From the literature survey it is concluded that a very less no of literatures are found for Estimation of Tamsulosin Hydrochloride in Pharmaceutical dosage form by RP-HPLC. Tamsulosin is increasingly finding use for the Treatmetn of Prostatic disease in Men. So the aim of present work is to develop a precise, accurate, simple and reliable RP-HPLC method for the estimation of Tamsulosin in Tamsulosin Pellets 0.2%.
The solubility of drug was checked by treating the different solvents with the working standard. So based on solubility nature finally the diluents is selected for preparation of standard and stock solutions which are used for the analysis.
Selection of wave length is important for the determination of drugs by instrumental methods. The choice of detection wave length is based on the absorption Spectrum of Tamsulosin. So in selection of wave length first the working Standard is dissolved in the Solvent and dilute to get 1ppm. Then scan the solution over the range of 200-400 nm against blank. So finally from the absorption spectrum the detection wave length 220 nm was selected for estimation of Tamsulosin by RP-HPLC.
Selection of Chromatographic mode:
The selection of chromatographic mode is based on the nature of the drug to be estimated i.e. Polar nature and Non-polar nature. If the nature of drug is non-polar the selected chromatographic mode is Normal Phase (Stationary phase is Polar in nature),if the nature of drug is polar it is Reverse phase for effective separation (Stationary phase is in Non-polar nature), so from the solubility studies it concludes that Tamsulosin is Polar in nature, so finally Reverse phase chromatographic mode is selected for best separation.
Method development Trails:
Trail - 1
The trail was done by taking phosphate (PO4) buffer at pH (7.0): Methanol in the ratio of 70:30 v/v at a flow rate 1.0 ml/min.
Observation:
The chromatograms of Tamsulosin were observed at the Retention time of 6.8 min and asymmetric peak shapes were observed with less peak area.
Conclusion:
So to reduce the retention time of drug the mobile phase composition was changed.
Trail - 2
The trail was done by using a mixture of mobile phase [Phosphate buffer (pH7.0): Methanol; 90:10%V/V] at a flow rate 1.0 ml/min.
Observation:
From the Chromatograms the retention time of Tamsulosin observed at 6.2 min with less Peak area
Conclusion:
To increase the peak area the injection volume was adjusted from 20µL to 50µL and the composition of mobile phase also changed.
Trail - 3
The trail was done by using the mobile phase [PO4 buffer (pH 7.0): ACN; 50:50 v/v]
Observation:
Based on the chromatographic data the tailing factor was more than 2.0 and peak shape was not good.
Conclusion:
To get the better chromatographic resolution the mobile phase ratio, pH of buffer and injection volume was changed.
Trail - 4
The trail was done by mixing of [PO4 buffer (pH 6.5): ACN; 60:40 v/v] at a flow rate of 1.0mL/min with an injection volume of 100 µL.
Observation:
The retention time of Tamsulosin was 5.6 min with symmetric peak shape .The tailing factor was found to be below 2.0.
Conclusion:
From the observation of chromatographic data of all the trails (Trail-1,2&3) the Trail - 4 shows less retention time with a good peak shape and peak area.
OPTIMIZED METHOD
Chromatographic conditions:
Column : Inertsil ODS-3V C 18, 250 x 4.6 mm, 5ïm
Flow Rate : 1.0 mL/min
Mobile Phase : Buffer: ACN (60:40% V/V)
Mode of separation : Isocratic
Detector wave length : 220nm
Column temperature : 250C
Injection volume : 20ïl
Run time : 10 min
Buffer Preparation:
Weigh accurately about 6.8 gms of potassium dihydrogen orthophosphate and 0.58 gms of sodium hydroxide in 1000ml volumetric flask add 500ml of water sonicate and dissolve the content and make up to the mark and adjust the pH to 6.50 +0.05 with dilute sodium hydroxide and dilute orthophosphoric acid .filter through 0.45m membrane filter paper and degas.
Preparation of mobile phase:
Mix the filtered and degassed Buffer and Acetonitrile in the volume ratio of (60:40%v/v)
Diluent: mobile phase
Preparation of placebo solution:
Weigh accurately about 499mg of placebo powder transfer in 100ml volumetric flask add 60ml of methanol, sonicate for 30 min and make up to the mark with diluents. Filter through 0.45ïm nylon filter.
Preparation of standard solution:
Weigh accurately about 25mg of Tamsulosin Hydrochloride and transfer in 50 ml volumetric flask add 20 ml of Methanol, sonicate to dissolve the content and make up to the mark with Methanol.
Transfer 2ml of above solution in to a 100 ml volumetric flask and make up to the mark with diluents. Filter through 0.45ïm nylon filter.
Preparation of Sample Solution:
Take not less than 20 capsules content, accurately weigh equivalent to 2mg of Tamsulosin Hydrochloride fine powder of Tamsulosin hydrochloride pellets in to 200 ml volumetric flask add 60 ml of Methanol, sonicate for 30 min and make up to the mark with diluents. Filter through 0.45 ïm nylon filter.
Procedure:
Transfer the individual blank, placebo, standard and sample solutions to the HPLC vails. Separately inject diluents, single injection of placebo solution, six replicate injections of standard solution add two injections of sample solution in to the chromatograph, record the chromatograms and measure the peak responses. Based on the chromatographic data, the fallowing formula is used to estimate the drug concentration.
Ru Std.wt 2 200 100
Tamsulosin HCl = ---------- x ---------- x ---------- x ---------- x ---------- x P
Rs 50 100 Spl.wt L.C.
Where,
Ru = Peak area of Tamsulosin Hydrochloride in sample solution
Rs = Average peak area of Tamsulosin Hydrochloride in standard solution
Std. Wt= Weight of Tamsulosin Hydrochloride standard
Spl.Wt = Weight of Tamsulosin Hydrochloride pellets
P = Potency of Tamsulosin Hydrochloride working standard (on as is basis)
L.C. = Label claim
VALIDATION OF THE ASSAY METHOD
The following experimental design is drawn in order to prove the test method is capable to yield consistent, reliable and reproducible results within the pre-determined acceptance limits.
The following parameters have been validated:
System suitability
Specificity
Linearity
Accuracy
Precision
Ruggedness
Robustness
Solution stability
Procedure
SYSTEM SUITABILITY:
Prepare a placebo solution and a standard solutions (contains 0.01 mg/ml) in diluents as per optimized methodology. These are injected in following way.
Table: 6
S.No.
Sample Name
No. of Injections
1
Diluent
1
2
Placebo solution
1
3
Standard solution
6
The system suitability parameters were evaluated from standard chromatograms by calculating the % RSD from retention times and peak areas from six replicate injections of Tamsulosin Hydrochloride.
Acceptance Criteria:
% RSD of six replicate standard injections is not more than 2.0%
The tailing factor of Tamsulosin Hydrochloride peak should be not more than 2.0
Theoretical plates of Tamsulosin Hydrochloride peak should be not less than 2000.
Specificity:
Prepare the placebo solution and known impurity solution with a concentration of 0.5% of the Impurity-D and Tamsulosin Hydrochloride at 100% working concentration. A solution of known impurity spiked with the Tamsulosin Hydrochloride solution also prepared. All these solutions are injected to HPLC by applying optimized Chromatographic conditions.
This study showed that the known impurity of Tamsulosin Hydrochloride and placebo were adequately resolved from the Tamsulosin Hydrochloride peak.
Acceptance Criteria:
The Tamsulosin Hydrochloride and its known impurity (Impurity-D), placebo solution should elute at different retention times.
The placebo and Impurity-D should be adequately resolved from the Tamsulosin Hydrochloride peak.
The peak purity of Analyte should be not more than 0.990
ACCURACY (RECOVERY):
The accuracy of the method was determined by spiking a series of known amount of Tamsulosin Hydrochloride to placebo at approximately 80%, 100% and 120% of the test concentration. Each solution was analyzed in triplicate. The average% recovery of Tamsulosin Hydrochloride was calculated by separately injecting the blank, placebo, standard and finally 80%, 100% and 120% in to the chromatograph.
Acceptance Criteria:
The recovery should be in the range of 98.0% - 102.0%
The percentage relative standard deviation should be not more than 2.0%
PRECISION:
System Precision: Placebo and standard solution prepared as per test method and arrange them in a sequence such that blank, placebo and standard and inject them in 1, 1, and 6 times individually.
Acceptance Criteria:
% RSD of six replicate standard injections is not more than 2.0%
The tailing factor of Tamsulosin Hydrochloride peak should be not more than 2.0
Theoretical plates of Tamsulosin Hydrochloride peak should be not less than 2000.
Method Precision: The method precision was performed by preparing six samples (0.01 mg/ml concentration) as per the test method representing a single batch.
Acceptance criteria:
The % relative standard deviation for the assay of six samples should be not more than 2.0
Linearity and Range:
The linearity of Tamsulosin Hydrochloride is established by preparing a series of solutions using working standard over the range of 80% to 120% inject these solutions in to HPLC system.
Measure the peak area of analyte by plotting a graph of concentration vs analyte peak area and evaluate the correlation coefficient between concentration and peak area.
Acceptance criteria:
The plot of concentration vs. analyte peak area at 80% to 120% level should be linear with correlation coefficient ® value should be not less than 0.9990.
Y-Intercept should be + of the response of 100% solution.
Solution stability:
A solution of Tamsulosin Hydrochloride pellets at 100% of working concentration as well as the standard solution was kept at room temperature. The solutions were analyzed at different time intervals (i.e. initial, 4 hrs, 8 hrs, 12 hrs, 16hrs, 20hrs and 24hrs).
No significant variation in the percentage of assay was observed up to 24hrs at room temperature for standard solution both the sample and standard solutions were found to be stable up to 24hours at 250C.
Acceptance criteria:
For a stable solution, assay results should not vary from the initial value by more than 1.0%.
Ruggedness:
Ruggedness is a parameter in validation, it is used to determine the reproducibility of the test results, which are obtained by the optimized method for variability's include, Instrument to Instrument, Reagent to Reagent, Column to Column, analyst to analyst and day to day.
Ruggedness is done by preparing standard and six samples representing the single batch as per optimized method and compared the chromatograms obtained by visibilities include, Instrument to Instrument, Reagent to Reagent, Column to column, analyst to analyst and Day to Day.
Acceptance Criteria:
The bias in assay determination for each parameter should be + 1.0%.
The percentage of relative standard deviation among the results obtained should be not more than 2.0.
ROBUSTNESS:
Generally robustness is an measure of its capacity to remain un affected by small changes in the method, but when deliberate variations in method parameters has occurred it provides indication of its reliability during its normal usage. By this it is verify that, the method performance is not affected by typical changes in normal experimental conditions.
Procedure of Robustness is done by applying the minor change to the optimized method by
Changing the column temperature to + 50 C (i.e. 200 C and 300 C)
Changing the Floe rate to + 0.1 ml/min (i.e. 0.9 ml/min and 1.1 ml /min)
Changing the pH by + 0.2 (6.3 and 6.7)
Changing the composition of Buffer by + 2% (i.e. 58% and 62%)
Acceptance Criteria:
% RSD of six replicate standard injections is not more than 2.0%
The tailing factor of Tamsulosin Hydrochloride peak should be not more than 2.0
Theoretical plates of Tamsulosin Hydrochloride peak should be not less than 2000.
