DEVELOPMENT AND VALIDATION OF STABILITY INDICATING RP-HPLC METHOD FOR ESTIMATION OF TADALAFIL AND DAPOXETINE HCL IN SOLID DOSAGE FORM

About Authors:
CHIRAG S. RAJPARA*1, JAWED AKHTAR1, AMIT KHANDHAR2
1. Department of Quality Assurance, School of Pharmaceutical Sciences, Jaipur National University, Jagatpura, Jaipur-302025, Rajasthan, India.
2. Zydus cadila pharma, moraiya,ahmedabad.
*chiragrajpara2601@gmail.com

ABSTRACT
* A simple, specific, accurate and stability-indicating reversed phase high performance liquid chromatographic method was developed for simultaneous estimation of Tadalafil and Dapoxetine HCL, Water Symmetry C-18 (150 x 4.6 mm),5 µ and a mobile phase composed of Buffer : Acetonitrile (65:35)

* The retention time of Tadalafil and Dapoxetine HCL were found to be10.08 and 4.45 min respectively. Linearity was established for Tadalafil and Dapoxetine HCL in the range of 8.0-60 μg/ml and 24.0-180.0 μg/ml respectively. The percentage recovery of Tadalafil and Dapoxetine HCL were found to be in the range of 99.7-100.6% and 98.07-99.09% respectively. The drug was subjected to acid and alkali hydrolysis, oxidation, dry heat and photolytic degradation. The degradation studies indicated, Tadalafil showed degradation in acid and alkali while it was found stable in H2O2, photolytic and in presence of dry heat and Dapoxetine showed degradation in thermal and peroxide while it was found stable in rest of parameters . The degradation products of Tadalafil and Dapoxetine HCL in acidic, alkaline conditions were well resolved from the pure drug with significant differences in their retention time values. This method can be successfully employed for the quantitative analysis of Tadalafil and Dapoxetine HCL in bulk drugs and formulations.

Reference Id: PHARMATUTOR-ART-1387

1.       INTRODUCTION
Tadalafil (6R-trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12a-hexahydro-  2-methy  pyrazino [1', 2':1,6] pyrido[3,4-b]indole - 1,4-dione(fig. 1.1), is a white powder, freely soluble in acetonitrile. It is A long acting PDE5 Inhibitor for the management of erectile dysfunction. It is indicated for the treatment of erectile dysfunction in men.[10, 11, 12].

Dapoxetine S)-N,N-dimethyl-3-(naphthalen-1-  yloxy)-1-phenylpropan-1-amine(fig. 1.2), is white crystalline powder, Freely soluble in water and  ethanol  and Acetonitrile. It is   Short acting serotonine reuptake inhibitor. It indicated for the treatment of premature ejaculation in men 18 to 64 years of age.[13,14]

Literature survey indicated that determination of Tadalafil and Dapoxetine HCL were done by RP-HPLC method with UV detection for routine analysis of Dapoxetine HCl in a pharmaceutical formulation. [8]The literature survey does not reveal any stability indicating assay method for simultaneous estimation of Tadalafil and Dapoxetine HCL in bulk and Pharmaceutical dosage form which gives information about the degradation products as well as separation of degradation products. The literature survey  indicated  that  no  stability  indicating  RP-HPLC method  was  proposed  for  Tadalafil and Dapoxetine HCL .

The International Conference on Harmonization (ICH) guideline entitled “Stability testing of new drug substances and products” requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance [4]. An ideal stability-indicating method is one that resolves the drug and its degradation products efficiently. Consequently, the implementation of an analytical methodology to determine Tadalafil and Dapoxetine HCL  in presence of its degradation products is rather a challenge for pharmaceutical analyst. Therefore, it was thought necessary to study the stability of Tadalafil and Dapoxetine HCL under acidic, alkaline, oxidative, photolytic and dry heat conditions. This paper reports validated stability-indicating HPLC method for simultaneous determination of Tadalafil and Dapoxetine HCL  in presence of their degradation products. The proposed method is simple, accurate, reproducible, stability-indicating and suitable for routine determination of Tadalafil and Dapoxetine HCL in its dosage form. The method was validated in compliance with ICH guidelines. [5, 6]

2.       MATERIALS AND METHODS:
Tadalafil and Dapoxetine HCL  of working standard grade was kindly supplied as gift sample by Zydus Cadila Healthcare Ltd., Ahmedabad, India and was certified to contain 99.30% (w/w) and 99.40%(w/w) respectively, on as is basis. Tadalafil and Dapoxetine HCL  Active Pharmaceutical Ingredient (API) was kindly supplied as gift sample by MSN Laboratories, Hyderabad, India and was certified to contain 99.30% (w/w) and 99.40%(w/w) respectively, on as is basis. Acetonitrile and Methanol used were of HPLC grade and were purchased from Spectrochem Pvt. Ltd. Mumbai, India. The tablet formulation containing 20 mg of Tadalafil and 60 mg of Dapoxetine HCL were procured from local market and used for analysis of marketed formulation. After several trials taken by changing mobile phase, columns, flow rate, column temperature, the following chromatographic conditions were finalised.

The HPLC system (Shimadzu Corporation, Japan), model Shimadzu VP, consisted of a system controller (CLASS-VP), on-line degasser (LC 2010C, Shimadzu), low pressure gradient valve  (LC 2010C, Shimadzu), solvent delivery module (LC 2010C, Shimadzu), auto injector (LC 2010C, Shimadzu), column oven (LC 2010C, Shimadzu), and CLASS – VP software version = SPI, binary pump, auto injector (SIL-10AD VP, Shimadzu), column oven (CTO-10AS VP, Shimadzu) and PDA detector (PDA-SPD-M10A VP, Shimadzu Diode Array Detector) and Chem station (software).

The wavelength of detection chosen was 285 nm for both the drug. Water Symmetry C-18 (150 x 4.6 mm),5 µ was used for the analysis. The mobile phase comprising of a mixture of-Butyl ammonium hydrogen sulphate and NaH2PO4 with organic mixture in ratio of 65: 35 % v/v. The buffer pH adjusted to 2.5 with H3PO4 .The acetonitrile was used as a organic mixture. The flow rate of mobile phase is 1.0 ml/min. The injection volume was 10 μl.

In addition, an electronic balance (Mettler Toledo), a pH meter (Metrohm), a sonicator (Flexit jour lab pvt. Ltd., model UCB 45), a hot air oven (Labhosp) was used in this study.

2.1    Preparation of Mobile Phase:
Dissolve 0.7 gm of Tetra-Butyl ammonium hydrogen sulphate and 3.7 gm NaH2PO4 in 2000 ml Milli-Q water and then adjust ph 2.5 with H3PO4. Mix the buffer solution and acetonitrile in proportion of 65:35 and sonicate it properly and filter the mobile phase through 0.45 µ HVLP milli-pore filter.

2.2 Preparation of Standard Solution:
The standard stock solution was prepared by transferring 40 mg of Tadalafil (A) and 60 mg of Dapoxetine (B) in a 100 ml volumetric flask separately.  Then pipette out 5ml (A) and 10ml (B) in to 50ml volumetric flask and make up the volume up to 50mlwith diluents which is having final concentration 40 µg/ml and 12040 µg/ml. The HPLC analysis was performed on reversed-phase high-performance liquid chromatographic system with isocratic elution mode using a mobile phase of Buffer: organic mixture (65:35 %v/v), on Water Symmetry C-18 (150 x 4.6 mm),5 µ with 1.0 ml/min flow rate at 285 nm using UV detector.

2.2    Analysis of Marketed Formulations:
Take tablets in to mortar pestle and crused it to make a fine powder and then take a powder drug equivalent to 2.5 tablets (50 mg tadalafil and 150 mg of dapoxetine) and transfer both drugs in to 250 ml of volumetric flask separately and make up the volume up to 100 ml with diluent and sonicated them for 20 min. then make up the volume up to 250 ml with diluent. Then pipette out 5 ml of both drugs and transfer in to 25ml of volumetric flask separately and make up the volume up to 25 ml with diluent. These give final concentration 40 μg /ml and 120 μg /ml of tadalafil and dapoxetine respectively. The solution was further filtered using 0.45μ PVDF filter.

2.4 Method Validation:
The method of analysis was validated as per the recommendations of ICH [7] and USP [3] for the parameters like accuracy, linearity, precision, specificity, detection limit, quantitation limit ruggedness, robustness and solution stability.

2.4.1 Linearity:
Every 10 μl of the working standard solution of Tadalafil and Dapoxetine HCL in the mass concentration range of 8.0-60.0 μg/ml and 24.0-180 μg/ml respectively, were injected into the chromatographic system. The chromatograms were developed and the peak area was determined for each concentration of the drug solution. Calibration curve of Silodosin was obtained by plotting the peak area ratio versus the applied concentrations of Tadalafil and Dapoxetine HCL (fig. 2.1 & 2.2).

2.4.2 Accuracy:
The accuracy of the method was determined by calculating percentage recovery of Tadalafil and Dapoxetine HCL . Recovery studies were carried out by applying the spiking method in which known amount of Tadalafil and Dapoxetine corresponding to 50, 100 and 150% of label claim had been added (standard addition method) placebo. At each level of the amount three determinations were performed and the results obtained were compared.

2.4.3 Precision:
The method precision was done by preparing six different sample preparations by one analyst under the same conditions and analysing them.

2.4.4 LOD and LOQ:
The limit of detection (LOD) and limit of quantitation (LOQ) were calculated using following formulae: LOD= 3.3(SD)/S and LOQ= 10 (SD)/S, where SD=standard deviation of response (peak area) and S= average of the slope of the calibration curve.

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