Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), Available online through

Transcription

1 Research Article ISSN: Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), Available online through Determination of Stability Indicating Assay Method for Capecitabine in Pharmaceutical Drug Substances a comparative study by UPLC and HPLC Hanumanturayudu. K* 1, Sreeramulu.J 1, Maheswara Reddy.M 1 1. Department of chemistry, Sri Krishnadevaraya University, Anantapur ,Andhra Pradesh, India. Received on: ; Revised on: ; Accepted on: ABSTRACT Capecitabine chemically denotes N 4 -Pentoxy Carbonyl 5-Deoxy 5-Fluorocytidine is an anticancer prodrug of 5-Fluoro Uracil. 5-FU (Fluoro Uracil) is used as an anticancer agent in the chemotherapy of solid tumor. A simple, rapid and selective RP-UPLC method was developed for the quantification of Capecitabine in active pharmaceutical ingredients (API S) using a simple mobile phase composition of solution A(0.1% of acetic acid in water : acetonitrile : methanol (in the ratio of 11:2:7) and solution B (0.1% of acetic acid in water : acetonitrile : methanol (in the ratio of 4:1:15) with a simple gradient program ( Time in min. / % of solution A : /100, 4.00/100, 4.80/45, 5.20/45, 6.00/100) at a flow rate of 0.70 ml/min. Poroshell C18 column of dimension 4.6 X 50 mm, 2.7 µm was elected for the analysis and the column temperature was maintained at 30 C. UV detector was monitored at 250nm.The retention time (RT) of Capecitabine was about 3.45 min and total run time for a single run is 6.0 min. the method development and validation was performed as per ICH guidelines. Finally a comparison of the method was done against conventional HPLC. The developed method was superior with respect to analysis time, efficiency and sensitivity. The runtime of Capecitabine in conventional hplc method was about 40.0 min. Compare to HPLC the developed method is cost effective and more rugged. The method can be used for routine analysis of assay in Quality Control laboratory on regular basis. Key words: Ultra performance liquid chromatography (UPLC), Capecitabine, assay, anticancer drug, method development and validation. INTRODUCTION Capecitabine physically white powder, odorless, freely soluble in dichloromethane, slightly soluble in ethanol, and practically insoluble in water. Capecitabine chemically is pentyl1-(5-deoxy-b-d-ribofuronosyl 5- fluoro-1, 2 dihydro-2-xoo-4-pyrimidine carbamate is an anticancer prod rug of 5-fluoro uracil which is designed to undergo preferential conversion of 5- FU with in tumor. It inhibits DNA synthesis and slows down the growth of tumor tissues. Its molecular weight is g/mol and molecular formula is C 15 H 22 FN The structure of Capecitabine and its related compounds A, B and C are shown in the figure-1, 2, 3 and 4 respectively. Figure-3: Structure of Related compound B Figure-1: Structure of Capecitabine Figure-4: Structure of Related compound C *Corresponding author. Hanumanturayudu Kuruba C/O. Prof. J.Sreeramulu, Department of chemistry, Sri Krishnadevaraya University, Anantapur, Andhra Pradesh, India Figure-2: Structure of Related compound A Literature survey reveals that several methods based on techniques viz. HPLC- UV, HPLC-MS, LC-MS, LC-MS-MS, GC-MS and MS-MS for the determination Capecitabine in human plasma and pharmaceutical dosage form. The method is simple, rapid, reproducible and economical. The present method is developed and validated according to ICH guidelines. Materials: Capecitabine API and its impurities were purchased from USP, Acetonitrile and Methanol was obtained from rankem and glacial acetic acid was obtained from Qualigens.

2 Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), Equipments: Waters Acquity UPLC system (Waters, USA), An Agilent HPLC system (Agilent, USA) equipped with binary gradient pump, auto sampler, column oven and photodiode array detector (PDA) was employed for analysis. Chromatographic data was acquired using Empower 2 software. Millipore Milli Q Plus water purification system were used for the analysis. Poroshell C18 column of dimension 4.6 X 50 mm, 2.7 µm and Mettler balance were used for the study. Chromatographic conditions: Analysis was performed on a high strength Agilent Poroshell C18 column of dimension 4.6 X 50 mm, 2.7µm under reverse phase condition. Solution A ( 0.1% acetic acid in water: acetonitrile : methanol in the ratio of 11:2:7 and solution B (0.1% acetic acid in water : acetonitrile : methanol in the ratio of 4:1:15 were used as mobile phase with a simple gradient program ( time in min/ %of solution A : /100, 4.00/100, 4.80/45, 5.20/45, 6.00/100) at a flow rate of 0.70 ml/min. Column and sample compartment temperature were maintained at 30 C and 25 C respectively. The detection was done at 250nm with an injection volume of 1.0 ul. Preparation of standard and sample solutions 30.0 mg of Capecitabine reference standard and samples were separately weighed and transferred quantitatively into a 50 ml volumetric flask and diluted to volume with diluent, filtered through 0.22um syringe to obtain a solution of 600 µg/ml. RESULTS AND DISCUSSIONS Method Development: Initially isocratic method was elected but it was observed that the co elution of impurities and peak symmetry was not good. To obtain a better resolution and peak symmetry, gradient program was selected for the analysis. Blank and system suitability solution chromatograms are shown in figure-1 and 2. Blank solution chromatogram was free of interferences and system suitability chromatogram met as per USP system suitability criteria. Sample chromatogram acquired by hplc and UPLC are given in figure-3 and figure-4 respectively. Chromatographic data of system suitability are shown in Table- I. A comparative chromatographic performance data of HPLC and UPLC are indicated in the Table-II. The elution of all the components in UPLC was observed to be 7-folds minimized to that of HPLC. The resolution and efficiency obtained for all the components selected in the study by UPLC showed comparatively better than HPLC. The tailing factors of all the components are less than Figure-1: Blank chromatogram CAPECITABIEN REL COMP A CAPECITABINE REL COMP B CAPECITABINE CAPECITABINE REL COMP C Figure-2: System suitability Chromatogram

3 Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), 0.45 CAPECITABINE Figure-3: Assay sample chromatogram by UPLC CPE Table-I: system suitability results Figure-4: Assay sample chromatogram by HPLC Table-II: Comparison of system suitability data by UPLC and HPLC S.No Name Retention RT USP USP USP Plate Time(min) Ratio Resolution Tailing Count 1 Related Compound-A Related Compound-B Capecitabine Related Compound-C S.No. Name of drug Retention USP USP USP component Time(min) Resolution Tailing Plate Count UPLC HPLC UPLC HPLC UPLC HPLC UPLC HPLC 1 Related Compound-A NA Related Compound-B Capecitabine Related Compound-C CAPECITABINE CAPECITABIEN REL COMP A CAPECITABINE REL COMP B CAPECITABINE REL COMP C Figure-5: Spiked sample Chromatogram

4 Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), 0.45 CAPECITABINE Figure-6: five replicated standard injection chromatogram Specificity: To conform the specificity of the method sample was spiked with three impurities.figure-5 represents spiked sample chromatogram.it was observed that the sample has no interference with the impurites and the peak purity angle is less than that of threshold which indicates the homogenity of the peak. Precision: The precision of the assay method was evaluated by carrying out five replicate injections of Capecitabine (600 µg ml -1 ) qualified reference standard. The percentage of RSD of five injections was calculated. The % RSD of areas was less than 0.5% which indicates the reproducibility of the system. Overlaid chromatogram of five replicate injections is given in figure-6. The tabulated data of precision are mentioned in Table-III. Table-III: precision results Injection No. Peak Name Retention Area Time (min) 1 Capecitabine Capecitabine Capecitabine Capecitabine Capecitabine Mean %RSD Linearity: Linearity solutions were prepared from stock solution at five concentration levels from 40% to 160% of analyte concentrations (240 to 960µg ml -1 ). The linear regression analysis of Capecitabine was constructed by plotting the peak area of the analyte (y) versus analyte concentration in (x) axis. The calibration curve was linear in the range of 240 to 960µg ml -1 for Capecitabine with a correlation coefficient of greater than The linearity plot is summarized in Figure -7 and the data are given in Table -IV. Figure -8 indicates the overlaid chromatogram of linearity. Table-IV: Linearity results S.No Name Retention Area Standard Time Level 1 Capecitabine % 2 Capecitabine % 3 Capecitabine % 4 Capecitabine % 5 Capecitabine % Correlation coefficient CAPECITABINE Figure-7: Linearity plot of Capecitabine. Figure-8: linearity standard injections chromatogram Robustness: The robustness of the method was determined by making deliberate changes in the chromatographic conditions, such as change in mobile phase composition, flow rate and column temperature. The flow rate of mobile phase is 0.70 ml/min. To study the effect of flow rate on resolution and retention time of main peak, 0.07 units changed i.e ml/min and 0.77 ml/min. The column temperature was studied at 25 o C and 35 o C instead of 30 o C. It was observed that in all above conditions, there are no marked changes in the chromatograms, which demonstrates that the developed RP-UPLC method is rugged and robust.

5 Hanumanturayudu. K et al. / Journal of Pharmacy Research 2012,5(12), Forced degradation studies: Stress testing of a drug substance can help to identify the likely degradation products, which in turn help to establish the degradation pathways and the intrinsic stability of the molecule. All stress decomposition studies (acid, base and peroxide) were performed at an initial drug concentration of 0.60 mg/ml. The results were tabulated in Table-V. In all the conditions the purity angle is less than that of the purity threshold. Table-V: Forced degradation results Stress Conc. Match Match Purity Purity Percent of condition µg/ml angle threshold angle Threshold Capecitabine Non stressed Acid hydrolysis Base hydrolysis Oxidation Heat & Humidity Photo stability Dry heat CONCLUSION: The developed method was specific, accurate and precise. The method is suitable for quantification of Capecitabine. The shorter run time demonstrates that the method is cost and time effective aiming towards green chemistry. Thus developed RP-UPLC method was specific and stability indicating. This method can be effectively transferred to quality control labs and can be used for carry out the quantitative analysis Capecitabine drug substance. REFERENCES: 1. F. Desmoulin, V. Gilard, M. Malet-Martino, R. Martino, Drug Metabolism Disposition, 2002, 30, I.R. Judson, P.J. Beale, J.M. Trigo, W.Aherne, T. Crompton, D. Jones, E. Bush, B.Reigner. Investigational New Drugs. 1999, Source of support: Nil, Conflict of interest: None Declared 3. A.H. Braun, W. Achterrath, H. Wilke, U.Vanhoefer, A. Harstrick, P. Preusser., Cancer, 2004, 100, T. Moriwaki, I. Hyodo, T. Nishina, K. Hirao, T. Tsuzuki, S. Hidaka, T. Kajiwara, S. Endo, J.Nasu, S. Hirasaki, T. Masumoto, A. Kurita., Cancer Chemotherapy and Pharmacology, 2005, 56, P. Compagnon, L. Thiberville, N. Moore, C. Thuillez, C. Lacroix., Journal of Chromatographia B, 1996, 677, E. Gamelin, M. Boisdron-Celle, A. Turcant, F. Larra, P. Allain, J. Robert., Journal of Chromatographia B, 1997, 695, F.P. LaCreta, W.M. Williams., Journal of Chromatographia B, 1987, 414, W. Wattanatorn, H.L. McLeod, J. Cassidy,K.E. Kendle., Journal of Chromatographia B, 1997, 692, M.J. Del Nozal, J.L. Bernal, A. Pampliega, P.Marinero, M. Pozuelo., Journal of Chromatographia B, 1994, 656, M.J. Moore, P. Bunting, S. Yuan, J.J.Thiessen., Therapeutic Drug Monitoring, 1993, 15, L.J. Schaaf, D.G. Ferry, C.T. Hung, D.G.Perrier, I.R. Edwards., Journal of Chromatographia, 1985,342, J. Escoriaza, A. Aldaz, E. Calvo, J. Giráldez., Journal of Chromatographia B, 1999,736, L. Zuf ýa, A. Aldaz, C. Castellanos, J.Giráldez. Therapeutic Drug Monitoring, 2003, 25, L. Zufia, A. Aldaz, J. Giraldez., Journal of Chromatographia B, 2004, 809, S.M. Guichard, I. Mayer, D.I. Jodrell., Journal of Chromatographia B, 2005, 826, Y. Xu, J. Grem., Journal of Chromatographia B, 2003, 783, C. Siethoff, M. Orth, A. Ortling, E. Brendel,W. Wagner-Redeker., Journal of Mass Spectrum, 2004, 39, Roos, L. Banken, M. Utoh, B. Osterwalder, Clinical Cancer Research, 1998, 4, B. Reigner, S. Clive, J. Cassidy, D. Jodrell, R.Schulz, T. Goggin, L. Banken, B. Roos, M. Utoh, T. Mulligan, E. Weidekamm., Cancer Chemotherapy and Pharmacology, 1999, 43, C. Siethoff, M. Orth, A. Ortling, E. Brendel,W. Wagner-Redeker., Journal of Mass Spectrum, 2004, 39, 884.