Development and Validation of RP-HPLC Method for the Quantitative ...

Research Article

Development and Validation of RP-HPLC Method for the Quantitative Estimation of Tolterodine Tartrate in Capsule Formulation C. Bala Kumar1, B. Lakshmi Narayanan2*, M. Chandrasekar1, P. Malairajan3, E. P. Kumar1 Department of Pharmaceutical Analysis, Karpagam College of Pharmacy, Othakkalmandapam, Coimbatore 641032, Tamilnadu, India 2 Department of Pharmaceutical Analysis, Aditya Bangalore Institute of Pharmacy Education and Research, # 12, Kogilu main road, Yelahanka, Bangalore–560 064, Karnataka, India 3 Department of Pharmaceutical Analysis, Rofel Shri G.M. Bilakhia College of Pharmacy, Vapi, Gujarat, India 1

ABSTRACT Simple and sensitive method for the quantification of tolterodine tartrate in capsule formulation by RP-HPLC has been developed. Isocratic separation of tolterodine tartrate was carried out using a reversed-phase column C18 (150 × 4.6 mm, 5μm) with mobile phase consisting of methanol and phosphate buffer (pH adjusted to 7) in the ratio of 40:60 (v/v) and quantified by UV detection at 220 nm. Analytical run time was 10 min with retention time of 5.8 min. The assay exhibited good linear relationship, accuracy and precision were over the concentration level from 50% to 150% w/v. This method can be used for routine analysis of tolterodine tartrate in capsule formulation. Keywords: RP-HPLC, Tolterodine tartrate, Capsule formulation, Validation.

INTRODUCTION Tolterodine tartrate is a competitive muscarinic receptor antagonist and is chemically R-N, N-di-isopropyl-3-(2-hydroxy-5-methylphenyl)3-phenylpropanamine-L-hydrogen tartrate. Zhang et al developed a selective and sensitive high performance liquid chromatographyelectrospray ionization-mass spectrometry (HPLC-ESI-MS) method for the determination of tolterodine tartrate in human plasma. Tolterodine tartrate was extracted from plasma with n-hexane:isopropanol (95:5, v/v) with oxybutynin as a internal standard. The organic layer was evaporated and the residue was redissolved in mobile phase comprised of acetonitrile:water (40:60, v/v). An aliquot of 10 μl was chromatographically analyzed on a pre-packed shimadzu shim-pack VP-ODS C18 column (150 mm × 2.0 mm) by means of selectedion monitoring (SIM) mode MS. The validated HPLC-ESI-MS method has been 58

used successfully to study pharmacokinetic, bioavailability and bioequivalence of tolterodine tartrate in healthy male volunteers.1 Rao et al developed HPLC method for the simultaneous determination of combination of tamsulosin hydrochloride and tolterodine tartrate with UV detection at 220 nm. The chromatographic system consisted of an Inertsil ODS 3V (4.6 mm × 150 mm) containing 5μm packing column. Gradient elution with mobile phase consists of perchloric acid and water has been used. Tamsulosin hydrochloride was eluted at 3.9 min and tolterodine tartrate at 13.6 min, there was no integrated peak from excipient used for preparation of dosage form. Based on the stability and basic nature of the drug, dissolution experiments were conducted in 900 ml of phosphate buffer of pH 6.8. The dissolution profile proved to satisfy the specification, hence the method was applied successfully for the determination

Received Date : 11-09-2013 Revised Date : 21-10-2013 Accepted Date : 22-10-2013

DOI: 10.5530/rjps.2013.3.8 Address for correspondence B. Lakshmi Narayanan, M. Pharm, (Ph D) Assistant Professor, Aditya Bangalore Institute of Pharmacy Education and Research, # 12, Kogilu main road, Behind Annapurneshwari Temple, Yelahanka, Bangalore – 560 064, Karnataka, India Email: [email protected] Ph: 09611363347, 09940965008

www.rjps.in

RGUHS J Pharm Sci | Vol 3 | Issue 3 | Jul–Sep, 2013

C. Bala Kumar, et al.: Development and Validation of RP-HPLC Method for the Quantitative Estimation of Tolterodine Tartrate in Capsule Formulation

of tamsulosin hydrochloride and tolterodine tartrate combination pellets.2 Madhavi et al developed and validated a new analytical method for the determination of related components in tolterodine tartarate using LC method. Efficient chromatographic separation was achieved on a C8 stationary phase (150 × 4.6 mm, 3.5μm particles) with a simple mobile phase combination delivered in an isocratic mode and quantitation was carried out using UV detection. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by ICH. Microwave (MW) assisted degradation procedure was employed for stress testing studies in addition to the conventional way of a refluxing method. The results of both studies were compared. In the developed LC method, the resolution between tolterodine and its three potential impurities was found to be greater than 2.0. Regression analysis shows an r value (Correlation coefficient) greater than 0.999 for tolterodine and for its three impurities. The validated method has shown good, consistent recoveries for tolterodine (98.9–101.6% w/v) and for its three impurities. The test solution was found to be stable in the diluent for 48 h. Microwave degradations were very fast and comparable to the conventional way of the refluxing method. Robustness studies were carried out and suggested that system suitability parameters were unaffected by small changes in critical factors. The validated method was successfully applied for the determination of tolterodine tartrate in drug substances and drug products.3 Simple, accurate, rapid and sensitive of two methods were developed for the estimation of tolterodine tartrate in bulk and tablet formulation by Shinde et al. In his first method, methanolic solution of tolterodine tartrate showed λmax at 283 nm and in second method, tolterodine tartrate forms blue color with folin ciacaltau reagent in 0.5 N sodium hydroxide which exhibited λmax at 720 nm. Molar absorptivity of the drug was found to be 2.1 × 104 and 3.021 × 104 for first and second method respectively. The Beer’s law was obeyed in the concentration range of 10–60μg/ml and 5–40μg/ml for both of these methods. The developed method has been successfully applied to the analysis of tolterodine tartrate in tablet formulation.4 Nanda et al reported simple, precise and economical of three UV methods for the estimation of tolterodine in pharmaceutical dosage form. Tolterodine has the λmax at 284 nm (Method A) and in the first order derivative spectra showed sharp peak at 234 nm (Method B). The employed method C has area under curve (AUC) in the wavelength range of 279–289 nm. Linearity was observed in the concentration range of 10–50μg/ml for all three methods. These methods were successfully applied RGUHS J Pharm Sci | Vol 3 | Issue 3 | Jul–Sep, 2013

for the simultaneous determination of tolterodine in commercial pharmaceutical preparations. The results of the analysis were validated by statistically and by recovery studies, it was found to be satisfactory.5 Radha Krishna et al reported a simple and accurate RPHPLC method for the determination of related substances, degradants and assay of tolterodine tartrate. The separation was achieved using a Water X-terra MS C18, 150 × 4.6, 3.5μm column with the mobile phase A (0.05% v/v TFA in water) and mobile phase B (0.05% v/v TFA in acetonitrile) using a binary gradient mode with flow rate at 1.0 ml/min and was detected at 220 nm. The resolution between the critical pair of peaks (Impurity-2 & 3) was found to be greater than 4.0. The validated method yielded a good result of precision, linearity, accuracy and robustness.6 A capillary solid-phase extraction (SPE) system coupled directly to ESI-MS for the quantification of free tolterodine and metabolite concentrations in plasma has been reported by Swart et al. The unbound fraction of these compounds was obtained by ultrafiltration of plasma and the ultrafiltrate was directly injected onto the SPE capillary (4 mm, 3200 mm, 5 mm, C18). After desalting and clean-up of the sample, the analytes were eluted in backflush mode with methanol:triethylamine (70:30, v/v), providing considerable solute focusing. The unresolved compounds eluted simultaneously with the remaining matrix compounds and were detected in a multiple-reaction monitoring (MRM) mode. The validated method has been successfully employed for the bio-analysis of tolterodine.7 Kumar S. Ashutosh et al developed a method and validation of Tolterodine Tartrate in bulk as well as in pharmaceutical formulation by using RP-HPLC.8 Paul Alok Kumar et al did a comparative analysis of detection of tolterodine tartrate with a HPLC-UV method using sodium, potassium and ammonium dihydrogen phosphate buffer in the mobile phase.9 Ramathilagam N et al developed a method and validation of HPLC method for the estimation of Tolterodine tartrate in tablets.10 Yanamandra Ramesh et al developed a new rapid and sensitive stability-indicating UPLC assay method for tolterodine tartrate. Application in pharmaceuticals, human plasma and urine samples.11 Development and validation of a simple, stabilityindicating high-performance liquid chromatographic method for analysis of tolterodine tartrate in the bulk drug and in its tablet formulation by Sinha V. R et al.12 The survey of literatures revealed that few analytical methods have been developed for the estimation of 59


tolterodine tartrate in single component or combination in formulations. It was thought of our interest to develop RP-HPLC method for the estimation of tolterodine tartrate in capsule formulation. MATERIALS AND METHODS Chemicals and reagents

The standard tolterodine tartrate and capsule formulation were obtained from Micro Labs Ltd, Hosur, Tamil Nadu, India. Ammonium dihydrogen phosphate, orthophosphoric acid (88.0%), methanol and acetonitrile of HPLC grade, triethylamine and tetrahydrofuran were of analytical grade purchased from Merck Ltd, Mumbai. Water for injection (0.22μ filtered) and double distilled water for analytical purpose was obtained from milli-Q R-O system, Karpagam College of Pharmacy, Coimbatore, Tamil Nadu, India. Instrumentation

The HPLC system consisted of AGILENT (1100-series), rheodyne injection port with 20μl sample loop and UVdetector (Shimadzu). Data collection, integration and calibration were accomplished using LC software data system. The optimized HPLC conditions

The chromatographic separation of tolterodine tartrate in capsule formulation was accomplished using Hypersil BDS, C18 (150 x 4.6 mm), 5μm reversed phase analytical column with isocratic separation of flow rate 1 ml/min at room temperature. The mobile phase consisting of methanol and phosphate buffer (pH adjusted to 7) in the ratio of 40:60 (v/v) and quantified by UV detection at 220 nm. Analytical run time was 10 min with retention time of 5.8 min. Before use, the mobile phase was filtered by passing it through a 0.45μm nylon membrane filter and the filtrate was degassed by using bath sonicator (Metler). All the procedures were performed at ambient temperature. Preparation of buffer

About 2.88 g of ammonium dihydrogen phosphate was transferred into 1000 ml volumetric flask and add 5 ml of triethylamine. Add 500 ml of milli-Q water. The pH was adjusted to 7.0 ± 0.05 using orthophosphoric acid then the volume was made up to 1000 ml with milliQ water and filtered through 0.45μ filter paper and degassed. Preparation of mobile phase

A mixture of methanol and buffer solution in the ratio of 40:60 (v/v) was prepared and filtered through 0.45μ-nylon membrane filter and sonicated to degas. 60

Preparation of diluents/blank

Mixture of tetrahydrofuran and methanol (15:85% v/v) was prepared and filtered through 0.45μ-nylon membrane filter and sonicated to degas. Preparation of standard solution

100 mg of tolterodine tartrate working standard was weighed accurately and transferred into a 100 ml volumetric flask. To this, 50 ml of diluent was added, sonicated to dissolve, allowed to cool at room temperature and the final volume was made up to 100 ml with diluent to produce the concentration of 1 mg/ml. Then 5 ml of the above solution (1 mg/ml) was pipette and diluted to 50 ml with diluent to obtain the concentration 100μg/ml. Preparation of test solution

The average weight of 20 capsules was determined. Accurately weighed the contents of capsule equivalent to 10 mg of tolterodine tartrate and transferred in to 100 ml volumetric flask. To this 30 ml of diluent was added and sonicated for 10 min with intermittent shaking. The solution was allowed to cool at room temperature and the final volume was made up to 100 ml with diluent. The contents were mixed well, filtered through 0.45μ membrane nylon filter, first few ml of filtrate was discarded. Thus the concentration obtained was found to be 100μg/ml. Assay validation

The RP-HPLC assay validation was done as per ICH Q2A and Q2B guidelines.13 These tests included system suitability, determination of accuracy, precision, linearity, ruggedness and robustness. System suitability

About 10μl of test solution (100μg/ml) was injected and recorded the suitability parameters. Accuracy

Samples were prepared by adding the active ingredients (Tolterodine tartrate) into the placebo at different concentrations (50%, 100%, and 150% w/v) each in triplicate. Each preparation was injected into the RP-HPLC system in duplicate. Precision

Precision is the measure of either the degree of reproducibility or repeatability of the analytical method under normal operating conditions. a) Repeatability (Intra-assay precision): Repeatability expresses the precision under the same operating conditions over a short interval of time. R epeatability RGUHS J Pharm Sci | Vol 3 | Issue 3 | Jul–Sep, 2013


is also termed intra-assay precision. Repeatability should be assessed using three concentrations and three replicates of each concentration or using a minimum of six determinations at 100% of the test Concentration b) Intermediate precision: Intermediate precision expresses within-laboratory variations, different days and different analysts or equipments. Intermediate Precision is demonstrated by carrying out the complete experiment by the different analyst on different days, on different instruments in the same laboratory. The six concentrations of 100% Test solution prepared by dissolving 665 mg of placebo and 10 mg of Tolterodine tartrate in 15 ml Tetrahydrofuran and made the volume to 100 ml with methanol.

RESULTS AND DISCUSSION Chromatography

Sensitive, rapid, specific and reproducible RP-HPLC method has been developed and validated for quantitative determination of tolterodine tartrate in capsule formulation. The representative chromatograms of tolterodine tartrate standard, sample and blank were shown in Fig. 1, Fig. 2 and Fig. 3 respectively. The retention time of tolterodine tartrate was 5.8 min and the peaks were sharp. There was good baseline separation observed. System suitability

The results of system suitability were shown in Table No. 1. System suitability parameters (Column efficiency,

Linearity

Linearity of an analytical method is the ability (Within a given range) to obtain test results that are directly proportional to the concentration (Amount) of analyte in the sample. The degree of linearity was estimated by calculating the correlation coefficient, regression coefficient (r2), Y-intercept, residual sum of squares and Y-intercept / response at 100% w/v of working concentration. Further a slope was drawn and reported by injecting each of the solutions in triplicate. A plot of data was established between analyte response and concentration. For the experiment, the linearity level in percentage has been measured from 50% to 150% w/v at five different concentration levels.

Figure 1: Chromatogram of standard tolterodine tartrate.

Ruggedness

The ruggedness of an analytical method is the degree of reproducibility of test result obtained by the analysis of the same sample under a variety of normal test condition such as different laboratories, different analyst, different instruments, and different lots of reagents, different elapsed assay times, different assay temperature and different days. Ruggedness is normally expressed as the lack of influence on test result of operational and environmental variables of the analytical method. Five sample injections were injected into the different RP-HPLC system (Preferably with different manufacturer or same manufacturer with different configuration) by using the same column and by the same analyst.

Figure 2: Chromatogram of tolterodine tartrate sample.

Robustness

Robustness of an analytical method is the measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. A variation in flow rate, wavelength and column temperature has been measured. RGUHS J Pharm Sci | Vol 3 | Issue 3 | Jul–Sep, 2013

Figure 3: Blank chromatogram. 61


plate count and tailing factor) were in compliance with the ICH guidelines. Accuracy

The results of accuracy were shown in Table No. 2. It was found that the accuracy (Recovery) for the average of triplicate from each concentration levels were within 50% to 150% w/v. Precision

Repeatability (Intra-assay precision) and System precision: The %RSD value for six test preparations was found to be 0.9. The results were shown in Table No. 3. Hence the method was precise for the estimation of tolterodine tartrate.

%RSD value found to be less than 2. Hence the test method was found precise and rugged for the estimation of tolterodine tartrate. The results were shown in Table No. 5 & Table No. 6. Robustness Changes in flow rate of mobile phase

Change the flow rate (1 ± 0.1 ml) of mobile phase, the %R.S.D was found to be less than 2%. The results were shown in Table No. 7. Changes in wavelength

Change the wave length (220 ± 5 nm), the %R.S.D was found to be less than 2%. The results were shown in Table No. 8.

Linearity

Changes in column temperature

The regression coefficient of tolterodine tartrate was found to be 0.9998. Statistical Y intercept was found to be 0.47. The results obtained were within the acceptance criteria (Table No. 4) and the linearity plot was shown in Fig. 4.

Change the column temperature (30 ± 5 OC), the % R.S.D was found to be less than 2%. The results were shown in Table No. 9.

Ruggedness

A simple, economical and reproducible RP-HPLC method for the estimation of tolterodine tartrate in capsule formulation has been developed by using methanol

Variability due to HPLC system, HPLC column and different analyst was determined and it was found that the

CONCLUSION

Table 1: System suitability of tolterodine tartrate S. No

Injections

RT

Area

USP Tailing

USP Plate count

1

Injection-1

5.439

1436539

1.39

3310

2

Injection-2

5.439

1440277

1.39

3350

3

Injection-3

5.439

1441348

1.39

3345

4

Injection-4

5.444

1443152

1.39

3333

5

Injection-5

5.443

1441320

1.40

3334

Figure 4: Linearity plot for tolterodine tartrate.

Mean area

14405227

%RSD

0.171

Table 2: Summary of accuracy parameter for tolterodine tartrate S. No

Target conc.(ppm)

Area of Injection 1

Area of Injection 2

Average area

Mg Recovery

%Recovery

1

50

769328

772084

770706

5.02

100.4

2

50

758192

758585

758389

4.94

98.8

3

50

765315

761970

763643

4.97

99.4

4

100

1424095

1455825

1439960

9.37

98.6

5

100

1451856

1491945

1471901

9.58

99.8

6

100

1489545

1444876

1467211

9.55

99.5

7

150

2226797

2214748

2220773

14.46

99.0

8

150

2145430

2151888

2148659

13.99

99.2

9

150

2177042

2186354

2181698

14.20

99.3

Overall recovery 62

Average

%RSD

99.5

0.8

99.3

0.6

99.2

0.2

99.3

0.5



Table 3: Precision of tolterodine tartrate S. No

Conc. (ppm)

Method precision (Mp)

1

100

2

100

3 4

Injections Injection 1

Injection 2

Average

Mp 1

1469036

1492026

1480531

Mp 2

1476389

1487617

1482003

100

Mp 3

1499175

1497260

1498218

100

Mp 4

1454337

1440348

1447343

5

100

Mp 5

1480574

1491698

1486136

6

100

Mp 6

1474378

1480641

1477510

Table 4: Summary of linearity parameter for tolterodine tartrate Injection 2

Table 5: Column variability of tolterodine tartrate Area counts

1

1

5.701

5167

1.3

643785

2

2

5.701

5188

1.3

644253

3

3

5.700

5181

1.3

643373

1820859

4

4

5.697

5158

1.3

642867

2304118

5

5

5.694

5140

1.3

643396

770765

772036

771401

80

1246710

1229061

1237886

3

90

1370605

1375012

1372809

4

100

1530175

1532836

1531506

5

110

1678332

1677625

1677979

6

120

1824259

1817458

Average

150

2305450

2302785

Correlation coefficient

0.9998

Slope (m)

15316.4

Intercept (Y)

8486.9605

Y- Intercept

0.47

Table 6: System variability of tolterodine tartrate USP Plate count

USP tailing

1

5.678

5187

1.3

634674

2

2

5.685

5183

1.3

633080

3

3

5.683

5147

1.3

634863

4

4

5.691

5177

1.3

632278

S. No 1 2

Flow rate

1.1 ml

MEAN

633241

1

215 nm

SD

1530

% RSD

0.24

2

225 nm

1.3

SD

517

% RSD

0.08

T Plates

Peak area

Mean area

1.12

3524

1632704

%R.S.D.

0.25

0.76

1.833

Mean area

1.12

2995

1390826

%R.S.D.

0.44

0.62

1.683

Table 8: Changes in wavelength

631311

5156

6433535

Asymmetry

0.9 ml

S. No

5.687

MEAN

Table 7: Changes in flow rate

Area counts

1

5

0.9

USP tailing

50

5

0.801

USP Plate count

2

RT

1478623

RT

1

Injections

%RSD

Injections

Conc. (ppm)

S. No

SD

S. No

S. No

7

Injection 1

Mean

Wave length

T Plates

Peak area

Mean area

Asymmetry 1.13

3283

1969512

%R.S.D.

0.29

0.39

0.459

Mean area

1.11

3206

1089505

%R.S.D.

0.14

0.36

0.237

Table 9: Changes in column temperature S. No

Column temp

1

25OC

2

35OC


Asymmetry

T Plates

Peak area

2998

1508605

Mean area

1.11

%R.S.D.

0.36

0.49

0.712

Mean area

1.11

3265

1521731

%R.S.D.

0.54

0.6

0.845

63


and buffer solution as a mobile phase. Results of analysis were validated by statistically and by recovery studies. It was found to be accurate, precise and sensitive. This method can be used for routine analysis of tolterodine tartrate in capsule formulation. ACKNOWLEDGEMENT The authors were grateful to the Chairman and Principal, Karpagam Group of Institutions for providing the necessary facilities to carry out the work. REFERENCES 1. Zhang B, Zhang Z, Tian Y, Xu F. High performance liquid chromatography-electro spray ionization mass spectrometric determination of tolterodine tartarate in human plasma. Journal of Chromatography B, 2005; 824: 92–98. 2. Rao MVB, Reddy BCK, Rao CKTS, Rao GV. Drug release method by HPLC for tamsulosin hydrochloride 0.2% and tolterodine tartrate 0.2% combination pellets. Int J Chem Eng Res. 2009; 1(2): 155–159. 3. Madhavi A, Reddy GS, Suryanarayana VM, Naidu A. A Development and validation of a new analytical method for the determination of related components in tolterodine tartarate using LC. Chromatographia. 2008; 86: 399–407. 4. Shinde DB, Sangshetti JN, Rane VP, Chaudhari PD, Kolsure PK. Spectrophotometric estimation of tolterodine tartrate in bulk and tablet formulation, Analytical Chemistry: An Indian Journal. 2007; 5: 1–6.

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