Development and Validation of UV Spectrophotometric Methods for Simultaneous Estimation of Amlodipine besylate and Theophylline

 

Ramesh L Sawant, Rahul B Kale, Amol S Ghodechor, Abhijit B Sherkar, Kunal R Sanklecha

Department of Pharmaceutical Chemistry, Dr. Vithalrao Vikhe Patil Foundation’s College of Pharmacy, Vilad Ghat, Ahmednagar,414111, India.

 

ABSTRACT:

The present study describes simple, specific, sensitive, rapid and economical U.V. spectrophotometric methods for simultaneous estimation of amlodipine besylate and theophylline. The absorbance maxima of the drug were found to be239 nm and 272 nm respectively. Method I is based on formation and solving of simultaneous equation known as Vierodt’s method. Method II is based on principle of Q-analysis known as absorbance ratio method. Beer’s law obeyed in concentration range of 5-30 μg/ml having regression line equation y = 0.041x + 0.017 and y = 0.064x + 0.052 with correlation coefficient 0.999 and.0.997 of amlodipine besylate and theophylline respectively. Different parameter such as linearity, precision, LOD and LOQ, slope and standard deviation were used for validation in both method.

 

 

 

INTRODUCTION:

Amlodipine besylate and theophylline have been extensively used antihypertensive and antiasthmatic agents [1]. According to medical literature review both drug are used in combination for the treatment of asthma in hypertension patient. Literature review shows that several methods are reported for quantification of amlodipine besylate and theophylline individually [2, 3] but no analytical method using UV spectroscopy for these combination is reported.

 

Amlodipine besylate, a calcium channel blocker is a dihydropyridine calcium antagonist that inhibits the trans-membrane influx of calcium ions into vascular smooth muscle and cardiac muscle. It is used as antihypertensive and antianginal drug [4, 5].

 

Figure 1: Structure of amlodipine besylate

 

Theophylline, an alkaloid found in the leaves of cameliai sinesis, is used clinically as a bronchodilator in the managements of chronic obstructive pulmonary diseases [6, 7].

 

Figure 2: Structure of theophylline

MATERIALS AND METHODS:

UV-Visible double beam spectrophotometer, Jasco model V-630 with spectral bandwidth of 2 nm, wavelength accuracy of ± 0.3 nm and a pair of 10 nm matched quartz cell were used.

 

Preparation of Standard Stock Solution and Calibration Curve:

The standard stock solution of amlodipine besylate and theophylline were prepared by dissolving 0.01 gm of each drug in distilled water and final volume was adjusted with the same solvent in 100 ml of volumetric flask to get a solution containing 100 μg/ml of each drug. These stock solutions were used to prepare series of standard solution with concentration 5-40 μg/ml of amlodipine besylate and theophylline respectively for both methods.

 

Working standard solution of 20μg/ml was scanned in the entire UV range of 400-200 nm to determine the λ_max. The absorbance of resulting solutions was measured at their respective λ_max and isobestic point and plotted a calibration curve to get the linearity and regression equation.

 

Method I (Simultaneous Equation Method):

Simultaneous equation method [8] of analysis is based on the absorption of drugs (amlodipine besylate and theophylline) at their wavelength maximum. Two wavelengths selected for the development of the simultaneous equation are 239 nm and 272 nm. The absorbances and absorptivity at these wavelengths were substituted in equation 1 and 2 to obtain the concentration of drugs.

 

C _x = A₂ ay₁- A₁ay₂ / ax₂ ay₁-ax₁ ay₂                           (1)

 

C y = A_{1 ax2-} A₂ ax₁ / ax₂ ay₁- ax₁ ay₂                                  (2)

 

A₁ and A₂ are the absorbance of the mixture at 239 nm and 272 nm respectively.

 

Method II (Absorbance Ratio Method):

Absorbance ratio method [8] of analysis is based on the absorbance at two selected wavelengths, one of which is an isobestic point and the other being the wavelength of maximum absorption of one of the two components. From overlain spectra (Figure 5) 254 nm (isobestic point) and 272 nm (λ_max of theophylline) are selected for the formation of Q absorbance equation (Eqn. 3 and 4).

Calibration curve was plotted at both wavelengths and concentrations of amlodipine besylate and theophylline were determined using equations 3 and 4.

 

C _x = {(Q_m – Q_y) / (Q_x – Q_y)} A₁/ ax₁                           (3)

 

C _y = {(Q_m – Q_x) / (Q_y – Q_x)} A₁/ ay₁                                   (4)

 

Where,

Q_m = A₂/ A₁,

Q_x = ax₂/ ax₁,

Q_y = ay₂/ay₁.

Where, A₁ and A₂ are absorbance’s of mixture at 239 nm and 272 nm respectively ax₁ and ax₂ are absorptivity of amlodipine besylate at λ₁ and λ₂ respectively and ay₁ and ay₂ are absorptivity of theophylline at λ₁ and λ₂ respectively. C_x and C_y are concentrations of amlodipine besylate and theophylline respectively.

 

Validation of Developed Methods:

Linearity:

For each drug, appropriate dilutions of standard stock solutions were assayed as per the developed method. The Beer law concentration range was found to be 5-30 μg/ml for both amlodipine besylate and theophylline.

 

Precision:

The intraday and interday precision was determined by assay of the sample solution on the same day and on different days at different time intervals respectively.

 

Limit of Detection (LOD):

The LOD of amlodipine besylate and theophylline by proposed methods were determined using calibration standards. LOD were calculated as 3.3 σ/S. Where S is the slope of the calibration curve and σ is the standard deviation of response.

 

Limit of Quantitation (LOQ):

The LOQ of amlodipine besylate and theophylline by proposed methods were determined using calibration standards. LOQ were calculated as 10 σ/S respectively. Where S is the slope of the calibration curve and σ is the standard deviation of response.

 

RESULTS AND DISSCUSSION:

Amlodipine besylateand theophylline showed maximum absorbance at 239 nm and 272 nm by UV spectrophotometric method (Fig 3 and 4). Overlain absorption spectra of amlodipinebesylate and theophylline showing isoabsorptive point at 254 nm (Fig 5). Based on the experimental data the standard calibration curve was plotted (Fig 6 and 7). Linearity range of amlodipine besylate and theophylline is 5-30 μg/ml at respective selected wavelengths. The coefficient of correlation foramlodipine besylate at 239 nm and for theophylline at 272 nm is 0.999 and 0.997 respectively. Both drugs show good regression values at their respective wavelengths (Table 1 and 2).

 

Precision was determined by studying the repeatability and intermediate precision. Repeatability result indicates the precision under the same operating condition over a short interval of time and interassay precision. The standard deviation, coefficient of variance and standard error was calculated foramlodepinebesylate and theophylline.

 

Intermediate precision study expresses within laboratory variation in different days. In intra and inter day precision study for both the methods % COV are not more than 2.0 % indicates good repeatability and intermediate precision (Table 3 and 4).

 

The LOD values were 0.241 and 0.360μg/ml while LOQ values were 00.7857 and1.094μg/ml for amlodipine besylate and theophylline respectively. Low values of LOD and LOQ indicates good sensitivity of proposed methods (Table 5).

 

Figure 3: Spectra of amlodipine besylate in distilled water

 

Figure 4: Spectra of theophylline in distilled water

 

Figure 5: Overlain absorption spectra of amlodipine besylate and theophylline showing isoabsorptive point (254 nm) in distilled water

 

Figure 6: Standard calibration plot of amlodipine besylate in distilled water

 

Figure 7:  Standard calibration plot of theophylline in distilled water

Table 1: Linearity

Amlo dipine besylate (µg/ml)	Absorbance		Theo phylline (µg/ml)	Absorbance
	239 nm	272 nm		239 nm	272 nm
5	0.2356	0.0637	5	0.4256	0.1535
10	0.4325	0.0799	10	0.7184	0.2157
15	0.6458	0.1332	15	1.0076	0.3210
20	0.8452	0.1524	20	1.3564	0.3520
25	1.0256	0.2035	25	1.6600	0.4748
25	1.2665	0.2097	30	1.9774	0.5121
35	1.1254	0.1932	35	1.8472	0.4872

 

Table 2: Regression Characteristics

Parameters	At 239nm	At 272nm
Beer’s Law Range	5-30 µg/ml	5-30 µg/ml
Regression Equation ( y = mx + c )	y = 0.041x + 0.017	y = 0.064x + 0.052
Slope (m)	0.041	0.064
Intercept (c)	0.017	0.052
Correlation Coefficient (R2)	0.999	0.997

 

 

Table 3: Intraday Precision studies

Sr.no.	Conc. (µg/ml)	Absorbance difference		SD		% RSD
		Amlodipine besylate	Theophylline	Amlodipine besylate	Theophylline	Amlodipine besylate	Theophylline
1	10 (n = 3)	0.4325 0.4368 0.4299	0.7184 0.7277 0.7215	0.0034	0.004	0.8036	0.5536
2	15 (n = 3)	0.6458 0.6397 0.6421	1.0076 1.0145 1.0006	0.003	0.006	0.4782	0.5955
3	20 ( n = 3)	0.8452 0.8414 0.8398	1.3564 1.3511 1.3043	0.0027	0.02	0.3206	1.4958

Limit: % RSD for area NMT 2.0 %

 

Table 4: Interday precision studies

Sr.no.	Conc. (µg/ml)	Absorbance difference		SD		% RSD
		Amlodipine besylate	Theophylline	Amlodipine besylate	Theophylline	Amlodipine besylate	Theophylline
1	10	0.4196 0.4220 0.4154	0.7223 0.7294 0.7364	0.003	0.0070	0.7161	0.9685
2	15	0.6360 0.6171 0.6198	1.1641 1.1633 1.1595	0.0024	0.0024	1.60	0.2064
3	20	0.8454 0.8399 0.8391	1.4129 1.3612 1.3951	0.026	0.026	0.4040	1.8709

Limit: % RSD for area NMT 2.0 %

 

Table 5: Limit of Detection and Limit of Quantification

Parameter	Amlodipine besylate		Theophylline
	At 239 nm	At 272 nm	At 272 nm	At 239 nm
LOD (µg/ml)	0.241	1.65	0.360	1.44
LOQ (µg/ml)	0.7857	0.731	1.093	1.073

 

CONCLUSION:

The developed method is sensitive, precise, cost effective, fast and reproducible for simultaneous estimation of amlodipine besylate and theophylline in bulk mixture.

 

REFERENCES:

1.       Tripathi K. D. Essentials of Medical Pharmacology, Jaypee Publisher, New Delhi. 6^thEdn: 2008:213,539.

2.       K. Nanda Gopal, K. Gangaiah, M. Anitha and J. U. M Rao, “UV Visible Spectrometric Estimation of amlodipine in P’ ceutical Dosage form” International Journal of Research and Reviews in Pharmacy and Applied Sciences.5(1);2015:1251-1556.

3.       Sawant Ramesh L., Bharat Anjali V., Tanpure Kallyani D., and Jadhav Kallyani A., “Spectroscopic methods for the Simultaneous estimation of theophylline and Furosamide” Scholars research Library.7(2);2015:199-205.

4.       Indian Pharmacopoeia, Government of India Ministry of Health and Family Welfare, Published by the Indian Pharmacopoeia Commission, GAZIABAD, Volume ІІІ, 2010:1092-1093.

5.       www.drugbank.com (https://www.drugbank.ca/drugs/DB00381)

6.       Indian Pharmacopoeia, Government of India Ministry of Health and Family Welfare, Published by the Indian Pharmacopoeia Commission, GAZIABAD, Volume ІІІ, 2010:1092-1093.

7.        www.drugbank.com (https://www.drugbank.ca/drugs/DB00277)

8.       Sawant R, Joshi R, “Handbook of experimental ultraviolet absorption spectroscopy” career publication, first edition 2016:23, 27.

 

 

 

 

 

Received on 06.04.2017       Accepted on 16.05.2017     

© Asian Pharma Press All Right Reserved