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DENSITOMETRIC EVALUATION OFSTABILITY – INDICATING HPTLC METHODFOR THE ANALYSIS OF DARIFENACINHYDROBROMIDE IN BULK AND IN TABLETDOSAGE FORMS. Kathirvel a , S. V. Satyanarayana b & G. Devalarao ca Department of Pharmaceutical Analysis, Hindu College ofPharmacy, Guntur, Indiab Department of Chemical Engineering, JNTU College of Engineering,Anantapur, Indiac Department of Pharmaceutical Analysis, K.V.S.R Siddhartha Collegeof Pharmaceutical sciences, Vijayawada, India

Available online: 30 Jan 2012

To cite this article: S. Kathirvel, S. V. Satyanarayana & G. Devalarao (2012): DENSITOMETRICEVALUATION OF STABILITY – INDICATING HPTLC METHOD FOR THE ANALYSIS OF DARIFENACINHYDROBROMIDE IN BULK AND IN TABLET DOSAGE FORM, Journal of Liquid Chromatography & RelatedTechnologies, 35:2, 280-293

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DENSITOMETRIC EVALUATION OF STABILITY – INDICATINGHPTLC METHOD FOR THE ANALYSIS OF DARIFENACINHYDROBROMIDE IN BULK AND IN TABLET DOSAGE FORM

S. Kathirvel,1 S. V. Satyanarayana,2 and G. Devalarao3

1Department of Pharmaceutical Analysis, Hindu College of Pharmacy, Guntur, India2Department of Chemical Engineering, JNTU College of Engineering, Anantapur, India3Department of Pharmaceutical Analysis, K.V.S.R Siddhartha College of PharmaceuticalSciences, Vijayawada, India

& A simple and sensitive thin-layer chromatographic method has been established for analysis ofdarifenacin hydrobromide in pharmaceutical dosage form. Chromatography on silica gel 60 F254plates using toluene:acetone:methanol (6:3:3 v=v=v) as the mobile phase furnished compact spotsat Rf 0.34� 0.02. Densitometric analysis was performed at 286 nm. To show the specificity of themethod, darifenacin hydrobromide was subjected to acid, base, neutral hydrolysis, oxidation,photolysis, and thermal decomposition. The drug underwent degradation only under oxidativecondition; also the degraded product was well separated from pure drug with significantly differentRf value. Linear regression analysis revealed a good linear relationship between peak area andamount of darifenacin hydrobromide in the range of 50–450 ng=spot. The minimum amount ofdarifenacin hydrobromide that could be authentically detected and quantified was 30.36 and90.79 ng=spot, respectively. The method was validated, in accordance with ICH guidelines forprecision, accuracy, and robustness. As the method could effectively separate the drug from itsdegradation products, it can be regarded as stability indicating.

Keywords darifenacin hydrobromide, degradation, ICH guidelines, specificity,stability-indicating, validation

INTRODUCTION

Darifenacin hydrobromide (Figure 1) is chemically (S)-2-f1-[2-(2,3–dihydrobenzofuran-5-yl)ethyl]-3-pyrrolidinylg-2,2-diphenylacetamide hydro-bromide and is utilized for the treatment of overactive bladder.[1] Overac-tive bladder is used to describe a collection of urinary symptoms composedof urgency, with or without urge incontinence, usually with frequency and

Address correspondence to G. Devalarao, Department of Pharmaceutical Analysis, K.V.S.RSiddhartha College of Pharmaceutical Sciences, Vijayawada, A.P., India. E-mail: [email protected]

Journal of Liquid Chromatography & Related Technologies, 35:280–293, 2012Copyright # Taylor & Francis Group, LLCISSN: 1082-6076 print/1520-572X onlineDOI: 10.1080/10826076.2011.597076

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nocturia, in the absence of proven infection or other obvious pathology. Asa selective antagonist of the M3 receptor (the major subtype that modulatesurinary bladder muscle contraction), darifenacin has a clinically significanteffect on bladder function and control.[2] In the literature, the analyticaltechniques for determination of darifenacin hydrobromide are scarce. Vis-ible spectrophotometric method has been reported for the determinationof darifenacin in formulation.[3] Vishnu Murthy et al. has published anHPLC method for darifenacin and its enantiomers.[4] However, no HPTLCmethod been reported so far for either the determination of darifenacinhydrobromide in pharmaceutical dosage form or its stability studies. In viewof the aforementioned fact, it is essential to develop a stability- indicatingHPTLC method that would serve as a rapid and reliable method for thedetermination of darifenacin hydrobromide in bulk and in tablet dosageform.

HPTLC has a potential that meets the demands of a routine analyticaltechnique due to its advantages of low operating cost, high sample through-put, and need for minimum sample clean up. The major advantage ofHPTLC is that several samples can be run simultaneously using a smallquantity of mobile phase, unlike HPLC, thus lowering the analytical runtimes and cost per analysis. In pharmaceutical laboratories, there is alwaysa need for faster, simpler, less expensive, and better performing analyticalmethods. Further, TLC and HPTLC, in an instrumentalized mode thatutilizes scanning densitometry, have been included as general methods inEuropean Pharmacopoeia, permitting the use of planar chromatographyfor quantification at different stages of pharmaceutical research, develop-ment, and production.[5] Furthermore, one of the main advantages ofplanar chromatography is its potential ability to facilitate separations, whichcan be successfully utilized to evaluate different drug molecules, theirimpurities, and metabolites. Generally, the separations are discrete and veryoften complementary to other classified techniques, such as HPLC. There-fore, HPTLC can be a viable alternative for impurity profiling and charac-terization of newer drugs and the unknown compounds. Hence, theobjective of the present study is to develop and validate an HPTLC methodfor the determination of darifenacin hydrobromide in bulk drug and itsdosage form. The proposed method was validated as per ICH guide-lines.[6,7] The optimization of the method separation, stability indicating

FIGURE 1 Structure of darifenacin hydrobromide.

HPTLC Method for Darifenacin Hydrobromide 281

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property, and quantification of darifenacin hydrobromide in bulk drug andits dosage form are reported in the following sections.

EXPERIMENTAL

Materials and Reagents

Darifenacin hydrobromide was obtained from Aurabindo Pharmaceuti-cals Ltd. (Hyderabad, India). It was used without further purification. Itspharmaceutical preparation, Vesigard, was purchased from a local drugstore; it contained 15mg active material. The other chemicals and reagentsused were of AR grade and procured from S.D. Fine-Chem (New Delhi,India).

HPTLC Instrumentation

Chromatography was performed on 10 cm� 10 cm aluminum foil platesprecoated with 0.2-mm layers of silica gel 60 F254 (E. Merck, Germany).Before use, the plates were prewashed by development with methanol thendried in a current of dry air and activated at 60�C for 5min. Samples wereapplied as bands 6mm wide, 15mm apart, by use of a Camag (Switzerland)Linomat 5 equipped with 100 microliter syringe. A constant application rateof 150 nL s�1 was used and the space between two bands was 10mm. The slitdimension was kept at 5� 0.45mm and a scanning speed of 20mm=sec wasemployed. The monochromator bandwidth was set at 20 nm; each track wasscanned three times and baseline correction was used. Toluene:acetone:methanol (6:3:3 v=v=v) was used as the mobile phase. Linear ascendingdevelopment was performed in a twin-trough glass chamber previouslysaturated with mobile phase vapor for 30min at room temperature (RT,25� 2�C) and relative humidity 60� 5%. The development distance wasapproximately 80mm. After development, the plates were dried in currentof air by use of an air dryer in wooden chamber with adequate ventilation.Densitometry scanning was performed with a Camag TLC scanner III in theabsorbance reflectance mode at 286 nm and operated by WINCATS soft-ware (V 1.4.3 camag) resident in the system. The source of radiation utilizedwas a deuterium lamp emitting a continuous UV spectrum between200–400nm and concentrations of the compound chromatographed weredetermined from the intensity of diffusely reflected light.

Calibration Plots of Darifenacin Hydrobromide

A stock solution containing 100 mg=mL of darifenacin hydrobromidewas prepared by dissolving an accurately weighed 10mg portion of the drug

282 S. Kathirvel et al.

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in methanol in a100-mL volumetric flask. Different volumes of stock sol-ution (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 mL) were spotted on an HPTLCplate in triplicate to obtain concentrations of 50, 100, 150, 200, 250, 300,350, 400, and 450ng=spot of darifenacin hydrobromide, respectively. Thedata of peak area versus drug concentration were treated by linearleast-squares regression to obtain the calibration graphs.

Method Validation

Validation of the optimized HPTLC method was carried out withrespect to the following parameters.

Precision

The precision of the method was verified by repeatability and inter-mediate precision studies. Repeatability studies were performed by analyz-ing three different concentrations (100, 200, and 300ng=spot) of the drugby six times on the same day. The intermediate precision of the method waschecked by repeating on three different days.

Robustness

The analytical conditions were deliberately changed, by introducing smallchanges in mobile phase composition (�2%), mobile phase volume (�2%),chamber saturation period (�10%), development distance (�10%), timefrom application to development (0, 10, 15, and 20min), and time fromdevelopment to scanning (0, 30, 60, and 90min) were carried out.

Limit of Detection and Limit of Quantification

The method was used to determine the limit of detection (LOD) andlimit of quantification (LOQ). Blank methanol was spotted six times, andthe SD (Sb) of the peak area of the blanks was calculated. The limits weredetermined from the slope (S) of the calibration plot and the SD of theresponse for the blank sample (Sb) by use of the formula:

LOD ¼ 3:3� Sb=S and LOQ ¼ 10� Sb=S

Accuracy

To check the degree of accuracy of the method, recovery studies wereperformed in triplicate by standard addition method at 50, 100, and


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150%. A known amount of standard darifenacin hydrobromide was addedto pre-analyzed samples and was subjected to the proposed HPTLCmethod. Six determinations were performed at each level of recovery.

Specificity

The specificity of the method was determined by comparing the resultsfor the standard drug and the sample. The peak purity of the sample wasassessed by comparing the spectra at peak start, peak apex, and peak endpositions of the spot.

Forced Degradation Studies of Darifenacin Hydrobromide

A stock solution containing 10mg darifenacin hydrobromide in 100mLmethanol (100mg=mL) was used for forced degradation to provide an indi-cation of the stability-indicating ability of the proposed method. Studies ofacid-induced decomposition were performed by exposing the solution ofthe drug to 2N hydrochloric acid by heating the solution under reflux at80�C for 8 hr. Studies of base-induced decomposition were performed in2N sodium hydroxide and the solution was heated under reflux for 8 hrat 80�C. The resulting solutions were applied on a TLC plate in triplicate(4 mL each, i.e., 400 ng=spot). The plate was chromatographed as describedin the previous section. Hydrogen peroxide-induced degradation was stud-ied by refluxing the drug solution to 30% hydrogen peroxide for 4 hr at80�C. The resulting solution was applied to TLC plates such that 400ngper spot was applied to the plate. The photochemical stability of the drugwas also studied by exposing the stock solution to direct sunlight for 24 hron a wooden plank and kept on terrace. The solution (4mL, equivalent to400 ng= spot) was then applied to TLC plates and densitograms wereobtained as described for acid and base induced degradation. Neutralhydrolysis was carried out by accurately weighing 25mg of the drug into25mL HPLC grade water, and the solution was refluxed for 12hrs at50�C. Appropriate aliquot was taken from the aforementioned solutionand diluted with methanol to obtain a final concentration of 100ng=mL.The resultant solution (4 mL) was applied to a TLC plate (400 ng=spot)and the chromatogram was run to evaluate the degradation effect. Simi-larly, dry heat degradation was also carried out by placing the standard drugin solid form in an oven at 100�C for 10hr. Darifenacin hydrobromide inthe amount of 1mg was accurately weighed and separately dissolved inmethanol in a 10-mL volumetric flask. The resultant solution was appliedto the TLC plate in such a way that final concentration achieved was400 ng spot. The densitogram was run in triplicate in all the conditionsto evaluate the degradative effect.


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Analysis of Darifenacin Hydrobromide in Tablet Dosage Form

To determine the darifenacin hydrobromide content of conventionaltablets, (Brand name: Vesigard, label claim: 15mg per tablet), twentytablets were weighed and powdered in a glass mortar. An amount of powderequivalent to 10mg darifenacin hydrobromide was transferred to a 100-mLvolumetric flask, extracted with methanol, sonicated for 30min, anddiluted to volume with the same solvent. The resulting solution was filteredthrough a 0.45-mm filter (Millifilter; Milford, MA; USA). The solution (4mL,400 ng=spot of darifenacin hydrobromide) was applied in triplicate on toan HPTLC plate for quantification using the proposed method. The possi-bility of excipient interference with the analysis was examined.

RESULTS AND DISCUSSION

Optimization of the Mobile Phase

Several solvent mixtures in different ratios were tested to obtain a com-pact band of darifenacin hydrobromide. Toluene:acetone:methanol(6:3:3 v=v=v) was found to give a compact band for darifenacin hydrobro-mide with an Rf value of 0.34� 0.02 (Figure 2). The selected mobile phase

FIGURE 2 A typical densitogram of darifenacin hydrobromide (Rf-0.34� 0.02) at 286 nm usingtoluene:acetone:methanol (6:3:3 v=v=v).


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composition gave a sharp and symmetrical peak (Figure 3), which is clearlyvisible under UV light and the same has been reproduced as a video image(Figure 4) of the developed TLC plate. This mobile phase gave good

FIGURE 3 Densitogram of standard darifenacin hydrobromide at different concentrations from50–450ng=spot). (Color figure available online.)

FIGURE 4 Video image of standard darifenacin hydrobromide. (Color figure available online.)


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resolution for the separation of darifenacin hydrobromide from the degra-dation product and was selected for the proposed stability-indicatingmethod. Thirty min was found to be sufficient for saturation of the devel-opment chamber with the mobile phase vapor in order to obtain separ-ation of the compounds. A 20-mL aliquot of mobile phase was used for a20-min development over a distance of 80mm.

Calibration Plot

The linear regression analysis data for the calibration plots showed agood linear relationship (r2¼ 0.9986� 0.000188) with respect to peak areain the concentration range of 50–450 ng=spot. The mean values of theslope and intercept were 2.01� 0.0041 and 20.77� 2.063, respectively, fordensitometric analysis at 286 nm (Table 1).

Method Validation

PrecisionThe results of the repeatability and intermediate precision experiments

are shown in Table 2. The developed method was found to be precise as theRSD values for repeatability and intermediate precision studies were <2%,respectively, as recommended by ICH guideline.

RobustnessThe low values of RSD obtained after introducing small, deliberate

changes in the mobile phase composition, mobile phase volume, chamber

TABLE 1 Linear Regression Data for the Calibration Curves (n¼ 6)

Parameters TLC Densitometry

Linear range 50–450ng=spotCorrelation coefficient (r)� SD 0.9986� 0.000188Slope� SD 2.01� 0.0041Intercept� SD 20.77� 2.063LOD (ng=spot) 30.36LOQ (ng=spot) 90.79

TABLE 2 Intra and Inter-Day Precision by HPTLC Method (n¼ 6)

Repeatability Intermediate Precision

Amount(ng=spot) Mean Area (AU)� SD RSD (%) Mean Area (AU)� SD RSD (%)

100 224.7� 8.250 1.83 235.2� 6.65 1.41200 424.8� 10.85 1.27 433.6� 13.6 1.56300 605.5� 17.1 1.41 616.7� 16.3 1.32


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saturation time, time from application to development, and time fromdevelopment to scanning in the developed HPTLC method indicated therobustness of the method (Table 3).

LOD and LOQLOD and LOQ were determined by the SD method and were found to

be 30.36 and 90.79ng=spot, respectively. The low values of LOD and LOQindicated the sensitivity of the method is adequate (Table 1).

AccuracyAccuracy of the method was obtained by recovery after spiking with 50,

100, and 150% of additional drug. Recovery of darifenacin hydrobromidein samples was 98.84–100.8% (Table 4).

SpecificityThe Rf value (0.34� 0.02) of the sample and standard was almost ident-

ical, and spectra of the sample and the standard were superimposable.These results indicated the specificity of the method (Figure 5).

Forced Degradation Studies of Darifenacin Hydrobromide

Typical densitograms presented in (Figures 6–10) indicating that theactive drug darifenacin hydrobromide remained stable in acid, basic,

TABLE 3 Robustness of the Method (n¼ 6)

Parameters TLC Densitometry

% Change in mobile phase 0.72� 0.01Chamber saturation time 0.74� 0.02Development distance 0.76� 0.01Time from application to development 0.75� 0.02Time from development to scanning 0.74� 0.01

TABLE 4 Recovery Studies of Darifenacin Hydrobromidea

Excess of Drug Added to the Analyte Amount of Drug Found (ng) Recovery (%) RSD (%)

0 197.6 98.84 0.23300 302.4 100.80 0.20400 398.6 99.66 0.20500 502.3 100.40 1.30

Matrix containing 200 ng of drug.an¼ 6.


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FIGURE 5 In situUV spectra of darifenacin hydrobromide standard and sample. (Color figure availableonline.)

FIGURE 6 HPTLC densitogram of acid degradation (400ng=spot using 2N HCl at 80�C for 8hr).Darifenacin Rf- 0.34 indicating that the drug is not degraded.


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FIGURE 7 HPTLC densitogram of base degradation (400ng=spot using 2N NaOH at 80�C for 8hr).Darifenacin Rf-0.34 indicating that the drug is not degraded.

FIGURE 8 HPTLC densitogram of the neutral degradation of darifenacin hydrobromide (400ng=spotusing HPLC grade water and reflux at 50�C for 12hr).


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FIGURE 9 HPTLC densitogram of the dry-heat degradation (400ng=spot in oven at 100�C for 10 hr).

FIGURE 10 HPTLC densitogram of photo degradation sample (400ng=spot in sunlight for 48hr).


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neutral, thermal and photolytic conditions, which was evident from theabsence of additional peaks. However the drug undergoes degradation inoxidative condition (Figure 11) as it is evident from the additional peakappeared at Rf 0.31, with a proportionate reduction in the area of the maindrug at Rf 0.34. The details of forced degradation studies of darifenacinhydrobromide are incorporated in Table 5.

Analysis of Darifencain Hydrobromide in Tablet Dosage Form

A single spot at Rf-0.34 was observed in the densitogram of the drugsamples extracted from tablets. There was no interference from the excipi-ents commonly present in the tablets. The drug content was found to be99.45%� 0.31 with a RSD of 0.73% for analysis in triplicate. It may, there-fore, be inferred that darifenacin hydrobromide had no quantifiableadditional impurities in the marketed formulation analyzed by use of thismethod. The successful performance of the method was indicative of itssuitability for routine analysis of darifenacin hydrobromide in pharmaceu-tical dosage forms.

FIGURE 11 HPTLC densitogram of oxidative degradation (400ng=spot using 30% H2O2 at 80�C for4 hr). Degradation product Rf-0.31; darifenacin Rf-0.34.


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CONCLUSION

Introducing HPTLC into pharmaceutical analysis represents a majorstep in terms of quality assurance. The developed HPTLC technique is pre-cise, specific, accurate, and stability indicating. Statistical analysis provesthat the method is suitable for the analysis of darifenacin hydrobromideas a bulk drug and in pharmaceutical formulation without any interferencefrom the excipients. This study is a typical example of a stability-indicatingassay established following the recommendations of ICH guidelines[6],which can also be used to determine the purity of the drug and the methodcan also be extended to quantitative analysis of the drug in plasma andother biological fluids. As the method quantifies and resolves its degradati-on products from the main active drug ingredient, it can be used as astability-indicating analytical method for darifenacin hydrobromide.

REFERENCES

1. Chapple, C. R. Darifenacin: A novel M3 Muscarnic selective receptor antagonist for the treatment ofoveractive bladder. Expert Opin. Investig. Drugs. 2004, 13, 1493–1500.

2. Ohtake, N.; Mase, T. J. Med. Chem. 2000, 43, 5017–5029.3. Saipraveen, P.; Jagathi, V.; Devalarao, G.; Sudhakar Sai Babu, G. Visible spectrophotometric methods

for the determination of darifenacin. Research J. Pharm. Biol. Chem. Sci. 2010, 1, 254–257.4. Vishnu Murthy, M.; Raghavacharyulu, K. S.V.; Krishnaiah, C. H.; Katkam Srinivas.; Mukkanti, K.;

Ramesh Kumar, N. A stereoselective, stability-indicating validated LC-assay method for the separationand quantification of darifenacin and its enantiomers. Anal. Chem. Indian J. 2009, 8, 491–493.

5. Renger, B. Planar Chromatographic Procedures in Pharmaceutical Analysis. JAOAC Int. 2001, 84 (4),1217–1218.

6. ICH, Q2A(R1) Harmonized Tripartite Guideline, Text on validation of Analytical Procedures,IFPMA. In: Proceedings of the International Conference on Harmonization, Geneva, March 1994.

7. ICH Harmonized Tripartite Guideline. Guidance on Analytical Method Validation. In Proceedings ofthe International Convention on Quality for the Pharmaceutical Industry, Toronto, Canada,September 2002.

TABLE 5 Degradation of Darifenacin Hydrobromide

Condition Time (hr) Recovery (%) (%) Value of Degraded Products

2N HCl (Reflux at 80�C) 8 100 None detected2N NaOH (Reflux at 80�C) 8 100 None detectedH2O2 30% v=v (Reflux at 80�C) 4 63.65 36.35Daylight 24 100 None detectedDry heat degradation 10 100 None detectedNeutral hydrolysis (Reflux at 50�C) 12 100 None detected


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