1. INTRODUCTION:

RP-HPLC involves the separation of compound. Good resolution that can be achieved under a wide range of chromatographic conditions. Chromatographic selectivity can be manipulated through changes in mobile phase characteristics and other important factors.

Ivabradine HCl is a novel medication used for symptomatic management of stable angina pectoris and symptomatic chronic heart failure. Ivabradine acts by reducing heart rate by selectively inhibiting the "funny" channel pacemaker current in the sinoatrial node in a dose-dependent fashion and thus more blood to flow to the myocardium.

Ivabradine HCl is marketed under the brand name Corlanor and Corlentor. Therefore, it is very important to have a specific, selective, reliable and economic method for determination of Ivabradine in bulk drug and pharmaceutical dosage form. In this paper we describe a very simple yet rapid, selective, and highly sensitive HPLC method, not requiring sample treatment, for determination of Ivabradine. We have also laid emphasis on the stability indicating assay method of the same by HPLC involving stress studies.

Chemical Structure of Ivabradine:

Figure:1: Ivabradine HCl

2. MATERIALS AND METHODS:

2.1. Materials:

Ivabradine HCl, Active Pharmaceutical Ingredient (API) and working standard was supplied by Alkem Laboratories (Taloja, Navi Mumbai). Tablets of Ivabradine as a sample for respected study were provided by the same firm.

2.2. Chemicals and Reagents:

1) Potassium Dihydrogen Phosphate: AR grade or equivalent.

2) Water: HPLC grade or equivalent

3) Methanol: HPLC grade or equivalent

4) Triethylamine: AR grade

5) Acetonitrile: HPLC grade

6) Orthophosphoric acid: AR grade

2.3. Apparatus and Equipment:

1) HPLC equipped with pump, injector and UV Detector, Make: Agilent 1100 Series

2) Analytical Balance: Make: Mettler Toledo

3) Hot air oven: Make: Thermolab

4) Photostability Chamber: Make: Neutronic

5) pH meter: LAB INDIA, model PICO⁺

6) Sonicator: Spectra lab, UCB 300D

2.4 Chromatographic conditions:

Chromatographic separation was performed on a reverse phase HexonC₁₈ shield. The mobile phase was a mixture of buffer pH 5.8: Acetonitrile: Methanol (65:25:10 % (v/v)).

Column: Octadecylsilane Column, 150mm x 4.6mm, 5µm

(Inertsil ODS -3V, C18 or equivalent)

Flow rate: 1.5 ml /minute

Wavelength: 286 nm

Injection volume: 50µl

Column Temperature: 35°C

Sample Compartment Temperature: 25°C

Run Time: 6 minutes

Retention Time: 3.9 minutes of Ivabradine HCl

a. Preparation of standard solution:

Accurately about 30 mg of Ivabradine HCl standard was weighed and transferred into 200 ml volumetric flask. About 100 ml of mobile phase was added, and then solution was sonicated to dissolve. Then volume of the solution was made up to the mark using mobile phase. Further 5 ml of this solution was diluted to 50 ml with mobile phase. Further the absorbance of resulting solution was taken at 286 nm.

b. Analysis of Marketed Formulation:

Assay was performed by using Tablets of Ivabradine for the preparation of the sample solution the 10 tablets were used. The powder equivalent to 247 mg of Ivabradine was weighed accurately and transferred to volumetric flask of 200 ml capacity. 100 ml of Methanol was added to dissolve the sample and sonicated for 30 minutes. Further the volume was made up to the mark with mobile phase. From this solution 5 ml solution was pipette out and placed into 50 ml of volumetric flask. The volume was made upto mark with mobile phase.

3. METHOD VALIDATION:

The method was validated for linearity, accuracy, precision, Specificity, robustness, filter paper study, solution stability and forced degradation study.

3.1. Accuracy:

Placebo was spiked with the known amount of Ivabradine HCl at 70%, 100% and 130% of test concentration as Ivabradine HCl SR Tablet (15mg Ivabradine HCl per Tablet). The amount of Ivabradine HCl was quantified as per the test method. The percentage recovery was calculated from the amount found and the actual amount added.

3.2. Precision:

The instrument precision was evaluated by determining the absorbance of the standard solution six times repeatedly. The results are reported in terms of relative standard deviation for the same. The intra-and inter-day variation for the determination was carried out in triplicate for the standard solution.

3.3. Linearity:

Linearity for Ivabradine HCl performed in the range of 2.119 mcg/ml to 3.935 mcg/ml (about 70% to 130%) test concentration. A graph was plotted with concentration (in mcg/ml) on x-axis and peak areas on y-axis. Slope, y-intercept, correlation coefficient (r-value) and residual sum of squares (RSS) were determined from the obtained results.

3.4. Specificity:

Blank (mobile phase), Placebo, standard and sample solution were injected into the HPLC system. There was no interference from the blank and placebo at the retention time of IvabadineHCl peak. Peak purity data reveals that Ivabradine HCl peak was homogeneous and there were no co-eluting peaks at the retention time of Ivabradine HCl peak.

3.5. Stability in analytical solution:

Stability of Ivabradine HCl peakin analytical solution was verified by analyzing the standard solutions and sample solutions, initially and also at different time intervals as mentioned below by storing in sample compartment of HPLC instrument at 25°C. Calculated the % assay for standard solution and sample solutions.

3.6. Filter paper selection study:

Selection of filter paper was evaluated by preparing the assay preparation in triplicate as per test method. Filtered the test solution through 0.45µm PVDF filter and 0.45µm Nylon filter analysed the samples against centrifuged a portion of sample solution. The % assay of Ivabradine HCl was calculated and compared the results with method precision results.

3.7. Robustness:

Robustness of the method was verified by deliberately varying the following instrumental conditions.

a By changing the temperature by ± 5°C

b By changing the flow rate by ± 10%

c By changing the organic content in mobile phase by ± 2% absolute.

3.8. Forced Degradation:

Forced degradation study was carried out by treating the sample under the following conditions along with control sample.

Table 1. Forced degradation conditions in detail:

Degradation Conditions	Condition details
Thermal	105º C for 12 Hrs
UV	254 nm for 48 Hrs
Acid	5 mL 0.1 N HCL at RT
Alkali	5 mL 0.1 N NaOH at RT
Oxidation	5 mL 0.3% H2O2 at RT
Humidity	25ºC / 90% RH for 24 Hrs

3.9. System Suitability:

1. % Relative standard deviation for five replicate injections of Standard preparation should be not more than 2.

2. Tailing factor for Ivabradinepeak should not be more than 2.0

4. RESULT AND DISCUSSION:

The methods given here werefound to be highly specific and linear according to the results. The peaks of the active ingredients and other additivesas well as the degradation products were separated out by the HPLC method. Therefore, HPLC method is highly specific and stability indicating for degradation products of the dosage form andformulation excipients. The method can be applied for routine analysis of Ivabradine HCl insolid dosage form. Developed HPLC method was found to be stability indicating and very specificfor estimation of Ivabradine HCl in presence of other degradation products and variousexcipients used in solid dosage form. Complete validation studies for the method proved it to be specific, linear,

robust and reproducible.

4.1. System Suitability:

System Suitability was daily performed during the entire validation to ensure the suitability of method.

The system suitability and system precision results of developed HPLC method, are given in

Table 2.

Table 2. System suitability and System Precision

Retention time (Mean ± SEM)	Theoretical plates (n)	Asymmetry/Tailing (T)
3.785	2725	1.5

4.2. Linearity:

Characteristics of the method from Standard calibration curve:

Table 3: Linearity specifications obtained from the Standard calibration curve:

Slope	359451
y-intercept	-52318
r-value	0.999162
RSS	277879747

Fig.2. Linearity: Standard calibration curve

4.3. Specificity:

The data demonstrate that there is no interference of blank, known impurity and placebo at retention time of Ivabradine HCl.

Table 4. Specificity parameters obtained from the test results:

Sample type	Retention Time (min)	Peak purity
Standard	3.89	999.349
Control sample	3.907	999.426

4.4. Stability indicating nature of the Developed Method-Forced degradation:

Placebo preparation of Ivabradine HCl, Ivabradine HCl API, and samples i.e., tablets of Ivabradine HCl were exposed to various stress conditions showed the peak purity as that of the normal condition. The degradation study clearly indicated that Ivabradine HCl degrades in alkali and hydrolytic condition and it also degrade in oxidative and humidity condition. The degradation results were shown in Table 5.

Table 5. Degradation data obtained from Forced degradation studies:

Conditions	% Assay	% Degraded	Peak Purity
Control	98.37	NA	999.349
Photolytic	98.93	-0.43	999.503
Heat degradation	99.68	-1.33	999.395
Humidity degradation	92.39	6.08	999.518
Acid degradation	99.15	-0.79	999.426
Base degradation	85.13	13.46	999.552
Peroxide degradation	94.43	4.01	999.440
Hydrolysis Degradation	76.79	21.94	999.636

4.5. Method Precision:

The method precision was obtained by determining the assay by preparing six-sample preparation. The low value of standard deviation i.e., >2 proved the method to be very precise and accurate.

Table 6. Results for the method precision test of 6 consequent samples:

Sr. No.	% Assay
1	98.73
2	99.34
3	101.47
4	98.50
5	99.50
6	101.81
Mean	99.89
SD	1.408
% RSD	1.41

4.6. Method Recovery:

To determine the reliability and accuracy of the method recovery studies were carried out In triplicate at 70%, 100% and 130% of target concentration. Results of accuracy study are found to be within the range of 98% to 102% and RSD < 1%.

Table.7. Accuracy details obtained with sample spiking at concentrations 70%-130%:

Levelno. (Spike level in %)	Mean (%)	SD	% RSD
Level – 1 (70%)	99.38	0.376	0.38
Level – 2 (100%)	100.54	0.899	0.89
Level – 3 (130%)	99.57	0.335	0.34
Over all mean		99.83
Over all SD		0.747
Over all % RSD		0.75

4.7. Method Robustness:

Robustness of the method was determined by small deliberate changes in some Important chromatographic conditions like flow rate, organic phase ratio, buffer pH and column oven temperature. Even after these changes content of the drug did not affect adversely. The low values of relative standard deviation indicating that the HPLC method is robust for this particular parameter.

Table 8. Critical parameters involved in Method robustness

Sr. No	Experiment (Actual Value)
I	Method Precision data
II	Plus Temperature (40°C)
III	Minus Temperature (35°C)
IV	Plus flow (1.65ml/min)
V	Minus flow (1.35ml/min)
VI	Plus organic content (2%)
VII	Minus organic content (2%)
VIII	Plus pH (6.0)
IX	Minus pH (5.6)

Table 9. Results for the method robustness by changing the above-mentioned parameters:

Sr. No.	I	II	III	IV	V	VI	VII
1	98.73	100.84	101.31	104.82	101.39	99.36	99.11
2	99.34	101.25	100.58	102.39	101.08	99.28	98.99
3	101.47	103.67	101.59	101.97	102.18	99.31	99.04
4	98.50	-	-	-	-	-	-
5	99.50	-	-	-	-	-	-
6	101.81	-	-	-	-	-	-
Over all Mean		100.57	100.20	102.91	100.44	99.70	99.61
Over all SD		1.689	1.254	0.871	1.417	1.150	1.191
Overall % RSD		1.68	1.25	0.85	1.41	1.15	1.20

4.8. Solution Stability:

Standard and sample solutions in HPLC method were evaluated at roomtemperature for 16Hours. The solutions were analysed after 4, 6, 8, 10, 12, 14 and 16 Hours therelative standard deviation was found to be below 2.0% in both the methods. It proves the Solution stability of standard and sample solution is at room temperature. The results of solutionstability HPLC method are shown in Table.

Table 10. Solution stability for standard solution:

Time in hours	% Assay	Difference
Initial	96.68	-
4	96.91	0.23
6	96.90	0.22
8	97.11	0.43
10	97.38	0.70
12	98.30	1.62
14	99.62	2.94
16	99.44	2.76

Table 11. Solution stability for sample solution:

Time in hours	% Assay	Difference
Initial	98.76	-
4	98.95	0.19
6	98.97	0.21
8	98.37	0.39
10	98.25	0.51
12	99.48	0.72
14	99.11	0.35
16	99.44	0.68

4.9. Assay:

The developed and validated method was successfully applied for the estimation of Ivabradine in tablet dosage form. The assay results were about 98-100%. The mean retention time was about 3.7. The results of assay by using this method indicate that the method is specific for the analysis of Ivabradine without interference from the excipients used to prepare and formulate these tablets.

5. CONCLUSION:

The above-developed method is suitable for determination of Ivabradine HCl in solid dosage form. The method was found to be highly sensitive, precise, and accurate for determination of Ivabradine HCl in pharmaceutical dosage form. These methods are quite useful and reasonably satisfy all the standards. Therefore, these methods can be used for Routine analysis of Ivabradine HCl in pharmaceutical dosage form i.e., sustained release tablet dosage form. More over the HPLC method has also proved to be stability indicating because it can separate degradation peaks from the main peaks and accurately quantifies it in the stability samples.

6. ACKNOWLEDGEMENT:

All authors are thankful to Alkem Laboratories, Taloja, Navi Mumbai, Maharashtra, India for providing reference standards and all facilities to complete this research work.

7. REFERENCES:

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Received on 17.05.2019 Accepted on 25.06.2019

Asian J. Pharm. Ana. 2019; 9(3):133-137.

DOI: 10.5958/2231-5675.2019.00025.5