Int J Pharm Pharm Sci, Vol 6, Issue 8, 604-609Original Article
STABILITY INDICATING RP HPLC METHOD FOR THE ESTIMATION OF ARMODAFINIL IN TABLET DOSAGE FORM
P. VIVEK SAGAR1*, NELOFER BAGUM1, S. SHOBHA RANI2
1Department of Pharmaceutical Analysis Care College of pharmacy, Oglapur (v), Atmakur (M), Warangal, Telangana, India, 2Department of Pharmaceutical Analysis Jawaharlal Nehru Technological University, Kukatpally, Hyderabad, Telangana, India.
Email: viveksagar.p111@gmail.com
Received: 12 Jun 2014 Revised and Accepted: 19 Jul 2014
ABSTRACT
Objective: To develop simple reverse phase HPLC method for the estimation of Armodafinil in tablet dosage form.
Methods: Chromatography was performed by isocratic elution on a Stainless steel Hibar C18 column with dimensions 4.6 x 250 mm, packed with octadecylsilane bonded to porous silica (C18) with particle size 5 micron. Acetonitrile and water in the ratio of 50:50 v/v is used as mobile phase. The flow rate is 1.0 ml/ min and effluent is monitored at 220 nm. Armodafinil was eluted at a retention time of 3.8 minutes.
Results: The standard curve of Armodafinil was linear over a working range of 1–700 µg/ml and gave an average correlation coefficient of 0.999. The limit of quantitation (LOQ) of the drug is 0.1 µg/ ml. Recovery studies were carried out by standard addition method and the recoveries are found satisfactory within the range of 99.3 to 101.5 %. The method is precise with % RSD below
Conclusion: The method is validated in terms of robustness and forced degradation studies were carried out and this method can be applied for routine degradation studies ans quantification in regular laboratories.
Keywords: Armodafinil, RP HPLC, Stability indicating assay, Validation.
INTRODUCTION
Armodafinil (Figure 1) (2-[(R)-(diphenylmethyl) sulfinyl] acetamide) is the R-enantiomer of modafinil, which is a racemic mixture of the R- and S-enantiomers. The molecular formula is C15H15NO2S and the molecular weight is 273.35. Used in treating narcolepsy and shift work sleep disorder (SWSD) and for adjunctive treatment of obstructive sleep apnea/ hypopnea syndrome (OSAHS). The drug is not official in any of the pharmacopoeia. Literature survey revealed that various analytical methods [1-4] were reported for determining the racemic mixture containing both R and S forms of Modafinil. An electrophoretic method [5] and one LC-MS/MS method [6] is reported for determining the R form of Modafinil i. e, Armodafinil. The present study focuses on development of simple, specific, precise, sensitive and economic stability indicating assay method for estimation of Armodafinil in tablet dosage form.
Fig. 1: Structure of Armodafinil
MATERIALS AND METHODS
Reagents and chemicals
Armodafinil tablets were procured form Orchid Pharma limited, Mumbai. Methanol, Acetonitrile, Hydrochloric Acid, Sodium Hydroxide are purchased from MERCK.
Stock solutions and standards
Stock solution of Armodafinil (1 mg/ml) was prepared by dissolving 25 mg of Armodafinil in 25 ml of volumetric flask containing 10 ml of mobile phase.
The volume was made up to the mark with mobile phase and the solution was sonicated for about 10 min. Working standard solutions of Armodafinil were prepared by taking suitable aliquots of drug solution from the standard stock solution, 1000µg/ml, and the volume was made up to 10 ml with mobile phase.
Apparatus and chromatographic conditions:
Quantitative HPLC was performed on Waters HPLC system equipped with waters 515 pump and Waters 2489 dual wavelength UV detector. Empower2 software is used for data acquisition. A Stainless steel Hibar column with dimensions 4.6 x 250 mm, packed with Octadecylsilane bonded to porous silica (C18) having particle size 5 micron.
Method development and optimization
To develop a suitable HPLC method for the determination of Armodafinil, trials were done with different mobile phases, using water, buffer(0.5 gm potassium dihydrogen phosphate)and acetonitrile in different pH with different compositions of mobile phases (40:60, 50:50, 60:40). The method was optimized finally using combination of Acetonitrile and water in the ratio of 50/50 v/v with a flow rate of 1.0 ml/ min. The drug was eluted at retention time around 3.8 min with symmetric peak shape. Run time was set for 8 minutes. Detection is performed at wavelength 220 nm.
System suitability
For performing system suitability studies, 100% test concentration under degradation conditions was selected. System suitability test was performed by injecting blank solution once and standard solution of 100% test concentration six times in to stabilized HPLC system. The system suitability was established by evaluating the system suitability parameters from the last peak obtained. System suitability parameters include retention factor (k’), repeatability, resolution (R), tailing factor (T) and theoretical plates (N). It was performed by using the concentration of 50µg/ml. The system suitability data was given in the table 1
Assay of Armodafinil marketed formulation
Twenty tablets of Armodafinil were weighed and powdered uniformly using mortar and pestle. An accurately weighed sample of powdered drug containing 25 mg of Armodafinil was dissolved with sufficient quantity of mobile phase in a 25 ml volumetric flask. The volume was made up to the mark finally using the same. The solution is sonicated for 5 minutes. This solution was filtered through 0.45 µm filter paper. The solution obtained was diluted with mobile phase so as to obtain a required concentration. The determinations were carried out in triplicate. The amount of Armodafinil present is calculated by comparing with the standard solution of Armodafinil. The representative chromatrograms are shown in figure 2 & 3. the peak areas were mentioned in the table 2.
Table 1: Assay results of Armodafinil
| S. No. | Peak areas | |
| Standard drug | Tablet formulation | |
| 1 | 6259784 | 6312897 |
| 2 | 6265017 | 6318229 |
| 3 | 6262995 | 6053611 |
| Mean | 6262599 | 6228245 |
| % Assay | 99.4% |
Fig. 2: chromatogram of Armodafinil standard solution (conc. 100µg/ ml)
Fig. 3: chromatogram of Armodafinil test solution
Validation of the assay method [7-9]
Linearity
Linearity solutions for assay method were prepared from stock solution at concentration levels from 1to 1000µg/ml of analyte concentration. The graph of peak area versus concentration was plotted by least-squares linear regression analysis.
The linear fit of the system was illustrated graphically. The linearity range was found to be 1 - 700 µg/ml. The samples were assayed using the method described above. The standard calibration curve for Armodafinil was constructed using the average peak-area versus the nominal concentrations of the analyte. Linear least-squares regression analysis was performed to assess the linearity.
Recovery and accuracy
The accuracy of the assay method was evaluated at five levels, i. e. 50, 75, 100, 125 and 150% levels (concentrations of 150, 225, 300, 375 and 450µg/ml respectively) in bulk drug sample. The percentages of recoveries were calculated from the slope and Y-intercept of the calibration curve obtained. Accuracy/recovery experiments were performed in triplicate. Accuracy was determined by standard addition method. Known amount of different concentrations of pure drug solutions were spiked with solution of pre analysed formulation of concentration 100 µg/ ml.
Precision
The precision was carried out at three levels, intra assay precision of injection, intermediate precision and reproducibility.
Intra assay precision was assessed using 9 determinations covering the range of 50,100 and 150% concentration levels of drug solution.
Intermediate precision (inter day precision) was assessed by inducing typical variations like different days and different columns.
Reproducibility was assessed by different analysts.
Robustness
Robustness of the method was studied under degradation conditions to study the effects of degradants on Armodafinil in changes method conditions. It was carried out by considering deliberate changes in detection wavelength, flow rate, mobile phase ratio. Robustness was carried out by changing detection wavelength by ±3 nm. Robustness was checked by changing the proportion of organic solvent in the mobile phase by ±4%. It was also checked for robustness by change in flow rate by ±0.2 ml/ min.
Forced degradation studies
To study the specificity of the method, pure drug was stressed under different degradation conditions. Degradation studies were carried out by exposing drug for acid hydrolysis, alkali hydrolysis, oxidative degradation, thermal degradation and photolytic degradation. Mobile phase is used as solvent for all degradation studies. All the solutions for degradation studies were prepared by dissolving Armodafinil drug in little amount of mobile phase and the volume was made up to the mark with 0.1N HCl, 0.1N NaOH, 1% H2O2. Acid hydrolysis is carried out by exposing the drug to 0.1N HCl. Alkali hydrolysis is carried out by exposing the bulk drug and powdered sample to 0.1N NaOH. Oxidative degradation is carried out by exposing the bulk drug to 1% H2O2. Thermal degradation is carried out by exposing the bulk drug in Hot air oven at 50 0C. Photolytic degradation is carried out by exposing the bulk drug to sun light. The degradation studies were carried at a time interval of 15 minutes. The drug solution was prepared at a concentration of 100µg/ ml.
Acid degradation
10mg of drug was dissolved in a few ml of mobile phase in a 10 ml volumetric flask. The volume was made up to the mark with 0.1N HCl, mixed thoroughly and kept aside. After 15, 30 minutes, solution was mixed and 1 ml of this solution was pipetted into another 10 ml volumetric flask. To this 1 ml solution, 1 ml of 0.1N NaOH was added to neutralize the acid and final volume was made upto the mark with mobile phase and its peak area was observed by injecting into HPLC.
Alkali degradation
10mg of drug was dissolved in a few ml of mobile phase in a 10 ml volumetric flask. The volume was made up to the mark with 0.1N NaOH, mixed thoroughly and kept aside. After 15, 30 minutes, solution was mixed and 1 ml of this solution was pipetted into another 10 ml volumetric flask. To this 1 ml solution, 1 ml of 0.1N HCl was added to neutralize the alkali and volume was made up to the mark with mobile phase and its peak area was observed by injecting into HPLC.
Photo degradation
Drug powder was exposed to sunlight. After 15, 30 minutes, 10mg of the exposed powder was dissolved in mobile phase in a 10 ml volumetric flask. From this solution, 1 ml was pippeted into another 10 ml volumetric flask and its volume was made upto the mark with mobile phase. The peak area of this solution was observed.
Thermal degradation
Drug powder was exposed to 500C in a hot air oven. After 15, 30 minutes, 10mg of the exposed powder was dissolved in mobile phase in a 10 ml volumetric flask. From this solution, 1 ml was pippeted into another 10 ml volumetric flask and its volume was made upto the mark with mobile phase. The peak area of this solution was observed.
Results
System suitability
The system suitability of Armodafinil was ascertained good under degradation conditions and didn’t show any significant change with %RSD less than 2. The degradants peak and Armodafinil peak has resolution greater than 2 in all the conditions. This shows no interference of degradant peak on Armodafinil peak. The tailing factor for Armodafinil was always less than 2.0 with plate count more than 2000.
Linearity
Linear calibration plot for assay method was obtained over the calibration ranges tested, i. e. 1- 700 µg/ml and the correlation coefficient obtained was greater than 0.999. The results show that an excellent correlation existed between the average peak area and concentration of the analyte(Table 3).
Table 2: System suitability data for Armodafinil
| Retention time (Rt) | 3.810 |
| Peak area | 4982685 |
| USP plate count(N) | 4677.0355 |
| USP tailing factor(T) | 1.186 |
| % RSD of(n= 6) | 1.3 |
Results of method validation experiments
Table 3: Linearity of Armodafinil
| Conc.(µg/ml) | Peak Area | Conc.(µg/ml) | Peak Area |
| 1 | 91760 | 300 | 12505210 |
| 5 | 307010 | 400 | 16251659 |
| 50 | 2989697 | 500 | 20323095 |
| 100 | 4837989 | 600 | 24402243 |
| 200 | 8843917 | 700 | 28711674 |
Recovery and accuracy
The percentage recovery of Armodafinil in bulk drug samples was ranged from99.3-101.5 which indicates that the method isaccurate (Table 4).
Precision
From the results shown in precision Tables5, 6, 7 & 8, it was found that pooled and % RSD was less than 2%; which indicates that the proposed method has good reproducibility.
Table 4: Accuracy data for Armodafinil
| Spiked Levels | Standard | Test peak area(conc. 100µg/ml) | Spiked | % Recovery | Mean % Recovery | ||
| Conc. (µg/ml) | Peak Area |
Conc. (µg/ml) | Peak Area |
||||
| 50% | 150 | 9829413 | 6358316 | 250 | 16263079 | 101.3 | 101.5 |
| 9698922 | 6371291 | 16153903 | 101.4 | ||||
| 9788409 | 6318229 | 16152568 | 101.9 | ||||
| 75% | 225 | 14928123 | 6372897 | 325 | 21324411 | 100.5 | 100.8 |
| 14259859 | 6214178 | 20650587 | 101.6 | ||||
| 14333226 | 6343288 | 20660537 | 100.3 | ||||
| 100% | 300 | 19488942 | 6325669 | 400 | 25776514 | 100 | 100.3 |
| 19661406 | 6213901 | 26130545 | 101.5 | ||||
| 19508556 | 6357126 | 25743117 | 99.6 | ||||
| 125% | 375 | 22164423 | 6153611 | 475 | 28395215 | 100.6 | 100.4 |
| 23975972 | 6229245 | 30391629 | 101 | ||||
| 23917241 | 6342612 | 30170864 | 99.8 | ||||
| 150% | 450 | 26011432 | 6139224 | 550 | 31665322 | 98.3 | 99.3 |
| 27629712 | 6373759 | 33829352 | 99.5 | ||||
| 28262352 | 6335217 | 34620376 | 100.2 |
Table 5: Intra assay precision data for Armodafinil
| % Level | Peak Area | Amount Obtained(µg/ml) | % Obtained |
| 50 | 9530370 | 152.5 | 101.7 |
| 9448723 | 151.3 | 100.9 | |
| 9551190 | 153 | 102 | |
| 100 | 18424051 | 296.1 | 98.7 |
| 18647226 | 299.7 | 99.9 | |
| 18817822 | 302.4 | 100.8 | |
| 150 | 27536746 | 442.9 | 98.4 |
| 27629712 | 444.4 | 98.7 | |
| 28262352 | 454.6 | 101 | |
| Pooled RSD | 1.3 |
Table 6: Intermediate precision data of Armodafinil for day 1 and day 2
| S. No. | Day 1 | Day 2 | ||
| Retention Time | Peak Area | Retention Time | Peak Area | |
| 1 | 3.810 | 4982685 | 3.832 | 4827119 |
| 2 | 3.822 | 5043391 | 3.869 | 4975624 |
| 3 | 3.827 | 4956253 | 3.817 | 4853891 |
| 4 | 3.832 | 4959394 | 3.859 | 4942266 |
| 5 | 3.863 | 4857086 | 3.846 | 4931394 |
| 6 | 3.878 | 4967299 | 3.813 | 4920865 |
| % RSD | 1.2 | % RSD | 1.1 |
Table 7: Intermediate precision data of Armodafinil for Column 1 and Column 2
| S. No. | Column 1 | Column 2 | ||
| Retention Time | Peak Area | Retention Time | Peak Area | |
| 1 | 3.845 | 22081819 | 3.915 | 22250028 |
| 2 | 3.848 | 22129248 | 3.917 | 22317532 |
| 3 | 3.869 | 21926638 | 3.919 | 22317228 |
| 4 | 3.872 | 21908561 | 3.912 | 22198769 |
| 5 | 3.874 | 21882375 | 3.921 | 22102983 |
| 6 | 3.878 | 22117781 | 3.917 | 22043224 |
| % RSD | 0.5 | % RSD | 0.5 |
Table 8: Reproducibility data for Armodafinil
| S. No. | Analyst 1 | Analyst 2 | ||
| Retention time | Peak area | Retention time | Peak area | |
| 1 | 3.901 | 25458196 | 3.900 | 25224810 |
| 2 | 3.903 | 25554856 | 3.903 | 26154098 |
| 3 | 3.903 | 25324321 | 3.904 | 25894457 |
| 4 | 3.904 | 25592739 | 3.906 | 26085712 |
| 5 | 3.920 | 25472533 | 3.906 | 26071259 |
| 6 | 3.921 | 25414751 | 3.907 | 25938737 |
| % RSD | 0.4 | % RSD | 1.3 |
Table 9: Robustness data for change in wavelength
| S. No | λ max -220 nm | λ max - 217 nm | λ max - 223 nm | |||
| Retention Time | Peak Area | Retention Time | Peak Area | Retention Time | Peak Area | |
| 1 | 3.806 | 3775757 | 3.875 | 3832275 | 3.798 | 3603294 |
| 2 | 3.817 | 3628570 | 3.894 | 3883298 | 3.807 | 3461902 |
| 3 | 3.820 | 3711842 | 3.867 | 3773320 | 3.812 | 3543550 |
| 4 | 3.837 | 3772145 | 3.909 | 3812237 | 3.829 | 3598687 |
| 5 | 3.842 | 3707342 | 3.929 | 3895566 | 3.834 | 3538528 |
| 6 | 3.846 | 3750440 | 3.962 | 3911544 | 3.838 | 3578669 |
| % RSD | 1.5 | - | 1.4 | - | 1.5 |
Table 10: Robustness data for change in mobile phase composition
| S. No. | Normal MP | ACN: -4%; Water: +4% | ACN: +4%; Water: -4% | |||
| Retention Time | Peak Area | Retention Time | Peak Area | Retention Time | Peak Area | |
| 1 | 3.932 | 3881751 | 4.103 | 3805520 | 3.852 | 3893374 |
| 2 | 3.919 | 3913373 | 4.114 | 3873667 | 3.892 | 3885448 |
| 3 | 3.874 | 3795148 | 4.117 | 3849376 | 3.879 | 3845856 |
| 4 | 3.937 | 3889966 | 4.124 | 3864902 | 3.895 | 3818582 |
| 5 | 3.930 | 3890511 | 4.134 | 3829262 | 3.869 | 3868084 |
| 6 | 3.953 | 3923926 | 4.142 | 3900418 | 3.881 | 3901546 |
| % RSD | 1.2 | - | 0.9 | - | 0.8 |
Robustness
The percent recovery of Armodafinil was good under most conditions and didn’t show any significant change when the critical parameters were modified under degradation conditions. The degradants peak and Armodafinil peak has resolution greater than 2 in all the conditions. This shows no interference of degradant peak on Armodafinil peak.
The tailing factor for Armodafinil was always less than 2.0 with plate count more than 2000 and with %RSD less than 2. The component was well eluted under all the changes carried out. Considering the modifications in the system suitability parameters, as well as carrying the experiment at room temperature may conclude that the method conditions were robust (Tables 9,10,11).
Table 11: Robustness data for change in flowrate
| S. No. | Flow rate-1 ml/min | Flowrate-0.8 ml/min | Flowrate-1.2 ml/min | |||
| Retention Time | Peak Area | Retention Time | Peak Area | Retention Time | Peak Area | |
| 1 | 3.816 | 3966950 | 4.813 | 5039838 | 3.225 | 3313690 |
| 2 | 3.806 | 3915545 | 4.824 | 5015856 | 3.237 | 3302692 |
| 3 | 3.821 | 3985012 | 4.857 | 5089164 | 3.231 | 3354139 |
| 4 | 3.822 | 3969882 | 4.823 | 4916595 | 3.257 | 3386897 |
| 5 | 3.832 | 3966345 | 4.842 | 5053443 | 3.262 | 3388427 |
| 6 | 3.863 | 3993207 | 4.885 | 5031944 | 3.282 | 3374513 |
| % RSD | 0.7 | - | 1.2 | - | 1.1 | |
| USP Plate count | 3219.21 | - | 3528.07 | - | 3753.59 | |
| USP Resolution | 8.613 | - | 10.495 | - | 7.997 | |
| USP Tailing | 1.108 | - | 1.112 | - | 1.129 |
Table 12: Stability data for Armodafinil
| Days | Retention time | Peak area | % Stability |
| 1 | 3.856 | 7184293 | 100 |
| 3.979 | 7169924 | 99.7 | |
| 2 | 3.981 | 7140816 | 99.3 |
| 3.904 | 7114240 | 99 | |
| 3 | 3.869 | 7096528 | 98.7 |
| 3.884 | 7053627 | 98.1 | |
| 4 | 3.882 | 7025064 | 97.7 |
Stability of drug solution:
The stability was checked by diluting the stock solution to 100µg/ ml. It was checked for 4 days at interval of 12 hours under normal laboratory conditions (25 ±1 0C). The drug was stable up to 36 hours and has no significant change in analyte composition and peak areas (Table 12).
Forced degradation studies:
Specificity was studied by exposing the sample solutions to stress conditions i. e. 0.1N HCl, 0.1N NaOH, photolytic degradation and thermal degradation.
Percentage degradation level of 10% was considered for degradation studies. Degradant peaks at 1.73 minutes was observed by acid hydrolysis, a degradant peak at 1.75 minutes was observed by alkali degradation, a degradant peak at 1.48 minutes was observed by photolytic degradation and a degradant peak at 1.68 minutes was observed by thermal degradation. From the results obtained (shown in tables 13, 14, 15, 16), it was found that the drug is stable for 30 minutes in acidic and basic medium. The drug has thermal and photolytic stability for one hour. By the chromatograms obtained during the degradation studies it was found that there is no interference of the degradant peak with the analyte peak. Hence the method is stability indicating.
Table 13: Degradation data for Armodafinil in acidic condition
| S. No. | Time of Exposure | Peak Area of Armodafinil | % of Stable Armodafinil | % Degraded |
| 1 | 0 hour | 6783903 | 100 | 0 |
| 2 | 30 min | 5445913 | 80.2 | 19.8 |
| 3 | 1 hour | 5316242 | 78.3 | 21.7 |
Table 14: Degradation data for Armodafinil in Alkaline condition
| S. No. | Time of Exposure | Peak Area of Armodafinil | % of Stable Armodafinil | % Degraded |
| 1 | 0 hour | 6783903 | 100 | 0 |
| 2 | 30 min | 5036633 | 74.2 | 25.8 |
| 3 | 1 hour | 4220732 | 62.2 | 37.8 |
Table 15: Photo degradation data for Armodafinil
| S. No. | Time of Exposure | Peak Area of Armodafinil | % of Stable Armodafinil | % Degraded |
| 1 | 0 hour | 6783903 | 100 | 0 |
| 2 | 30 min | 6032936 | 88.9 | 11.1 |
| 3 | 1 hour | 5851660 | 86.2 | 13.8 |
CONCLUSION
The proposed method was found to be simple, rapid, sensitive, precise, robust and accurate for determination of Armodafinil in formulation. The proposed method was very simple as the preparation of mobile phase is simpler. The method is very sensitive as the LOQ concentration was very low. The method was found linear over wide range of concentration.
The method can be used for routine analysis of Armodafinil in the presence of degradants.
ACKNOWLEDGEMENTS
The authors are thankful to the management and authorities of Department of pharmaceutical Analysis, Care college of pharmacy for providing the necessities required for carrying out the present research work and also MSN laboratories, Hyderabad for providing the gift sample of Armodafinil.
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