Development and Validation of a RP-HPLC Method for Estimation of Darifenacin Hydrobromide in Bulk and in Tablet Dosage Form.

Kathirvel S.¹, Satyanarayana S.V.² and Devalarao G.³*

¹Department of Pharmaceutical analysis, Hindu College of Pharmacy, Amaravathi road, Guntur, A.P, India.

²Department of Chemical Engineering, JNTU College of Engineering, Anantapur, A.P, India.

³Department of Pharmaceutical Analysis, K.V.S.R Siddhartha College of Pharmaceutical sciences, Vijayawada, A.P, India.

*Corresponding Author E-mail: devalarao2007@gmail.com

ABSTRACT:

The present work describes a simple, precise and accurate HPLC method for estimation of darifenacin hydrobomide in bulk and in tablet dosage form. The separation was achieved by using Waters Sunfire C ₁₈ (4.6 X 250 mm) 5µm particle size column. The mobile phase consisted of 0.02M potassium dihydrogen phosphate buffer pH 7 adjusted with triethylamine: acetonitrile: methanol in the ratio of 40:30:30(v/v/v). Detection was carried out at 280 nm. The retention time of darifenacin hydrobromide was found to be 4.2 min respectively. The Limit of detection and limit of Quantification was found to be 2.112 µg/mL and 6.400 µg/mL respectively. The accuracy and reliability of the proposed method was ascertained by evaluating various validation parameters like linearity (10-100 µg/mL), precision, accuracy and specificity according to ICH guidelines. The proposed method provides an accurate and precise quality control tool for routine analysis of darifenacin hydrobromide in bulk and in tablet dosage form.

KEYWORDS: Darifenacin hydrobromide, HPLC, method development, validation.

INTRODUCTION:

Darifenacin hydrobromide (Fig 1) is chemically (S)-2-{1-[2-(2, 3 –dihydrobenzofuran-5-yl) ethyl]-3-pyrrolidinyl}-2, 2-diphenylacetamidehydrobromide, indicated for the treatment of overactive bladder ⁽¹⁾. Overactive bladder is used to describe a collection of urinary symptoms composed of urgency, with or without urge incontinence, usually with frequency and nocturia, in the absence of proven infection or other obvious pathology. As a selective antagonist of the M₃ receptor (the major subtype that modulates urinary bladder muscle contraction), darifenacin has a clinically significant effect on bladder function and control ⁽²⁾. In the literature, the analytical techniques for determination of darifenacin hydrobromide are scarce. In view of the above fact it is felt necessary to develop a liquid chromatographic (LC) procedure which would serve as a rapid and reliable method for the determination of darifenacin hydrobromide in bulk and in tablet dosage form.

EXPERIMENTAL:

Equipment:

The HPLC system consisted of a Waters 600 controller pump; Waters 717 Auto sampler and Waters 486 tunable absorbance detector and temperature control device with operating software Millenium 32 were used during the study. A Sunfire C₁₈ column (250x4.6 mm, 5 µm particle size) was used as stationary phase.

Chemicals and Reagents:

Darifenacin hydrobromide working standard was obtained from Aurabindo Pharmaceuticals, Hyderabad, India. Darifenacin hydrobromide tablets of two different manufacturers were purchased from the local market. The HPLC grade solvents were used of E-Merck (India) Ltd., Mumbai, and HPLC grade water was prepared using Millipore purification system (Millipore, Molshein, France, Model Elix-10)

Chromatographic Conditions:

Column : Waters Sunfire C ₁₈ (4.6 X 250 mm) 5µm particle size

Mobile phase: 0.02M potassium dihydrogen phosphate buffer pH 7 adjusted with triethylamine: acetonitrile: methanol in the ratio of 40:30:30(v/v/v).

Flow rate: 1.0mL /min

Injection volume : 20 mcL

Detection wavelength : 280

Run time : 7 min

Fig: 1 Structure of Darifenacin hydrobromide

Standard preparation:

About 10 mg of Darifenacin hydrobromide standard was weighed accurately and transferred to 100ml volumetric flask. The volume was made up to the mark with the mobile phase to obtain a concentration of 100µg/mL. Further dilutions were made to obtain the concentration in the range of 10-100 µg/mL of darifenacin hydrobromide. 20 mcL of each of the solution was injected.

Sample preparation:

Twenty tablets were weighed and pulverized with the help of a pestle-mortar. The powder equivalent to 10 mg of darifenacin hydrobromide was accurately weighed and transferred in to a 100 ml volumetric flask. The volume was made up to mark with the mobile phase to obtain a concentration of 100 µg/mL. Further dilutions were made to obtain a concentration of 40 µg/mL and filtered through 0.45 µm membrane filter.

RESULTS AND DISCUSSION:

The system suitability was checked by injecting 20 µL of standard solution and found the results within the range. The relative standard deviation on five replicate injections was found to be 0.6%. The tailing factor and the number of theoretical plates were found to be 1.25 and 5278 respectively

Method validation:

The described method has been validated ³ for the assay of darifenacin hydrobromide using following parameters.

Linearity:

Linearity was studied by preparing standard solution at different concentration levels. The linearity range was found to be 10-100 µg/mL. The standard calibration curve was generated using regression analysis with Microsoft excel. The assay was judged to be linear as the correlation coefficient was geater than 0.995 by the least- square method as shown in Table 1.

Table 1. Validation parameter of the proposed method

Parameter

Darifenacin hydrobromide

Linearity range (µg/mL)

Correlation coefficient

Regression equation (y = mx + c)

Slope (m)

Intercept (c)

Limit of detection (LOD) (µg/mL)

Limit of quantitation (LOQ) (µg/mL)

10-100 µg/mL

0.9988

y = 8.7831x + 35.849

8.7831x

35.849

2.112

6.400

Precision:

Precision was studied to find out intra and inter day variations in the test methods of darifenacin hydrobromide in the concentration range of 40-60 µg/mL for three times on the same day and different day. Precision was determined by analyzing corresponding standard daily for a period of three days. The inter-day and intra-day precision obtained was %RSD (<2) indicated that the proposed method is quite precise and reproducible as shown in Table III.

Accuracy:

Recovery studies of the drug were carried out for the accuracy parameters at three different concentration levels i.e. multiple level recovery studies. A known amount of darifenacin hydrobromide was added in to pre-analysed sample and subjected to the proposed HPLC method. Percentage recovery was found to be within the limits as listed in Table II.

Table 2. Recovery studies of darifenacin hydrobromide in Tablet

Label claim (mg / tablet)	Total amount added (mg)	Amount recovered* (mg) ± SD	% Recovery ± SD
15	12	26.94 ± 0.06	99.77 ± 0.06
15	15	29.91 ± 0.01	99.70 ±0.02
15	18	32.85 ± 0.12	99.54 ± 0.13

*Each value is a mean ± standard deviation of six determinations.

Specificity:

Specificity was studied for the examination of the presence of interfering components. Darifenacin hydrobromide standard solution of 40 µg/mL was injected and none of the impurities were interfering in its assay. The retention time obtained by HPLC for darifenacin hydrobromide is about 4.2 min as shown in Fig.2.

Fig: 2 A typical chromatogram of Darifenacin hydrobromide showing retention time at 4.2 min

Table 3. Intra-day and Inter-day precision of darifenacin hydrobromide (n=3)

Nominal Concentration (µg / mL)	Day 1			Day 2			Day 3
Nominal Concentration (µg / mL)	Mean	SD	%RSD	Mean	SD	%RSD	Mean	SD	%RSD
40	40.86	0.11	1.14	39.95	0.15	0.81	39.89	0.14	0.83
50	49.91	0.84	1.10	50.03	0.64	0.57	49.98	0.58	0.97
60	59.45	0.67	0.55	59.69	0.77	0.54	59.62	0.67	0.55

Robustness:

This study was done by small deliberate changes in the chromatographic conditions at different levels and retention time is noted for darifenacin hydrobromide. The factors that were selected were small changes in flow rate, detection wavelength range and mobile phase composition. The method exhibited good robustness because the changes made in chromatographic conditions did not influence the analytical results. Darifenacin hydrobromide solution was found to be stable during the procedure.

Limit of Detection and Limit of Quantitation:

Limit of Detection and Limit of Quantitation were calculated by the method which was based on the standard deviation (SD) of the response (S) of the calibration curve at levels approximately the LOD and LOQ, LOD = 2.112 µg/mL and LOQ = 6.400 µg/mL respectively.

CONCLUSION:

A simple, accurate, fast and precise isocratic reversed phase high performance liquid chromatographic method has been developed for the determination of darifenacin hydrobromide in bulk and in tablet dosage form. The developed method was found to be simple and have short run time which makes the method rapid. The results of the study indicated that the proposed HPLC method is simple, precise, accurate and less time consuming.

ACKNOWLEDGEMENTS:

The authors are grateful to Hindu College of Pharmacy, Guntur and Management of Siddhartha College of Pharmaceutical sciences, Vijayawada, for their continuous support and encouragement and for providing the necessary facilities

REFERENCES:

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2. Ohtake, N, Mase, T, J.Med.Chem. 43, 2000, p. 5017-5029

3. Khan RH, Talegaonkar S, Singh RM, Mathur SC, Shiv R, Singh G N. A simple HPLC method for quantitation of Ripaglinide in tablet dosage form. Indian Drugs. 44 ; 2007: 428-433.

Received on 23.03.2011 Modified on 04.04.2011

Asian J. Research Chem. 4(6): June, 2011; Page 946-948