Development and Validation of a New Analytical Method for the Determination of Related Components in Tolterodine Tartarate Using LC

2008, 68, 399407

A. Madhavi1,2,&, G. S. Reddy1, M. V. Suryanarayana1, A. Naidu2

1 2

Dr. Reddys Laboratories, Active Pharmaceutical Ingredients, Unit-III, Bollaram, Hyderabad 502 325, India; E-Mail: madhavi_devarakonda@yahoo.com Department of Chemistry, Jawaharlal Nehru Technological University, Kukatpally, Hyderabad 500 072, India

Received: 21 January 2008 / Revised: 2 May 2008 / Accepted: 13 June 2008 Online publication: 8 August 2008

Abstract
A novel liquid chromatographic method has been developed, and validated for the determination of tolterodine tartarate, for its potential three impurities in drug substances and drug products. Efcient chromatographic separation was achieved on a C8 stationary phase (150 9 4.6 mm, 3.5 lm particles) with a simple mobile phase combination delivered in an isocratic mode at a ow rate of 0.8 mL min-1 and quantitation was carried out using ultraviolet detection. Microwave assisted degradation procedure was employed for stress testing studies in addition to the conventional way of a reuxing 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 coefcient) greater than 0.999 for tolterodine and for its three impurities. This method was capable to detect all three impurities of tolterodine at a level below 0.0038% with respect to a test concentration of 0.5 mg mL-1 for a 10 lL injection volume. The inter- and intra-day precisions for all three impurities and for tolterodine were found to be within 1.1% RSD at its specication level. The method has shown good, consistent recoveries for tolterodine (98.9101.6%) and for its three impurities (94.5103.0%). The test solution was found to be stable in the diluent for 48 h. The drug was subjected to stress conditions of hydrolysis, oxidation, photolysis and thermal degradation, as prescribed by ICH. Degradation was found to occur in alkaline stress condition, while the drug was stable to water hydrolysis, acid hydrolysis, oxidative stress, photolytic and thermal stress. The assay of stressed samples was calculated against a qualied reference standard and the mass balance was found close to 99.5%. Microwave degradations were very fast and comparable to the conventional way of the reuxing 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 tartarate in drug substances and drug products.

Keywords
Column liquid chromatography Microwave assisted degradation Validation Tolterodine tartarate

Introduction
Tolterodine [(R)-N,N-diisopropyl-3-(2hydroxy-5-methylphenyl)-3-phenylpropanamine] (Fig. 1) is a new muscarinic receptor antagonist intended for the treatment of urinary urge incontinence

Full Short Communication DOI: 10.1365/s10337-008-0735-y 0009-5893/08/09

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Tolterodine tartarate:
OH H H 3C H N . HO COOH H COOH OH

(R)-N,N-Diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenyl)-3-phenyl propanamine L-hydrogen tartarate Imp-1:

OH

OH O OH OH

N H

N-Isopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropylamine-L-hydrogen tartarate Imp-2:

H3C

OCH2C6H5

CH-CH2-CH2-N(CH2-CH2-CH3)2

N,N-Diisopropyl-3-(2-benzyloxy-5-methylphenyl)-3-phenylpropylamine Imp-3:

OH

HO

N-Isopropyl-bis-(3-(2-hydroxy-5-methyl)phenyl)-3-phenylpropylamine
Fig. 1. Chemical structures and label of tolterodine tartarate and impurities. Tolterodine tartarate: (R)-N,N-diisopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenyl)-3-phenyl propanamine L-hydrogen tartarate, Imp-1: N-isopropyl-3-(2-hydroxy-5-methylphenyl)-3-phenylpropylamineL-hydrogen tartarate, Imp-2: N,N-diisopropyl-3-(2-benzyloxy-5-methylphenyl)-3-phenylpropylamine, Imp-3: N-isopropyl-bis-(3-(2-hydroxy-5-methyl)phenyl)-3-phenylpropylamine

and other symptoms related to unstable bladder [1, 2]. It is a follow up to terodiline, which was withdrawn due to side eects. Tolterodine has a safety prole in humans with no side eects. In the search for new drugs with improved side eect proles for treatment of an overactive bladder, a family of phenyl polyamines as muscarainic receptor antagonists has been investigated and very recently, (+)-N,N-diisopropyl-3(2-hydroxy-5-methylphenyl)-3-phenyl)-3phenylpropanamine [(+)(R)-tolterodine, brand names Detrol or Detrugitol] was launched and marketed world wide with success as (R,R)-tartaric acid salt. A convenient synthesis for this drug was cited in the literature [3] starting from 1-[(2-hydroxy-5-methyl)phenyl]ethylene, accessible in high yield by alumina-promoted ortho-alkenylation of p-cresol with phenyl acetylene. Accordingly, the aim of the present study was to establish inherent stability of tolterodine tartarate through stress studies under a variety of ICH recommended test conditions [46] to develop a stability-indicating assay method for the determination of tolterodine tartarate and its potential three impurities [79]. Extensive literature survey did not reveal any simple, sensitive and stability indicating method for the determination of related components of tolterodine tartarate in drug substances and drug products. Among the reported liquid chromatographic methods the use of UV detection for quantication of enantiomers of tolterodine using chiral LC [10], is included. The other reported methods are applicable for biological matrices using complex analytical instruments such as mass spectrophotometer [1115]. To our present knowledge no stability indicating LC assay method has yet been developed and validated for the determination of related components of tolterodine tartarate in drug substances and drug products. More intensive stress studies were carried out on tolterodine tartarate in a conventional way of reux procedure and by using a microwave oven. Accordingly, a stability-indicating

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method was developed, which could separate various degradation products. In the present paper, we describe a new analytical method for the determination of related components in tolterodine tartarate, namely impurity-1, impurity-2 and impurity-3 using reverse phase liquid chromatography (RP-LC). Specicity of the proposed method was established by conducting stress degradation studies on the sample according to ICH prescribed conditions [4]. The developed method was successfully adopted for the determination of related components in tolterodine tartarate bulk drug and dosage forms.

Analytical Method Development

In order to develop a selective and sensitive method, primary concern during the development was to achieve resolution between imp-1 and tolterodine peaks, and symmetry of the tolterodine peak. Dierent buer types and pH media such as phosphate buer (pH 3 7), citrate buer (pH 35), ammonium acetate buer (pH 35) and acetic acid buer (pH 35) were studied in combination with acetonitrile and methanol (30, 50 and 70%). The eect of various organic modiers such as acetonitrile, methanol and their combinations, on peak properties (peak height and symmetry) and resolution between tolterodine and other impurities (imp-1, imp-2 and imp-3) were investigated. Various ion pairing agents such as triuoroacetic acid and triethylamine were tested to improve peak properties such as peak height, peak symmetry among others. In addition chromatographic parameters such as retention factor (k), number of theoretical plates (N) were also recorded. Further, a robustness study was conducted during the nal phase of method development.

were prepared by dissolving appropriate amounts. Acetonitrile: water (1:1, v/v) was used as diluent. Working solutions of 500 and 100 lg mL-1 were prepared from the above stock solutions for related substances determination and assay determination, respectively. A stock solution of impurities (mixture of imp-1, imp-2 and imp-3) at 0.5 mg mL-1 was also prepared in diluent.

Preparation of Sample Solution

Twenty-ve tablets were weighed and the content transferred into a clean and dry mortar, grinded well. Then an equivalent to 50 mg of the drug was transferred to a 100 mL volumetric ask. A measure of 30 mL of diluent was added and kept on a rotatory shaker for 10 min to disperse the material completely then sonicated for 10 min and diluted to 50 mL (500 lg mL-1). The resulting solution was centrifuged at 3,000 rpm for 25 min (supernatant solution was used for purity evaluation). Supernatant solution measuring 10 mL was taken and diluted to 100 mL with diluent (100 lg mL-1). This solution was ltered using a 0.45 l nylon 66-membrane lter for the assay analysis.

Experimental
Chemicals
Samples and standard were supplied by Gensen Laboratories, Mumbai, India; commercially available 2 mg tolterodine tartarate tablets were purchased. LC grade acetonitrile, analytical reagent grade sodium dihydrogen phosphate monohydrate, triethylamine and phosphoric acid were purchased from Merck, Darmstadt, Germany; high pure water was prepared by using Millipore Milli-Q plus water purication system.

Chromatographic Conditions
The chromatographic column used for separation purpose was a Zorbax SB C-8, 150 mm 9 4.6 mm i.d. with 3.5 lm particles. Mobile phase contains a mixture of buer and acetonitrile in the ratio of 60:40 (v/v). Buer consists of 50 mM sodium dihydrogen phosphate monohydrate and 5 mL of triethylamine, pH adjusted to 2.5 using diluted phosphoric acid (1 mL in 10 mL of Milli-Q water). The ow rate of the mobile phase was 0.8 mL min-1. The column temperature was maintained at 27 C and the detection was monitored at a wavelength of 210 nm. The injection volume was 10 lL.

Analytical Method Validation

The developed chromatographic method was validated for specicity, linearity, range, precision, accuracy, sensitivity, robustness and system suitability.
Specicity

Apparatus
The LC system used was a Waters 2695 binary pump plus autosampler and a 2996 photo diode array detector. The output signal was monitored and processed using empower software on a pentium computer (Digital Equipment Co.). Water baths equipped with MV controller (Julabo, Seelbach, Germany) were used for hydrolytic studies. Stability studies were carried out in a humidity chamber (Thermo Lab Humidity Chamber, India) and photo stability studies were carried out in a photo stability chamber (Sanyo Photo Stability Chamber, Leicestershire, UK). Thermal stability studies were performed in a dry air oven (Mach Pharmatech, Hyderabad, India).

Preparation of Stock Solutions

Stock solutions of tolterodine tartarate standard and sample (5.0 mg mL-1)

Specicity of the developed method was assessed by performing forced degradation studies. The terms selectivity and specicity are often used interchangeably. Specicity is the ability of the method to measure the analyte response in the presence of its potential impurities [7]. According to ICH [4] stress testing of the drug substance can help the intrinsic stability of the molecule and validate the stability indicating power of the analytical procedures used. The nature of the stress testing will depend on the indi-

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Table 1. System suitability report

Compound USP resolution (RS) 3.1 28.0 11.6 USP tailing factor No. of theoretical plates USP tangent method (N) 11,666 6,373 16,750 19,945

Imp-1 Tolterodine Imp-2 Imp-3

1.1 1.3 1.0 1.0

ucts] was calculated. Assay was also calculated for bulk samples and drug products by spiking all three impurities (imp-1, imp-2 and imp-3) at the specication level (i.e. 0.15% of analyte concentration which is 500 lg mL-1).
Linearity and Range

vidual drug substance and the type of drug product involved. Stress testing is likely to be carried out on a single batch of the drug substance. It should include the eect of temperatures [in 10 C increments (e.g., 50, 60 C, etc.) above that for accelerated testing], humidity (e.g., 75% RH or greater) where appropriate, oxidation and photolysis on the drug substance. The testing should also evaluate the susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or suspension. Photostability testing should be an integral part of stress testing. The standard conditions for photostability testing are described in ICH Q1B. The specicity of the developed LC method for tolterodine tartarate was determined in the presence of its impurities namely imp-1, imp-2, imp-3 and degradation products. Forced degradation studies were performed on tolterodine tartarate to provide an indication of the stability indicating property and specicity of the proposed method [8, 9]. In the present study stress studies were carried out in a conventional reux method, moreover a fast and eective microwave based degradation technique was designed and employed for the forced degradation study. Both studies were carried out as per ICH. The detailed studies using both techniques are addressed below. All stress decomposition studies were performed at an initial drug concentration of 0.5 mg mL-1 (500 lg mL-1). The stress conditions employed for the degradation study includes light (carried out as per ICH Q1B), heat (60 C), acid hydrolysis (1 N HCl, 80 C for 12 h), base hydrolysis (1 N NaOH, RT 24 h), water hydrolysis (80 C 12 h) and oxidation (6% H2O2, RT 48 h). For heat and light studies, the study period was 10 days. Peak purity of the stressed samples of tolterodine tartarate was

checked by using a 2996 photo diode array detector of Waters (PDA). The purity angle within the purity threshold limit obtained in all stressed samples demonstrates the analyte peak homogeneity. All stressed samples of tolterodine tartarate were analysed for an extended run time of 100 min to check the late eluting degradants. Assay studies were carried out for stress samples against qualied reference standard and the mass balance [assay percentage + percentage of impurities + percentage of degradation products] was calculated. Assay was also calculated for bulk samples and drug products by spiking all three impurities (imp-1, imp-2 and imp-3) at the specication level (i.e. 0.15% of analyte concentration which is 500 lg mL-1).
Microwave Assisted Degradations

A new, fast and eective microwave based degradation technique was designed and employed for the hydrolytic forced degradation study. All stress decomposition studies were performed at an initial drug concentration of 0.5 mg mL-1. The stress conditions employed for the degradation study includes, acid hydrolysis (1 N HCl, 5 min), base hydrolysis (1 N NaOH, 5 min), water hydrolysis (5 min) and oxidation (6% H2O2, 5 min). For hydrolytic treatments, 2 mL of either 1 N HCl or 1 N NaOH was added in each ask containing 5 mg of the drug. Prepared solutions were vortex mixed and subjected to microwave radiation (15 s/cycle, 2.45 GHz, 300 w) for 5 min. After the treatment, samples were allowed to cool and neutralized. Assay studies were carried out for stress samples against qualied reference standard and the mass balance [assay percentage + percentage of impurities + percentage of degradation prod-

To establish linearity of the assay method, calibration solutions were prepared from the stock solution at ve concentration levels from 50 to 150% of the assay analyte concentration (50, 75, 100, 125 and 150 lg mL-1); for the chromatographic purity method, nine concentration levels from LOQ to 200% (with respect to the test concentration of 500 lg mL-1) (LOQ, 25, 50, 75, 100, 125, 150, 175 and 200%) were prepared by diluting the impurity stock solution to the required concentrations. Average peak area at each concentration level was subjected to linear regression analysis with the least squares method. Calibration equation obtained from regression analysis was used to calculate the corresponding predicted responses. The residuals and sum of the residual squares were calculated from the corresponding predicted responses. The %y-intercept for both, assay and chromatographic purity method, was calculated. Analytical range of the method was established from the analysis of sensitivity curves. Upper and lower levels of range were also established.
Precision

The precision of the related substance method was checked by injecting six individual preparations of tolterodine tartarate (500 lg mL-1) spiked with 0.15% of each impurity. The %RSD for percentage of imp-1, imp-2 and imp-3 was calculated. For repeatability (intra-day) six individual preparations of tolterodine tartarate (500 lg mL-1) spiked with 0.15% each imp-1, imp-2 and imp-3 were injected. Assay method precision was evaluated by carrying out six independent assays of test samples of tolterodine tartarate against qualied reference standard. The %RSD of six assay values obtained was calculated. Full Short Communication

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Table 2. Summary of forced degradation results and microwave-assisted degradation results

Stress condition Time Degradation (approx., %) Assay of active substance (%) Mass balance (%assay + %impurities + %degradation products)

Forced degradation results Acid hydrolysis (1 N HCl, 80 C) Base hydrolysis (1 N NaOH, 80 C) Oxidation (6% H202) Water hydrolysis (80C) Thermal (60 C) Light (photolytic degradation) Microwave assisted degradation results Acid hydrolysis (1 N HCl) Base hydrolysis (1 N NaOH) Oxidation (6% H202) Water hydrolysis

12 12 48 12 10 10 5 5 5 5

h h h h days days

0.5 34.7 0.6 0.2 0.0 0.0 0.2 32.3 0.3 0.6

99.1 64.7 99.0 99.5 99.6 99.7 99.2 67.0 99.0 99.1

99.6 99.5 99.7 99.8 99.7 99.8 99.5 99.4 99.4 99.8

min min min min

The intermediate precision of the assay method was evaluated by a dierent analyst and by using a dierent instrument from the same laboratory.
Sensitivity

Sensitivity was determined by establishing the LOD and LOQ for imp-1, imp-2 and imp-3, estimated at a signal-to-noise ratio of 3:1 and 10:1, respectively, by injecting a series of dilute solutions with known concentration. The precision study was also carried out at the LOQ level by injecting six individual preparations of imp-1, imp-2 and imp-3 and calculating the %RSD for the areas of each impurity.
Accuracy

resolution, 0.2 units changed it from 0.6 to 1.0 mL min-1. The eect of pH on the resolution of impurities was studied by varying 0.1 pH units (at 2.4 and 2.6 buer pH). The eect of column temperature on the resolution was studied at 22 and 32 C instead of 27 C. In all the above varied conditions, the components of the mobile phase were held constant.
Solution Stability and Mobile Phase Stability

The content of imp-1, imp-2 and imp-3 was checked in the test solutions.

Results and Discussion

Analytical Method Development
In preliminary studies, peak properties and response functions were optimised by changing the type of organic modier, organic to aqueous ratio, buer type, buer strength and pH. Peak shape was not symmetric when triethylamine was not used in the mobile phase (tailing >3.5); increasing triethylamine and buer concentrations improved the peak shape (tailing *1.2). Changing the pH from basic to acidic improved the peak shape and resolution between imp-1 and tolterodine. When methanol was used in the mobile phase (buer:methanol, 60:40 v/v) the retention time of tolterodine is high (about 20 min) and the peak tailing is also high (*3.2). Using acetonitrile in the mobile phase instead of methanol improved the retention time and peak symmetry. In the optimised conditions tolterodine, imp-1, imp-2 and imp-3 were well separated with a resolution >3. Ecient results were achieved on a Zorbax SB C-8, 150 mm 9 4.6 mm i.d. with 3.5 lm particles, by using the mobile phase mixture of buer and acetonitrile in the ratio of 600:400 (v/v). Buer consisted of 50 mM sodium dihydrogen phosphate monohydrate and

Recovery study was carried out by spiking analysis. A known amount of the impurity stock solution was spiked to the previously analysed sample at 50, 75, 100, 125 and 150% of the analyte concentration (500 lg mL-1). The percentage of recoveries for imp-1, imp-2 and imp-3 was calculated. Each concentration level was prepared three times.
Robustness

By deliberate change in experimental conditions the resolution between tolterodine, imp-1, imp-2 and imp-3 was evaluated. The ow rate of the mobile phase was 0.8 mL min-1. To study the eect of ow rate on the

The solution stability of tolterodine tartarate in the assay method was carried out by leaving the test solution of the samples in tightly capped volumetric asks at room temperature for 48 h. The same sample solutions were assayed in 6-h intervals up to the study period against freshly prepared standard solution. The mobile phase stability was also carried out by assaying the freshly prepared sample solutions against freshly prepared reference standard solutions in 6-h intervals up to 48 h. Prepared mobile phase was kept constant during the study period. The solution stability of tolterodine tartarate and its impurities in the purity method was carried out by leaving spiked sample solutions in tightly capped volumetric asks at room temperature for 48 h. The content of imp-1, imp-2 and imp-3 was determined at every 6-h interval up to the study period. Mobile phase stability was also carried out for 48 h by injecting the freshly prepared sample solutions at every 6-h interval.

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0.18 0.16 0.14 0.12

(a)-i

Toltirodine - 5.219

0.20

Tolterodine tartarate

0.10 0.08 0.06 0.04 0.02 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time (min)
0.20 0.18 0.16 0.14 0.12

(a)-ii

Tolterodine tartarate spiked with impurities

0.10
Imp-1 - 4.297

0.08 0.06 0.04 0.02 0.00 2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

18.00

20.00

22.00

24.00

Time (min)
0.10

(b)-i
0.08 0.06

Tolterodine tartrate stressed with 1N HCl reflux for 12h.

0.04 0.02 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
3.703 4.018 4.145 4.320 4.580

5 mL of triethylamine, pH adjusted to 2.5 with phosphoric acid. The ow rate of the mobile phase was 0.8 mL min-1. At 27 C column temperature, the peak shape of tolterodine was found to be symmetrical. Acetonitrile:water (1:1, v/v) was used as blank; there was no interference of the blank with impurities (imp-1, imp-2 and imp-3) and tolterodine. The interference of excipients (colloidal anhydrous silica, calcium hydrogen phosphate dihydrate, cellulose microcrystalline, hypromellose, magnesium stearate, sodium starch glycolate (pH 3.05.0), stearic acid, and titanium dioxide) was also checked by injecting sample solutions of excipients. There was no interference of excipients with impurities (imp-1, imp-2, imp-3) and the tolterodine peak. The typical retention times of imp-1, tolterodine, imp-2 and imp-3 were about 4.3, 5.0, 15.1 and 21.4 min, respectively. The system suitability result is given in Table 1. The developed LC method was found to be specic for tolterodine tartarate and its three impurities namely imp-1, imp-2 and imp-3 (Table 2). Stability study results as per ICH Q1A (R2) for tolterodine tartarate [6] were generated (long term stability 12 months and accelerated stability 6 months) and the results are well within the limits.

AU

AU

Toltirodine - 4.936

Imp-2 - 15.155

AU

Toltirodine - 5.202

10.756

6.835

Imp-3 - 21.490

Analytical Method Validation

Time (min)

Specicity

(b)-ii

Toltirodine - 5.183

0.10 0.08 0.06

Tolterodine tartrate stressed with 1N HCl in microwave oven for 5 min.

0.04 0.02 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time (min)

Fig. 2. a Typical chromatogram of tolterodine tartarate (a-i) and stressed tolterodine tartarate samples (a-ii). b Comparison of acid degradation study under reux (b-i) and in a microwave oven (b-ii). c Comparison of peroxide degradation study at room temperature (c-i) and in a microwave oven (c-ii). d Comparison of base degradation study under reux (d-i) and in a microwave oven (d-ii). e Comparison of water degradation study under reux (e-i) and in a microwave oven (e-ii)

Minor degradation was observed in tolterodine tartarate stressed samples that were subjected to acid (*0.5%), water hydrolysis (0.2%), oxidative conditions (0.6%) and no degradation was observed under light and heat study. The degradation of drug substance was observed under base hydrolysis (*35%) (Fig. 2). Peak purity test results, derived from PDA detector, conrmed that the tolterodine peak was homogeneous and pure in all the analysed stress samples. Peak purity results for stressed tolterodine tartarate samples, using PDA detector, conrm that tolterodine, imp1, imp-2 and imp-3 peaks were homogeneous and pure. No degradation

AU

3.713 4.145 4.321 4.583 4.724

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19.730

10.774

6.309 6.846

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products peaks were observed after 30 min in the extended runtime of 100 min for all the tolterodine tartarate stressed samples. The mass balance of stressed samples was close to 99.5% (Table 2). The assay of tolterodine tartarate was unaected in the presence of imp-1, imp-2 and imp-3 and its degradation products conrm the stability indicating power of the developed method.
Results of Microwave-Assisted Forced Degradation Studies

(c)-i

0.08 0.06

Toltirodine - 5.212

0.10

Tolterodine tartrate stressed with 6% H2O2 for 48 h

AU
0.04
4.023 4.484

0.02 0.00 2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time (min)

(c)-ii

Minor degradation was observed in tolterodine tartarate stressed samples that were subjected to acid (*0.2%), water hydrolysis (*0.6%) and oxidative conditions (*0.3%). The degradation of drug substance was observed under base hydrolysis (*32%) (Fig. 2). Peak purity results for stressed tolterodine tartarate samples, using PDA detector, conrm that tolterodine peak, as well as imp-1, imp-2 and imp-3 peaks were homogeneous and pure in the analysed stress samples. No degradation products peaks were observed after 30 min in the extended runtime of 100 min for all the tolterodine tartarate stressed samples. The mass balance of stressed samples was close to 99.4% (Table 2). The assay of tolterodine tartarate was unaected in the presence of imp-1, imp-2 and imp-3 and its degradation products conrm the stability indicating power of the developed method.
Linearity and Range

Toltirodine - 5.182

0.10 0.08 0.06

Tolterodine tartrate stressed with 6% H2O2 in a microwave oven for 5 min

AU

0.04 0.02 0.00 2.00

4.029 Imp-1 - 4.482 4.652

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

9.995

7.518

Time (min)

3.267

(d)-i

Toltirodine - 5.216

0.10 0.08 0.06

Tolterodine tartrate stressed with 1N NaOH for 12 h at 80C

AU
0.04
3.801 3.585 4.471 4.972

0.02 0.00
2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

9.648

Calibration curve obtained by the least squares regression analysis between average peak area and the concentration showed linear relationship with a regression coecient of 0.999 over the calibration ranges tested, i.e. 50 150 lg mL-1 for assay calculation. The best-t linear equation obtained was y = 31114x - 57240. At all concentration levels, standard deviation of peak area was signicantly low and RSD was below 0.7%. Analysis of residuals indicated that residuals were scattered within 2% with respect to 100% concentra-

Time (min)

3.255

(d)-ii

Toltirodine - 5.158

0.10 0.08 0.06

Tolterodine tartrate stressed with 1N NaOH in a microwave oven for 5 min

AU

3.590 4.061 Imp-1 - 4.471

0.04 0.02 0.00

2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Fig. 2. continued

Time (min)

15.163

15.167

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(e)-i
0.08 0.06

Toltirodine - 5.206

0.10

Tolterodine tartrate stressed with water reflux for 12 h

0.04 0.02 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Time (min)

summarised that there was no signicant interference of excipients and the method was found to be accurate. The recovery study indicated that the method was suitable for the determination of tolterodine tartarate and its three related impurities in drug substances and drug products. An LC chromatogram of the spiked sample at 0.15% level of three impurities in tolterodine tartarate bulk drug sample are shown in Fig. 2.
Robustness

AU

(e)-ii

Toltirodine - 5.220

0.10 0.08 0.06

Tolterodine tartrate stressed with water in a microwave oven for 5 min

15.217

3.749 3.998 4.283 4.544

0.04 0.02 0.00 2.00 4.00 6.00 8.00 10. 00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
4.318 4.436 - 4.557 Imp-1

Time (min)

In all the deliberate varied chromatographic conditions carried out (ow rate, pH mobile phase organic ratio and column temperature), the resolution between closely eluting impurities, namely imp-1, tolterodine peaks, imp-2 and imp3 was >2, illustrating the robustness of the method.
Solution Stability and Mobile Phase Stability

AU

Fig. 2. continued

3.665

tion response. The %Y intercept was also within the limit (2% with respect to 100% area response). The points in the sensitivity graph were scattered within 2% with respect to 100% concentration response. Linear calibration plot for the related substance method was obtained over the calibration ranges tested, i.e. LOQ to 0.3% for imp-1, imp-2 and imp-3. The correlation coecient obtained was greater than 0.999. The best-t linear equation obtained for imp-1 was y = 499.46x - 127.34, for imp-2 y = 531.69x + 334.53 and for imp-3 y = 539.17x + 235.96.
Precision

The %RSD for the repeatability study with six individual assay preparations was 1.6%. For the intermediate precision the %RSDs were within 1.2%, conrming the ruggedness of the method.
Sensitivity

For repeatability (intra-day) six individual preparations of tolterodine tartarate (500 lg mL-1) spiked with 0.15% each, of imp-1, imp-2 and imp-3 were injected; the measured response demonstrated that the method was repeatable with RSDs below 0.8%. In the intermediate precision, %RSD for imp-1, imp-2 and imp-3 in the related substance method precision study were within 1.1% and the resolution between imp-1 and tolterodine is >3.0 conrming the good precision of the method.

The limit of detection for imp-1, imp-2 and imp-3 were 0.0045, 0.009 and 0.0019 lg (of analyte concentration, i.e. 500 lg mL-1), respectively, for 10 lL injection volume. The limit of quantication for imp-1, imp-2 and imp-3 were 0.0175, 0.036 and 0.0755 lg (of analyte concentration, i.e. 500 lg mL-1), respectively, for 10 lL injection volume. Upon repeated injections at quantitation limit, the peak properties (retention time, peak area) were not aected and mean absolute recovery was consistently high (98 103%) and acceptable with low %RSD (0.8%) Thus, the method was found to be highly sensitive.
Accuracy

The %RSD of the assay of tolterodine tartarate during solution stability and mobile phase stability experiments was within 1%. No signicant changes were observed in the content of imp-1, imp-2 and imp-3 during solution stability and mobile phase stability experiments when performed using the related substances method. The solution stability and mobile phase stability experiments data conrms that sample solutions and mobile phase used during assay and related substance determination were stable up to 48 h of the study period.

Conclusion
A new, sensitive and stability indicating isocratic RP-LC method was successfully developed for quantitative and related substance determination of tolterodine tartarate in both bulk drugs and pharmaceutical dosage forms. For the rst time, microwave stress technique was successfully used to demonstrate forced degradation study. The new microwave-assisted degradation technique was found to be fast, eective and accurate as compared to the conventional reuxing method of forced deg-

The method showed a consistent and high absolute recoveries spiking method. The percentage recovery of imp-1, imp-2 and imp-3 in bulk drugs samples ranged from 94.5 to 103.0. Thus, it can be

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radation. The method was found to be accurate and precise with good and consistent recoveries at all levels studied. Further, it has demonstrated high resolution from degradation products, impurities and formulation excipients. The validated method may be used for the routine analysis of tolterodine tartarate from bulk drugs, pharmaceutical preparations and other quality control samples of product development.

Acknowledgments
The authors wish to thank the management of Gensen Laboratories for supporting this work.

References
1. Nilvebrant L, Glas G, Jonsson A, Sparf B (1994) Neurourol Urodyn 13:433435

2. Nilvebrant L, Stahl M, Andersson K-E (1995) Neurourol Urodyn 14:523524 3. Botteghi C, Corrias T, Maschetti M, Paganelli S, Piccolo O (2002) Org Process Res Dev 6:379383. doi:10.1021/op0 20014k 4. International Conference on Harmonization (2003) Stability testing of new drug substances and products Q1A (R2). International Conference on Harmonization, IFPMA, Geneva 5. Singh S, Bakshi M (2000) Pharm Tech Online 24:114 6. International Conference on Harmonization (1996) Photo stability testing of new drug substances and products Q1B. International Conference on Harmonization, IFPMA, Geneva 7. Bakshi M, Singh S (2002) J Pharm Biomed Anal 28:10111040. doi:10.1016/ S0731-7085(02)00047-X 8. Singh S, Singh B, Bahuguna R, Wadhwa L, Saxena R (2006) J Pharm Biomed Anal 41:10371040. doi:10.1016/j.jpba.2006. 01.030 9. Carstensen JT, Rhodes CT (eds) (2002) Drug stability principles and practices, 3rd edn. Marcel Dekker, New York 10. Ravindra Kumar Y, Ramulu G, Vevakanand VV, Vaidyanathan G, Keesari Srinivas, Kishore Kumar M, Mukkanti

11. 12. 13. 14.

15.

K, Satyanarayana Reddy M, Venkatraman S, Suryanarayana MV (2004) J Pharm Biomed Anal 35:12791285. doi: 10.1016/j.jpba.2004.03.026 Zhang B, Zhang Z, Tian Y, Xu F (2005) J Chromatogr B 824:9298. doi:10.1016/j. jchromb.2005.07.010 Bjorkman HT, Edlund P-O, Jacobsson SP (2002) Anal Chim Acta 468:263274. doi:10.1016/S0003-2670(02)00569-X Swart R, Koivisto P, Markides KE (1999) J Chromatogr B 736:247253. doi: 10.1016/S0378-4347(99)00462-4 Palmer L, Andersson L, Andersson T, Stenberg U (1997) J Pharm Biomed Anal 16:155165. doi:10.1016/S0731-7085(97) 00023-X Swart R, Koivisto P, Markides KE (1998) J Chromatogr A 828:209218. doi: 10.1016/S0021-9673(98)00788-2

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