RP-HPLC METHOD DEVELOPMENT FOR THE QUANTIFICATION OF GABAPENTIN IN FORMULATIONS
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1 RP-HPLC METHOD DEVELOPMENT FOR THE QUANTIFICATION OF GABAPENTIN IN FORMULATIONS ABSTRACT A simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of Gabapentin in tablet dosage form. Isocratic elution at a flow rate of 1 ml/min was employed on a symmetry Zodiac C18 (250mmx4.6mm,I.D., 5µm particle size) at ambient temperature. The mobile phase consisted of Methanol: Acetonitrile: Ortho phosphoric acid 65:33:2 % (V/V/V). The UV detection wavelength was 216nm and 20µl sample was injected. The retention time for Gabapentin was 3.7 2mins. The %RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per the ICH guidelines. The method was successfully applied for routine analysis of Gabapentin in tablet dosage form and bulk drug. KEY WORDS: Gabapentin, RP-HPLC, UV detection, recovery, precise, 216 nm, 84
2 INTRODUCTION Gabapentin (figure 1) is a cyclic GABA [gamma - amino butyric acid] analogue (Fig 1). Although, it is structurally related to GABA, gabapentin has no direct GABA mimetic effect. Gabapentin is originally developed for the treatment of epilepsy. It is widely used to relieve pain, especially neuropathic pain. EXPERIMENTAL Figure 1: Structure of Gabapentin Materials :Working standard of Gabapentin was obtained from well reputed research laboratories. Acetonitrile, Methanol,OPA was purchased from E. Merck (Mumbai, India). Apparatus :A Series HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system, Rheodyne manual sample injector with switch (77251), Analytical column Zodiac C18. (250mm 4.6mm i.d.,5µm particle size),electronic balance-denver (SI-234), a manual Rheodyne injector with a 20 μl loop was used for the injection of sample. PEAK LC software was used. UV 2301 Spectrophotometer was used to determine the wavelength of maximum absorbance Determination of wavelength of maximum absorbance:the standard solutions of Gabapentin were scanned in the range of nm against mobile phase as a blank. Gabapentin showed maximum absorbance at 216 nm. So the wavelength selected for the determination of Gabapentin was 216 nm. Chromatographic equipment and conditions: The development and validation of the assay was performed on a Series 200 HPLC system PEAK LC7000 isocratic HPLC with PEAK 7000 delivery system Rheodyne manual sample injector with switch (77251), Analytical column column Zodiac C18. (250mm 4.6mm i.d.,5µm particle size),manual injector rheodyne valve) with 20μL fixed loop, PEAK LC software was used.the mobile phase consisted of a Methanol: Acetonitrile: OPA 65:33:2 (v/v/v). Injections were carried out using a 20 μl loop at room temperature ( C) and the flow rate was 1mL/min. Detection was performed at 216 nm with 10 mins runtime. Standard and sample solutions:10 mg of Gabapentin reference substance was accurately weighed and dissolved in 10 mobile phase in a 10mL volumetric flask to obtain 1000 µg/ml concentrated solution. From standard solution, by serial dilution the required concentrations including standard concentration of 120µg/mL was prepared. A composite of 20 tablets was prepared by grinding them to a fine, uniform size powder. 10 mg of Gabapentin was accurately weighed and quantitatively transferred into a 100mL volumetric flask. Approximately 25mL mobile phase was added and the solution was sonicated for 15 mins. The flask was filled 85
3 to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted with mobile phase to a concentration of 120 µg/ml. Optimization of Chromatographic conditions:chromatographic conditions as optimized above were shown in Table 1.These optimized conditions were followed for the determination of Gabapentin in bulk samples and its combined tablet Formulations. The chromatograms of standard and sample were shown in Figure 2. Table 1: Optimized chromatographic conditions for the estimation of Gabapentin Mobile phase Pump mode ph Diluent Column Column Temp Wavelength : Methanol: Acetonitrile: Orthophosphoric acid (65:33:2, v/v/v) : Isocratic : 4.0 (adjusted with 0.1% Formic acid) : Mobile phase : Zodiac C 18 (250 mm 4.6mm I.D., 5 μm particle size) : Ambient : 216nm Injection Volume : 20µL Flow rate Run time : 1.0mL/min : 10 mins Typical t R of Gabapentin : 3.72mins Figure.2 Chromatogram of standard solution 86
4 Method Validation:The proposed method was validated as per ICH guidelines. The parameters studied for validation were specificity, linearity, precision, accuracy, robustness, system suitability, limit of detection, limit of quantification, and solution stability. Linearity:Standard curves were constructed using six standard concentrations of 30, 60, 90, 120, 150 and 180 µg/ml of Gabapentin. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equation was y = 7236.x (correlation coefficient= 0.999). Linearity values are shown in Table 2. Table 2 : Linearity result of Gabapentin Level Concentration of Gabapentin (µg/ml) Mean peak area Level Level Level Level Level Level Slope 7236 Intercept Correlation Coefficient
5 Peak Area Linearity graph of Gabapentin y = 7236.x R² = Concentration µg/ml Figure.2 Linearity plot of Gabapentin Precision:To study precision, six replicate standard solutions of Gabapentin (120 µg/ml) were prepared and analyzed using the proposed method. The percent relative standard deviation (% RSD) for peak responses was calculated and it was found to be within the acceptance criteria of not more than 2.0%. Table.3 : Precision results INJECTION INTRA DAY INTER DAY %RSD Intermediate Precision or Ruggedness: Ruggedness was performed by using six replicate injections of standard (120 µg/ml) and sample solutions which were prepared and analyzed by different analyst on three different days over a period of one week.. Ruggedness is also expressed in terms of percentage relative standard deviation. 88
6 Table 4: Ruggedness results Sample Area %RSD 0.44 Limit of Detection and Limit of Quantification:To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injected until peak was disappeared. After 2.5 µg/ml dilution Peak was not clearly observed, based on which 2.5 µg/ml is considered as Limit of Detection and Limit of Quantification is 8.25 µg/ml. Robustness:Typical variations in liquid chromatography conditions were used to evaluate the robustness of the assay method. Table. 5:Robustness results of Gabapentin Condition %Assay %Difference Unaltered 100 Flow rate at 0.8 ml/min Flow rate at 1.2 ml/min Mobile phase: Methanol(67):acetonitrile(33) v/v Methanol (63):acetonitrile(37) v/v ph of buffer at ph of buffer at
7 Accuracy:Recovery test was performed at 3 different concentrations i.e. 90µg/mL, 120µg/mL, 150µg/mL. Results are given in table.6. Table.6: Recovery results Level Target Concentration (µg/ml) Spiked concentration (µg/ml) Concentration obtained %Recovery 50% 100% % Stability test:to perform the Stability test the standard solution of 120µg/ was stored at ambient temperature (±10 0 C) for two days. After this these storage solutions and freshly prepared solution were tested with proposed method. It is noticed that assay of these results did not decrease below 98%. The results of stability test were shown in Table7. Table.7: Solution stability of Gabapentin Standard solution Sample solution Time (hours) Peak area %variation Time (hours) Peak area %variation Initial Initial System suitability :System suitability was studied under each validation parameters by injecting six replicates of the standard solution. The system suitability parameters are given in Table 8. Table.8:System suitability parameters Parameter Tailing factor Theoretical plates Specificity study Linearity study Precision study
8 Assay of formulation of Gabapentin: Table.9: Assay Result S.NO Tablet Dosage Sample conc Sample estimated % of Drug Estimated in Tablet 1 Neurontin 100mg 120µg/mL CONCLUSION The proposed method for the assay of Gabapentin in tablets or capsules is very simple and rapid. It should be emphasized it is isocratic and the mobile phase do not contain any buffer. The method was validated for specificity, linearity, precision, accuracy and robustness. Although the method could effectively separate the drug from its products, further studies should be performed in order to use it to evaluate the stability of pharmaceutical formulations. REFERENCES 1. C.P. Taylor, N.S. Gee, T.Z. Su, J.D. Kocsis, D.F.Welty, J.P. Brown,D.J. Dooley, P. Boden, and L. Singh. A summary of mechanistic hypotheses of Gabapentin pharmacology. Epilepsy Re., 29: (1998). 2. Y.P. Maneuf, Z.D. Luo, and K. Lee. α 2δ and the mechanism of action of gabapentin in the treatment of pain. Semin. Cell Dev. Biol., 17: (2006). 3. R.L. Terry, S.M. McDonnell, W. van Eps, L.R. Soma, Y. Liu, C.E. Uboh, P.J. Poate, and B. Driessen. Pharmacokinetic profile and behavioral effects of Gabapentin in the horse. J. Vet. Pharmacol. Ther.,33: (2010). 4. H. Rosner, L. Rubin, and A. Kestenbaum. Gabapentin adjunctive therapy in neuropathic pain states. Clin. J. Pain. 12: (1996). 5. N.V. Ramakrishna, K.N. Vishwottam, M. Koteshwara, S. Manoj. M. Santosh. J. Chidambara, B. Sumatha, D.P.Varma. Rapid quantification of Gabapentin in human plasma by liquid chromatography/ tandem mass spectrometry. J. Pharm. Biomed. Anal. 40: (2006). 6. G.L. Lensmeyer, T. Kempf, B.E. Gidal, and D.A. Wiebe. Optimized method for determination of Gabapentin in serum by highperformance liquid chromatography. Ther. Drug Monit.,17: (1995). 7. Z. Zhu and L. Neirinck. High-performance liquid chromatographic method for the determination of Gabapentin in human plasma. J. Chromatogr. B., 779: (2002) 8. G. Forrest, G.J. Sills, J.P. Leach, and M.J. Brodie. Determination of Gabapentin in plasma by highperformance liquid chromatography. J. Chromatogr. B., 681: (1996) 91
9 9. N. Wad and G. Krämer. Sensitive high-performance liquid chromatographic method with fluorometric detection for the simultaneous determination of Gabapentin and Vigabatrin in serum and urine. J. Chromatogr. B., 705: (1998). 10. N. Ratnaraj and P.N. Patsalos. A high-performance liquid chromatography Micro method for the simultaneous determination of Vigabatrin and Gabapentin in serum. Ther. Drug Monit.,20: (1998). 11. Q. Jiang and S. Li. Rapid high-performance liquid chromatographic determination of serum Gabapentin. J. Chromatogr. B.,727: (1999). 12. P.H. Tang, M.V. Miles, T.A. Glauser, and T. DeGrauw. Automated microanalysis of Gabapentin in human serum by high-performance liquid chromatography with fluorometric detection. J. Chromatogr. B., 727: (1999). 13. D.F. Chollet, L. Goumaz, C. Juliano, and G. Anderegg. Fast isocratic high-performance liquid chromatographic assay method for the simultaneous determination of Gabapentin and vigabatrin in human serum. J. Chromatogr. B., 746: (2000). 14. B. Uday Kumar Rao, F. Maqdoom and A. Pratimanikalje. Determination of Gabapentin in bulk and pharmaceutical dosage form by HPLC method. J. Chil. Chem. Soci., 54: (2009). 15. G. Rajendra Singh and S.V. MagurlHaque. Development and validation of new HPLC method for the determination of Gabapentin. Int. J. Biomed. Sci., 5: (2009). 16. S.T. Ulu and E. Kel. Highly sensitive determination and validation of Gabapentin in pharmaceutical preparations by HPLC with 4-fluoro-7-nitrobenzofurazon. J. Chromatogr. Sci., 49: (2009). 92
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