Stability-Indicating LC Method for Determination of Tadalafil in Bulk Drug and Pharmaceutical Dosage Form

Chem. Anal. (Warsaw), 54, 679 (2009) Stability-Indicating LC Method for Determination of Tadalafil in Bulk Drug and Pharmaceutical Dosage Form by Mad...
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Chem. Anal. (Warsaw), 54, 679 (2009)

Stability-Indicating LC Method for Determination of Tadalafil in Bulk Drug and Pharmaceutical Dosage Form by Madhuri M. Mathpati, Jaipraksh N. Sangshetti1, Vipul P. Rane1,2, Kiran R. Patil1,2 and Devanand B. Shinde1* 1

Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India 2 Wockhardt Research Centre, Aurangabad-431210 (MS), India

Keywords:

Column liquid chromatography; Forced degradation; Tadalafil; Stabilityindicating method; Method validation

A novel stability-indicating LC assay method for quantitative determination of tadalafil in bulk drug and pharmaceutical dosage form in the presence of forced-degradation products was developed and validated. An isocratic reversed phase LC method was developed to separate the drug from its degradation products using a Zorbax SB-C18 column and water–acetonitrile mixture as a mobile phase. Detection was carried out at the wavelength of 235 nm. Tadalafil was subjected to stress conditions in order to perform its hydrolytic (acid, base), oxidative, photolytic, and thermal degradation. Degradation of tadalafil was observed in the presence of acid, base, and 30% H2O2. The drug was found to be stable under other stress conditions. The signals of degradation products were well-resolved from the main peak of tadalafil. Percentage recovery of tadalafil in pharmaceutical dosage form ranged from 98.89% to 101.25%. The developed method was validated with respect to linearity, accuracy (recovery), precision, specificity, and robustness. Forced degradation studies proved the stability-indicating power of the method. Opracowano i zwalidowano now¹ metodê iloœciowego oznaczania tadalafilu za pomoc¹ chromatografii cieczowej, w obecnoœci produktów rozk³adu, pozwalaj¹c¹ na ocenê stabilnoœci surowca i formy farmaceutycznej. Zastosowanie kolumny Zorbax SB-C18 i izokratycznej mieszaniny woda –acetonitryl pozwala³o na rozdzielenie leku od produktów rozk³adu. Detekcjê przeprowadzono przy d³ugoœci fali 235 nm. Tadalafil poddano ekstremalnym warunkom w celu uzyskania hydrolitycznych (kwas, zasada), oksydatywnych, fotolitycznych i termicznych produktów rozk³adu. Rozk³ad tadalafilu obserowwoano w obecnoœci kwasów, zasad i 30% H2O2. Lek okaza³ siê trwa³y w pozosta³ych ekstremalnych warunkach. Sygna³y produktów rozk³adu by³y dobrze oddzielone od g³ównego piku tadalafilu. Odzysk w przy* Corresponding author. E-mail: [email protected]

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padku badania postaci farmaceutycznej wynosi³ od 98,89% do 101,25%. Opacowan¹ metodê zwalidowano w zakresie liniowoœci, dok³adnoœci (odzysku), precyzji, specyficznoœci i odpornoœci na zmiany warunków otoczenia. Badania rozk³¹du w warunkach ekstremalnych wykaza³y przydatnoœæ opracowanej metody do oceny stabilnoœci leku.

Chemical name of tadalafil (Fig. 1) is (6R,12Ar)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylene dioxyphenyl) pyrazino (1’,2’:1,6) pyrido-(3,4-b) indole1,4-dione. An oral treatment for erectile dysfunction is based on selective inhibition of cyclic guanosine monophosphate (cGMP) – specific phosphodiesterase type 5 (PDE5). Through the inhibition on PDE5, tadalafil causes an increase of the concentration of cGMP, what results in smooth muscles relaxation and increased blood flow to the corpus cavernosum, and thereby enhanced erectile response following appropriate sexual stimulation.

Figure 1. Chemical structure of tadalafil

A literature survey has revealed that several methods for determination of tadalafil in pharmaceutical formulations have been reported, including HPLC [1–3] and capillary electrophoroses with UV detection [4, 5]. Many validated analytical methods for quantitation of tadalafil in human plasma using LC–MS–MS have been published [6]. However, only one stability-indicating HPLC method for determination of tadalafil in bulk and pharmaceutical dosage forms has been reported in the literature [7]. In this method, an ion-pairing reagent was used in a mobile phase; retention time of tadalafil was about 18 min. In this paper we report on the development and validation of a stability-indicating HPLC method for determination of tadalafil in bulk drug and pharmaceutical dosage form. This procedure allows for separation of the drug signal from the signal of its degradation products formed under stress conditions suggested by ICH (hydrolysis, oxidations, photolysis, and thermal stress) [8–10]. The method is rapid, robust, and

Stability-indicating LC method for determination of tadalafil

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economical. It does not require the presence of an ion-pairing reagent. It is useful for the routine analysis in the quality control labs due to the short run time. The developed procedure is stability-indicating and can be used for assessing the stability of tadalafil in bulk drugs and pharmaceutical dosage forms. The developed method has been validated with respect to linearity, accuracy, precision, LOD, LOQ, and robustness.

EXPERIMENTAL Material and reagents Tadalafil bulk drug (purity 99.24%) was obtained from Lupin Pharmaceutical Ltd. (Mumbai, India). Tadalafil tablets (20 mg) were purchased at the local market. Hydrochloric acid was obtained from Qualigens Fine Chemicals Ltd, India. Acetonitrile, hydrogen peroxide, sodium hydroxide were purchased from Rankem Laboratories, India. All chemicals and reagents used were of analytical or HPLC grade. UV cabinet was produced by Kumar (India). Milli–Q-water was used throughout. Chromatographic conditions An HPLC system (Jasco 2000 series) equipped with a PU2080 pump, a UV2075 detector, a rheodyne injector, and Borwin software was used. A Zorbax SB-C18 (octadecyl silane) 4.6 × 250 mm (5 μm) stainless steel chromatographic column (Agilent Technologes, Palo Alto, CA) was used. Sensitivity and flow rate were set at 0.001 AUFS (absorbance unit full scale) and 1 mL min–1, respectively. Injection volume was 10 µL. Detection wavelength was adjusted to 235 nm. Mobile phase Mobile phase consisted of water and acetonitrile in proportion 70:30, v/v. pH of water was adjusted to 3.5 with acetic acid. Mobile phase was mixed, filtered through a 0.45 µm nylon filter, and degassed. Preparation of standard stock solution All solutions were prepared by weighting appropriate amounts of the chemicals.. Standard solution of tadalafil was prepared by dissolving the drug in a diluent and completing the volume to the required concentration. Diluent A composed of methanol and acetonitrile in the ratio 50:50 v/v and diluent B composed of water and acetonitrile in the ratio 50:50 v/v. 50 mg of tadalafil were accurately weighed, transferred into a 50 mL volumetric flask, and dissolved in 50 mL of diluent A to obtain stock solution. 5 mL of the stock solution were transferred to 50 mL volumetric flask and diluted to the mark with diluent B. Final concentration of tadalafil in the obtained solution was 100 µg mL–1.

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Sample solution (tablets) 20 tablets of tadalafil (20 mg) were finely ground using an agate mortar and a pestle. The ground material equivalent to 100 mg of the active drug component was extracted with diluent A in a 100 mL volumetric flask applying vortex mixing followed by sonication and volume completion with diluent B. The solution was filtered through a 0.45-mm filter. Appropriate concentration of tadalafil (100 µg mL–1 for assay) in the final solution was adjusted with diluents at the time of analysis. Specificity/selectivity Specificity is the ability of the method to assess unequivocally the analyte in the presence of other components, which may be expected to be present in a sample. Typically, these might include degradation products, matrix, etc. [11]. Specificity of the developed LC method for tadalafil was investigated in the presence of its degradation products. Stress studies were performed using a bulk drug; their aim was to obtain an indication of tadalafil stability and thus to check the specificity of the proposed method. Intentional degradation was performed under stress condition by exposing the analyte to an acid (0.5 mol L–1 HCl), a base (0.1 mol L– NaOH), an oxidant (30% H2O2), heat (80°C), and UV light (wavelengths: 254 nm and 366 nm) in order to evaluate the ability of the proposed method to separate tadalafil from its degradation products. For photostability studies, an UV–cabinete was used. It was equipped with two sources of short and long UV light. Photostability studies lasted 10 days. Stress studies in the presence of acid, base, or hydrogen peroxide lasted 48 h. Peak purity of tadalafil was checked using a PDA detector. Assay of tadalafil was carried out against its reference standard; the results were calculated on the basis of the mass balance (% assay + % sum of all impurities + % sum of all degradation products). The mixture of excipients present in tadalafil tablets was injected under the optimised conditions to show specificity of the method towards tadalafil in its formulation. Procedure for forced degradation of tadalafil Acidic degradation. About 20 mg of tadalafil were accurately weighed and dissolved in 10 mL of diluent A. Then, 10 mL of 0.5 mol L–1 HCl were added and the solution was kept at 30°C for about 48 h in a water bath. After that time the solution was allowed to assume ambient temperature, then neutralised with 0.1 mol L–1 NaOH to pH 7, and finally its volume was made up to 50 mL with diluent B. Basic degradation. About 20 mg of tadalafil were accurately weighed and dissolved in 10 mL of diluent A. Then, 10 mL of 0.1 mol L–1 NaOH were added and the solution was kept at 30°C for about 48 h in a water bath. After that time the solution was allowed to assume ambient temperature, then neutralised with 0.1 mol L–1 HCl to pH 7, and finally its volume was made up to 50 mL with diluent B. Oxidative degradation. About 20 mg of tadalafil were accurately weighed and dissolved in 10 mL of diluent A. Then, 10 mL of 30% H2O2 solution were added and the sample was kept at the room temperature for about 48 h. Finally, its volume was made up to 50 mL with diluent B. Thermal degradation. About 50 mg of the drug were kept at 80°C for 48 h. Then, the drug was used to prepare tadalafil solution of the final concentration of 100 µg mL–1.

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UV degradation. About 50 mg of the drug were exposed for 48 h to the UV irradiation of short (254 nm) and long (366 nm) wavelength. Then, the drug was used to prepare tadalafil solution of the final concentration of 100 µg mL–1.

RESULTS AND DISCUSSION Optimisation of chromatographic conditions The primary target in developing the stability-indicating HPLC method was to achieve satisfactory resolution between the signals of tadalafil and its degradation products. Separation of tadalafil and its degradation products was achieved using a Zorbax SB column (250 mm × 4.6 mm, 5 µm) packed with C18 stationary phase and water (pH 3.5)–acetonitrile (70:30 v/v) mobile phase; column temperature was 30°C. The obtained tailing factor was lower than 2 and retention time was about 7.0 min for the main peak and less than 5 min for the peaks of degradation products. These parameters allowed one to reduce the total run time and ultimately increase the productivity, as well as to reduce the cost of analysis per sample. The forced degradation study showed that the proposed method is highly specific and that the signals of degradation products are well resolved from the main peak. The developed method was found specific; it was validated according to the ICH guidelines. Results of forced degradation experiments Tadalafil was found to be stable under the influence of heat and light. Degradation was observed under hydrolytic (acid and alkali) and oxidative stress conditions. The chromatograms of degradation products of tadalafil obtained after acidic, basic, and oxidative degradation are shown in Figures 2a, 2b and 2c, respectively. The accepted criterion for the stability of tadalafil was 20–80% degradation in the forced degradation study [12]. The conditions of forced degradation were adjusted to achieve degradation in the range of 20–80%. Tadalafil peak purity was greater than 99% indicating its homogeneity under all stress conditions tested. Mass balance of tadalafil in the samples under stress was close to 100%. Moreover, undisturbed results of tadalafil assay in tablets confirmed its stability and indicated the power of the method used. The summary of forced degradation studies is given in Table 1a.

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Time, min Figure 2a. Chromatogram of tadalafil after acidic degradation (product – 8.78, tadalafil – 6.99)

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Figure 2b. Chromatogram of tadalafil after basic degradation (product – 5.59, tadalafil – 6.95)

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Time, min Figure 2c. Chromatogram of tadalafil after oxidative degradation (product – 3.94, 5.04, 6.02, tadalafil – 7.00) Table 1a. Results of forced degradation study performed using the proposed method

Method validation Precision. Intra-day precision of the method was evaluated by carrying out six independent assays of test samples of tadalafil against the reference standard. Intermediate (inter-day) precision of the method was evaluated by two different analysts using different HPLC systems on different days in the same laboratory. Percentage RSD and six assayed values obtained by two analysts were 0.95, 0.98, 0.54 and 0.88, 0.99, 0.87 respectively.

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Accuracy (recovery test). Accuracy of the method was studied in recovery experiments. These experiments were performed by spiking blank samples with the known amounts of the drug. Recovery was performed at five concentration levels, which corresponded to 50%, 75%, 100%, 125%, and 150% of the declared content of tadalafil in one tablet (20 mg). The samples for recovery studies were prepared according to the aforementioned procedure. Then, 5 mL-in-volume aliquots of tadalafil solutions were transferred into 50 mL volumetric flasks and their volumes were completed with diluent B. Three samples for each recovery level were prepared. Finally, the solutions were analysed and percentage recoveries were calculated from the calibration plot. The results are shown in Table 1b. Table 1b. Results of recovery tests of tadalafil

Linearity. Linearity of the response of tadalafil was verified at seven concentration levels, ranging from 10 to 200% of the target level (100 µg mL–1) and selected from the concentration range of the standard solution, namely 10–200 µg mL–1. Solution of each concentration was injected in triplicate. Calibration graph was obtained by plotting the peak areas versus the concentration data and then applying the least squares method. The obtained linear regression equation for tadalafil is: y = 5267.5x [µg mL–1] – 4505.5; r = 0.999. The calibration plot was linear in the above concentration range. RSD values for the slope and intercept are 1.1 and 0.92, respectively. Limit of detection and limit of quantification (LOD and LOQ). For determination of LOD and LOQ, a special calibration plot was constructed using the samples containing tadalafil in the concentration ranges of LOD and LOQ. The LOD and LOQ values for tadalafil in the HPLC method were 0.021 and 0.066 µg mL–1, respectively.

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Precision at the limit of quantification was checked by analysing six test solutions prepared at the LOQ level and calculating the percentage RSD of the peak area, which was less than 2.8%. Robustness. To determine the robustness of the developed method, experimental conditions were purposely altered and the resolution between the signals of tadalafil and its basic degradation product was evaluated. The flow rate of the mobile phase was 1.0 mL min–1. To study the effect of the flow rate on resolution, it was changed from 0.8 to 1.2 mL min–1 each 0.2 unit. The effect of the content of the organic modifier on the signals’ resolution was studied by varying the concentration of acetonitrile in the mobile phase by ± 10 %. The effect of column temperature on the signals’ resolution was studied at 25°C and 35°C at the constant composition of the mobile phase. Resolution between the signals of tadalafil and basic degradation product was not less than 5 in all cases. Stability of analytical solution. Stability of the standard and sample solutions of tadalafil was tested. Stability was determined by comparing the results for the solutions stored for 24, 48 and 72 h with the values obtained for freshly prepared standard solutions. RSD of the assay results was 0.77% for the solutions stored for up to 72 h, and within 1.5% for the solutions stored for longer than 72 h. The results indicate that the solutions were stable for 72 h at the ambient temperature. Determination of tadalafil in tablets. The validated LC method was applied to the determination of tadalafil in tablets. Three batches of tablets were assayed and the results are shown in Table 1c. Table 1c. Assay result for tadalafil in tablets (20 mg per tablet)

The determined values were in the range of 90–110% of the declared content of tadalafil. The chromatogram of the tablet sample is shown in Figure 2d.

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Time, min Figure 2d. Chromatogram of tadalafil in tablet (7.01)

CONCLUSION The developed HPLC method is stability-indicating and can be used for assessing the stability of tadalafil, as well as for its determination, in bulk drugs and pharmaceutical dosage forms. It is specific, selective, robust, reproducible, and precise. It can be more conveniently used for assessing stability, assay, related substances and dissolution of tablets containing tadalafil in quality control laboratory than previously reported method. Acknowledgements The authors are grateful to the Lupin Ltd. (Mumbai, India) for gift samples of tadalafil and to the Department of Chemical Technology, Dr. Babasaheb Ambedkar Marathawada University, Aurangabad, India for providing laboratory facilities.

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5. Rodriguez Flores J., Brezas Nevado J.J., Castaneda Penalvo G. and Mora Diez N., J. Chromatogr. B, 811, 31 (2004). 6. Ramakrishna N.V.S., Viswottam K.N., Puran S., Koteshwara M., Manoj S., Santosh M., Chidambara J., Wishu S. and Sumatha B., J. Chromatogr. B, 809, 243 (2004). 7. Subba Rao D.V., Radhakrishnanand P. and Himabindu V., Chromatograhia, 1, 183 (2007). 8. Bakshi M., Singh B., Singh A. and Singh S., J. Pharm. Biomed. Anal., 26, 1011 (2001). 9. Stability Testing of New Drug Substances and Products (Q1AR2), ICH Harmonised Tripartite Guideline. 10. Validation of Analytical Procedures: Methodology (Q2B), ICH Harmonized Tripartite Guidelines. 11. Text on validation of analytical procedures (Q2A), ICH. 12. Singh S. and Bakshi M., Pharm. Tecnol., 26, 24 (2000).

Received November 2008 Revised May 2009 Accepted June 2009

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