Theory:

We can find out concentration of both the drug from combination mixture using the simultaneous equation method. In this method using the absorbance of both the drug and mixture at their wavelength and put this value in following equation and we can find out the concentration of drugs present in combination.⁽⁵⁾

where,

C_x= Concentration of Atenolol

C_y = Concentration of Ivabradine HCl

A₁= Absorbance of test at λ₁(λ_maxATN)

A₂ = Absorbance of test at λ₂(λ_max of IVA)

ax₁= Absorptivity of x drug(ATN) at λ₁

ax₂ = Absorptivity of x drug(ATN) at λ₂

ay₁= Absorptivity of y drug(IVA) at λ₁

ay₂ = Absorptivity of y drug(IVA) at λ₂

MATERIAL AND METHOD:

Instrument:

Spectroscopic Analysis was carried out on a UV/VISIBLE 2450 and UV/VISIBLE 1800 (Shimadzu) double beam UV-Visible spectrophotometer with software of UV Probe version 2.34. A Semi-micro analytical balance (Sartorius CD2250, Germany) was used for weighing purpose.

Material:

Analytically pure Ivabradine HCl raw material was received as gift sample (from Torrent Pharmaceutical, Ahmedabad)

Atenolol raw material as gift sample (from Meridian Enterprise Pvt. Ltd., Navsari).

Methanol AR Grade (from FINAR Pvt. Ltd., Ahmedabad)

Distilled Water (from SDPC research laboratory, Surat)

NaOH AR Grade (from RANKEM Pvt. Ltd., Ahmedabad)

HCl AR Grade (from ASTRON Pvt. Ltd., Ahmedabad) were used for development purpose.

Preparation of stock solution:

A standard solution of Atenolol and Ivabradine HCl were prepared by dissolving 100mg in 25ml of Methanol in 100 ml volumetric flask, dilute up to mark with Methanol to make stock solution. The absorbance value of resulting solution was then measured at 276 nm and 286.50 nm respectively.

Preparation of Linearity Curve:

To construct Beer’s law of Atenolol and Ivabradine HCl different aliquots from 100µg/ml stock of atenolol (2,4,6,8,10 ml) with different concentration (20, 40, 60, 80, 100µg/ml) and Ivabradine HCl (0.2, 0.4, 0.6, 0.8, 1ml) with different concentration (2,4, 6, 8, 10µg/ml) respectively. Mixed standard solution were prepared from standard stock solution of the two drugs. Than absorbance of solution was measured at 276nm for Atenolol and 286.50 for Ivabradine HCl. Both drug obeyed beer law individually and mixture with the concentration range (20-100 µg/ml) of Atenolol and (2-10 µg/ml) of Ivabradine HCl in 1:10.

Preparation of test solution and estimation of Atenolol and Ivabradine HCl in synthetic mixture:

For analysis of combined drug in synthetic mixture of formulation, mixture given in Table 1 from US patent ⁽³⁾accurately weighed equivalent to 10 mg of Ivabradine HCl and 100mg of Atenolol which was transferred into 100 ml volumetric flask, solubilize in 25 ml methanol and make up to 100ml with methanol, Filtered and further diluted with methanol to get the concentration within linearity range of respective drug and measured absorbance for both drug respectively Fig. 4. Then the amount of drug present in the formulation was calculated using calibration curve Fig. 5. Calibration curve of both drug in respective range taken at maximum absorbance.

Figure 4: Zero order linearity spectra of both drug

Recovery Studies:

The recovery studies were carried out at three level i.e. 80%, 100%, 120%. To ensure the reliability of the above method, recovery studies were carried out by mixing a known amount quantity of sample formulation in Table 2. It reanalyzed, % recovery found in Table 3.

Table 1 Synthetic formulation for combined dose of Atenolol and Ivabradine HCl

INGREDIENT	QUANTITIY
Ivabradine HCl	100 mg
Atenolol	1000 mg
Starch	120 mg
Magnesium Stearate	40 mg
MCC	1000 mg
Lactose	1732 mg
Hydrophobic colloidal silica	8 mg
Total	4000 mg (4 gm)

Figure 5: Calibration curve and regression data of both drug

Table 2 Quantity taken of synthetic mixture for accuracy study

Level of recovery	Amt. of Synthetic mixture taken (mg)	Amt. of Drug in synthetic mixture (mg)		Spiked Amt. of API in mixture (mg)		Total Amt. (mg)
Level of recovery	Amt. of Synthetic mixture taken (mg)	IVA	ATN	IVA	ATN	IVA	ATN
0%	400	10	100	-	-	10	100
80 %	400	10	100	8	80	18	180
100 %	400	10	100	10	100	20	200
120 %	400	10	100	12	120	22	220

Table 3 % Recovery study data

Level of recovery	Total Conc. (µg/ml)		Result of recovery study
	Total Conc. (µg/ml)		Total Quantity Found (µg/ml)				% Recovery
	IVA	ATN	IVA	%RSD	ATN	%RSD	IVA	% RSD	ATN	% RSD
0%	4	40	4.04	0.754	40.25	0.074	101.13	0.794	100.62	0.074
80 %	7.2	72	7.23	0.319	72.19	0.244	101.03	0.714	100.61	0.548
100 %	8	80	8.02	0.190	80.05	0.019	100.66	0.379	100.14	0.036
120 %	8.8	88	8.82	0.17	88.05	0.039	100.48	0.319	100.11	0.070
Mean of 3 Determination							100.82	0.390	100.37	0.182

Table 4 Data of precision study

Wavelength	Conc.(µg/ml) Ivabradine Hydrochloride: Atenolol	Precision
		Intraday		Interday
		Mean (n=3)	% RSD	Mean (n=3)	% RSD
286.5 nm	2 : 20 μg/ml	0.068	0.844	0.068	0.844
	6 : 60 μg/ml	0.188	0.931	0.186	0.941
	10 : 100 μg/ml	0.303	0.190	0.305	0.500
276 nm	2 : 20 μg/ml	0.127	0.452	0.128	0.781
	6 : 60 μg/ml	0.335	0.172	0.335	0.620
	10 : 100 μg/ml	0.555	0.674	0.555	0.580

Validation Parameter:

Linearity:

Six-point calibration curves were obtained in the concentration range of 20-100 μg/ml for Atenolol and 2-10 μg/ml for Ivabradine HCl. The response of drug was found to be linear in investigation range and the regression equations was found to be y = 0.0054x - 0.0405 for Atenolol (n=5) and y = 0.015x + 0.0065for Ivabradine HCl (n=5), with the correlation coefficient 0.9990 and 0.9993 (n=5) respectively, is listed in Figure 4 and 5.

Precision:

The precision of the method was evaluated in terms of inter-day and intra-day by carrying out independent assays of three concentrations chosen from range of the standard curves (20, 60, 100) and (2, 6, 10) μg/ml of Atenolol and Ivabradine HCl respectively) and the % RSD of assay (inter-day and intra-day) was calculated. The results of study are shown in Table 4.

Limit of Detection and Limit of Quantification:

The limit of detection (LOD) and limit of quantization (LOQ) of the method were evaluated by standard deviation of response and slope method. LOQ and LOD were calculated by the equation LOD = 3.3 × N/B and LOQ = 10 × N/B, where “N” is standard deviation of the absorbance, and “B” is the slope of the corresponding calibration curve. It presented in Table 5.

Table 5 Data of LOD (limit of detection) and LOQ (limit of quantitation)

Parameter	IVA	ATN
LOD (µg/ml)	0.283	0.315
LOQ (µg/ml)	0.857	0.956

ROBUSTNESS AND RUGGEDNESS:

Robustness was done by different instrument and difference in wavelength. And ruggedness done by different wavelength, solvent (±2 nm, ±2% water in solvent of selected wavelength and solvent for study). The result was decided by %RSD which is in the limit which is mentioned in Table 6 and 7.

Table 6 Data of robustness study

Condition	Conc. (µg/ml) IVA: ATN	Different Instrument				Different Analyst
		UV-2450		UV-1800		A		B
		Mean (n=3)	% RSD	Mean (n=3)	% RSD	Mean (n=3)	% RSD	Mean (n=3)	% RSD
286.50 nm	4 : 40	0.131	0.876	0.127	0.453	0.131	0.876	0.133	0.854
276.00 nm	4 : 40	0.256	0.224	0.257	0.896	0.256	0.896	0.253	0.710

Table 7 Data of ruggedness study

Condition	Conc. (µg/ml) IVA : ATN	Different Solvent				Change in Wavelength ± 2 nm
		2 % water in Methanol		5 %water in Methanol		284.50 nm		288.50 nm
		Mean (n=3)	%RSD	Mean (n=3)	%RSD	Mean (n=3)	%RSD	Mean (n=3)	% RSD
286.50 nm	4 : 40	0.131	0.876	0.095	0.605	0.213	0.540	0.104	0.553
						274.00 nm		278.00 nm
276.00 nm	4 : 40	0.242	0.376	0.332	0.694	0.256	0.224	0.180	0.639

Table 8 Summary data of all validation parameters

SR. NO.	PARAMETER	IVABRADINE HYDROCHLORIDE	ATENOLOL
1	Wavelength Max.	286.50nm	276.00nm
2	Linearity (µg/ml) (n=6)	2-10 µg/ml	20-100 µg/ml
3	Regression equation	y = 0.015x + 0.0065	y = 0.0054x - 0.0405
4	Correlation coefficient (R²)	0.9993	0.9990
5	Accuracy(%Recovery) (%RSD)	100.82 ± 0.390	100.37 ± 0.182
6	Precision Intra-day (%RSD) (n=3) Inter-day (%RSD) (n=3)	0.190 - 0.931 0.311 - 0.844	0.172 – 0.452 0.180 - 0.781
7	LOD(µg/ml) (n=10)	0.283	0.315
8	LOQ (µg/ml) (n=10)	0.857	0.956
9	Robustness Different Instrument (%RSD) (n=3) Different Analyst (%RSD) (n=3)	0.453 - 0.876 0.854 - 0.876	0.224 – 0.896 0.896 – 0.710
10	Ruggedness Different Solvent (%RSD) (n=3) Change in Wavelength (%RSD) (n=3)	0.605 – 0.876 0.540 – 0.553	0.376 – 0.694 0.224 - 0.639
11	Assay	101.13%	100.62%

Application of the proposed method for analysis of Atenolol and Ivabradine HCl in synthetic mixture

A zero order spectrum of the sample solution containing 40µg/ml of Atenolol and 4 µg/ml of Ivabradine HCl was recorded and the absorbance at 276.00nm and 286.50nm were noted for estimation of Atenolol and Ivabradine HCl respectively. The concentration of Atenolol and Ivabradine HCl in mixture was determined using the corresponding calibration graph. The results from the analysis of synthetic mixture containing Ivabradine Hydrochloride (10 mg) and Atenolol (100mg) in combination were presented in Table 1.

The percent assay shows that there is no interference from excipients and the proposed method can successfully have applied to analysis of commercial formulation containing Atenolol and Ivabradine HCl. The % assay value for Atenolol found to be 100.62% and for Ivabradine HCl 101.13%.

RESULT AND DISCUSSION:

From the optical characteristics of the proposed method, it was found that the drug obeys linearity within concentration range of 20-100µg/ml for Atenolol and 2-10µg/ml for Ivabradine HCl. From the result %assay was found 100.62% for Atenolol and 101.13% for Ivabradine HCl respectively and % RSD is less than 2 % which indicate that the method has good reproducibility. From the result shown in accuracy table it was found that the percentage recovery of pure drug from reanalysed solution of formulations were in between 98.00%- 101.00%, which indicate that the method is accurate and which reveals that commonly used excipient and additives present in the synthetic formulation did not interfere in proposed method. The proposed method was simple, sensitive and reliable with good precision and accuracy. Hence this method can be used for the routine determination of Atenolol and Ivabradine HCl in synthetic mixture.

CONCLUSION:

A convenient and rapid UV method has been developed for simultaneous estimation of Atenolol and Ivabradine HCl in synthetic mixture. The assay provides a linear response across a wide range of concentration. Low intra-day and interday %RSD coupled with excellent recoveries. Hence, this method can be easily and conveniently adopted for routine analysis of Atenolol and Ivabradine HCl in pure and its synthetic mixture.

CONFLICT OF INTEREST:

The authors confirm that this article content has no conflict of interest.

ACKNOWLEDGMENT:

I am very much thankful Shree Dhanvantary pharmacy college, Kim, Surat for giving permission to carry out my research work. I am very thankful to my guide Dr. Hasumati Raj (QA Department), Co-guide Dr. Gautam Sonara and principle Dr. Noolvi for guidance, help and encouragement at every step during the progress of my work.

REFERENCE:

1. Lookchem for Drug profile of Atenolol. http://www.lookchem.com/Atenolol/, 2015 (accessed 12.9.015)

2. Eurotrade world Commerce, S.L. for Drug profile of Ivabradine HCl. http://www.sigmaaldrich.com/catalog/product/sigma/sml0281?lang=enandregion=IN, 2015 (accessed 11.9.015)

3. Guy Lerebours-Pegeonniere, Levallois-Perret and Jean-Henri Calvet. Drugs comprising combination of Sinus node if current channel blocker with β-blocker. United States Patents US 8217030 B2, 2009.

4. International Conference on Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and Methodology, ICH Q2(R₁), 2005.

5. Davidson AG., Beckett AH., Practical Pharmaceutical Chemistry, CBS Publisher, New Delhi 2002, pp 275-300.

Received on 13.03.2016 Accepted on 08.04.2016

Asian J. Pharm. Ana. 2016; 6(2): 109-114.

DOI: 10.5958/2231-5675.2016.00016.8