A RP-HPLC Method Development and Validation for the Estimation of Tadalafil in Bulk and Pharmaceutical Dosage Forms

 

Saroj Kumar Raul¹*, B.V.V Ravi kumar², Ajaya Kumar Pattnaik³

¹M. R. College of Pharmacy, Phool-Baugh, Vizianagaram, A.P, India.

²Roland Institute of Pharmaceutical Sciences, Berhampur, Orissa, India.

³Departments of Chemistry, Ravenshaw University, Cuttack, Orissa, India.

 

ABSTRACT:

A simple, selective, linear, precise and accurate RP-HPLC method was developed and validated for rapid assay of Tadalafil in pharmaceutical dosage form. Isocratic elution at a flow rate of 1.0 mL min -1 was employed on a Symmetry C18 column at ambient temperature. The mobile phase consisted of acetonitrile: buffer 50:50 (v/v) and the detection wavelength was at 284 nm. Linearity was observed in concentration range of 20-70 μg/mL. The retention time for Tadalafil was 2.98 min. The method was validated as per the ICH guidelines. The proposed method can be successfully applied for the estimation of Tadalafil in pharmaceutical dosage forms.

 

 

INTRODUCTION:

Tadalafil is chemically (6R-trans)-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a hexahydro-2-methyl-pyrazino [1', 2':1,6] pyrido [3,4-b]indole-1,4-dione (Figure-1). It is used for the treatment of male penile erectile dysfuctions (ED) through selective inhibition of enzyme phosphodiesterase to decrease the metabolism of cyclic guonosine monophosphate (cGMP) which induces smooth muscle relaxation in the corpus cavereosaus with onset around 30-45 min. It is not official in any of the pharmacopoeias but listed in the Merck Index¹ and Martindale^2.

 

Literature survey reveals that few spectrophotometric methods^3-4   HPLC methods^5-10 has been reported for the estimation of Tadalafil. The aim of the present study is to develop a simple, precise and accurate reversed-phase HPLC method for the estimation of Tadalafil in pharmaceutical dosage form as per ICH guidelines¹¹.

 

Figure1: Chemical structure of Tadalafil

 

MATERIALS AND METHOD:

Instrumental and analytical conditions

The HPLC analysis was carried out on Waters HPLC system (2695 module) equipped with 2487 dual lambda detector with auto Sampler and running on Waters Empower software. The column used is Symmetry C₁₈ (150 × 4.6 mm, packed with 5 µm) and detection was performed at 284 nm. The injection volume of sample was 20 µL and the run time was 6 minutes. An isocratic mobile phase containing acetonitrile and acetic acid buffer   at 50: 50 (v/v) carried with the flow rate at 1.0mL min^-1. The mobile phase was filtered through 0.45µm membrane filter and degassed before use.

 

Reagents and chemicals

Tadalafil working standard was kindly gifted by pharmatrain, Hyderabad. Tablets were purchased from local pharmacy manufactured by Macleods Pharmaceuticals Ltd (Megalis). Ultra pure water was obtained from a millipore system. HPLC grade acetonitrile was obtained from Merck (India) limited. All other chemicals used were AR grade. The optimum chromatographic conditions were summarized in table 7.

 

Preparation of mobile phase

Two mL of Glacial acetic acid was transferred in to 1000 mL beaker dissolve and volume adjusted with water and mixed, then sonicated to degas the buffer. Transferred 500 volumes of acetonitrile and 500 volumes of buffer into a 1000 volumes mobile phase bottle and mixed.

 

Figure 2: Linearity curve of Tadalafil

 

 

Then sonicated up to 15 minutes for degas the mobile phase and filtered through 0.45 μm filter under vacuum. The same mobile phase was used as diluent.

 

Preparation of Standard Solution

Accurately weighed about 10 mg of Tadalafil and transferred into a 10mL volumetric flask and 7 mL of diluent was added and sonicate to dissolve it completely and the volume was adjusted with the mobile phase to get stock solution of 1000 µg/mL. Then 0.4 mL of stock solution is transferred into 10 ml volumetric flask and make up to volume with mobile phase and filter through 0.45 μm filters, which gives a solution of strength 40 µg/mL. 

 

Preparation of sample solution

Weigh 20 tablets of Tadalafil and calculate the average weight. Accurately weigh and transfer the sample equivalent to 50 mg of Tadalafil into a 50 ml volumetric flask. Add about 25mL of diluent, sonicate to dissolve it completely and make volume up to the mark with diluent. Mix well and filter through 0.45 μm filter. Further pipette 0.4 ml of the above stock solution into a 10 mL volumetric flask and dilute up to the mark with diluent. Mix well and filter through 0.45 μm filter.

 

Method Validation

The objective of the method validation is to demonstrate that the method is suitable for its intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision, accuracy, specificity, limit of detection, limit of quantification, robustness and system suitability.

 

Linearity

From the standard stock solution, the various dilutions of Tadalafil in the concentration of 20, 30, 40, 50, 60 and 70 µg/mL were prepared. The solutions were injected using 20 μL injection volumes in to the chromatographic system at the flow rate of 1.0 mLmin^-1 and the effluents were monitored at 284 nm, chromatograms were recorded. Calibration curve of Tadalafil was obtained by plotting the peak area ratio versus the applied concentrations of Tadalafil, given in table 1. The linear correlation coefficient was found to be 0.999, shown in figure 2.

 

 

Table 1: Linearity of Tadalafil

Precision

Repeatability of the method was checked by injecting replicate injections of 40 μg/mL of the solution for six times on the same day as intraday precision study of Tadalafil and the % RSD was found to be 0.25, given in table 2.

 

Table 2: Precision of Tadalafil

 

Accuracy

Tadalafil reference standards were accurately weighed and added to a mixture of the tablets excipients, at three different concentration levels (50%, 100% and 150%). At each level, samples were prepared in triplicate and the recovery percentage was determined and presented in table 3.

 

Table 3: Accuracy of Tadalafil

% Conc	Amount added (mg)	Amount found (mg)	% Recovery	Mean Recovery
50%	5.0	4.9	98%	99.44 %
100%	10.0	10.1	101%
150%	15.0	14.9	99.33 %

Table 4: Robustness parameter of Tadalafil

Parameters	Adjusted to	Average Area	Rt	SD	% RSD
Flow rate as per method 1.0mL/min	0.8 mL/min	1369987	2.991	3618.92	0.26
	As it is	1378876	2.988	3593.62	0.26
	1.2ml/min	1379823	2.898	3897.36	0.28
Mobile phase composition Acetonitrile: Buffer (50:50)	Acetonitrile: Buffer (45:55)	1379221	2.956	5972.18	0.43
	As it is	1374635	2.987	4987.56	0.36
	Acetonitrile: Buffer (55:45)	1380231	2.977	10235.12	0.74

 

 

Specificity

Spectral purities of Tadalafil chromatographic peaks were evaluated for the interference of the tablet excipients as per the methodology. In the work, a solution containing a mixture of the tablet excipients was prepared using the sample preparation procedure to evaluate possible interfering peaks and no interference peaks were observed.

 

Robustness

To determine the robustness of the method, two parameters (flow rate, composition of mobile phase) from the optimized chromatographic conditions were varied. Statistical analysis showed no significant difference between results obtained employing the analytical conditions established for the method and those obtained in the experiments in which variations of parameters were introduced. Thus the method showed to be robust which is shown in table 4.

 

Ruggedness

Inter day variations were performed by using six replicate injections of standard and sample solutions of concentrations which were prepared and analyzed by different analyst on three different days over a period of one week. Ruggedness also expressed in terms of percentage relative standard deviation and statistical analysis showed no significant difference between results obtained employing different analyst.

 

Detection and quantitation limits

According to the determined signal-to-noise ratio, Tadalafil Presented limits of detection of 0.2 μg/mL and limits of quantitation of 0.92μg/mL, where the compounds proportion found in the sample solutions injected on to the chromatograph. However, the objective of the method is the quantitation of Tadalafil so that the values obtained should be considered as the limit of method sensitivity.

 

System Suitability

System suitability tests were carried out on freshly prepared standard stock solutions of Tadalafil and it was calculated by determining the standard deviation by injecting standards in six replicates at 6 minutes interval and the values were recorded and the system suitability parameters are shown in table 5.

 

Assay of Tadalafil tablet

Three different batches of Megalis were analyzed using the validated method. For the analysis, six replicates of each batch were assayed. Twenty tablets were weighed and finely powdered. An accurately weighed portion of the powder, equivalent to about 50mg of Tadalafil was transferred to a 50 ml volumetric flask followed by the addition of 25 ml of mobile phase. The solution was sonicated for 3 minutes and volume adjusted with the mobile phase then filtered through 0.45 μm membrane filter. Further dilutions were made to get the final concentration equivalent to 40 µg/mL of Tadalafil. The mean peak area of the drug was calculated and the drug content in the tablets was quantified and the results were presented in table 6.

 

Table 5: System Suitability of Tadalafil

 

All the analyzed batches presented Tadalafil were very close to the labeled amount. The Tadalafil content in the tablets samples varied from 98.8 to 99.8%.

 

Table 6: Contents of Tadalafil in tablets (n=6)

Sample tablet	Batch	Labelled amount(mg)	Amount found (mg)±SD	%Amount found
Megalis (10mg)	1 2 3	10 10 10	9.98±0.12 9.88±0.08 9.96±0.14	99.8 98.8 99.6

S.D=Standard Deviation

 

RESULTS AND DISCUSSION:

The nature of the sample, its molecular weight and solubility decides the proper selection of the stationary phase. The drug Tadalafil was preferably analyzed by reverse phase chromatography and accordingly C₁₈ column was selected. The elution of the compound from the column was influenced by polar mobile phase. The ratio of the acetonitrile to acetic acid buffer was optimized to give symmetric peak with short run time. Different mobile phases were tried but satisfactory separation, well resolved and good symmetrical peaks were obtained with the mobile phase of acetonitrile: buffer at the ratio of 50:50 (v/v).The retention time of Tadalafil was found to be 2.98 min, which indicates a good base line. The RSD values for accuracy and precision studies obtained were less than 2% which revealed that developed method was accurate and precise. The system suitability parameters are given in Table 5. Developed chromatographic method was applied for the determination of Tadalafil in tablet formulation, given in table 7. A typical chromatogram showing the separation of Tadalafil is shown in figure 3.

 

Table7: Developed Chromatographic Conditions

Parameters	Method
Stationary phase (column)	Symmetry C18 (150 × 4.6 mm, packed with 5 µm)
Mobile Phase	50:50 (Acetonitrile : Acetic acid Buffer)
pH	3.5 ± 0.02
Flow rate (ml/min)	1.0
Run time (minutes)	6.0
Column temperature (°C)	Ambient
Volume of injection loop (ml)	20
Detection wavelength (nm)	284
Drugs Rt (min)	2.98

 

Figure 3: Standard Chromatogram of Tadalafil.

 

CONCLUSIONS:

A validated RP-HPLC method has been developed for the determination of Tadalafil in tablet dosage form. The proposed method is simple, rapid, accurate, precise and specific. Therefore, it is suitable for the routine analysis of Tadalafil in pharmaceutical dosage form.   

 

ACKNOWLEDGEMENTS:

The authors are thankful to Pharmatrain, Kukatapally, Hyderabad for providing gift sample of Tadalafil and for providing necessary facilities to carry out the research work.

 

REFERENCES:

1.        O Neil MJ. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals. Merck and Co. Inc. Whitehouse station, NJ. 2001; 13^th Edition: pp 218.

2.        Sweetman SC. Martindale, The complete Drug Reference. The Pharmaceutical press, London. 2002; 34th Edition: pp875.

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4.        Ganjan A, Bhavin C, Ashutosh P, Jagdish K,Dharmendrasinh B. Development and validation of dual wavelength UV Spectrophotometric method for simultaneous estimation of Tadalafil and Dapoxetine hydrochloride in their Combined tablet dosage form. International journal of pharmaceutical research and bio-science. 1(2); 2012:247-255.

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8.        Kannappan N, Yada D, Shashikanth, Mannavalan R. Method development and validation of stability indicating methods for assay of Tadalafil and sildenafil citrate by HPLC. International Journal of Chem Tech Research. 2(1); 2010: 329-333.

9.       9.   Chandran M,   Kannan K.   A Validated RP-HPLC Method for Simultaneous Estimation of Tadalafil and Dapoxetine in Tablet Dosage Form. Journal of Scientific Research in Pharmacy. 1(2); 2012: 36-39.

10.     Abha DG, Vidhya KB, Sunil RD. Validated HPLC Method for Simultaneous Quantitation of Tadalafil and Dapoxetine Hydrochloride In Bulk Drug and Formulation. International Journal of Pharmacy and Pharmaceutical Sciences. 4(2); 2012: 654-658.

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Received on 23.11.2012          Modified on 26.11.2012

Accepted on 29.11.2012         © RJPT All right reserved