Method Development and Validation of Tamsulosin in Bulk and Orally Disintegrating Extended Release Tablets Pharmaceutical Dosage Form by Using RP HPLC

Abstract

A simple, accurate and precise method was developed for the estimation of Tamsulosin in bulk and Orally Disintegrating Extended release Tablets pharmaceutical dosage form by RP-HPLC technique. Chromatogram was run through Hypersil BDS C18 150 mm × 4.6 mm, 5 μ. Mobile phase pH 2.0 Phosphate buffer: Acetonitrile: Methanol in the ratio of 40:30:30 and flow rate was maintained at 1.0 ml/min. Column temperature was set to 40ºC. Optimised wavelength selected was 225 nm. Retention time of Tamsulosin was found to be 6.651. The correlation coefficient for the linearity concentration levels range of 25% to 200% was 0.999. The method was found to be precise as the repeatability; intermediate precision values were 0.9, 0.4%. Recovery was obtained as 100.3%. Hence the suggested method can be adopted in routine analysis in industries.

Keywords

Tamsulosin; RP-HPLC; Chromatogram; Disintegration; Tablets

Introduction

Tamsulosin is a selective alpha 1A and alpha 1B adrenoceptor antagonist that exerts its greatest effect in the prostate and bladder, where these receptors are most common. It is indicated for the treatment of signs and symptoms of benign prostatic hypertrophy. Antagonism of these receptors leads to relaxation of smooth muscle in the prostate and detrusor muscles in the bladder, allowing for better urinary flow. The specificity of tamsulosin focuses the effects to the target area while minimizing its effects on other areas.

Tamsulosin is indicated for the treatment of signs and symptoms of benign prostatic hyperplasia. Tamsulosin is also used off label for the treatment of ureteral stones, prostatis, and female voiding dysfunction [1]. Tablets Chemical Structure of Tamsulosin has been given in Figure 1.

Figure 1: Tablets chemical structure of Tamsulosin.

An attempt has been made to develop and validate simple, precise, accurate economical RP-HPLC method as per ICH guidelines for the estimation of Tamsulosin in Bulk and Pharmaceutical dosage form.

Materaials and Methods

Chemicals and reagents

Acetonitrile (HPLC grade), orthophosphoric acid (HPLC grade), methanol (HPLC grade), sodium hydroxide (AR grade), Hydrochloric acid (AR grade) was purchased from Rankem Ltd, India. All active pharmaceutical ingredients (APIs) of Tamsulosin reference standards were procured from Neuheit pharma technologies private limited, Hyderabad, India.

Instruments and chromatographic conditions

Electronic Balances-Radwag, pH meter-Labindia, sonicator-PCI analytics, Centrifuge-Eltek, Water bath-VLS, WATERS HPLC 2695 system equipped with quaternary pumps, photo diode array detector 2996, UV 2487 and Auto sampler integrated with Empower 2 Software UV-VIS spectrophotometer Shimadzu 1800 integrated with UV-probe 3.2.1 Software was used for measuring absorbance’s of Tamsulosin solution. The mobile phase used was pH 2.0 buffer: Acetonitrile: Methanol (40:30:30) at a flow rate of 1.0 ml/min, samples were analysed at 225 nm detector wavelength and at an injection volume of 100 μL using Hypersil BDS C18 (150 × 4.6 mm, 5 μm) with run time of 15 mins [2].

Diluent

Mobile phase is used as diluent.

Buffer

1.37 g of potassium di hydrogen phosphate is dissolved in 900 ml of water and adjust pH to 2.0 ± 0.5 with ortho phosphoric acid and was further diluted to 1000 ml with milli Q water.

Internal standard solution preparation

Weighed about 10 mg of methyl paraben and transferred to 250 ml volumetric flask. Added about 150 ml of diluent and sonicated for 10 minutes to dissolve, made upto the volume with diluent.

Standard stock solution preparation

Accurately weighed 10 mg of Tamsulosin, transferred to 100 ml volumetric flasks and 70 ml of diluent was added to these flasks and sonicated for 10 mins. Flask was made up with diluent and labeled as standard stock solution (100 μg/ml) [3].

Standard working solution preparation

10 ml of internal standard solution, 2 ml from standard stock solution was pipetted out and taken into a 100 ml volumetric flask, and made up with diluent (2 μg/ml).

Preparation of 0.2 M hydrochloric acid

Dissolve 1.8 ml of concentrated hydrochloric acid to 100 ml of purified water and mix well.

Preparation of 0.05 M sodium hydroxide

Weigh and transfer 1.0 g of sodium hydroxide to 500 ml of volumetric flask and make up the volume with purified water.

Sample stock solution preparation

5 tablets were accurately weighed and were transferred into a 100 ml volumetric flask, add 10 ml of water and 5 g of glass balls, sonicate for 5 minutes. Then add 40 ml of 0.05 M sodium hydroxide solution and kept on water bath at 50ºC for 10 minutes, cool and kept on orbital rotary shaker at 100 rpm for 30 minutes. The add 20 ml of acetonitrile, 10 ml of 0.2 M HCL ad make up to volume with mobile phase and mix well. Centrifuge the solution at 5000 rpm for 5 minutes [4].

Sample working solution preparation

5 ml of supernatant filtered sample stock solution was transferred to 25 ml volumetric flask, add 2.5 ml of internal standard solution and made up with diluent (2 μg/ml). Filter the solution through a 0.45 μm nylon syringe filter. By discarding initial 5 ml of filtrate, collect the solution for analysis [5].

Method validation

As per ICH guidelines the method was validated and the parameters like Linearity, Specificity, Accuracy, Precision, Limit of Detection (LOD) and Limit of Quantitation (LOQ) were assessed.

Specificity: To check the interference in an optimized method, we should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So, this method was said to be specific.

Linearity: At different levels Stock solution of tamsulosin is taken into 6 different volumetric flasks and added 10 ml of internal standard solution, diluted to 100 ml with diluents [6]. Linearity solutions are prepared such that 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 3.0 ml, 4.0 ml.

Accuracy: Preparation of Standard stock solutions: Accurately weighed 10 mg of Tamsulosin and transferred to 100 ml volumetric flask, 70 ml of diluent was added and sonicated for 10 minutes. Flask was made up with diluent and labeled as Standard stock solution. (100 μg/ml) Preparation of 50%, 100%, 150% spiked solution: Weigh and transfer equivalent weight of placebo sample to 100 ml of each volumetric flasks respectively. To above add 1 ml, 2 ml and 3 ml of Tamsulosin stock solution respectively. Add 10 ml of water and 5 g of glass balls. Sonicate for 5 minutes. Then add 40 ml of 0.05 M sodium hydroxide solution and kept on water bath at 50ºC for 10 minutes, cool and kept on orbital rotary shaker at 100 rpm for 30 minutes. The add 20 ml of acetonitrile, 10 ml of 0.2 M HCL, 10 ml of Methyl paraben add make up to volume with mobile phase and mix well. Centrifuge the solution at 5000 rpm for 5 minutes. Supernatant solution was filtered through 0.45 μm nylon syringe filter [7].

Robustness: Small deliberate changes in method like flow rate, mobile phase ratio, pH and temperature are made but there was no recognized change in the result and are within range as per ICH guide lines.
Robustness conditions like Flow minus (0.8 ml/min), Flow plus (1.2 ml/min), mobile phase minus (37:33:30), mobile phase plus (43:30:27), pH plus (1.8), pH minus (2.2), temperature minus (35ºC) and temperature plus (45ºC) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. % RSD was within the limit [8].

LOD and LOQ values were found to be 0.1 and 0.4.

System suitability parameters: The system suitability parameters were determined by preparing standard solution of Tamsulosin (2 ppm) the solution was injected six times and the parameters like peak tailing, resolution and USP plate count were determined [9].

The % RSD for the area of six standard injections results should not be more than 2%.

Assay

Assay of the marketed formulation (Veltam 0.4 mg) was carried out by injecting sample corresponding to equivalent weight into HPLC system [10].

Results and Discussion

Optimization of chromatographic conditions

To develop and establish a suitable RP-HPLC method for estimation of Tamsulosin in bulk and tablet dosage forms, different preliminary tests were performed and different chromatographic conditions were tested and optimized chromatographic conditions were developed which were given in Table 1 [11]. The final analysis was performed by using pH 2.0 buffer: Acetonitrile: Methanol (40:30:30) at a flow rate of 1.0 ml/min, samples were analysed at 225 nm detector wave length and at an injection volume of 100 μL using Hypersil BDS C18 4.6 × 150 mm, 5 μm with run time of 15 min. The proposed method was optimized to give sharp peak with good resolution and minimum tailing effect for Tamsulosin the optimized chromatogram was obtained as shown in Figure 2 [12].

Table 1. Optimised chromatographic conditions.

Figure 2: Chromatogram of Tamsulosin.

Validation

Linearity was established for Tamsulosin at six different concentrations and were injected in a duplicates and average areas were determined and linearity equation was obtained and correlation coefficient (R2) was determined as 0.999. The Linearity calibration curves were plotted as shown in Figure 3. Retention time of Tamsulosin was 6.651 min where no interfering peaks in blank and placebo were found in this method. So, this method holds its specificity. Three levels of Accuracy samples 50%, 100%, 150% were prepared and triplicates of injections were given for each level of accuracy and mean % Recovery was obtained as 100.3% for Tamsulosin given in Table 2. % RSD was calculated from the corresponding peaks obtained by injecting six times a known concentration and the % RSD for Repeatability, Intermediate precision was obtained as 0.4, 0.9. Low % RSD value indicates that the method developed was precise as shown in Table 3. The LOD and LOQ values were evaluated based on Relative standard deviation of response and slope of the calibration curve of the two drugs. The detection limit value was obtained as 0.12 and Quantitation limit was found to be 0.42 as given in Table 4 [13]. Robustness conditions like Flow minus (0.8 ml/min), Flow plus (1.2 ml/min), mobile phase minus (37:33:30), mobile phase plus (43:30:27), pH minus (1.8), pH plus (2.2), temperature minus (35ºC) and temperature plus (45ºC) were maintained and samples were injected in duplicate manner Table 5. System suitability parameters were not much affected and all the parameters were passed. % RSD was within the limit (Tables 6 and 7). Tamsulosin pure drug (API) was obtained from Neuheit Pharma technologies private limited. Assay was performed with the Veltam marketed formulation. Average % Recovery obtained for Tamsulsoin was 100.3% and the chromatogram of standard drugs and pharmaceutical dosage forms were shown in Figures 4 and 5 respectively. Assay values 99.8% are within the acceptance limits of 94 to 106% (Figures 4 and 5) [14].

Table 2. Accuracy results of Tamsulsoin.

Table 3. Precision results of Tamsulosin.

Table 4. LOD and LOQ values of Tamsulosin.

Table 5. Robustness data of Tamsulsoin.

Table 6. System suitability parameters for Tamsulosin.

Table 7. Assay results of Tamsulsoin.

Figure 3: Linearity curve of Tamsulosin.

Figure 4: Standard chromatogram of Tamsulosin.

Figure 5: Sample chromatogram of Tamsulsoin.

Conclusion

Chromatographic conditions used are stationary phase Hypersil BDS C18 (150 mm × 4.6 mm 5 μ) Mobile phase pH 2.0 buffer: Acetonitrile: Methanol (40:30:30) and flow rate was maintained at 1.0 ml/min, detection wave length was 225 nm, column temperature was set to 40ºC. Conditions were finalized as optimized method. System suitability parameters were studied by injecting the standard six times and results were well under the acceptance criteria. Linearity study was carried out between 25% to 200% levels, R2 value was found to be 0.999. Precision was found to be 0.4 (repeatability), 0.9 (intermediate precision). LOD and LOQ are 0.1, 0.4. By using above method assay of marketed formulation was carried out 99.8%. The method developed was simple and economical that can be adopted in regular Quality control test in industries.

References

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