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Volume : 2 Issue : 8 Aug 2013 ISSN - 2250-1991
A Stability Indicating Rp-Hplc Method for the
Estimation of Tadalafil in Oral Jelly Dosage
* M. Sankar ** S. Arulantony
* Post Graduate and Research Department of Chemistry, Presidency College,
Wallajah Road, Chepauk, Chennai - 600 005, TamilNadu, India
A simple, selective, precise and stability indicating RP-HPLC method for the determination of tadalafil was developed and validated in oral jelly dosage forms. The chromatographic parameters comprised of Zorbax C18 column (250X4.6 mm, 5µ) and mixture of buffer: acetonitrile (55: 45 v/v) as mobile phase. The detection was observed at 225nm with 1.5 ml/min flow rate. The developed method has been validated according to ICH and USP guidelines. The linearity studies showed a good correlation over the range of 10 to 75 µg/ml with correlation coefficient (r2) of 0.998. The drug was subjected to forced degradation analysis on varied conditions of acidic, basic, peroxide, thermal, light and UV radiation. All the results have proved that the method was selective and reproducible for the determination of tadalafil. The proposed stability indicating RP-HPLC method can be effectively employed for the determination of tadalafil in routine drug analysis of oral jelly dosage forms. Keywords : Tadalafil, HPLC, stability indicating, and validation
with HPLC water.
Tadalafil ((6R-trans)-6-(1,3-benzodioxol-5-yl)- 2,3,6,7,12,12 a-hexahydro-2-methyl-pyrazino [1', 2':1,6] pyrido [3,4-b] in- 2.4 Preparation of standards
dole-1,4-dione or C H N O ) (Figure 1) is a phosphodiester- Transfer accurately weighed quantity of 25 mg of Tadalafil ase type 5 (PDE5) inhibitor used to treat the erectile dysfunc- standard in 100 ml volumetric flask, and add 50 ml of mobile tion (ED) by increasing the levels of cGMP1. It is yellowish phase, sonicate for 15 mins using PCI Analytical sonicator, white crystalline powder, and ease of soluble in water. The Mumbai to dissolve it and make upto the mark with mobile literature survey reveals various methods like Spectropho- phase. Take 5 ml of the resulting solution into 25 ml volumet- tometric2-4, HPLC5-17, LC/MS18, 19 have been studied for tada- ric flask, and make upto the mark with the mobile phase.
lafil. So far, there is no stability indicating method has not revealed. Hence, an attempt has been made to confirm the 2.5 Preparation of sample
stability indicating method for the determination of tadalafil in Transfer the weight of 25 mg equivalent of Tadalafil oral jelly pharmaceutical dosage forms. It was optimized and validat- sample in a 100 ml volumetric flask, and add 50 ml of mobile ed according to International Conference on Harmonization phase, sonicate for 15 mins using PCI Analytical sonicator, (ICH) guidelines 20, 21 and USP 22 guidelines. The present study Mumbai to dissolve it and make upto the mark with mobile was aimed to develop a simple, rapid, precise, accurate, sta- phase. Take 5 ml of the resulting solution into 25 ml volumet- ble and selective reversed phase chromatographic method ric flask, and make upto the mark with the mobile phase.
along with the stability indicating studies to estimate the tada- lafil in oral jelly dosage forms.
2.6 Preparation of placebo
Transfer 6.25g of Tadalafil oral jelly placebo in a 100 ml volu- 2. MATERIALS AND METHODS
metric flask, add 50ml of mobile phase, sonicate for 15 mins using PCI Analytical sonicator, Mumbai to dissolve it and The HPLC grade of methanol and acetonitrile (Qualigens) make upto the mark with mobile phase. Take 5 ml of the re- was used for the analysis. The gift sample of tadalafil oral sulting solution into 25 ml volumetric flask, and make upto the jelly and its placebo was obtained from Caplin Point Laborato- mark with the mobile phase.
ries Limited, Chennai. Commercially available gel formulation Tadaga oral jelly, Cialis was used for verifying the effective- 2.7 Forced / Stress degradation studies
ness of the method. The experimental procedure was carried The Tadalafil standard was exposed to various stress condi- out completely using Pall Cascada AN water, New York. All tion like Acidic hydrolysis (1 N and 2 N HCl), Basic Hydrolysis other chemicals were used with their respective grade.
(1 N and 2 N NaOH), Peroxide (5% and 10% H O ), and the thermal degradation of tadalafil was carried out by using hot 2.2 Instrumentation and Analytical Conditions
air oven and monitored at the temperature of 50 ºC, 60 ºC The chromatographic method was carried out using the Ag- and 70 ºC after 2 hours exposure. The light and UV exposure ilent 1260 equipped with DAD and VWD detector. The soft- study carried out for 15 days.
ware of open lab was used to monitor the data acquisitions and other proceedings. The freshly prepared mobile phase Take 25mg of tadalafil standard in a 100 ml flask and add 5ml was vaccum filtered through a 0.45 μm Millipore nylon filter, of 1N NaOH and heat it for one hour at 60°C .Cool to room used for the entire study. temperature, neutralize and transfer the whole contents ex- actly into a 100ml volumetric flask, make upto the mark with 2.3 Preparation of 0.05 M Phosphate Buffer
mobile phase. Take 5ml from the above solution and transfer Weigh accurately about 7.098 grams of Disodium hydrogen into a 25ml volumetric flask, make upto the mark with mobile orthophosphate into the 1000 ml volumetric flask and dissolve it using 100 ml of HPLC water and then made up the mark 19 X PARIPEX - INDIAN JOURNAL OF RESEARCH Volume : 2 Issue : 8 Aug 2013 ISSN - 2250-1991
Perform the same manner for rest of the acidic (1N and 2N 3.2 Method Validation
HCl), basic (2N NaOH) and peroxide (5% and 10% H O ) treatments for standard tadalafil. Perform and continue the The results of the specificity were showed there was no inter- degradation study with the sample and placebo of tadalafil ference and co-elution of any other peaks with the retention oral jelly in the same manner. Run the chromatogram upto 25 of tadalafil. The peak purity of tadalafil and sample of oral jelly minutes for all stress study samples analysis to monitor the found within the limit which proved that there was no interfer- degraded or matrix peaks in higher retention time.
ence between the blank and placebo peaks. 3. RESULTS AND DISCUSSION
The study was aimed to develop a stability indicated RP- The linear calibration plot was constructed by analyzing the HPLC method for the determination of tadalafil in tadalafil concentrations over the range of 10-75 μg/ml. The sample oral jelly dosage form. The initial trails were conducted based volume of 20 µl was injected three times into the column and upon the peak symmetry and time reduction in the chromato- it was used for the determination of peak area. The standard graphic analysis. The C column was selected to conduct the graph has drawn taking the concentration samples of drug on method development study based on the polarity of tadalafil. x-axis and peak area of absorption on y-axis and showed in All the trails were done using Zorbax C (250 X 4.6mm, 5 µm) Figure 9. The results are interpolated using the linear regres- column as stationary phase. The mobile phase was assessed sion correlation method. The correlation coefficient obtained after conducting the various trails using the solvents of meth- from the linearity studies showed a good linear response with anol, acetonitrile and phosphate buffer. The mobile phase limits of correlation between the peak area and concentration of Phosphate buffer: methanol (50: 50 v/v) was used and it of the analytes. shows a poor peak shape with long retention time. Hence, decided to modify the mobile phase on the basis of the po- larity chosen the solvents of phosphate buffer: acetonitrile. The intra-day and inter-day precision was determined by in- The different proportions of mobile phase was mixed and jecting the six replicates of standard concentration into the analyzed in the HPLC system which include 40:60 v/v, 45:55 HPLC system. The % RSD was calculated from the peak area v/v, 50:50 v/v, 55: 45 v/v, 60:40 v/v, 70:30 v/v, and 75:25 v/v. responses of the concentration on the same day and it on When the content of acetonitrile proportion was decreased in consecutive days (n=3). The result showed the % RSD was the mobile phase, the result showed a good peak shape. The found to be less than 2.0 and it indicates the precise method. ratio of mobile phase buffer: acetonitrile (60: 40 v/v) showed The summarized results were showed in Table 2 and 3.
a concise peak due to decreased organic content. The ratio of the organic modifier was adjusted to 55: 45 v/v showed a good peak shape with limits of system suitability. The meth- The accuracy was estimated by using the standard addition od was furtherly proceeded using the mobile phase of buffer: method. It was determined by spiking the concentration with acetonitrile (55: 45 v/v) and it showed in Figure 2. The results levels of 50%, 80%, 100%, 120% and 150%. The known found to be specific and it shows there is no interference and quantity of tadalafil standard concentration was spiked with co-elution of any other peaks with the retention of tadalafil. placebo. The results showed a good recovery and showed The optimized conditions are showed below.
Optimized Chromatographic Conditions:
3.2.5 Limit of Detection and Limit of Quantification
: Zorbax C [250 X 4.6mm, 5 µm] The LOD and LOQ were evaluated on the basis of the line- : Buffer: Acetonitrile [55: 45 v/v] arity curve. The LOD and LOQ of tadalafil were found to be 0.42 µg/ml and 1.28 µg/ml respectively. The results showed that the method can be efficiently employed for the estimation of tadalafil.
The robustness was conducted by deliberate changing in the optimized chromatographic conditions. The mobile phase var- 3.1 Stress Degradation Studies
iation (± 3% v/v), flow rate (±0.2 ml/min), wave length (±2 nm), The stress degradation studies are conducted in the condi- column oven temperature (±5 ºC), were slightly changed. The tions of acidic, basic, peroxide, thermal, light and UV radia- reproducible results were obtained which proves the robust tion. The chromatograms obtained after stress degradation method and the summarized data was showed in Table 5.
studies of tadalafil are showed in Figure 3-8. The results are compared with the untreated standard and summarized in 3.2.7 Stability of the solution
The stability of the solution was performed for 24 hours peri- od at each interval of 4 hours. The percentage stability was 3.1.1 Degradation in acid hydrolysis
showed in Table 6.
It was performed using 1 N and 2 N HCl at 60 ºC for 1 hour. The results showed a significant degradation.
3.2.8 Analysis of Commercial Formulation
The method was verified using the commercial formulation of 3.1.2 Degradation in basic hydrolysis
TADAGA Oral Jelly which it was injected six replicate prepa- The analysis conducted using of 1 N and 2 N NaOH at 60 ºC ration of samples into the LC system and the results found for 1 hour. The result showed a moderate degradation.
between 100.16 to 100.80% and summarized in the Table 7. 3.1.3 Peroxide Conditions
The degradation was carried out using the 5% and 10% H O A simple, selective, accurate, precise and stability indicating and the result showed the prominent degradation.
RP-HPLC method has been developed for the determination of tadalafil in tadalafil oral jelly. The method was validated 3.1.4 Thermal Conditions
successfully with significant results according to ICH and The tadalafil exposed at different temperature conditions us- USP guidelines. The present study can be effectively em- ing the thermostat at 50ºC, 60ºC and 70ºC for 2 hours and the ployed for the determination of tadalafil in oral jelly dosage results found to be less moderate degradation.
form for routine drug analysis.
3.1.5 Light and UV radiation
The sample of tadalafil was exposed for 15 days and the re- The authors are thankful to Caplin Point Laboratories Limited, sults showed to be stable.
Chennai, India and Presidency College, Chepauk, Chennai, 20 X PARIPEX - INDIAN JOURNAL OF RESEARCH Volume : 2 Issue : 8 Aug 2013 ISSN - 2250-1991
Tamil Nadu, India for providing the necessary facilities for the completion of work successfully.
120 658425874 60.11 Table 1: Summarized data of forced degradation studies
150 818542713 74.73 % Assay Peak Area % Assay Acidic degradation Table 5: Robustness study of tadalafil standard
Basic degradation Condition Retention 1 N NaOH 511459785 96.99 2 N NaOH 499658217 94.05 Thermal Degradation after 2 Hours Table 6: Stability data of tadalafil standard and sample
dosage form
Table 2: Summarized data of Repeatability sample
Repeatability of sample Difference Peak Area Table 7: Summarized data of Tadaga oral jelly
Repeatability of sample Table 3: Summarized Inter-mediate Precision data of
standard and sample dosage forms
545548712 100.25 545574892 100.30 545589652 100.65 546489245 100.20 549325404 100.75 545362547 100.36 544879562 99.90 545487914 100.05 543254892 100.04 547546894 100.55 545992200 100.30 544879546 100.45 546154879 100.00 546022324 99.95 548754821 99.69 545584970 100.30 544269225 100.05 547754895 100.02 Mean 546034043 100.20 546111993 100.23 545932726 100.20 Table 4: Accuracy results of tadalafil sample
Recovery Recovery 100 547895426 50.02 Figure 1: Structure of tadalafil
21 X PARIPEX - INDIAN JOURNAL OF RESEARCH Volume : 2 Issue : 8 Aug 2013 ISSN - 2250-1991
Figure 2: Standard Chromatogram of tadalafil
Figure 6: Chromatogram of tadalafil using Thermal deg-
Figure 3: Chromatogram of tadalafil using 1N HCl
Figure 7: Chromatogram of tadalafil using Light
Figure 8: Chromatogram of tadalafil using UV radiation
Figure 4: Chromatogram of tadalafil using 1N NaOH
Figure 5: Chromatogram of tadalafil using 10% H O
Figure 9: Linearity of tadalafil
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