INTRODUCTION:

Tolterodine tartrate (TOL) is described chemically, 2-[(R)-[3-[bis (1-methylethyl) amino]-1-phenylpropyl]-4-methyl phenol tartrate. It is a muscarinic receptors antagonist and used in the treatment of overactive urinary bladder. Tamsulosin hydrochloride (TAM) belongs to a selective α₁- adrenergic receptor blockers, used for the treatment of benign prostatic hyperplasia and chemically, (R)-5-(2{[2-(o-ethoxyphenoxy) ethyl}] aminopropyl]-2-methoxybenzene sulphonamide hydrochloride¹.

Assay of both the drugs in bulk is official in Indian Pharmacopoeia 2014² and British Pharmacopeia 2016³. Literature survey reveals UV-Visible spectrophotometry^4-11, spectrofluorimetric method¹², HPLC^13-24, LC−MS/MS²⁵ and HPTLC^26-27 methods for both the drugs in alone and combination. However, no stability indicating RP-HPLC method has been reported the simultaneous estimation of TOL and TAM in the formulation. The objective of present research work to develop stability indicating RP-HPLC method for the simultaneous estimation of TOL and TAM in capsule formulation. The developed method was validated according to International Conference on Harmonization (ICH) Q2 (R1) guidelines²⁸. The chemical structure of TOL and TAM were shown in (Figure 1).

Figure 1: Chemical structure of (A) Tolterodine tartrate and (B) Tamsulosin hydrochloride

MATERIALS AND METHOD:

Chemicals and Reagents:

TOL and TAM was procured as a gift sample from Aurobindo Pharma Ltd., Hyderabad, India and Sun Pharmaceutical Ltd., Gujarat, India respectively. The capsule of TOL and TAM was procured from local drug store. Methanol and water of HPLC grade was purchased from S D Fine Chemicals, Mumbai, India and potassium dihydrogen orthophosphate and sodium hydroxide pellets of AR grade was purchased from Suvidhinath Laboratories, Vadodara, India.

Instrument and software:

The HPLC chromatograph (1220 Infinity, Agilent Technologies Ltd., Japan) equipped with gradient pump, auto sampler, diode array detector, thermostatic control column oven and Chem station software was used to perform this study. The drugs were separated on a Hypersil octadecyl silane C₁₈ (250 × 4.6mm, 5μ) column. An electronic balance (AX200, Shimadzu, Ltd., Japan), an ultrasonic cleaner (EIE 808, EIE Instruments Pvt. Ltd., India), a digital pH meter (Toshcon Industries Pvt. Ltd., India) was used in the research work. The calibrated glassware was used in this study.

Preparation of stock and working standard solutions:

Accurately weighed 100mg each of TOL and TAM was transferred to 50ml volumetric flasks separately, dissolved and diluted up to 100mL with mobile phase to obtained 1000μg/mL concentration of each drug. The stock solutions were further diluted with mobile phase to obtained working standard solutions of 100μg/mL of each drug.

Preparation of mobile phase:

Accurately weighed 2.04gm of potassium dihydrogen orthophosphate, transferred and dissolved into 1000mL volumetric flask with water. The pH 7.0 of solution was adjusted with dilute sodium hydroxide. The solution was filtered through 0.45μm nylon filter and degassed. The mobile phase comprised of a mixture of methanol and 0.05 M potassium dihydrogen orthophosphate in the ratio of 90:10 V/V.

Chromatographic conditions:

The RP-HPLC method was carried out with isocratic mode using a mobile phase compositions of methanol: 0.05 M potassium dihydrogen orthophosphate, pH 7.0 (90:10 V/V). The separation of drugs was carried out on Hypersil octadecyl silane C₁₈ (250 × 4.6mm, 5μ) column at room temperature with 1mL/min flow rate and eluents were monitored at 255nm using photodiode array detector. The run time for chromatogram was kept 10 min.

Forced degradation studies:

The forced degradation of each bulk drug and capsule formulation was performed under various stressed conditions like, hydrolysis, oxidative hydrolysis, photolysis and thermolysis. The photolysis and thermolysis degradation of bulk drug and formulation was carried out in solid state. The degraded drug samples were dissolved and diluted with mobile phase to achieved concentration of 20μg/ml of TOL and 2μg/ml of TAM (as per label claim of capsule). Then fixed volume of degraded solutions were injected into chromatograph and analysed under the optimized chromatographic conditions described earlier. Acid hydrolysis of bulk drug and formulation in solution state was performed in 0.1N hydrochloric acid at room temperature for 2 h. Alkali hydrolysis of bulk drug and formulation in solution state was carried out in 0.1N sodium hydroxide at room temperature for 4 h. For oxidative hydrolysis, sample solutions of bulk drug and formulation were performed in 3% hydrogen peroxide at room temperature for 6 h. For thermolysis degradation, samples of bulk drug and formulation were kept in hot air oven at 80ºC for 4 h. The photolytic degradation of solid bulk drug and formulation was carried out under ultra violet light (short wavelength) for 24 h.

Validation of RP - HPLC method:

The developed RP – HPLC method for the simultaneous estimation of TOL and TAM in capsule dosage form was validated as per ICH Q2 (R1) guideline. The method validation parameters like, linearity, range, accuracy, precision, detection limit, quantitation limit and robustness were studied.

Linearity and range:

Fixed standard aliquots of TOL and TAM were transferred into five different volumetric flasks separately and diluted up to 10ml with mobile phase to achieve concentrations in the range of 10-50µg/mL of TOL and 1-5µg/mL of TAM. The fixed volume of sample was injected into the chromatograph with auto sampler and chromatograms of each sample were recorded. Calibration curves were prepared by plotting peak areas versus concentration (n=3) and correlation coefficient and regression equations were calculated for TOL and TAM.

Accuracy:

The recovery experiment was conducted for the determination of accuracy of developed method by spiking known amount of standard drug into pre-quantified sample of 20µg/mL of TOL and 2µg/mL of TAM at three different levels of 50, 100 and 150%.

Precision:

Repeatability of method was checked by injecting the solution of 30µg/mL of TOL and 3μg/mL of TAM into chromatograph for six times (n=6) and analyzed under the chromatographic conditions described earlier and % RSD was calculated. Intraday precision was checked by determining three different concentrations of TOL (10 µg/mL, 30µg/mL and 50µg/mL) and TAM (1µg/mL, 3 µg/mL and 5µg/mL) for three times on a same day and interday precision was checked by determining the same concentrations for three times on a three consecutive days and %RSD was calculated for both the drugs.

Limit of detection and limit of quantitation:

Limit of detection (LOD) and limit of quantitation (LOQ) for RP-HPLC method was calculated by using the equation given in ICH Q2 (R1) guidelines. LOD = 3.3 x σ/S, LOQ = 10 x σ/S; Where, σ = Standard deviation of y intercepts of regression line obtained from calibration curve, S = Mean slope of calibration curve.

Robustness:

Robustness of RP-HPLC method was checked by making small deliberate changes in three chromatographic conditions - change in flow rate of mobile phase, composition of mobile phase and pH of mobile phase. The samples were analysed on chromatograph using optimized chromatographic conditions discussed earlier. The peaks area was measured and the %RSD was calculated for changes in each condition.

System suitability parameters:

The system suitability parameters were carried out on freshly prepared standard solution of drugs. The standard solutions of TOM (10-50µg/mL) and TAM (1-5µg/mL) was injected under optimized chromatographic condition and parameters such as plates, capacity factor, resolution and peak asymmetry were studied to evaluate the suitability of the system.

Assay of marketed formulation:

Total twenty capsules (each capsule containing 4 mg of TOL and 0.4mg of TAM) were weighed, removed capsule shells and finely powdered in the glass mortar. From the fine powder of capsules, the quantity of powder equivalent to 40mg of TOL (equivalent to 4mg of TAM) was transferred into 100ml glass volumetric flask, dissolved and diluted to 100ml with mobile phase to make the solution of 400µg/mL of TOL (40µg/mL of TAM). The stock solution was filtered through 0.45µ nylon filter paper. The filtrate was appropriately diluted to make the final solution of 20µg/mL of TOL (2µg/mL of TAM). The sample solutions were injected into HPLC chromatograph and analysed under optimized chromatographic conditions.

RESULTS AND DISCUSSION:

RP-HPLC method development and optimization:

In the present work, the selected drug substances having different physicochemical parameters such as solubility, polarity and dissociation constants. The physiochemical parameters of individual drug molecules were studied and suitable analytical wavelength for the simultaneous quantitative estimation of TOL and TAM was determined by using diode array detector. Based on literature and data of physicochemical parameters, various compositions of mobile phases were tried for effective separation of drugs in the presence of degradants. The effect of chromatographic variables such as detection wavelength, stationary phase, mobile phase compositions, pH of mobile phase and flow rate were studied to optimized chromatographic conditions. The well separation of both drugs in presence of its degradants were obtained with mobile phase consisting of methanol and 0.05 M phosphate buffer pH 7.0 (90:10 V/V) at a flow rate of 1.0 mL/min. The both drugs absorbed significantly at 255 nm hence it was selected as a detection wavelength for analysis of drugs. Typical chromatograms of optimized chromatographic conditions of standard drugs were shown in (Figure 2).

Figure 2: Typical HPLC chromatogram of placebo and standard mixture of drugs (Retention time of TAM at 4.15±0.2 min and TOL at 8.42±0.2 min)

Validation of RP-HPLC method:

The developed method was validated as per ICH (Q2-R1) guidelines in terms of linearly and range, precision, accuracy, limit of detection, limit of quantitation, robustness, specificity.

Linearity and range:

Linearity of the method was evaluated by measuring five different concentrations of both the drugs separately. The calibration curve was constructed by plotting concentration against peak areas and the regression analysis was performed. The summary of regression parameters is shown in (Table 1).

Table 1: Regression analysis data for the proposed method

Parameter	TOL	TAM
Detection wavelength (nm)	255	255
Linearity (μg/ml)	10 - 50	1 – 5
Regression equation y = mx + c	y = 38.2x + 295.95	y = 46.653x + 13.043
Slope (m)	38.198	46.653
Intercept (c)	296.952	13.043
Correlation coefficient (r²)	0.9981	0.9988

Accuracy:

Accuracy of developed RP-HPLC was determined by performing percent recovery studies. The % recovery was found within the range indicates that the developed stability indicating RP-HPLC method is accurate. The result of accuracy was tabulated in (Table 2).

Precision:

The mean % RSD for repeatability was found to be 0.45 % and 0.91% for TOL and TAM, respectively. The % RSD value within limits for peaks area indicates that developed method is repeatable. The %RSD value of intra-day precision was found to be 0.66% - 0.92% for TOL and 0.41% - 1.01% for TAM. The %RSD of inter-day precision was found to be 0.67% - 0.97% for TOL and 0.65% - 0.81% for TAM. The % RSD value less than 2 % indicate that the method is precise.

Specificity:

The chromatograms of forced degradation studies and assay results of marketed formulations confirmed that there was no interference of drugs with degradants and excipients of formulation. The 3D chromatograms and peak purities of the stressed samples were performed using photodiode array detector and peak purity data of samples indicate the developed analytical method is specific.

Limit of detection and limit of quantitation:

Limits of detection and quantitation were based on the standard deviation and the slope of the calibration plots. The estimation of LOD and LOQ considered acceptable signal-to-noise ratios 3:1 and 10:1, respectively. LOD of TOL and TAM were found be to 0.147μg/ml and 0.062 μg/ml respectively. LOQ of TOL and TAM were found to be 0.445μg/ml and 0.189μg/ml respectively.

Robustness:

Robustness of the developed method was evaluated by making small deliberate changes in chromatographic variables. Here the changes were made in flow rate, mobile phase composition and pH of mobile phase. The % RSD of TOL was found to be 0.33% – 0.78% for flow rate, 0.46% - 0.80% for mobile phase composition and 0.45% ̶ 0.98% for pH of mobile phase.The % RSD of TAM was found to be 0.17% – 0.91% for flow rate, 0.33 % ̶ 0.68% for mobile phase composition and 0.23% ̶ 0.63% for pH of mobile phase. No significant changes observed in selected variables proved that the developed RP-HPLC-DAD is robust.

System suitability test parameters:

The system suitability of method was checked by evaluating the parameters like, resolution, tailing factor, theoretical plates and reproducibility. The results of system suitability parameters were observed within the specified limits according to ICH guidelines. Results of validation parameters for system suitability are summarized in (Table 3).

Table 2: Data of accuracy study of the proposed method

Analyte	Target concentration (μg/ml)	Amount added (μg/ml)	Total Amount (μg/ml)	% Recovery (Mean ± SD) (n=3)
TOL
50% level	20	10	30	99.59 ± 0.15
100% level	20	20	40	98.76 ± 0.28
150% level	20	30	50	98.54 ± 0.13
TAM
50% level	2	1	3	99.46 ± 0.56
100% level	2	2	4	101.05 ± 0.72
150% level	2	3	5	100.55 ± 0.21

n = average of three determinations: S. D. = standard deviation

Table 3: System suitability data of the proposed method

Parameters	Tam (Mean + SD) (n = 3)	Tol (Mean + SD) (n = 3)
Retention time (min)	4.18 ± 0.0330	8.539 ± 0.081
Theoretical plates (N)	5854 ± 62.385	6608.667 ± 72.14
Tailing factor (T)	0.67 ± 0.01	0.58 ± 0.01
Resolution (α)	5.536 ± 0.094

n = average of three determinations: S. D. = standard deviation

Forced degradation studies:

The forced degradation studies show that both the drugs were susceptible to acid, alkali, oxidation, photolytic and thermolytic conditions. The chromatograms of degraded samples show well separation of drugs from degradants (Figure 3). The 3D chromatograms of different stressed conditions of sample indicate that the drug substances were resolved from impurities as well as degradants (Figure 4). The percent content of drugs after degradation were calculated by comparing the areas of the drugs in each condition with the corresponding peak areas of both the drugs under control condition. The summary of stressed degradation studies was shown in (Table 4).

Figure 3: Chromatograms of acid degradation (A); alkali degradation (B); oxidative degradation (C); Photolytic degradation (D) and thermal degradation (E), where retention time of TAM at 4.15±0.2 min and TOL at 8.42±0.2 min

Figure 4: 3D HPLC chromatograms of acid degradation (A); alkali degradation (B); oxidative degradation (C); Photolytic degradation (D) and thermolytic degradation (E)

Table 4: Summary of forced degradation studies

Description of stress condition	Percent degradation(n=3)		Peak purity index
Description of stress condition	Tam	Tol	TAM	TOL
Acidic/0.1N HCl/2 hrs/solution	17.15	8.12	0.99992	0.99994
Alkali/1N NaOH/ 4 hr/ solution	9.79	17.10	0.99999	0.99999
Photo/under UV light/24 hrs/solid	12.10	18.14	0.99995	0.99995
Oxidative/3% H₂O₂/ 6 hrs/ solution	11.67	9.06	0.99999	0.99995
Thermal/90 °C/ 3 hrs/solid	14.49	16.78	0.99998	0.99992

Assay of marketed formulation:

Application of developed analytical method was checked by analysing the marketed capsule formulation of drugs. The mean percent content of TOL and TAM from calibration curves were found within the range according to specification limits. None of excipients of tablet formulation interfered with the analyte peaks of drug molecules. The %RSD for all method parameters was observed within limits, which indicate the validity of method and percent content of drugs obtained by this method are in limits. The summary of results was shown in (Table 5).

Table 5: Results of analysis of marketed formulation

Analyte	Amount taken (μg/ml)	Amount found (μg/ml) (Mean ± SD) (n=3)	Percent assay (Mean ± SD) (n-3)	% RSD
TOL	20	19.71 ± 0.12	98.57 ± 0.62	0.73
TAM	2	1.99 ± 0.15	99.82 ± 0.78	0.68

n = average of three determinations; SD = standard deviation; RSD = Relative standard deviation

Stability of solution:

The sample and mobile phase stability studies were checked at intervals of 3 hrs, 6 hrs and 2 hrs at room temperature. The peak area of drug sample was measured at particular intervals by using optimized chromatographic conditions. Results show that the sample solution and mobile phase was stable for more than 12 hrs at room temperature.

CONCLUSION:

A specific, accurate and precise stability-indicating RP-HPLC-DAD method was developed for the determination of Tolterodine tartrate and Tamsulosin hydrochloride in the combined pharmaceutical dosage form. The ICH guidelines for analytical method validation parameters suggested that proposed method successfully separated two drugs with its degradants by using photo diode array detector. The developed stability-indicating RP-HPLC-DAD method could be routinely used in the quality control department for the simultaneous quantitation of two drugs.

ACKNOWLEDGEMENTS:

The authors thank Aurobindo Pharma Ltd., Hyderabad, Telangana, India and Pharmaceutical Ltd., Vadodara, Gujarat, India for providing gift sample of Tolterodine tartrate and Tamsulosin hydrochloride, respectively.

CONFLICTS OF INTEREST:

Conflicts of interest is nil from authors.

REFERENCES:

1. Tripathi K. D., Editors. Essentials of Medical Pharmacology, Jaypee Brothers Publishers Ltd, New Delhi, 2003, 6th ed., pp. 135-136.

2. Indian Pharmacopoeia. Govt. of India Vol-III. Ghaziabad: Ministry of Health and Family Welfare, Published by The Indian Pharmacopoeia Commission. 2007; pp. 2891-2892.

3. British Pharmacopoeia; London, British Pharmacopoeia Commission, 2016, 8th ed; pp 1082-1084.

4. Shetty SK, Shah K. New spectrophotometric method for estimation of Tolterodine in bulk and pharmaceutical formulation. International Journal of Pharmaceutical Sciences and Research. 2011; 2(6): 145514-61.

5. Bari SB, et Al. Application of UV-spectroscopy and first order derivative method for determination of tamsulosin hydrochloride in bulk and tablets. Pharmaceutica Analytica Acta. 2011; 2(2): 11-16.

6. Rani GD, at Al. Development and Validation of Ultraviolet-Spectrophotometric Method for the Determination of Tamsulosin. Indian Journal of Advances in Chemical Science. 2015; 3(4): 323-327.

7. Jadhav RS, Bharad JV. Analytical Method Development and Validation for Estimation of Tamsulosin Hydrochloride by UV-Spectroscopic method. International journal of chem tech Research. 2017; 10(5): 740-747.

8. Nanda, RK, et Al. Estimation of Tamsulosin and Tolterodine in its pharmaceutical dosage form by Spectrophotometric method. International journal of chemtech Research. 2009; 1(3): 420-423.

9. Viplava U, et Al. Assay of Tolterodine Tartrate in Bulk and Capsule Formulations by using Simple and Affordable Visible Spectrophotometric Methods. Research Journal of Pharmaceutical Dosage Forms and Technology. 2012; 4(4): 211-215.

10. Prajakta D, et Al. Implementation UV-Spectrophotometry approach for Determination of Tamsulosin HCl in tablets using area under Curve Technique. Asian Journal of pharmaceutical Analysis. 2020; 10(2): 86-90.

11. Susmitha K., et Al. Extractive Spectrophotometric Methods for Determination of Tamsulosin HCl in Pharmaceutical Formulations Using Acidic Triphenyl Methane Dyes. Asian Journal of Research in Chemistry.2011: 4(7): 1114-1118.

12. Mohammed WN, et Al. Spectrofluorimetric determination of tolterodine tartarate in pure form and pharmaceutical preparation. International Journal of Pharmaceutical Science and Research. 2013; 4(10): 3845-3849.

13. Shetty SK, Shah K. Development and validation of tolterodine by RP-HPLC method in bulk drug and pharmaceutical dosage forms. International Journal of Pharmaceutical Research. 2011; 3: 1083-1087.

14. Saxena V, et Al. Stability indicating HPLC determination of tolterodine tartrate in pharmaceutical dosage form. Indian Journal of Chemical Technology. 2006; 13: 242-246.

15. Shah U, et Al. Stability Indicating RP-HPLC Method Development and Validation of Tolterodine Tartrate and its Degradants in its Capsule Dosage Form. Journal of Pharmaceutical Science and Research. 2017; 9(6): 860-866.

16. Laha TK, et Al. RP-HPLC Method for the Determination of Tamsulosin in Bulk and Pharmaceutical Dosage Forms. Asian Journal of Chemistry. 2011; 23(1): 11-14.

17. Mhamunkar SM, et Al. RP-HPLC method development and validation for the simultaneous estimation of Tamsulosin HCl and Tolterodine tartrate in pharmaceutical dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 2012; 4(5): 319-322.

18. Nanda RK, et Al. Simultaneous RP-HPLC estimation of Tamsulosin and Tolterodine in its pharmaceutical dosage form. Journal of Pharmacy Research. 2009; 2(11): 1786-1788.

19. S. Ashutosh Kumar, et Al. Stability Indicating RP-HPLC Method for the Determination of Tolterodine Tartrate in Bulk as well as in Pharmaceutical Formulation. Asian Journal of Research in Chemistry. 2013: 6(7): 694-701.

20. Joshi HV, et Al. Development and Validation of Stability Indicating Method for the Simultaneous Estimation of Tamsulosin HCl and Tolterodine Tartrate in Pharmaceutical Dosage form. Asian Journal of Pharmaceutical Analysis. 2019; 9(4): 205-209.

21. Nidhi K. Patel, Shailesh K. Koradia. Development and Validation of stability indicating RP-HPLC method for the simultaneous estimation of Beclomethasone dipropionate and Formoterol fumarate in their combined pharmaceutical dosage form. Asian Journal of Pharmaceutical Analysis. 2016; 6(2): 83-90.

22. SS Chitlange, et Al. Stability Indicating RP- HPLC Method for Simultaneous Estimation of Valsartan and Amlodipine in Capsule Formulation. Asian Journal of Research in Chemistry. 2008; 1(1): 15-18.

23. Deepa R. Patel, et Al. Stability Indicating HPLC Method for Simultaneous Determination of Repaglinide and Metformin Hydrochloride in Pharmaceutical Dosage Form. Asian Journal of Research in Chemistry. 2011; 4(3): 500-505.

24. Kinjal V. Vekariya, et Al. Development and Validation of Stability Indicating RP-HPLC method for simultaneous estimation of Thiocholchicoside and Lornoxicam in Combined Tablet Dosage Form. Asian Journal of Research in Chemistry. 2012; 5(8): 980-984.

25. Mohammed EA, et Al. A Validated LC-MS/MS Method for the Determination of Tamsulosin Hydrochloride in Six Brands; Applications to Content Uniformity and Dissolution Studies. Reseach Journal of Pharmacy and Technology. 2011; 4(12): 1885-1890.

26. Mishra A, et Al. Stability-indicating HPTLC method for analysis of Tolterodine in the bulk drug. Journal of Planner Chromatography. 2011; 24(2): 150-153.

27. Shah A, et Al. Simultaneous estimation of Tolterodine tartrate and Tamsulosin HCl by validated HPTLC assay method from combination capsule form. Journal of Chemical and Pharmaceutical Research. 2015; 7(5): 81-88.

28. ICH Harmonized Tripartite Guideline; Validation of Analytical Procedures: Text and Methodology, Q2(R1); International Conference on Harmonization, IFPMA, Geneva, Switzerland. 2005.

Received on 28.04.2020 Modified on 19.09.2020

Research J. Pharm. and Tech. 2021; 14(9):4561-4566.

DOI: 10.52711/0974-360X.2021.00793