INTRODUCTION:

Tolterodine, is chemically 2-[(1R)-3-[bis(propan-2-yl)amino]-1-phenylpropyl]-4-methylphenol. Tolterodine is a potent and competitive muscarinic receptor antagonist used for the treatment of urinary incontinence (incontinence in detrusor instability) and other overactive bladder symptoms, such as urgency and high micturition frequency. The drug also increases functional bladder volume. The drug blocks muscarinic receptors, which can be found on the muscle cell of the bladder wall. Stimulation of these receptors causes the bladder to contract and empty when these receptors are blocked the muscle of the bladder wall contracts less. Tolterodine acts on M1, M2, M3, M4 and M5 subtypes of muscarinic receptors whereas modern anti muscarinic treatments for overactive bladder only act on M3 receptors making them more selective[1]. Some analytical methods which include UV[2], visible spectrophotometric[3,4], HPLC[5-12] and UPLC[13], have been reported in the literature for the determination of Tolterodine in biological fluids and in pharmaceutical preparations. The developed method was validated as per ICH guidelines[14].

MATERIALS AND METHODS:

The spectrophotometric measurements were carried out using a Shimadzu UV-1700 UV/Vis spectrophotometer with 1cm matched quartz cell and Shimadzu ELB 300 analytical balance, Tolterodine pure drug (99.95%) was obtained as a gift sample from Dr. Reddy’s Laboratories Pvt Ltd (Hyderabad, India). All chemicals and reagents used were of analytical grade. Formulation used for studies was developed by Dr. Reddy’s Laboratories Pvt Ltd.

Preparation of Standard solution:

Standard drug of Tolterodine was proposed by dissolving 10mg pure Tolterodine in 0.1N NaOH and transferred into 10ml volumetric flask to obtain 1000μg/ml of stock solution. The standard solution of Tolterodine having concentration of 100μg/ml was scanned in UV range (200-400nm) in 1.0 cm cell against in solvent as blank and spectrum was obtained.

Determination of λmax:

100μg/ml of Tolterodine was prepared and scanned in UV range of 200-400nm and spectrum was obtained. The λmax was found to be at 280nm wavelength where absorbance was found maximum at this wavelength. Hence it is considered as absorbance maxima (λmax).

Preparation of calibration curve:

Standard stock solution was suitably diluted with 0.1N NaOH to obtain concentrations ranging from 40-180μg/ml. Absorbance of these solutions was measured at 280nm. Calibration curve was obtained by plotting graph between concentration and absorbance.

Preparation of test solution:

20 Tablets were weighed and its average weight was determined. An accurately weighed tablet powder equivalent to 10mg of Tolterodine transferred into 100ml volumetric flask dissolved in 0.1N NaOH, sonicated for 10min and volume was made up to the mark. Solution was filtered using whattman filter paper (No.41) to obtain 40μg/ml stock solution.

METHOD VALIDATION:

Linearity:

The absorbances were observed from 40 to 180μg/ml and were shown in Table-1. Linearity was obtained between 40 to 180μg/ml. Concentration graph was plotted for concentration and absorbance. The equation of calibration curve obtained was y = 0.006x – 0.003. The correlation coefficient (r) was found to be 0.999.

Table 1: Calibration data for analysis of Tolterodine in 0.1N NaOH at λmax = 280nm

S.No.	Concentration (µg/ml)	Mean Absorbance (+ SD)*
1	40	0.242 (+ 0.0006)
2	60	0.363 (+ 0.001)
3	80	0.479 (+ 0.0015)
4	100	0.593 (+ 0.0012)
5	120	0.719 (+ 0.0015)
6	140	0.846 (+ 0.0021)
7	160	0.967 (+ 0.0015)
8	180	1.088 (+ 0.0012)

*n=3 (Average of 3 determinations)

Accuracy:

To determine the accuracy of the method recovery was performed by standard addition method. To pre-analyzed sample known amount of standard Tolterodine was spiked in different concentrations. The recovery was performed at three levels 80%, 100% and 120% of standard Tolterodine. The solutions were prepared in triplicate and the accuracy was indicated by % Recovery.

Precision:

Intraday and Interday Precision:

The concentration of 80μg/ml, 100μg/ml and 120μg/ml of Tolterodine (on label claim basis) was taken. The absorbance of the final solution was read after 0hr, 12hr and 24hr in 1.0 cm cell at selected wavelength. Similarly the absorbance of the same solutions was read on 1^st, 2^nd and 3^rd day. All the solutions are prepared triplicate and analyzed.

Ruggedness:

It was carried out by analyzing the sample by two analysts and estimation of drug by proposed methods. The % RSD was calculated.

RESULTS AND DISCUSSIONS:

Attempt has been made to develop rapid, sensitive, economic, precise and accurate analytical method for Tolterodine in pure and pharmaceutical dosage form. The proposed method is based on UV Spectrophotometric absorption in UV region using 0.1N NaOH as solvent. Maximum absorbance was found to be at 280nm and is shown in Figure: 1. Beer’s law was obeyed in concentrations ranging from 40 to 180μg/ml is incorporated in Table: 1. The correlation coefficient values were above 0.999 which shows that absorbance was linear with concentration (Figure: 2). The %Recovery studies were performed at 80%, 100% and 120% and was represented in Table: 2. The assay of the two brands were performed and the results incorporated in Table: 3. LOD and LOQ were found to be 0.715μg/ml and 2.167μg/ml (Table: 4). Precision of the method was confirmed by Intraday and Interday analysis, %RSD values were represented in Table: 5 and 6. The ruggedness parameter was performed between two analysts and %RSD was found 0.27 and 0.09 (Table: 7). The optical characteristics such as Beer’s law limit, correlation coefficient, slope, intercept, molar absorptivity, scandell’s sensitivity were calculated and validated (Table: 8). The method was validated and found to be simple, sensitive, accurate and precise. Hence the proposed method could be effectively adopted for routine quality control of Tolterodine in bulk and formulated tablet dosage form.

Fig - 1: UV Spectrum of Tolterodine in 0.1N NaOH at λmax = 280nm

Fig - 2: Calibration curve of Tolterodine in 0.1N NaOH showing linearity relationship

Table-3: Results of analysis of laboratory samples (Assay)

Sample	Label	Amount found	% Purity + RSD*
Brand-1 Tolterodine	4mg	4.01mg	99.79 + 0.77
Brand-2 Tolterodine	4mg	4.02mg	99.90 + 0.59

*n=3 (Average of 3 determinations)

Table-4: Lowest Limit of detection and Lowest Limit of quantification

LOD (µg/ml)	LOQ (µg/ml)
0.715	2.167

Table-5: Results of Intraday Precision of Tolterodine in 0.1N NaOH

Parameter	% Recovery Estimated (Mean + RSD)*
Parameter	80 (µg/ml)	100 (µg/ml)	120 (µg/ml)
At 0 hr	99.26 + 0.12	99.09 + 0.17	99.41 + 0.12
At 12 hr	99.33 + 0.10	99.29 + 0.24	99.23 + 0.07
At 24 hr	99.39 + 0.22	99.24 + 0.15	99.47 + 0.07

*n=3 (Average of 3 determinations)

Table-6: Results of Inter-day Precision of Tolterodine in 0.1N NaOH

Parameter	% Recovery Estimated (Mean + RSD)*
Parameter	80 (µg/ml)	100 (µg/ml)	120 (µg/ml)
Day-1	99.46 + 0.12	99.18 + 0.34	99.37 + 0.19
Day-2	99.73 + 0.22	99.29 + 0.08	99.51 + 0.08
Day-3	99.48 + 0.23	99.13 + 0.24	99.32 + 0.23

*n=3 (Average of 3 determinations)

Table-7: Results of Ruggedness of Tolterodine in 0.1N NaOH

Ruggedness	% Purity + RSD*
Analyst – 1	100.07 + 0.27
Analyst – 2	99.97 + 0.09

*n=3 (Average of 3 determinations)

Table-8: Validation Parameters

Parameters	Results
Absorption maxima λmax (nm)	280
Beer’s law limit (µg/ml)	40-180
Molar Absorptivity (L mole^-1, cms^-1)	1.9302 x 10³
Sandell’s sensitivity (µg/cm2/0.001)	0.1686
Correlation coefficient	0.999
Regression equation	y = 0.006x - 0.003
Limit of detection	0.715
Limit of quantification	2.167
Precision(% RSD)	0.22

CONCLUSION:

The spectrophotometric method being reported for the assay of Tolterodine in pure form and also in its formulations is simple and inexpensive. The proposed method was found to be simple, sensitive, accurate and with good precision. Thus, this approach could be considered for the analysis of this drug in the quality control laboratories.

ACKNOWLEDGEMENTS:

The authors are thankful to Dr. Reddy’s Laboratories Pvt. Ltd. for providing standard drug samples and also to Malla Reddy College of Pharmacy, for providing the facilities to carry out the work.

REFERENCES:

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Received on 07.08.2013 Accepted on 05.09.2013

Asian J. Pharm. Ana. 3(3): July-Sept. 2013; Page 102-104

Ingredient	Amount of drug from formulation	Amount of standard added	Percentage added	Amount added (µg/ml)	Amount found (µg/ml)	% Recovery (Mean + RSD)*
Tolterodine	100µg	80µg	80%	79.60	80.28	100.85 + 0.21
Tolterodine	100µg	100µg	100%	99.49	99.39	99.89 + 0.10
Tolterodine	100µg	120µg	120%	119.38	119.81	100.35 + 0.36