DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TAMULOSIN AND DUTASTERIDE IN A BULK AND TABLET DOSAGE FORM

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF TAMULOSIN AND DUTASTERIDE IN A BULK AND TABLET DOSAGE FORM

Birender Singh and Devender Chauhan *

Gateway College of Pharmacy, Delhi-NCR, Sonipat, Haryana, India.

ABSTRACT: Chromatography is a separation process that is achieved by distributing the substances to be separated between a moving phase and a stationary phase. HPLC is high resolution, high pressure and high-speed liquid chromatography. It has several resolving powers than open column liquid chromatography hence it is used for speedy resolution of complex mixture, separation and determination of species in a variety of organic, inorganic and biological materials. The developed HPLC method was validated for various parameters like accuracy, precision, specificity, LOD, LOQ, linearity, range and robustness as per ICH guidelines. The results obtained were well within the acceptance criteria for all the parameters. The proposed method was applied for simultaneous estimation of Tamsulosin and Dutasteride formulation. The HPLC system used was WATERS Alliance series System with Rheodyne injector 20 μL and the column Inertsil ODS 250 x 4.6 mm, 5 μm. The mobile phase comprised of Sodium phosphate buffer and Acetonitrile in the ratio of 30:70 v/v and flow rate of 1.0 ml/min with UV detection at 233 nm produced peaks of Tamsulosin and Dutasteride in the chromatogram which were well resolved with retention time of 2.939 min and 5.572 min respectively.

Keywords: Chromatography, Validation, Tamulosin, Dutasteride, Accuracy, Precision

INTRODUCTION: Chromatography is unique in the history of analytical methodology and is probably the most powerful and versatile technique available to the modern analyst. In a single procedure it can separate a mixture into its individual components and simultaneously determine quantitatively the amount of each component present ¹.

Principle of Chromatographic Separation: Chromatography is a separation process that is achieved by distributing the substances to be separated between a moving phase and a stationary phase. Those substances distributed preferentially in the moving phase pass through the chromatographic system faster than those that are distributed preferentially in the stationary phase. As a consequence, the substances are eluted from the column in reverse order of their distribution coefficient with respect to the stationary phase. The process is diagrammatically depicted below ².

Classification of Chromatographic Techniques: All chromatographic separations are carried out using a mobile and a stationary phase.

As a result of this prerequisite, the primary classification of chromatography is based on the physical nature of the mobile phase. Thus, all separation processes that utilize a gas as the mobile phase are classed as gas chromatography. Conversely, all separation processes that utilize a liquid as the mobile phase are classed as liquid chromatography.

1. Adsorption Chromatography

• Gas-solid chromatography
• High performance liquid chromatography
• Thin layer chromatography

1. Partition Chromatography

• Liquid-liquid chromatography
• Paper chromatography
•  High performance liquid chromatography

1. Ion exchange chromatography
2. Permeation chromatography

• Size exclusion chromatography

1. Affinity chromatography

• DNA Affinity chromatography

1. Electrophoresis

• Capillary electro chromatography ^{3, 4}.

Fundamental Parameters:

Retention Time: It is the time in between the sample is injected and chromatographic peak is recorded. The total retention time (t_R1 or t_R2) is the time which is needed by sample component to migrate from column inlet (sample injection) to the column end (detector). The net retention time (t¢_R1 or t¢_R2) is the difference between total retention time and dead time i.e. the time the sample component remains in the stationary phase.

Capacity Factor (K¢): It is the measure of the position of a sample peak in the chromatogram. It is specific for a given substance. k¢ depends on the stationary phase, the mobile phase, the temperature, quality of the packing etc.

Relative Retention (a): Also known as separation factor is the ratio between two capacity factors. Relative retention describes the ability of a system of stationary and mobile phase to discriminate between two compounds. Impurities in the mobile phase (e.g. water content) strongly influence relative retention.

Resolution (RS): It is a measure of quality of separation of adjacent bands; obviously overlapping bands have small Rs values. It is calculated from width and retention time of two peaks.

Number of Theoretical Plates (N): Characterize the quality of a column packing and mass transfer phenomena. The larger n, the more complicated sample mixtures can be separated with the column.^{5, 6}

High Performance Liquid Chromatography: HPLC is high resolution, high pressure and high-speed liquid chromatography. It has several times resolving power than open column liquid chromatography hence it is used for speedy resolution of complex mixture, separation and determination of species in a variety of organic, inorganic and biological materials ⁷.      

TABLE 1: COMMONLY USED METHODS IN HPLC ⁷

Method Development in HPLC:  In developing HPLC method for the quantitative analysis of multicomponent formulation the following general requirements should be fulfilled.

1. The identity of the component to be analyzed should be established.
2. Separation of specific components should be achieved.
3. Sample preparation should be reproducible.
4. Standard of known purity should be available, and accuracy will be directly related to the degree of purity of standards used in determination.
5. A stationary phase that separates the component in a reproducible manner.
6. Sample application or injection should be reproducible.

Analytical Method Validation: Validation is “Action of proving accordance with the principle of good manufacturing practice, that any material, activity or system actually leads to expected result”. Method validation is the documented successful evaluation of any analytical method that provides a high level of assurance that such method will consistently yield results that are accurate with in previously established specification ⁸.

Type of Validation: The following are frequently required to be validated on pharmaceutical process; Equipment, Environment, Materials, Methods, Control, Process, Personnel’s Facilities and Operation procedure. Based on these the validation program comprises ⁸.

Analytical Method Validation: Method validation is a process to confirm that the analytical procedure employed for a specific test is suitable for its intended use. Analytical testing of pharmaceutical products is necessary to ensure their purity, stability, safety, and efficacy. Analytical method validation is an integral part of the quality control system. Although a through validation cannot rule out all potential problems, the process of method development and validation should address the most common ones ⁹.

Steps Involved in Quantitative Analysis:

Sampling and Sample Preparation: The sample should be homogeneous. It should be completely soluble, and the solvent used to dissolve the sample should be initial mobile phase or any solvent miscible with mobile phase.

Chromatographic Separation: After achieving a resolution with an optimized solvent system, obtaining reproducible results following criteria must be satisfied.

• Monitoring flow rate.
• Keeping the solvent composition intact.
• Solvent system must be covered before storage.
• Monitoring column temperature.

Detection: The response obtained from a given detector will vary according to the nature of solute molecules. With a UV detector the response is related to both concentration and molecular extinction coefficient of the component at the wavelength of detection ¹⁰.

Measurement and Calibration: The various approaches used for quantitative analysis:

Peak Height Method: Peak height measurements represent a simple, satisfactory method for calculating detector response in the absence of mechanical or electronic signal integrators. In practice, a baseline is drawn from the leading edge of the chromatography peak to the trailing edge. The vertical distance from the peak apex to the predetermined baseline represents the peak height. For quantitative purpose

1. The peak of interest must be symmetrical
2. All parameters that affect peak width must be held constantly.

Peak Area Method: Computing electronic integrators are the simplest and most popular method for the determination of chromatographic peak areas. Quantitative measurements based on peak areas can be performed by several methods such as,

The Internal Standard Method: The highest precision for quantitative chromatography is obtained by use of internal standards because the uncertainties introduced by sample injection are avoided.  In this procedure, a carefully measured quantity of an internal standard is added to each standard and sample. And the ration of analysis to internal standard peak areas (Heights) serves as the analytical parameter.

External Standard Method: Separate injections of fixed volume of sample and standard solution are given. Peaks are integrated and concentration of sample calculated by direct comparison ^12-15.

METHODOLOGY: A method was developed for simultaneous estimation of Tamsulosin and Dutasteride on HPLC by selecting the appropriate λ max, optimum mobile phase and flow rate which gives peaks with good resolution, HETP, Tailing factor and other related System suitability parameters.

Selection of Solvent: The ideal property of a solvent should be that the drug should be completely soluble in the solvent used. Drug should be stable in the solvent used and should be economical. Various solvents were studied for the solubility of Tamsulosin and Dutasteride. Both the drugs were very soluble in methanol and freely soluble in acetonitrile and slightly soluble in other non-polar solvents. Therefore, methanol was selected as a solvent of choice for both drugs. Both the drugs were stable in methanol ¹⁶.

Preparation of Standard Stock Solutions:

For UV: The stock solution of Tamsulosin and Dutasteride was prepared separately by dissolving accurately 20 mg in 20 ml of methanol mix well & sonicate. Take 0.1ml from above stock solution dilute with 10 ml methanol to obtain a final concentration of 10 mg/ml ¹⁷.

For HPLC: The stock solution of Tamsulosin was prepared by dissolving accurately weighed 4 mg in 10 ml of methanol mix well & sonicate after that 10ml transferred into the 100ml volumetric flask & make up the volume up to 100ml by methanol to obtain a final concentration of 40µg/ml. The stock solution of Dutasteride was prepared by dissolving accurately weighed 5 mg in 10 ml of methanol mix well & sonicate after that 10ml transferred in to the 100ml volumetric flask & make up the volume up to 100ml by methanol to obtain a final concentration of 50µg/ml.  This stock solution is used for system suitability study.

Mixed Stock Solution: The stock solution of Tamsulosin and Dutasteride was prepared by dissolving accurately weighed 4 mg of Tamsulosin & 5 mg of Dutasteride in 10 ml of methanol mix well & sonicate after that 10 ml of standard stock solution of Tamsulosin and Dutasteride was taken and transferred it into a 100ml volumetric flask and diluted to 100 ml with the diluents to get a concentration of 40µg/ ml of Tamsulosin and 50µg/ ml of Dutasteride.

Selection of Wavelength: The wavelength at which Tamsulosin and Dutasteride showed maximum absorption was selected by obtaining overlay spectra of Dutasteride and Tamsulosin of 10 µg/ml concentration in methanol.

Selection of Mobile Phase: Tamsulosin and Dutasteride are marketed as combined dosage formulation. The proposed  method  for estimation  of  Tamsulosin and Dutasteride required  adequate  resolution  between  the  two  drug  peaks  in  the  chromatogram. Several solvent systems were tried to obtain optimum resolution ¹⁸.

Determination of Retention Time: The time at which Tamsulosin and Dutasteride showed peaks with good resolution was determined by injecting 4µg/ml and 5µg/ml solution into a chromatogram at the flow rate of 1.0 ml/min, UV detection at 253 nm.

Determination of Retention Time for Tamsulosin:

Standard Stock Solution of Rosuvastatin: Accurately, 4 mg of Tamsulosin was weighed into a clean and dry 10mL volumetric flask, dissolved with sufficient volume of mobile phase. The volume was then made up to 10 ml with mobile phase to get a concentration of 400µg/ml (Stock I).10 ml of the stock solution was further diluted to 100 ml with mobile phase to get a concentration 40 µg/ml (Stock II).

System Suitability Parameters: The system suitability parameters are an integral part of many analytical procedures. The tests are performed to ascertain the suitability of the proposed method with mobile phase.

A solution of 4μg/ml of Tamsulosin and 5 µg/ml of Dutasteride was prepared by diluting suitably with mobile phase and was injected into the chromatograph, the peak area, chromatogram and various system suitability parameters such as Theoretical Plates, HETP (Height Equivalent to Theoretical Plate), Peak Asymmetry (As) and Resolution (Rs) ^20-22.

System suitability parameters can be calculated using the following equations.

Theoretical Plates: Measurement of the column efficiency. A measure of peak band spreading determined by various methods, some of which are sensitive to peak asymmetry ²³.

N = 5.54(t _R /W)²

Where, ‘W’ is the peak width at 50% peak height and t _R is the retention time.

HETP (Height Equivalent to Theoretical Plate): A measure of column’s efficiency. For a typical well packed HPLC column with 5µm particles, HETP values are usually between 0.01 and 0.03 mm

HETP = L/N

Where, L: Length of the column in millimeters. N: Number of Theoretical Plates

Asymmetric Factor: Factor describing the shape of a chromatographic peak. Theory assumes a Gaussian shape and that peaks are symmetrical. A value >1 is a tailing peak, while a value <1 is a fronting peak.

Af = b/a at 10% peak height.

Where, a = peak from at 10% to drop line, b = drop line to peak tail at 10%

Resolution (Rs): Ability of a column to separate chromatographic peaks.

RS = (2(t ₂-t ₁) / (W₁ +W₂)

Where, t₁ and t₂ are the retention time of the two components and W1 and W₂ are the width at the 50% of peak height ^24-27.

Validation of Analytical Method for the Assay of Tamsulosin and Dutasteride: Validation of an analytical method is a process to establish that the performance characteristics of the developed method meet the requirements of the intended analytical application.

The developed HPLC method for simultaneous estimation of Tamsulosin and Dutasteride using Universal inertsil ODS column Enable 250mm x 4.6mm, 5µm, mobile phase Sodium phosphate buffer: Acetonitrile (30:70), detection wavelength at 233 nm, at a flow rate of 1.0 ml/min was validated for typical analytical parameters like, Accuracy, Precision, Specificity, and Detection ²⁸.

Standard STOCK SOLUTION of Tamsulosin and Dutasteride: Accurately weighed 4mg of Tamsulosin and 5mg  of Dutasteride  was transferred into a clean and dry 10mL volumetric flask and dissolved with sufficient volume of mobile phase, and then volume was made up to 10mL with mobile phase to obtain concentration of 400 µg/ml and 500 µg/ml .

Standard Stock Solution of Tamsulosin and Dutasteride: Accurately weighed 4mg of Tamsulosin and 5mg of Dutasteride  was transferred into a clean and dry 10mL volumetric flask and dissolved with sufficient volume of mobile phase, and then volume was made up to 10 ml with mobile phase to obtain concentration of 400 µg/ml and500 µg/ml .

Working Solution: 10 ml of the stock solution was further diluted to 100 ml with mobile phase to get a concentration 40µg/ml and 50µg/Mr.

Standard Stock Solution of Tamsulosin and Dutasteride: Accurately weighed 4mg of Tamsulosin and 5mg of Dutasteride was transferred into a clean and dry 10mL volumetric flask and dissolved with sufficient volume of mobile phase, and then volume was made up to 10mL with mobile phase to obtain concentration of 400 µg/ml and 500 µg/mL ²⁹.

Working Solution: 10mL of the stock solution was further diluted to 100 ml with mobile phase to get a concentration 40µg/ml and 50µg/Mr.

Procedure: 20µl of mixed working standard solution were injected in different laboratories, by different analysts and chromatograms are recorded.

Acceptance Criteria: the relative standard deviation should be within 2% w/v.

Specificity: Specificity is the ability to assess unequivocally that the analyze in the presence of components which may be expected to be present; typically, these might include impurities, degradation products and matrix components.

Limit of Detection (LOD): Limit of detection is the lowest concentration of the analysis that can be detected by injecting decreasing amount, not necessarily quantity by the method, under the stated experimental conditions.

Linearity and Range: The linearity of an analytical procedure is its ability to obtain test results which are directly proportional to the concentration of analysis in the sample. Each working standard solution of Tamsulosin and Dutasteride were injected into the chromatograph. The peak areas and chromatograms were recorded. A linearity graph was plotted with concentration variable on x – axis and peak areas on y – axis for Tamsulosin and Dutasteride. The Correlation coefficient and Percentage curve fittings were calculated ^30-34.

Standard Stock Solution of Tamsulosin and Dutasteride: Accurately weighed 4mg of Tamsulosin and 5mg  of Dutasteride was transferred into a clean and dry 10mL volumetric flask and dissolved with sufficient volume of mobile phase, and then volume was made up to 10 ml with mobile phase to obtain concentration of 400 µg/ml and 500 µg/ml .

Working Standard Solution: Aliquots from standard stock solution were withdrawn in the volumes of 2.5, 5, 7.5, 10, 12.5 and 15mL in separate 100 ml volumetric flasks. The volume was made up with the mobile phase to get concentrations ranging from 10-60 µg/ml for Tamsulosin and 12.5-75 µg/ml for Dutasteride.

Procedure:  20 µl of each working standard solutions were injected into the chromatograph, the peak area and chromatogram obtained were recorded and are presented in Table 18 and Fig. 16 & Fig. 17. The linearity parameters were calculated and the results obtained are presented in Table 19.

Robustness: The robustness of an analytical method is a measure of its capacity to remain unaffected by small but deliberate variations in method parameters and provides an indication of its reliability during normal usage. The evaluation of robustness should be considered during the development phase and depends on the type of the procedure under study. It should show the reliability of an analysis with respect to deliberate variations in method parameters, like Influence of variations in flow rate, Influence of variations in Buffer Ratio ³⁵.

Standard Stock Solution of Tamsulosin and Dutasteride: Accurately weighed 4mg of Tamsulosin and 5mg of Dutasteride  was transferred into a clean and dry 10mL volumetric flask and dissolved with sufficient volume of mobile phase, and then volume was made up to 10mL with mobile phase to obtain concentration of 400 µg/ml and 500 µg/ml .

Working Solution: 10 ml of the stock solution was further diluted to 100 ml with mobile phase to get a concentration 40µg/ml and 50µg/Mr.

Change in Flow Rate: For the method developed, flow rate of 1mL/min was used. For Robustness study, flow rates of 0.9 and 1.1mL/min were selected. Three injections of 20 μl of each working standard solution of Tamsulosin and Dutasteride at flow rate of 0.9 and 1.1 ml/min were injected into the chromatograph, the peak area and chromatogram obtained were recorded and the % assay was calculated.

Change in Buffer Ratio: 20µl of mixed working standard solution were injected in different chromatographic condition of buffer ratio and chromatograms are recorded. The results obtained are presented

TABLE 2: ACCEPTANCE CRITERIA FOR DIFFERENT VALIDATION PARAMETERS

Application of the Developed HPLC Method for Simultaneous Estimation of Tamsulosin and Dutasteride in Marketed Formulation: The developed and validated method has been applied for Simultaneous Estimation of Tamsulosin and Dutasteride in marketed formulation ³⁶.

TABLE 3: CHROMATOGRAPHIC CONDITIONS FOR SAMPLE ANALYSIS

Preparation of Sample Stock Solution: Weighed 20 Tablet; take Avg. wt. of it powdered equivalent to 4 mg of Tamsulosin and 5 mg of Dutasteride was accurately weighed and transferred into clean, dry 10mL volumetric flask. The powder was first dissolved in few ml of mobile phase by sonication, the volume was made up to 100mL and then filtered through a Whatmann filter to obtain the concentration 400µg/ml and 500µg/ml for Tamsulosin and Dutasteride respectively (Stock I) ³⁸.

Working Sample Solutions: From the above stock, volumes of 10mL were transferred into 100mL volumetric flasks and volume made up to 100mL with the mobile phase to get the concentration of 40µg/ml for Tamsulosin and 50µg/ml for Dutasteride respectively.

Procedure: 20 µl solution of the working sample solutions were injected repeatedly into the chromatograph, at a flow rate of 1 ml/min and detection at 233 nm, the chromatograms obtained were recorded.

The amount of drug per Tablet & % assay is calculated by following formula:

Amount /Tablet = (sample peak avg.) / (std peak avg.) × (std wt) / (sample wt) × (std factor) /(sample factor) X avg. wt × (std purity) /100

% Assay = (amount per Tablet) / (label claim) × 100

RESULTS: A method was developed for simultaneous estimation of Tamsulosin and Dutasteride on HPLC by selecting the appropriate λ max, optimum mobile phase and flow rate which gives good and sharp resolution.

Selection of Wavelength: Standard solutions of Tamsulosin and Dutasteride (10 µg/ml)  were scanned  in  the  wavelength  range  of  200-400 nm using Methanol  as  diluents  and  UV  spectrum obtained  is  presented below.

FIG. 1: OVERLAY SPECTRUM OF TAMSULOSIN AND DUTASTERIDE

Report: The 10μg/ml of Tamsulosin and Dutasteride solutions showed λ max at 233.20 nm which was selected as wavelength maxima for determination of Tamsulosin and Dutasteride

Selection of Mobile Phase: Several solvent systems were tried to get optimum resolutions of Tamsulosin and Dutasteride in the present method. The observations obtained with various mobile phases in different ratios given in table in methodology chapter are given below.

FIG. 2(A): CHROMATOGRAM FOR COMB. F METHANOL AND WATER (70:30)

FIG. 2(B): CHROMATOGRAM FOR COMB. OF ACETONITRILE AND WATER (50:50)

FIG. 2(C): CHROMATOGRAM FOR COMB. OF ACETONITRILE AND MIX PHOSPHATE BUFFER (70:30)

FIG. 2(D): CHROMATOGRAM FOR COMB. OF ACETONITRILE AND MIX PHOSPHATE BUFFER (65:35)

FIG. 2(E): CHROMATOGRAM FOR COMB. OF ACETONITRILE AND MIX PHOSPHATE BUFFER (60:40)

FIG. 2(F): CHROMATOGRAM FOR COMB. OF SODIUM PHOSPHATE BUFFER: ACETONITRILE (20:80)

FIG. 2(G): CHROMATOGRAM OF TAMSULOSIN AND DUTASTERIDE PEAKS SHOWING GOOD RESOLUTION WITH MOBILE PHASE SODIUM PHOSPHATE BUFFER: ACETONITRILE (30:70)

Report: In this trail I got proper resolution i.e. 10.94 also proper retention time is 2.949 for Tamsulosin and 5.572 for Dutasteride and plate count are within limit so I finalized this trial as optimized method for Tamsulosin and Dutasteride drug

Determination of Retention Time: The optimum retention time at which Tamsulosin and Dutasteride showed good resolution was selected for the proposed method. The chromatograms obtained individually for Tamsulosin and Dutasteride and in combination are presented below.

(a) Determination of Retention Time for Tamsulosin:

FIG. 3: CHROMATOGRAM FOR RETENTION TIME OF TAMSULOSIN

Report: The Retention time for Tamsulosin was found to be 2.915 min.

Determination of Retention Time for Tamsulosin and Dutasteride:

FIG. 4: CHROMATOGRAM FOR RETENTION TIME OF TAMSULOSIN AND DUTASTERIDE IN COMBINATION

Report: The retention time for Tamsulosin  was found to 2.949 min and the retention time for Dutasteride  were found to be 5.672 min indicating no change in retention time for Tamsulosin and Dutasteride in combination.

System Suitability: This  parameter  ensures  that  the  analytical  system  is  working  properly  and  can  give accurate  and  precise  results.

FIG. 5: CHROMATOGRAM OF SYSTEM SUITABILITY PARAMETERS FOR TAMSULOSIN AND DUTASTERIDE

TABLE 4: SYSTEM SUITABILITY DATA OF PARAMETERS FOR TAMSULOSIN AND DUTASTERIDE

Report: The obtained system suitability parameters for Tamsulosin and Dutasteride were found to be within the acceptance criteria.

Validation of analytical method for the assay of Tamsulosin and Dutasteride: The HPLC method developed was validated by performing the various method validation parameters like specificity, LOD, LOQ, linearity, range, precision, robustness, accuracy, and system suitability parameters as per ICH guidelines.

This is to ensure that the performance characteristics of the HPLC method developed meets the requirements for the intended analytical applications.

Accuracy: This parameter is performed to determine the closeness of test results with that of the true value which is expressed as % recovery.

These studies were performed at three different levels (50%, 100% and 150%) and the % recovery of Tamsulosin and Dutasteride was calculated. 20μL of three different solutions of Tamsulosin and Dutasteride were injected repeatedly into chromatograph, the peak area and chromatogram were recorded and are presented below.

TABLE 5: RECOVERY STUDY DATA FOR TAMSULOSIN AND DUTASTERIDE

TABLE 6: RECOVERY STUDY DATA FOR TAMSULOSIN IN 50%, 100%, 150% SPIKE

TABLE 7: RECOVERY STUDY DATA FOR DUTASTERIDE IN 50%, 100%, 150% SPIKE

FIG. 6: CHROMATOGRAM FOR RECOVERY STUDIES AT 50% LEVEL

FIG. 7: CHROMATOGRAM FOR RECOVERY STUDIES AT 100% LEVEL

FIG. 8: CHROMATOGRAM FOR RECOVERY STUDIES AT 150% LEVEL

Report: The mean percentage recovery for Tamsulosin and Dutasteride at three different levels was found to be between 98.03-100.11% and 99.29-99.47% respectively, which are well within the limit and hence the method was found to be accurate.

Precision: Precision of an analytical method is usually expressed as the standard deviation or relative standard deviation (coefficient of variation) it is performed to see the closeness of agreement between the series of measurements. It is determined by assaying sufficient number of samples and their relative standard deviation is determined.

System Precision: This method validation parameter was performed to ensure the closeness of results between true value and experimental value. The peak area was recorded and is presented below.

TABLE 8: SYSTEM PRECISION DATA FOR TAMSULOSIN AND DUTASTERIDE

FIG. 9: CHROMATOGRAM OF TAMSULOSIN AND DUTASTERIDE FOR SYSTEM PRECISION

Report:  The % RSD values of peak area for six replicate injections of Tamsulosin and Dutasteride were found to be 0.35 and 0.27 respectively which are well within the acceptance criteria limit of NMT 2%.

Method Precision (Repeatability): The method precision was performed to standardize methodology i.e. to check whether the developed method is precise i.e. whether the method is giving consistent results.

The % RSD for peak area and was calculated.

TABLE 9: METHOD PRECISION DATA TAMSULOSIN AND DUTASTERIDE

FIG. 10: CHROMATOGRAM OF TAMSULOSIN AND DUTASTERIDE FOR METHOD PRECISION

Report:  The % RSD values of concentration for six replicate injections of Tamsulosin and Dutasteride were found to be 0.18 and 0.13 respectively which are well within the acceptance criteria limit of NMT 2%.

Specificity: Specificity was performed to assess and ensure that the impurities, degraded products do not interfere with peaks of analyses.

FIG. 11: CHROMATOGRAM FOR SPECIFICITY (BLANK)

FIG. 12: CHROMATOGRAM FOR SPECIFICITY (STANDARD)

FIG. 13: CHROMATOGRAM FOR SPECIFICITY (ALKALI)

Observation: The analyses do not show any interference with the degraded components.

Report:  As there is no interference with the degraded components at retention time of 2.979 (Tamsulosin) and 5.305 min (Dutasteride), hence the proposed method was specific for the detection of Tamsulosin and Dutasteride in combination product.

Limit of Detection (LOD): Limit of detection is the lowest concentration of the analyze that can be detected by injecting decreasing amount, not necessarily quantity by the method, under the stated experimental conditions. The minimum concentration at which the analyze can be detected is determined from the standard deviation of the response and the slope by applying the formula.

Report: The lowest concentration of Tamsulosin that can be detected, was determined from standard curve was 1.151µg/Mr. The lowest concentration of Dutasteride that can be detected was determined from standard curve was 4.498µg/mL.

Limit of Quantification (LOQ): Limit of quantification is the lowest concentration of the analysis in a sample that can be estimated quantitatively. By injecting decreasing amount of drug, with acceptable precision and accuracy under the stated experimental conditions of the method. Limit of quantification can be obtained from the standard deviation of the response and the slope by applying the following formula.

Limit of quantification =     σ/S   x 10

Report: The lowest concentration at which peak can be quantified is called LOQ, which was found to be 3.593µg/ml for Tamsulosin. The lowest concentration at which peak can be quantified is called LOQ, was found to be 14.041µg/ml for Dutasteride.

Linearity and Range:  The linearity was performed  to  ensure  that  the  test  results  are  directly  proportional to the concentration of analyze sample. The results obtained are tabulated below.

TABLE 10: LINEARITY RANGE DATA FOR TAMSULOSIN AND DUTASTERIDE

FIG. 14: LINEARITY RANGE GRAPH OF TAMSULOSIN                            

FIG. 15: LINEARITY RANGE GRAPH OF DUTASTERIDE

TABLE 11: LINEARITY REPORT FOR TAMSULOSIN AND DUTASTERIDE

Report: The linearity in response for Tamsulosin and Dutasteride was observed in the concentration range of 10 to 60µg/ml and 12.5 to 75µg/ml respectively, with percentage curve fittings found to be well within the limits of acceptance criteria (99%).

Robustness: Robustness was performed to check the ability of the system to give unaffected results for small deliberate changes in system parameters and method parameters.

 (a) Change in Flow Rate: 

TABLE 12: ROBUSTNESS DATA FOR CHANGE IN FLOW RATE

FIG. 16: CHROMATOGRAM FOR CHANGE IN FLOW RATE (0.9ML/MIN)

FIG. 17: CHROMATOGRAM FOR CHANGE IN FLOW RATE (1.1ML/MIN)

Report: The % assay were found to be 101.37%, 104.63% for Tamsulosin and Dutasteride    when the flow rate was deliberately changed to 0.9 ml and 1.1mL respectively, indicating that the method was found to be robust with deliberate change in flow rate.

CONCLUSION: Stability indicating HPLC were developed for the simultaneous estimation of Tamsulosin and Dutasteride in marketed formulation. The HPLC system used was WATERS Alliance series System with Rheodyne injector 20 μL and the column Inertsil ODS 250 x 4.6 mm, 5 μm. The mobile phase comprised of Sodium phosphate buffer and Acetonitrile in the ratio of 30:70 v/v and flow rate of 1.0 ml/min with UV detection at 233 nm produced peaks of Tamsulosin and Dutasteride in the chromatogram which were well resolved with retention time of 2.939 min and 5.572 min respectively.

The developed HPLC method was validated for various parameters like accuracy, precision, specificity, LOD, LOQ, linearity, range and robustness as per ICH guidelines. The results obtained were well within the acceptance criteria for all the parameters. The proposed method was applied for simultaneous estimation of Tamsulosin and Dutasteride in marketed formulations (Tablet). The assay results conformed to the label claim of the formulation. Hence the proposed method can be used for the routine analysis of Tamsulosin and Dutasteride in their marketed tablet dosage formulations.

ACKNOWLEDGEMENT: The authors are thankful to Department of Pharmacy, Hindu College of Pharmacy, Sonipat and thankful to Devender chauhan from Gateway College of Pharmacy, Delhi-NCR, Sonipat for good guidance.

CONFLICT OF INTEREST: Authors declare no conflict of interest.

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    How to cite this article:

    Singh B and Chauhan D: Development and validation of RP-HPLC method for simultaneous estimation of tamulosin and dutasteride in a bulk and tablet dosage form. Int J Pharm Sci & Res 2025; 16(7): 2063-82. doi: 10.13040/IJPSR.0975-8232.16(7).2063-82.

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