Assay of Tolterodine Tartrate in Bulk and Capsule Formulations by using Simple and Affordable Visible Spectrophotometric Methods

U. Viplava Prasad¹, M. Syam Bab¹*, B. Kalyana Ramu²,

¹Department of Organic Chemistry and Analysis of Foods Drugs and water Laboratories, AU College of Science and Technology, Andhra University, Visakhapatnam -530003 Andhra Pradesh, India

²Department of Chemistry, Maharajah’s College (Aided and Autonomous), Vizianagaram-535002 (AP) India.

ABSTRACT:

Two simple, sensitive and affordable visible spectrophotometric methods (M₁ and M₂) have been developed for the estimation of tolterodine tartrate (TT) in bulk and dosage forms. Method M₁ involves Internal salt formation of aconitic anhydride, dehydration product of citric acid [CIA] with acetic anhydride [Ac₂O] to form colored chromogen with an absorption maximum of 565 nm and the method M₂ is based on the formation of green colored coordination complex by the drug with cobalt thiocyanate which is quantitatively extractable into nitro benzene with an absorption maximum of 625 nm. Beer’s law obeyed in the concentration range of 8-24µg/ml for method M₁ and 16-48 µg/ml for method M₂. Commercial available capsules were analyzed and the results are statistically compared with those obtained by the reference UV method and validated by recovery studies. The results are found satisfactory and reproducible. These methods are applied successfully for the estimation of the tolterodine tartrate in the presence of other ingredients that are usually present in dosage forms. These methods offer the advantages of rapidity, simplicity and sensitivity and normal cost and can be easily applied to resource-poor settings without the need for expensive instrumentation and reagents.

Keywords: Assay, Acetic anhydride, Citric acid, Cobalt thiocyanate, Beer’s Law, Nitro benzene, Capsules.

INTRODUCTION:

Tolterodine tartrate (TT), chemically, (R)-N,N-diisopropyl-3-(2-hydroxy-5-methyl phenyl)-3-phenyl-propanamine L-hydrogen tartrate (Fig.1) 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 (TLD) 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. The drug exists in two isomeric forms (R) and (S) and its empirical formula and molecular weight are C₂₆H₃₇NO₇ and 475.6 respectively. TT is a white crystalline powder, soluble in water, methanol, slightly soluble in ethanol and practically insoluble in toluene. The drug is listed in the Merck Index ¹ but the drug is not yet official in any Pharmacopoeia.

Figure 1: Showing the Chemical structure of TT

Tolterodine has a high affinity and specificity for muscarinic receptors in vitro and exhibits the selectivity for the urinary bladder over salivary glands in vivo, so it has the advantageous tolerability profile in terms of the low frequency of bothersome dry mouth. The drug undergoes immediate and extensive first-pass hepatic metabolism, mainly by way of CYP 2D6-mediated oxidation and CYP 3A4-mediated N-dealkylation After oral administration, TLD is metabolized in liver by way of cytochrome P450 2D6 (CYP 2D6)-mediated oxidation, resulting in the formation of the 5- hydroxymethyl derivative, a major pharmacologically active metabolite. It is the product of the predominating CYP 3A4 pathway and is pharmacologically equipotent with TLD.

Some analytical methods which include HPLC^2-9, HPLC-ESI-MS¹⁰, UPLC¹¹, GC-MS¹², LC- MS-MS ^13-16, UV ¹⁷ and visible spectrophotometric ^18-19 have been reported in the literature for the determination of TT in biological fluids and in pharmaceutical preparations. The main purpose of the present study was to establish a relatively simple, sensitive and validated visible spectrophotometric method for the determination of TT in pure form and in pharmaceutical dosage forms, since most of the previous methods involve sophisticated equipments which are costly and pose problems of maintenance. The authors have made some attempts in this direction and succeeded in developing two methods based on the reaction between the drug and citric acid-acetic anhydride reagent ²⁰ (M₁) or drug and cobalt thiocyanate ²¹ (M₂). These methods can be extended for the routine assay of TT formulations.

MATERIALS AND METHODS:

Apparatus and chemicals:

A Milton Roy UV/Visible spectrophotometer model-1201 with 10mm matched quartz cells was used for all spectral measurements. Systronics model-362 pH meter was used for all the pH measurements. All the chemicals used were of analytical grade. Citric acid monohydrate (Prepared by dissolving 1.2 grams of (1.2%, 6.245X10^-2M) Citric acid in 5 ml methanol initially followed by dilution up to 100ml with acetic anhydride) and Acetic anhydride (SD Fine chemicals), CTC (2.50x10^-1M, solution prepared by dissolving 7.25 g of cobalt nitrate and 3.8 g of ammonium thiocyanate in 100ml distilled water), Citrate buffer pH(2.0) (prepared by mixing 306ml of 0.1M tri sodium citrate with 694ml of 0.1M HCl and pH was adjusted to 2.0) were prepared .

Preparation of Standard and sample drug stock solution:

An accurately weighed quantity of TT (pure or tablet powder) equivalent to 100mg was mixed with 5ml of 10% Na₂CO₃ solution and transferred into 125ml separating funnel. The freebase released was extracted with 3x15ml portion of chloroform and the combined chloroform layer was brought up to 100ml with the same solvent to get 1mg/ml TT drug stock solution in free base form. This free base stock solution was further diluted step wise with the same solvent to get the working standard solution concentrations [M₁-200 µg/ml, M₂-400 µg/ml].

Determination of wavelength maximum (λ _max)

Method M₁:

The 3.0 ml of working standard solution of TT (200µg/ml) (free base form) in chloroform was taken in 25ml standard flask and gently evaporated in a boiling water bath to dryness. To this, 10ml of citric acid- Acetic anhydride reagent was added and the tubes were immersed in a boiling water bath for 30 minutes then the tubes were cooled to room temperature and made up to the mark with acetic anhydride and sonicated for 1 min. to get a concentration of 24µg/ml. In order to investigate the wavelength maximum, the above standard stock solution was scanned in the range of 400-660nm by UV-Visible spectrophotometer. From the spectra (Fig.2), it was concluded that 565nm is the most appropriate wavelength for analyzing TT with suitable sensitivity.

Fig.2: Absorption spectra of TT-CiA/Ac₂O

Method M_2:

The 3.0 ml of working standard solution of TT (400µg/ml) (free base form) in chloroform was taken in 125 ml separating funnel. Then 2.0ml of buffer solution (pH 2.0) and 5.0ml CTC solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. To separating funnel 10.0ml of nitrobenzene was added and contents were shaken for 2 minutes, to get a concentration of 48µg/ml. The two phases were allowed to separate In order to investigate the wavelength maximum, the parrot green colored nitro benzene solution was scanned in the range of 400-700nm by UV-Visible spectrophotometer. From the UV spectra (Fig.3), it was concluded that 625nm is the most appropriate wavelength for analyzing TT with suitable sensitivity.

Fig.3 : Absorption spectra of TT-CTC

Preparation of calibration curve:

Method M_1:

Aliquots of standard TT drug solution [1.0-3.0ml;200µg/ml in free base form] in chloroform were taken into a series of 25ml graduated tubes and gently evaporated in a boiling water bath to dryness. To this, 10ml of citric acid- Acetic anhydride reagent was added and the tubes were immersed in a boiling water bath for 30 minutes then the tubes were cooled to room temperature and made up to the mark with acetic anhydride. The absorbance of the colored solutions was measured after 15minutes at 565 nm against the reagent blank (within the stability period of 15-60min.The amount of TT was computed from its calibration graph (Fig-4 showing Beer’s law plot).

Fig.4: Beer’s Law Plot of TT-CiA/Ac2O

Method M₂:

Aliquots of standard TT solution (1.0ml - 3.0ml, 400µg/ml in free base form) were delivered into a series of 125ml separating funnels. Then 2.0ml of buffer solution (pH 2.0) and 5.0ml CTC solution were added. The total volume of aqueous phase in each separating funnel was adjusted to 15.0ml with distilled water. To each separating funnel 10.0ml of nitrobenzene was added and contents were shaken for 2 minutes. The two phases were allowed to separate and absorbance of nitrobenzene layer was measured at 625nm against a similar reagent blank .The colored product was stable for 1 hour. The amount of TT in the sample solution was computed from its calibration graph (Fig-5 showing Beer’s law plot).

Fig.5: Beer’s Law Plot of TT-CTC

RESULTS AND DISCUSSION:

In developing these methods, systematic studies of the effects of various parameters were undertaken by varying one parameter at a time and controlling all others fixed (OVAT method). The effect of various parameters such as time, volume and strength of reagents, pH buffer solution and order of addition of reagents, stability period and solvent for final dilution of the colored species were studied and the optimum conditions were established. Among the various water immiscible organic solvents (C₆H₆, CHCl₃, dichloro methane, nitro benzene, chloro benzene and CCl₄) tested for the extraction of colored coordinate complex into organic layer, nitrobenzene was preferred for selective extraction of colored complex from organic phase in method M₂. Different solvents like acetic anhydride, acetic acid, methanol, ethanol and isopropanol were also used as diluents but acetic anhydride was found to be ideal for final dilution in method M₁. The ratio of organic to aqueous phase was found to be 1:1.5 by slope ratio method for method M₂. The optical characteristics such as Beer’s law limit, Sandell‘s sensitivity, molar absorptivity, percent relative standard deviation, (calculated from the six measurements containing 3/4^th of the amount of the upper Beer’s law limits ) were calculated and the results are summarized in Table-1.

Table - 1 Optical characteristics, precision and accuracy of the proposed methods

Parameters	Method A	Method B
λ max(nm)	565	625
Beer’s law limit (µg/ml)	8- 24	16-48
Sandell’s sensitivity (µg/cm^2/0.001 abs. unit)	0.002612245	0.00522449
Molar absorptivity (Liter/mole/cm)	182065.625	91032.8125
Regression equation (Y) ^*= a +b x
Intercept (a)	-0.075	-0.084
Slope(b)	0.02	0.010
%RSD	1.28	1.27
% Range of errors (95% Confidence limits) 0.05 significance level 0.01 significance level	1.34	1.33
	2.10	2.08

*Y = a + b x, where Y is the absorbance and x is the concentration of TT in µg/ml

Commercial formulations containing TT were successfully analyzed by the proposed methods. The values obtained by the proposed and reference methods for formulations were compared statistically by the t-and F-test and found not to differ significantly. As an additional demonstration of accuracy, recovery experiments were performed by adding a fixed amount of the drug to the pre analyzed formulations at three different concentration levels. These results are summarized in Table-2. Recovery experiments indicated the absence of interference from the commonly encountered pharmaceutical excipients present in formulations. The proposed methods are found to be simple, sensitive and accurate and can be used for the routine quality control analysis of TT in bulk and dosage forms.

Chemistry of colored species:

In method M₂ the green color species formation is the coordination complex of the drug (electron donor) and the central metal of cobalt thiocyanate, which is extractable into nitro benzene from aqueous solution and in method M₁ red-violet color internal salt of aconitic anhydride is formed when TT was treated with CTC or CIA/Ac₂O reagents. The formations of colored species are due to the presence of the tertiary amino group in it. It is based on the analogy of tertiary amine as given in scheme (Fig-6).

Fig. 6: Probable Schemes for methods M₁ and M₂

Table-2 Analysis of TT in pharmaceutical formulations:

Method	*Formulations	Labeled Amount (mg)	Found by Proposed Methods			Found by Reference Method ± SD	#% Recovery by Proposed Method ± SD
			**Amount found ± SD	t	F
M1	Batch-1	2	1.98±0.0093	1.22	4.00	1.99 ±0.005	99.30±0.46
	Batch-2	4	3.97±0.016	2.0	4.63	3.99 ± 0.0073	99.25±0.39
M2	Batch-1	2	1.97±0.009	2.93	3.83	1.99 ±0.005	98.76 ± 0.45
	Batch-2	4	3.99 ± 0.008	1.92	1.26	3.99 ± 0.0073	99.78 ± 0.21

* Batch-1 and Batch- 2 extended release capsules of two different companies (TEROL LA-2 of Cipla Ltd and TORQ SR 4 of Dr Reddy’s)

**Average ± Standard deviation of six determinations, the t- and f-values refer to comparison of the proposed method with UV reference method. Theoretical values at 95% confidence limits t =2.57 and f = 5.05. # Recovery of 10mg added to the pre analyzed sample (average of three determinations). Reference method (reported UV method) using distilled water (גּ _max=281.5nm).

CONCLUSION:

The reagents utilized in the proposed methods are normal cost, readily available and the procedures do not involve any critical reaction conditions or tedious sample preparation. The proposed visible spectrophotometric methods are validated as per ICH guide lines and possess reasonable precision, accuracy, simple, sensitive and can be used as alternative methods to the reported ones for the routine determination of TT depending on the need and situation.

ACKNOWLEDGEMENT:

The authors (MS Bab and BKR) are thanks to the University Grants Commission, New Delhi for providing financial assistance under teacher fellow ship and also thanks to University authorities for providing necessary facilities in this work.

REFERENCES:

1. The Merck Index, 13^th ed. Merck White House Station, 2001:1699.

2. Vinay S, Zahid Z, Mazhar F. Stability indicating HPLC determination of tolterodine tartrate in pharmaceutical dosage form. Indian J chem. Technol. 2006; 13(3): 242-246.

3. Krishna SR, Rao BM, Rao NS. A validated stability-indicating HPLC method for the determination of related substance and assay of tolterodine tartrate. Rasayan J Chem 2009; 2(1): 144-150.

4. Dwibhashyam VS, Keerthi P, Ratna JV, Nagappa AN. RP-HPLC method for the determination of tolterodine tartrate in routine quality control sample. PDA J Pharm. Sci. Technol. 2009; 63(3): 234-239.

5. Xia ZL, Chen ZY, Yao TW. An enantio specific HPLC method for the determination of (S)-enantiomer impurities in (R)-tolterodine tartrate. Pharmazie 2007; 62: 170-173.

6. Kumar YR, Ramulu G, Vevakanand VV, Vaidyanathan G, Srinivas K, Kumar MK, Mukkanti K. A validated chiral HPLC method for the enantiomeric separation of tolterodine tartrae. J Pharm. Biomed Anal 2004; 35: 1279-1285.

7. Madhavi A, Reddy GS, Suryanarayana MV, Naidu A. Development and validation of a new analytical method for the determination of related components in tolterodine tartrate using LC. Chromatographia 2008; 68: 399-407.

8. Sinha VR, Jindal V, Kumar RV, Bhinge JR, Goel H. Development and validation of a simple stability-indicating HPLC method for analysis of tolterodine tartrate in the bulk drug and in its tablet formulation. Acta Chromatogr 2011; 23: 133-143.

9. Shetty SK, Shah A. Development and validation of tolterodine by RP-HPLC method in bulk drug and pharmaceutical dosage forms. Int. J Pharm. Tech Res 2011; 3: 1083-1087.

10. Zhang B, Zhang Z, Tian Y, Xu F. HPLC-ESI-MS determination of tolterodine tartrate in human plasma. J Chromatogr B. 2005; 824(1-2): 92-98.

11. Ramesh Y, Chandra Sekhar V, Umamaheshwar P, Balaram P, Murthy YLN, Atchuta Ramaiah P. A new rapid and sensitive stability-indicating UPLC assay method for tolterodine tartrate: Application in pharmaceuticals, human plasma and urine samples. Scientia Pharmaceutica 2012; 80: 101-114.

12. Palmer L, Anderson L, Anderson T, Stenberg U. Determination of tolterodine and the 5-hydroxymethyl metabolite in plasma, serum and urine using gas chromatography-mass spectrometry. J Pharm. Biomed Anal 1997; 16: 155-165.

13. Swart R, Koivisto P, Markides KE. Capillary solid-phase extraction-tandem mass spectrophotometry for fast quantification of free concentrations of tolterodine and two metabolites in ultra filtered plasma samples. J Chromatogr B 1999; 736: 247-253.

14. Swart R, Koivisto P, Markides KE. Column switching in capillary liquid chromatography-tandem mass spectrometry for the quantitation of pg/ml concentrations of the free basic drug tolterodine and its active 5-hydroxy methyl metabolite in microlitre volumes of plasma. J Chromatogr A 1998; 828: 209-218.

15. Manish Yadav VU, Chauhan V, Solanki G, Jani A, Baxi GA, Singhal P, Shrivastav PS. LC-MS-MS separation and simoultaneous determination of tolterodine and its active metabolite, 5-hydroxymethyl tolterodine in human plasma. Chromatographia 2010; 72(3/4): 255-264.

16. Jan Macek, Pavel Ptacek, Josef Klima. Determination of tolterodine and its 5-hydroxymethyl metabolite in human plasma by hydrophilic interation liquid chromatography-tandem mass spectrometry. Journal of Chromaography B 2009; 877: 968-974.

17. Shetty SK, Shah A. New spectrophotometric method for estimation of tolteridone in bulk and pharmaceutical formulation. International Journal of Pharmaceutical Sciences and Research 2011; 2(6); 1456-1458.

18. Mohammed Ishaq B, Vanitha Prakash K, Manjula B, Hari kumar C, Usha Rani G. New Aurum coupling reaction for visible spectrophotometric determination of tolterodine in pharmaceutical preparations. International Journal of Chemical and Analytical Science 2010; 1(7): 165-167.

19. Walash MI, Belal F, EI-Enany N and Elmansi E. Determination of tolterodine tartrate in pharmaceutical preparations using Eosin, Application to stability study. International Journal of Pharmaceutical Sciences and Research 2011; 2(11): 2849-2855.

20. Massart DL, Vandegingtc BGM, Perming SM, Michotte Yand Kaufman L, Chemo metrics, A text book, Elsevier, Amsterdam, 1988, 283.

21. Zarapker SS, Rele RV, and Doshi VJ, A simple extractive colorimetric determination of three drugs from pharmaceutical preparations. Indian drugs, 24 (12); 1987: 560-564

Received on 11.06.2012

Accepted on 09.07.2012

© A&V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(4): July-Aug. 2012, 211-215