INTRODUCTION:

Darifenacin is chemically known as 2-[(3S)-1-[2-(2,3-dihydro-1-benzofuran-5-yl)ethyl]pyrrolidin-3-yl]-2,2-diphenylacetamide with molecular formula of C₂₈H₃₀N₂O₂(Fig.-1). It is a muscular antagonist. Darifenacin works by blocking the M₃ muscarinic acetylcholine receptor^[1], which is primarily responsible for bladder muscle contractions and is used to treat symptoms of overactive bladder, such as frequent or urgent urination, and incontinence (urine leakage). It was approved by FDA on December 23 2004. Formulation used was Vesigard 7.5mg tablets (Cipla Navi Mumbai).

Literature survey revealed several analytical methods for the determination of darifenacin, which employ techniques such as UV^{[2] [3]}, visible spectrophotometry^[4], LC assay method^[5] and HPTLC^[6]. Several pharmacological reviews in human plasma^[7-9] were also revealed. According to literature survey, it reveals that, Darifenacin is not official in any of the pharmacopoeia like IP, BP, USP and European pharmacopoeia.

The HPLC method is widely employed in quality control assessment of drugs because of their sensitivity, repeatability and specificity. Hence an attempt has been made to develop a simple, efficient and selective method for the determination of darifenacin in pharmaceutical dosage forms. In this study HPLC instrumentation with UV detection, which is readily available in most analytical and pharmaceutical laboratories, was used. The method requires no extraction or derivatization steps reducing the total analysis run time to less than 10 min. The method developed fulfilled the requirements of analytical quality necessary to be applied to the content uniformity tests indicated by Indian Pharmacopoeia and hence can be successfully applied for routine quantitative estimation of darifenacin in pure and pharmaceutical dosage forms.

Fig 1 chemical structure of darifenacin

MATERIALS AND METHODS:

Chemicals

Darifenacin pure drug was obtained as a gift sample from Pharma Train Laboratories. Tablets of Darifenacin (7.5mg) were purchased from local market for analysis. HPLC grade Methanol and Acetonitrile and Analytical grade potassium dihydrogen phosphate purchased from Merck (Merck Limited, Shivasagar estate, Worli, Mumbai – 40018, India) was used. Milli-Q water (prepared from Milli-Q Direct) was used in all experiments.

Instrumentation and Analytical conditions

Chromatography was performed using Water series (2487) with Empower Software with autosampler and DAD (or) UV detector. Symmetry BDS C₁₈ (4.6 x 100mm, 3.5 mm) analytical column was used. The mobile phase consists of Phosphate buffer adjusted to PH 7 using sodium hydroxide solution and Acetonitrile in the ratio of 35:65%v/v. The flow rate was maintained at 0.8 ml/min with injection volume of 20μl and the absorbance was measured at 286 nm. The column and the HPLC system were kept in ambient temperature.

Preparation of standard and quality control samples

Preparation of Phosphate buffer

7.0 grams of Potassium dihydrogen phosphate was weighed and transferred into a 1000ml beaker, dissolved and diluted to get 1000ml with HPLC water. Adjusted the pH to 7 with Sodium Hydroxide solution.

Preparation of mobile phase

A mixture of phosphate buffer 350ml (35%) and 650 ml of Acetonitrile HPLC (65%) was mixed and degassed in ultrasonic water bath for 5 minutes and filtered through 0.45 µ filter under vacuum filtration.

Preparation of standard stock solution

10mg of Darifenacin pure was accurately weighed and transferred into a 10ml volumetric flask. About 7 ml of mobile phase was added and sonicated to dissolve it completely and the volume was made up to the mark with the same mobile phase (1mg/ml) (1000µg/ml).

Preparation of Sample Solution (Analysis of formulation)

Five tablets of Darifenacin were weighed and the average weight was calculated. Tablet powder equivalent to 10 mg of Darifenacin was transferred into a 10 ml volumetric flask. About 7 ml of mobile phase was added and sonicated to dissolve it completely and made upto the volume with the same (1mg/ml) (1000µg/ml) and filtered through 0.45µm filter.

Method validation

Quantification of formulation (Assay)

From the prepared sample stock solution 0.7 ml was pipetted out and transferred into six separate 10ml volumetric flasks and made upto the volume with mobile phase (70μg/ml). 20µl of each solution was injected and the chromatogram was recorded. The peak area was determined and the procedure was repeated for six times. The standard solution was prepared in the same manner. By using the following formula the percentage purity of Darifenacin was calculated.

AT WS DT P Avg. Wt

-------------- x ----------x --------- x ----------x------------------ X 100

AS DS WT 100 Label Claim

Where:

AT = Peak Area of Darifenacin obtained with test preparation

AS = Peak Area of Darifenacin obtained with standard preparation

WS = Weight of working standard taken in mg

WT = Weight of sample taken in mg

DS = Dilution of Standard solution

DT = Dilution of sample solution

P = Percentage purity of working standard

Calibration graph (or) Linearity

Aliquots of standard stock solution of Darifenacin (0.5 - 0.9 ml of 1000µg/ml) was transferred into 10ml standard flask and made upto the volume with mobile phase. 20µl of the standard solutions were injected and the chromatograms were recorded. The calibration was done by external standard calibration method. Linearity was observed between 50 -90µg/ml. Calibration graph was obtained by plotting peak area versus concentration.

Limit of detection and Limit of quantification

The limit of detection (LOD) and the limit of quantification (LOQ) for the procedure were performed on samples containing very low concentration of analyte. Both were calculated based on the signal to noise ratio. LOD was expressed by establishing the minimum level at which the analyte can be reliably detected. LOQ was considered as the lowest concentration of analyte in standards that can be measured with acceptable accuracy and precision.

Accuracy

Accuracy of the method was checked by recovery studies of addition of standard drug solution to the prepared sample solution at different concentration levels (50%, 100% and 150%) within the range of linearity of the drug. The results of analysis of recovery studies were obtained by method validation by statistical evaluation.

Precision

The precision of the developed method was determined in terms of repeatability and intermediate precision and reported as %RSD for a statistically significant number of replicate measurements. Stock solution of 70µg/ml of the pure drug was prepared. Five replicate injections were given and the %RSD values for both precision and intermediate precision were calculated.

Robustness

The robustness of the method was evaluated by analysing the system suitability standards and evaluating system suitability parameter data after varying individually, the HPLC pump flow rate and organic composition of the mobile phase.

RESULTS AND DISCUSSION:

Method development and optimisation

The optimisation was done by changing the mobile phase, mobile phase ratio, flow rate and column. Eight trials were performed. The drug was found to be soluble in methanol. The mobile phase should be selected in such a way that it dissolves the analyte upto the concentration suitable for detection. Hence first five trials were performed using phosphate buffer and methanol by changing the mobile phase ratio, flow rate and column. But the peaks obtained had a broad shape together with tailing and bumping of peaks. The next three trials were performed by using phosphate buffer and Acetonitrile by changing the mobile phase ratio and flow rate. The peaks obtained had good shape, bumping was eliminated and tailing reduced. With phosphate buffer (pH 7) and Acetonitrile in the ratio 35:65% v/v, flow rate of 0.8ml/min and symmetry c₁₈BDS column, showed better separation, sharp peak without tailing. The chromatogram thus obtained was selected as the optimised chromatogram and the conditions were selected as the optimised chromatographic conditions. The optimised chromatogram was shown in figure 2. The various trials performed and the optimised chromatographic conditions were shown in table 1.The retention time in the optimised chromatogram was found to be 3.359min.

Fig 2: optimised Chromatogram of Darifenacin

Method validation

When a method has been optimised it must be validated before practical use. By following the ICH guidelines ^[10]for analytical method validation, Q2 (R1), the validation characteristics were addressed.

System suitability parameters

The system suitability parameters like tailing factor, number of theoretical plates, %RSD, HETP and capacity factor were calculated and these values were compared with the standard limit as per USP^[11] .The result was shown in Table 2

Table 1 Method development and optimisation

Sr. No	Mobile phase	Column	Flow rate
Trial 1	Buffer 7: methanol (35:65)	C8	0.6ml/min
Trial 2	Buffer 7: methanol (30:70)	C8	0.6ml/min
Trial 3	Buffer 7: methanol (30:70)	C8	0.8ml/min
Trial 4	Buffer 7: methanol (30:70)	C8	0.7ml/min
Trial 5	Buffer 7: methanol (35:65)	C18	0.7ml/min
Trial 6	Buffer 7: Acetonitrile (30:70)	C18	0.7ml/min
Trial 7	Buffer 7: Acetonitrile (30:70)	C18	0.8ml/min
Trial 8	Buffer 7: Acetonitrile (40:60)	C18	0.8ml/min
Optimised chromate gram	Buffer 7: Acetonitrile (35:65)	C18	0.8ml/min

Table 2

System suitability parameters for darifenacin

Sr. No	System suitability Parameters	Darifenacin report	USP limit
1	Tailing factor	1.59	Less than 2
2	Plate count	2179.7	Greater than 2000
3	HETP	0.068	-
4	Capacity factor	2.359	Greater than 2
5	% RSD	0.13	Less than 2

Calibration curve (Linearity)

Darifenacin was found to obey the Beers law in the concentration range of 50-90µg/ml. The calibration curve was plotted using concentration against peak area. The correlation coefficient was found to be 0.9992 which indicates that darifenacin has good linearity. The calibration curve was shown in figure 3.The regression analysis data was given in table 3.

Fig 3: calibration curve of darifenacin

Table 3 Regression analysis of Darifenacin

Parameter	Darifenacin results
λmax	286nm
Concentration range	50-90µg/ml
Correlation coefficient	0.9992
Slope	7919.1
Intercept	-44020
LOD	0.026g/ml
LOQ	0.8g/ml

LOD and LOQ

The limit of detection and the limit of quantification were determined based on the signal to noise ratio. The limit of detection was found to be 0.026µg/ml and the limit of quantification was found to be 0.8µg/ml. the results were shown in table 3

Quantification of formulation (Assay)

The tablet formulation of Darifenacin was selected for analysis and the percentage purity was found to be 100.8% .The procedure was repeated for six times to validate the method. The method was validated according to ICH guidelines. The %RSD^[12,13] was found to be less than 2% which indicates that the method had good precision.

Accuracy

Accuracy of the method was confirmed by the recovery studies. A known quantity of the raw material was added to the previously analysed test solution and the amount of drug recovered was calculated. The %recovery was found to be 101.5%. the procedure was repeated for three times. The %RSD values were found to be 0.23%. The low %RSD value indicates that there is no interference due to excipients used in the formulation. The analysis of results were shown in table 4

Precision

Precision study was done with darifenacin standard. 70µg/ml solution was prepared and injected five times and the areas of five replicate injections were recorded in HPLC. The %RSD for the area of five replicate injections was found to be 0.13 which was found to be within limits. The low %RSD values reveals that the proposed method was precise. It shows that the drug was having good precision and it was shown in table 5.

Intermediate precision

Intermediate precision study was done with darifenacin standard. 70µg/ml solution of darifenacin was prepared and injected five times and the areas of five injections were recorded in HPLC. The %RSD was found to be 0.67% which was found to be within the limits. It shows that the intermediate precision was within the specified limit which was shown in table 5.

Robustness

Robustness was performed by changing the flow rate and by changing the organic composition of the mobile phase. It shows that there is no change in the values even after

making deliberate change in the analytical procedure. The data are given in table 6.

Table 5 Precision and Intermediate precision

Injection	Precision	Intermediate precision
Injection-1	476526	491013
Injection-2	477003	491517
Injection-3	477028	491577
Injection-4	478139	491612
Injection-5	77394	498790
Average	477218	492902

Table 6 Robustness

Parameters		USP plate count	USP tailing
Variation in flow rate	0.7ml/min	2262.2	1.8
Variation in flow rate	0.9ml/min	2109.6	1.7
Change in organic Composition of mobile phase	10% less	2247.4	1.8
Change in organic Composition of mobile phase	10% more	2170.7	1.7

CONCLUSION

The proposed RP-HPLC method is simple, reliable and selective providing satisfactory accuracy and precision with lower limits of detection and quantification. The recovery achieved was also good. Moreover the shorter duration of analysis for darifenacin make the reported method suitable for routine quantitative analysis in pharmaceutical dosage forms.

REFERENCES:

1. Newgreen DT and Naylor AM. Characterisation of functional muscarinic receptors in human bladder. British Journal of Pharmacology. 1996: 45.

2. Sridharan D, Umarani, Thenmozhi A, Pavan Kumar L and Aswani Dutt Chintalapati. Development and Validation of UV Spectrophotometric Method of Darifenacin Hydrobromide in Bulk and Tablet Dosage Form. Asian Journal of pharmaceutical analysis 1(3); 2011: 43-45.

3. Kathirvel S, Satyanarayana S.V and Devalarao. A Validated Method for Development of Darifenacin Hydrobromide as Active Pharmaceutical Ingredient and Tablet Dosage form by UV- Spectroscopy. Research Journal of Pharmacy and Technology, 4(7), 2011: 1115-1117.

4. Sai Praveen P, Jagathi V, Devala Rao G and Sudhakar Saibabu. Visible spectrophotometric methods for the determination of darifenacin. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 1(2); 2010: 254.

5. Meneghini LZ, Junqueira C, Andrade AS and Salazar FR. Chemometric Evaluation of Darifenacin Hydrobromide Using a Stability-Indicating Reversed-Phase LC Method. Journal of Liquid Chromatography and Related Technologies 34(18); 2011: 2169-2184.

6. Sathish NK Chethan IA, Pradeep V Khandelwal and Ashwinee. Development and validation of rapid HPTLC method for determination of Darifenacin Hydrobromide in bulk drug and pharmaceutical dosage form. Pelagia Research Library, Der Chemica Sinica, 2 (3); 2011: 57-65.

7. Kay GG and Ebinger U. Preserving cognitive function for patients with overactive bladder: evidence for a differential effect with darifenacin. International Journal of Clinical Practice, 62(11); 2008: 1792-1802.

8. Thomas Kerbusch, Peter Milligan and Mats O. Karlsso. Assessment of the relative in vivo potency of the hydroxylated metabolite of darifenacin in its ability to decrease salivary flow using pooled population pharmacokinetic–pharmacodynamic data. British Journal of Clinical Pharmacology, 21; 2003: 170–180.

9. Linda Cardozo and Andrea Dixon. Increased Warning Time With Darifenacin, A New Concept in the Management of Urinary Urgency. The Journal of Urology, 173(4); 2005: 1214-1218.

10. Anonymous. ICH Harmonised Tripartite Guidelines, Text on Validation of Analytical procedures: Text and Methodology Q2 (R1). 2005 Geneva; 1-8.

11. Anonymous. United States Pharmacopoeia, National Formulary. The United States Pharmacopoeial Convention: MD; 21; 2003:1223.

12. Gupta S.P. Statistical methods. Sultan Chand and Sons, New Delhi. 1991: E10.1 – 10.61

13. Beckett AH and Stenlake J B. Practical Pharmaceutical Chemistry, Part-2, 4th edn; CBS Publishers and Distributors, New Delhi. 2007: 85, 86, 92

Received on 24.02.2012 Modified on 12.03.2012

Research J. Pharm. and Tech. 5(4): April 2012; Page 518-522

% conc.	Area	Mean area	Amount Added(mg)	Amount Found(mg)	%recovery	Mean recovery	Standard Deviation	%RSD
50%	255218	255070	5.17	5.27	367.4701	101.5%	0.67451	0.23%
	254652
	255341
100%	491761	492122	10.0	10.1	283.303
	492453
	492152
150%	736523	736384	15.1	15.2	202.258