Novus International Journal of Analytical Innovations 2012, Vol. 1, No. 3
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1 Novus International Journal of Analytical Innovations 2012, Vol. 1, No. 3 ISSN Accepted on October 22, 2012 RP-HPLC method for simultaneous estimation of Avitriptan and Eletriptan in pharmaceutical dosage forms N. Usha Rani 1*, R. Sreenivasa Rao 1, K. Saraswathi 2, T. E. G. K. Murthy 3 1* Department of Chemistry, B. C. A. S., Bapatla, Guntur (D.T), A.P., India 2 S. V University, Tirupati, A. P., India 3 Bapatla College of Pharmacy, Bapatla, Guntur (D.T), A.P., India ABSTRACT A simple, selective, accurate high Performance Liquid Chromatographic (HPLC) method was developed and validated for the analysis of Avitriptan and Eletriptan in the bulk and pharmaceutical dosage form. The method was validated as per ICH and FDA guidelines. Chromatographic separation achieved isocratic ally on a C-18 column [Use Inertsil C-18, 5m, 150 mm x 4.6 mm] utilizing a mobile phase of Methanol: Acetonitrile: 0.1% Orthophosphoric acid in the ratio of 30:40:30(v/v/v)) [P H adjusted to 4.1 with triethylamine]at a flow rate of 1.0 ml/min with UV detection at 241nm. Linearity was observed in the range of ppm. The percentage recoveries of Avitriptan and Eletriptan were found to be in the range of % and %, respectively. The low values of %RSD indicated that the method is precise. Limit of detection (LOD) values of Avitriptan and Eletriptan were experimentally verified to be 0.06 ppm and 0.12 ppm, respectively. Limit of quantization (LOQ) values of Avitriptan and Eletriptan were found to be 0. 2 ppm and 0.4 ppm respectively. KEY WORDS: Avitriptan, Eletriptan, RP-HPLC, method development, validation INTRODUCTION *Corresponding author: N. Usha Rani 1 Department of Chemistry, B. C. A. S., Bapatla, Guntur (D.T), A.P., India nannapaneniusharani73@gmail.com Avitriptan: Avitriptan (BMS-180,048) is an anti-migraine drug of the triptan class. [1] It acts as a 5- HT 1B and 5-HT 1D receptor agonist. [1] Systematic name Avitriptan is 1-[3-[3-[4-(5- methoxypyrimidin-4-yl) piperazin-1-yl] propyl] -1H-indol-5-yl] -N-methyl-methane sulfonamide and is has contain molecular mass of g/mol. Formula of Avitriptan is C 22 H 30 N 6 O 3 S. Eletriptan: Eletriptan is a second generation trip tan drug intended for treatment of migraine headaches. It is used as an abortive medication, blocking a migraine attack which is already in progress. Systematic name Eletriptan is(r)-3-[(-1-methylpyrrolidin-2-yl) methyl]-5-(2- phenylsulfonylethyl) - 1H-indole and is has contain molecular mass of 382. (R)-3-[(-1- Novus International Journal of Analytical Innovations 2012, 1(3) 19
2 methylpyrrolidin-2-yl) methyl]-5-(2-phenylsulfonylethyl) - 1H-indole g/mol. Formula of Eletriptan is C 22 H 26 N 2 O 2 S Figure 1: Structure of Avitriptan Figure 2: Structure of Eletriptan Literature survey revealed about the reported individual triptolidine drugs. However, RP- HPLC method employing UV visible detection has been reported in the combination of other triptolidine drugs. So, a RP-HPLC method was developed and validated for linearity, precision, accuracy, robustness, limit of detection and limit of quantification with combined dosage form of Avitriptan and Eletriptan in pharmaceutical dosage form. MATERIALS AND METHODS Reagents Used Avitriptan and Eletriptan as pure standard reference drugs were purchased from Reddy s Laboratory, Hyderabad and pharmaceutical formulation from local market were used for this present study. HPLC grade Water, Methanol, acetonitrile, Orthophosphoric acid LR-grade is procured from Merck, India. Preparation of Mobile Phase Methanol, acetonitrile, and 0.1% Orthophosphoric acid of HPLC grade were separately filtered through membrane filters (0.45µ). The filtered solvents were mixed in such a way that the final volume consisted of these in the ratio 30:40:30 respectively and degassed by subjecting to sonication for 15 min. ph was adjusted to 4.1 by adding triethylamine drop by drop and the resulting solution was used as mobile phase. Preparation of Diluents Methanol (HPLC grade) was used as diluents Novus International Journal of Analytical Innovations 2012, 1(3) 20
3 Instrumentation To develop a High Pressure Liquid Chromatographic method for quantitative estimation of Avitriptan and Eletriptan an isocratic peak HPLC instrument with Chromosil C18 column (250 mm x 4.6 mm, 5μ) was used. The instrument is equipped with a LC 20AT pump for solvent delivery and variable wavelength programmable LC 7000 UV-detector. A 20μL Rheodyne inject port was used for injecting the samples. Peak software is used for analyzing the data. UV-2306 Spectrophotometer was used for wavelength checking. Denver analytical Balance was used to weigh the drug. Preparation of mixed standard solution Avitriptan and Eletriptan (1mg/ml) standard stock solutions were prepared using methanol as a solvent. Aliquots of mixed standard solutions of Avitriptan and Eletriptan were diluted with methanol to get a final concentration of ppm. Sample Preparation Standard stock solution (1mg/ml) of Avitriptan and Eletriptan were prepared by dissolving 25 mg of drug in 25 ml of methanol, separately to get 1mg/ml (1000 µ g/ml). These solutions were suitably diluted with mobile phase to get further dilutions. A composite of 20 tablets (Relpax-20mg) (Avitriptan -75mg) was prepared by grinding them to a fine, uniform size powder. 10 mg of, Avitriptan and Eletriptan was accurately weighted individually and quantitatively transferred into a 10ml volumetric flask and the solution was sonicated for 15 min. The flask was filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted with mobile phase to required concentration. Experimental Condition (Chromatographic conditions) A High Performance Liquid Chromatography (5-8) system, with LC solutions data handling system (Peak-LC2010) with an auto sampler was used for the analysis. The data was recorded using LC 2010 solutions software. The purity determination performed on a stainless steel column 150 mm long, 4.6 mm internal diameter filled with Octadecyl silane chemically bonded to porous silica particles of 5mm diameter (Inertsil C18, 5m, 150 mm x 4.6 mm, make: Shimadzu ltd, Japan) with the mobile phase containing Methanol: Acetonitrile: 0.1% Orthophosphoric Acid in the ratio of 30:40:30(v/v/v) ph 4.1 at ambient temperature. Flow rate was kept at 1.0 ml/min and the elution was monitored at 241 nm. Recording of chromatograms After stabilization of the base line with the optimized chromatographic conditions, standard solutions containing ppm of Avitriptan and Eletriptan were injected and the corresponding chromatograms were recorded. Retention time of Avitriptan and Eletriptan are found to be and 5.25 min. respectively. Similarly the sample chromatograms were also recorded. VALIDATION PARAMETERS Method validation The method developed for the determination of Avitriptan and Eletriptan was validated as per ICH and FDA guidelines 14, 15. Linearity The linearity of the method was determined at seven concentration levels ranging from 90 to 240 μg/ml for both Avitriptan and Eletriptan. The calibration curve was constructed by plotting response factor against concentration of drugs. Novus International Journal of Analytical Innovations 2012, 1(3) 21
4 System suitability The system suitability studies were carried out to determine theoretical plate (N), resolution factor, asymmetric factor and tailing factor. The results were given in the Table. The values obtained demonstrated the suitability of the system for the analysis of this drug combinations, system suitability parameters may fall within ±3% standard deviation range during routine performance of the method. Precision The precision of the method was demonstrated by inter day and intraday variation studies. In the intraday studies, six repeated injections of standard solutions were made and the response factor of drug peaks and percentage RSD were calculated. In the inter day variation studies, six repeated injections of standard solutions were made for three consecutive days and response factor of drug peaks and percentage RSD were calculated. From the data obtained, the developed HPLC method was found to be precision. Accuracy Accuracy of the method was determined by applying the proposed method for the estimation of Avitriptan and Eletriptan from pharmaceutical dosage form. The accuracy of the standard solutions was conducted by adding them to pre-analyzed sample solution at different levels i.e. 25%, 50%, and 100% separately and corresponding chromatograms were recorded. Specificity Specificity was performed to exclude the possibility of interference with excipients in the region of elution of Avitriptan and Eletriptan. The specificity and selectivity of the method was tested under normal conditions and the results of the tests proved that the components other than the drug did not produce a detectable signal at the retention place of Avitriptan and Eletriptan. Limit of detection (LOD) and limit of quantification (LOQ) LOD and LOQ were detected from standard deviation of y-intercept of regression line and slope as per ICH guidelines. LOD and LOQ values were calculated with the following formula: LOD (ppm) = 3.3 x Standard deviation/slope LOQ (ppm) = 10 x Standard deviation/slope Robustness The drug solution was subjected to small deliberate changes like Wavelength, p H and mobile phase ratio. Variation in wavelength: Wavelength of detection varied from 241 to 236nm. Variation in p H : P H of mobile phase varied from 4.2 to 3.9. Variation in mobile phase ratio: Mobile phase (Methanol: Acetonitrile: 0.1% Orthophosphoric acid) ratio varied from 35:35:30 to 25:45:30 respectively. Novus International Journal of Analytical Innovations 2012, 1(3) 22
5 Ruggedness Ruggedness were performed by using six replicate injections of 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. Stability of the Solution 180ppm of sample solution was injected in every 6 hours interval and observed for change in the results. After 24 hours tailing and area of peak were disturbed means the sample solution is stable for 24 hours after preparation in method condition Analysis of a formulation Experimental results of the amount of Avitriptan and Eletriptan tablets, expressed as a percentage of label claims were in good agreement with the label claims thereby suggesting that there is no interference from any of the excipients which are normally present. The drug content was found to be 99.73% for Avitriptan and % for Eletriptan. Two different lots of Avitriptan and Eletriptan tablets were analyzed using the proposed procedures. RESULTS AND DISCUSSION The chromatographic parameters were initially evaluated using Phenomenex Chromosil C 18 column by adopting various mobile phases viz; methanol, acetonitrile and 0.1% Orthophosphoric acid at P H 4.1 in the ratio of 35:35:30 and 25:45:30. The system suitability parameters such as theoretical plates count and tailing factor observed from the chromatograms obtained with the mobile phase containing methanol, acetonitrile and 0.1% Orthophosphoric acid at P H 4.1 in the ratio of 35:35:30 and 25:45:30. The desired system suitability parameters were obtained with the mobile phase containing methanol, acetonitrile and 0.1% Orthophosphoric acid at P H 4.1 in the ratio of 35:35:30 and 25:45:30. The retention times of Avitriptan and Eletriptan were found to be 3.7 min and 5.2 min respectively. The typical chromatogram of standard drug mixture was shown in fig: 3. Calibration Curve of Avitriptan and Eletriptan showed good linearity in the range of ppm (Table: 2) A linear correlation was found between the chromatographic peak areas and concentration of Avitriptan and Eletriptan (Table: 3) and their corresponding regression equations are shown below: y=5036*x y= 4638*X ± 7857 The system parameters such as number of theoretical plates, tailing factor (Table: 4) were calculated and found to be within the limits. The precision of an analytical method is the degree of agreement among the individual test results when the method is applied repeatedly to multiple sampling of homologous sample. Results from determination of repeatability and intermediate precision, expressed as RSD (%). The low values of %RSD indicated that the method is precise. The reproducibility results were shown that, there were no significant differences between %RSD values for intra-day and inter-day precision, which indicated that the method, is reproducible (Table: 5 & 6). LOD and LOQ values were calculated (Table: 4) the obtained lower values of LOD and LOQ showed better sensitivity of the proposed method. The recovery values obtained from the accuracy varied from % and % and were within acceptable limits at different concentration levels (Table: 7). Robustness of the method was performed by deliberate changes such as flow rate variation, wavelength variation and P H variation and the corresponding results are showed in( Table: 8). Ruggedness were performed by using six replicate injections of solutions of concentrations which were prepared and analyzed by different analyst on three different days Novus International Journal of Analytical Innovations 2012, 1(3) 23
6 over a period of one week and the corresponding results are showed in (Table: 9). The result obtained were unaffected by small variations in these parameters indicating the robustness of the method. The proposed liquid chromagraphic method was applied for the determination of Avitriptan and Eletriptan in tablet formulation in (Table: 10). The result for the Avitriptan and Eletriptan was comparable with the corresponding labeled amount. The absence of additional peaks indicates no interference of the excipients used in the tablets.the % RSD of the peak area was found to be less than 2% which indicate suitability for routine analysis of Avitriptan and Eletriptan in pharmaceutical dosage form. The stability of the solution is confirming by observing the peak area contributed by the injection of 20ppm standard drug solution collected at 0, 6, 12, 18, 24 and 30 hrs after the preparation and maintained at room temperature. The validated HPLC method was adopted for the quantification of Avitriptan and Eletriptan in their combined pharmaceutical dosage form. The contents of the pharmaceutical dosage form were found to be in the range of 100±2% with %RSD less than 2% which indicate suitability for routine analysis of Avitriptan and Eletriptan in pharmaceutical dosage. Table 1: Optimized chromatographic conditions for estimation of Avitriptan and Eletriptan Mobile phase Methanol : Acetonitrile: 0.1% Orthophosphoric acid) 30:40:30 (v/v/v) Pump mode Isocratic A.P.I Concentration. Avitriptan 180 ppm Eletriptan 180ppm ph 4.1 Diluents Mobile phase Column C18 column (250 X 4.6 mm, 5μ) Column Temp Ambient Wavelength 241 nm Injection Volume 20 μl Flow rate 1.0 ml/min Run time Retention Time 10 minutes Avitriptan 3.74 min Eletriptan min Table 2: Linearity result S.No Concent ration Avitriptan Eletriptan Linearity Graph of Avitriptan: Eletriptan Novus International Journal of Analytical Innovations 2012, 1(3) 24
7 Table 3: Regression analysis of the calibration curve Parameters Avitriptan Eletriptan Calibration range (ppm) ppm ppm Slope Intercept Correlation coefficient (r 2 ) Table.4: System suitability and validation parameters Parameters Avitriptan Eletriptan Theoretical plates (N) Retention time(min) Tailing factor LOD(ppm) LOQ(ppm) min 5.25 min % RSD 0.94(intra day) 0.41(inter day) 0.06 ppm 0.12 ppm 0.12 ppm 0.4 ppm 0.35( intra day) 0.39(inter day) Table 5: Intraday Precision S.No Concentration Avitriptan Eletriptan ppm ppm ppm ppm ppm ppm Result % RSD Table 6: Interday Precision S.No Concentration Avitriptan Eletriptan ppm ppm ppm ppm ppm ppm Result % RSD Novus International Journal of Analytical Innovations 2012, 1(3) 25
8 Table 7: Recovery of Avitriptan and Eletriptan %of Recovery 25% 50% 100% Concentration in ppm Concentration Obtained Avitriptan % of Assay Concentration Obtained Eletriptan % of Assay Chromatographic Condition Table: 8 Robustness evolution of reverse phase HPLC method Parameter Avitriptan Peak Area Eletriptan Peak Area Avitriptan % of change Eletriptan % of change Wave Length 241 nm nm Mobile phase PH Methanol: Acetonitrile: 0.1% Orthophosphoric Acid :45:30 (v/v/v) Mobile phase ratio Methanol: Acetonitrile: 0.1% Orthophosphoric Acid. 25:45:30 (v/v/v) Table 9: Ruggedness S.No Concentration Area Avitriptan Eletriptan ppm ppm ppm ppm ppm ppm Result % RSD Novus International Journal of Analytical Innovations 2012, 1(3) 26
9 Table 10: Formulation results Drug Formulation Dosage Concentration Amount % Assay found Avitriptan Avitriptan 75mg 180 ppm Eletriptan Relpax 20mg 180 ppm Table 11: Stability Time Avitriptan Eletriptan (hours) Area Tailing factor Area Tailing factor Figure 3: Typical chromatogram of standard Avitriptan and Eletriptan Novus International Journal of Analytical Innovations 2012, 1(3) 27
10 Figure 4: O hours Figure 5: 6 hours Figure 6: 16 hours Figure 7: 18 hours Figure 8: 24 hours Figure 9: 3O hours Novus International Journal of Analytical Innovations 2012, 1(3) 28
11 CONCLUSION The developed RP-HPLC method was accurate, precise, reproducible, robust and ruggedness. The developed method has been found to be better, because of its wide range of linearity, use of a readily available mobile phase, lack of extraction procedure and low retention times. All these factors make the proposed method suitable for the quantification of Avitriptan and Eletriptan in bulk drugs and in table dosage form. The method can be successfully used for the routine analysis of Avitriptan and Eletriptan in pharmaceutical dosage forms without interference. REFERENCES 1. Saxena PR, De Varies P, Wang W, et al. (February 1997)."Effects of avitriptan, a new 5-HT 1B/1D receptor agonist, in experimental models predictive of ant migraine activity and coronary side-effect potential". Naunyn-Schmiedeberg's Archives of Pharmacology FDA Access Data entry for Eletriptan Hydro bromide, accessed March 10, U.S. Patent no , John E. Macor & Martin J. Wythes, Indole Derivatives, August 13, U.S. Patent no , Valerie Denise Harding, et al., Salts of an anti-migraine indole derivative, August 29, Lindsay, S.; Kealey, D. (1987). High performance liquid chromatography. Wiley. from review Hung, L. B.; Parcher, J. F.; Shores, J. C.; Ward, E. H. (1988). "Theoretical and experimental foundation for surface-coverage programming in gassolid chromatography with an absorbable carrier gas". J. Am. Chem. Soc. 110 (11): Displacement Chromatography. Sacheminc.com. Retrieved on A recent book provides a comprehensive treatment of the theory of highperformance gradient chromatography: Lloyd R. Snyder and John W. Dolan (2006). High-Performance Gradient Elution: The Practical Application of the Linear- Solvent-Strength Model. Wiley Interscience.. 8. Majors, Ronald E. ( ) Fast and Ultrafast HPLC on sub-2 μm Porous Particles Where Do We Go From Here? LC-GC Europe. Novus International Journal of Analytical Innovations 2012, 1(3) 29
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