopic study was done on FTIR (Bruker), and chromatographic separation was done on HPLC (AGILENT TECHNOLOGIES)

Preparation of mobile phase:

The optimized mobile phases consist of Acetonitrile: Methanol (20:80 v/v). The solvents of HPLC grade are used. Mobile phase are filtered through membrane filter 0.45μm and fill into solvent reservoir.

Preparation of standard stock solution

Standard stock solution of IvabradineHcl was prepared by accurately weighing 20 mg of IvabradineHcl to 20 ml volumetric flask containing mobile phase. The drug solution was sonicated and volume was made up to mark with mobile phase to get the concentration of 1000μg/ml.

Selection of analytical wavelength (λmax):

Aliquot portion of standard stock solution was appropriately diluted with mobile phase to obtain final concentration of 50μg/ml of IvabradineHcl. The solution was scanned using double beam UV-Visible Spectrophotometer-1800 in the spectrum mode between the wavelength ranges of 400 nm to 200 nm against mobile phase as blank. The wavelength selected was 286 nm as IvabradineHcl showed significant absorbance at this wavelength. Typical chromatogram obtained is shown in Figure. (Figure B)

Selection of concentration range and preparation of calibration curve:

Aliquots portion 0.25, 0.5, 1, 2, 5 ml were pipetted out from the standard stock solution of 100μg/mL and transferred to series of 10 ml volumetric flask and volume was made with mobile phase to get the concentration range from 2.5-50μg/ml. The observed chromatogram was measured three times for each concentration at wavelength 286 nm.

Analysis of Tablet formulation:

Twenty tablets were weighed and finely powdered. Equivalent to 10 mg of Ivabradine HCL was weighed and transferred to a 100 mL volumetric flask containing 60 ml methanol, and sonicated for 20 minutes with intermittent shaking. The solution was filtered through 0.45μm membrane filter and volume was made up to mark with and mixed to get 100μg/ml. An aliquot portion of tablet stock solution 1 mL was transferred to 10 mL volumetric flask and volume was made up to mark with methanol to get concentration of 10μg/mL of Ivabrad. Solution was injected and chromatogram and area was observed.

Method validation:

According to ICH Q2 (R1) guidelinesthe developed method was validated to assure the reliability of results of the analysis for different parameters like linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ),specificity, and robustness.¹⁴

RESULT AND DISCUSSION:

Linearity:

The correlation between peak area & concentration of IvabradineHcl was studied under the developed experimental conditions. A virtuous linear relationship is specified by correlation coefficient value. Five solutions of IvabradineHcl at concentrations are ranging from 2.5-50 μg/ml (Figure C). The straight line equation and correlation coefficient for IvabradineHcl calibration curve way Y=3939334 x + 1998632 and r²= 0.9997 respectively.

Analysis of tablet formulation (Recovery study):

Line equation obtained from calibration plot was used to calculate label claim of marketed formulation of Ivabradine HCL. (TableI and Table II)

Accuracy:

The accuracy of an analytical procedure expresses the results obtained by that method to the true value. The accuracy of the developed method was determined on the basis of recovery studies. The recovery tests were performed by adding known quantity of pure standard drug into the solution of tablet powder. The sample was then spiked with standard at levels 80%, 100% and 120% of tests concentrations (Table III). The resulting spiked sample solutions were analysed in triplicate.

Table I: The chromatographic method

Column	ZorbaxEcips Plus C18 (250mm × 46mm, 5.0µ)
Detector	PDA
Injection Volume	10μl
Flow Rate	1 ml/min
Temperature	30℃
Run Time	10 min
Mobile Phase	Methanol: Acetonitrile (80:20)
Diluent	Methanol
Retention Time	5.8 min

Table II: Data of Tablet analysis of Ivabradine HCL

Drug Name	Mean*	SD	%RSD
Ivabrad	100.26	0.3270	0.3261

*Average of six determinations

SD=Standard Deviation, RSD=Relative Standard Deviation.

Precision:

The Precision study of analytical method validation express the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions. The precision of methods can studied as interday precision and intraday precision. Repeatability study of the system precision and interday precision of IvabradineHcl were carried out by determining the response on same day and next day in triplicate for three different concentrations of IvabradineHcl. Results of precision study for intraday andinterday is shown in tables. (Table IV and Table V)

Robustness:

Robustness is the measure of a method unaffected by small, deliberate changes in method parameters like flow rate. The small but deliberate variations in the optimized method parameters were done to evaluate the robustness of the proposed method. The study was conducted to determine the effect of variation in flow rate. Standard solutions of IvabradineHcl was prepared and injected into the HPLC system by keeping flow rates 0.8 ml/min and 1.2 ml/min (Table VI). The effect of variations in flow rate was evaluated.

Table IV: Data for intraday precision of Ivabradine HCL by HPLC method

Sr. No.	Conc. (μg/mL)	Mean	SD	% RSD
1	5	21726625	25755	0.0009706
2	10	41542521	37158	0.0894
3	20	81151825	34248	0.04220

*Average of three determination

Table V: Data for interday precision of Ivabradine HCL by HPLC method

Sr. No.	Conc. (μg/mL)	Mean*	SD	% RSD
1	5	21697120.33	7550	0.0348
2	10	41485312	16525	0.0398
3	20	80906492	47766	0.05903

*Average of three determination

Table VI: Data for Robustness study of Ivabradine HCL by HPLC method

Sr. No

Parameter

Flow Rate

mL/min

Conc (μg/ml)

Mean*Area

% RSD

Change in

0.8

21751203

69095

0.3176

Flow rate

1.2

41580451

6283

0.0151

*Average of three determination

Ruggedness:

Ruggedness is the degree of reproducibility of the results obtained under a variety of conditions. Ruggedness study was performed to examine effect of non-procedure related factors such as instruments and analyst. Ruggedness study of IvabradineHcl was carried out by using two different analyst under the similar operational and environmental conditions. %RSD of ruggedness study was found to be less than 2 (Table VII)

Table VII: Data for ruggedness study of Ivabradine HCL by HPLC method

Sr. No

Analyst

Conc.

(μg/ml)

Mean

area*

% RSD

Analyst-Ι

2.5

11311729

94137

0.8322

Analyst-ΙΙ

4118488

56444

0.1372

*Average of three determination

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

The LOD and LOQ of IvabradineHclwere found to be 0.0216μg/ml and 0.06537μg/ml respectively. The low LOD and LOQ values for IvabradineHcl indicate the sensitivity of the method.

Specificity:

Specificity study is the ability to asses unequivocally the analyte in the presence of component which may be expected to be present. For the specificity study of proposed method the sample may be spiked with excipients or possible interfering components. Results of specificity study were found in analytical limits shown in table. (Table VIII)

Table VIII: Data for specificity study of Ivabradine HCL by HPLC method

Level of addition	Tablet drug conc. (μg/ml)	Exepients(μg/ml)	Total conc. (μg/ml)	SD	% RSD
80%	6	9	15	2796.32	0.0934
100%	9	9	18	6267.09	0.0169
120%	12	9	21	18451.91	0.00376

CONCLUSION:

From the above experimental parameter and results, it was concluded that the proposed RP-HPLC method is novel, sensitive, accurate, precise and cost effective and time effective with short column retention time of 5.8 min indicates to less solvent consumption. The results of analysis reveal that virtually no interference of the attitudes present in marketed formulations. This proposed method can be intended for the routine analysis in industry for IvabradineHcl in bulk and tablet dosage form.

CONFLICT OF INTEREST:

None

REFERENCES:

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11. Mitesh H. Motisariya, KalpanaGovindbhai Patel, Purvi A. Shah Anand, et al. Validated Stability-Indicating HighPerformance Thin Layer Chromatographic Method for Determination Of Ivabradine Hydrochloride In Bulk And Marketed Formulation: An Application To Kinetic Study. Bulletin of Faculty of Pharmacy, Cairo University. 2013; 51: 233–241.

12. Bhosale Swati D, Vanjari Suvarna S. And Jagtap Nikhil S., et al. 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.

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Received on 12.01.2019 Accepted on 18.02.2019

Asian J. Pharm. Tech. 2019; 9(2):89-92.

DOI: 10.5958/2231-5713.2019.00015.1

Level of addition	Tablet drug conc. (μg/ml)	Standard added (μg/ml)	Total conc. (μg/ml)	% Mean recovery*	SD	% RSD
80%	6	9	15	100.58	0.2486	0.2471
100%	9	9	18	99.79	0.1336	0.1340
120%	12	9	21	100.14	0.1331	0.1329