1. INTRODUCTION:

Tamsulosin hydrochloride:

5-[(2R)-2-{[2-(2-ethoxyphenoxy)ethyl]amino}propyl]-2-methoxybenzene-1-sulfonamide hydrochloride.

Approximately 70% of the alpha1-receptors in human prostate are of the alpha-1A subtype. Blockage of these receptors causes relaxation of smooth muscles in the bladder neck and prostate.

Tamsulosin is a selective antagonist at alpha-1A and alpha-1B-adrenoceptors in the prostate, prostatic capsule, prostatic urethra, and bladder neck. At least three discrete alpha1-adrenoceptor subtypes have been identified: alpha-1A, alpha-1B and alpha-1D, their distribution differs between human organs and tissue. Approximately 70% of the alpha1-receptors in human prostate are of the alpha-1A subtype. Blockage of these receptors causes relaxation of smooth muscles in the bladder neck and prostate, and thus decreases urinary outflow resistance in men.

Tolterodine tartrate:

(2R,3R)-2,3-dihydroxybutanedioic acid; 2-[(1R)-3-[bis(propan-2-yl)amino]-1-phenylpropyl]-4-methylphenol Tolterodine is an antimuscarinic drug that is used to treat urinary incontinence. Tolterodine acts on M2 and M3 subtypes of muscarinic receptors.

Tolterodine act as competitive antagonists at muscarinic receptors. This antagonism results in inhibition of bladder contraction, decrease in detrusor pressure, and an incomplete emptying of the bladder.

2. MATERIALS AND METHODS:

A. Instrument and Apparatus:

Ÿ Melting point apparatus name: Analab

Ÿ HPLC-1260 - Infinity II, Agilent Technologies Software: Open Lab CDS Chemstation

Ÿ HPLC Column- Zorbax SB-C18

Ÿ Detector-UV

Ÿ Ultrasonic Water Bath-Labman

Ÿ pH meter- CL54+

Ÿ Analytical Balance- ATX-224

Ÿ FT-IR: Model- IR Affinity, LC solutions (6200), Shimadzu

Ÿ UV-VIS - UV-VIS Spectrophotometer, Cary - 60Serial No.: MYB510013, Shimadzu

B. Reagents and material:

Ÿ Tamsulosin HCl (Mehta API Pvt. Ltd, Mumbai)

Ÿ Tolterodine tartrate (MSN Labs, Hyderabad)

Ÿ Methanol-HPLC Grade- (Finar, Ahmedabad)

Ÿ Acetonitrile- HPLC Grade- (Finar, Ahmedabad)

Ÿ Potassium Dihydrogen Phosphate- HPLC Grade- (Merck limited, Mumbai, India)

Ÿ Water- HPLC Grade- (Finar, Ahmedabad)

C. Chromatographic conditions:

Stationary phase	:	Hypersil BDS C18 (25cm × 4.6mm)
Mobile phase	:	0.05 M Phosphate Buffer (pH-4): Methanol (60:40 %v/v)
Flow rate	:	1ml/min
Detection wavelength	:	305nm
Injection volume	:	20μl
Column temperature	:	30˚C
Detector	:	UV detector

3. EXPERIMENTAL:

3.1 Preparation of Stock solutions:

Tamsulosin HCl standard stock solution: (4ppm)

A 40mg of Tamsulosin HCl was weighed and transferred into 100ml volumetric flask. Volume was made up to the mark with methanol. Further take 1ml from this solution and transfer to 100ml volumetric flask and make up the volume with methanol.

Tolterodine tartrate standard stock solution: (40ppm):

A 40mg of Tolterodine tartrate was weighed and transferred into 100ml volumetric flask. Volume was made up to the mark with methanol. Further take 10ml from this solution and transfer to 100ml volumetric flask and make up the final volume with methanol.

3.2 Preparations of sample solutions:

Take capsule Powder equivalent to 40mg of Tolterodine tartrate, and 4mg of Tamsulosin HCl were transferred into 100ml volumetric flask, add 60ml of Mobile phase and Shake for 15min and made up volume with Mobile phase. The solution was filtered through what man filter paper.

Pipetted out 1ml standard stock solution and transferred to 10ml volumetric flask and made up volume with the mobile phase.

4. METHOD VALIDATION:

The analytical procedures were validated according to ICH Q2 R1 guidelines in order to determine system suitability, linearity, precision, accuracy, specificity, and robustness of analysis.

4.1 Specificity:

The Chromatograms of Tamsulosin HCl and Tolterodine tartrate standards and Tamsulosin HCl and Tolterodine tartrate sample show no interference with the Chromatogram of Tamsulosin HCl and Tolterodine tartrate Blank, so the Developed method is Specific.

4.2 Linearity:

The linearity for Tolterodine tartrate and Tamsulosin HCl were assessed by analysis of combined standard solution in range of 20-60μg/ml and 2-6μg/ml respectively, 5, 7.5, 10, 12.5,15ml solutions were pipette out from the Stock solution of Tolterodine tartrate (400 μg/ml) and Tamsulosin HCl (40μg/ml) and transfer to 100ml volumetric flask and make up with mobile phase to obtain 20, 30, 40, 50 and 60μg/ml, and 2, 3, 4, 5 and 6 μg/ml for Tolterodine tartrate and Tamsulosin HCl respectively

In term of slope, intercept and correlation co-efficient value, the graph of peak area obtained verses respective concentration was plotted.

Correlation co-efficient for calibration curve Tolterodine tartrate and Tamsulosin HCl was found to be 0.999 and 0.998 respectively.

The regression line equation for Tolterodine tartrate and Tamsulosin HCl are as following:

For Tolterodine tartrate y = 123.05x - 0.8074 and For Tamsulosin HCl: y = 210.55x + 0.6068

4.3 Precision:

I. Repeatability:

The data for repeatability of peak area measurement for Tolterodine tartrate (40μg/ml) and Tamsulosin HCl (4 μg/ml) based on six measurements of same solution of Tolterodine tartrate (40μg/ml) and Tamsulosin HCl (4 μg/ml). The % RSD for Tolterodine tartrate and Tamsulosin HCl was found to be 0.528 and 0.374 respectively.

II. Intraday precision:

Standard solution containing (2,4,6μg/ml) of Tamsulosin HCl and (20,40,60μg/ml) of Tolterodine tartrate were analyzed three times on the same day and % R.S.D was calculated.

III. Interday precision:

Standard solution containing (2,4,6μg/ml) of Tamsulosin HCl and (20,40,60μg/ml) of Tolterodine tartrate were analyzed three times on the different day and % R.S.D was calculated

4.4 Accuracy:

To confirm the accuracy of the proposed method, recovery experiments were performed by the standard addition technique. In this method, a known quantity of a pure drug was added at three different levels, i.e. 80%, 100% and 120% to pre-analyzed sample solutions and the recovery of Tamsulosin HCl and Tolterodine tartrate was calculated for each concentration. The results are given in Table.

4.5 LOD and LOQ:

Calibration curve was repeated for five times and the standard deviation (SD) of the intercepts was calculated.

Then LOD and LOQ were calculated as follows:

LOD = 3.3 * SD/slope of calibration curve

LOQ = 10 * SD/slope of calibration curve

Where, SD = Standard deviation of intercepts

Table IV: Results of Recovery study (Accuracy) n=3

Amt. of sample (µg/ml)		Amt. of std. added (µg/ml)		Amt. recovered Mean ± S.D (µg/ml)		% Recovery
TAM	TOL	TAM	TOL	TAM	TOL	TAM	TOL
2	20	1.6	16	1.615	16.109	100.572	99.679
2	20	2	20	2.002	19.976	100.326	100.539
2	20	2.4	24	2.396	23.913	99.986	99.589

Table V: Results of Robustness study.

Condition	Variation	Tamsulosin HCl		Tolterodine tartrate
Condition	Variation	S.D.	% RSD	S.D.	% RSD
Flow Rate (1.0±0.2 mL/min)	0.8 ml/min	2.964	0.339	11.902	0.232
Flow Rate (1.0±0.2 mL/min)	1.2 ml/min	46.147	1.976	46.170	1.386
Mobile phase Buffer: Water (60:40 ±2 % v/v)	58:42 %v/v	6.443	0.753	5.855	0.117
Mobile phase Buffer: Water (60:40 ±2 % v/v)	62:38%V/V	4.709	0.558	32.066	0.651
pH (± 0.2)	pH 3.8	0.511	0.059	21.360	0.422
pH (± 0.2)	pH 4.2	3.098	0.362	5.854	0.117

Table VI: Results of Assay (n=3) of marketed formulation

Capsule

Roliflo-OD-4

Label claim

Tamsulosin HCl (0.4mg)

Tolterodine tartrate

(4mg)

Assay (% of label claim*) Mean ± S. D.

97.402±0.885

97.214±1.180

4.6 Robustness:

Following parameters were changed one by one and their effect was observed on system suitability for standard preparation.

1 Flow rate of mobile phase was changed (±0.2 ml/min) 0.8 ml/min and 1.2 ml/min.

2 pH of Mobile phase was changed (± 0.2) 5.2 and 4.8.

3 Ratio of Mobile phase was changed (±2) Buffer: Methanol (52:48) and Buffer: Methanol (48:52)

5. RESULTS AND DISCUSSION:

The degradation behaviour of Tamsulosin HCl and Tolterodine tartrate was investigated under different stress degradation (Hydrolytic, oxidative, photolytic and thermal) conditions recommended by International Conference on Harmonization (ICH) using HPLC. A stability indicating RP-HPLC method was developed that could separate drug from degradation products formed under various stressed conditions.

RP-HPLC method was developed for simultaneous estimation Tamsulosin HCl and Tolterodine tartrate. In RP-HPLC method, good resolution and separation of two drugs was achieved. Phosphate buffer (pH 4.0): Methanol (60:40) was used as mobile phase. Retention time of Tamsulosin HCl and Tolterodine tartrate were found to be 3.440 min and 5.693 min respectively with a flow rate of 1 ml/min. and Detection wavelength 305nm. The proposed method was accurate and precise. Therefore proposed method can be used for routine analysis of Tamsulosin HCl and Tolterodine tartrate in capsule.

Validation parameters like Linearity, Accuracy, Precision, Robustness, System suitability, Specificity were tested.

Observation of all these parameters leads to the point that developed RP-HPLC method is linear, accurate, precise, specific and robust.

It can be successfully adopted for routine quality control analysis of Tamsulosin HCl and Tolterodine tartrate in Combined dosage form without any interference from common excipients and impurity.

ASSAY OF MARKETED FORMULATION:

Take Tablet Powder equivalent to 4 mg of Tamsulosin HCl, and 40 mg of Tolterodine tartrate was transferred to a 100 ml volumetric flask, Add 60 ml Mobile phase and Shake for 15 min and make up volume with Mobile phase. The solution was filtered through Whatman filter paper no. 42.

Tamsulosin HCl

Tolterodine tratrate

Figure 1: Chemical structures of Tamsulosin HCl and Tolterodine tartrate

Drug	Retention Time	Area	Tailing factor	Theoretical plate	Resolution
Tamsulosin HCl	3.440	1387.644	1.348	7016	10.468
Tolterodine tartrate	5.963	2609.246	1.243	7316	10.468

Sr. No	Concentration (µg/ml)	Area
1	20	2517.111
2	30	3636.106
3	40	4923.711
4	50	6082.409
5	60	7446.404

Sr. No	Concentration (µg/ml)	Area
1	2	430.105
2	3	621.961
3	4	847.982
4	5	1040.391
5	6	1273.655

Drug	Inter-day Precision (% RSD)	Intra-day Precision (%RSD)
Tamsulosin HCl	0.438-1.049 % RSD	0.255-0.414 %
Tolterodine tartrate	0.500-1.277%	0.150-0.417%