Validated LC Method for the Estimation of

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Oranje WA, Wolffenbuttel BH. Lipid peroxidation and atherosclerosis in type II diabetes. J Lab Clin Med 1999;134:19-32. Bonnefont-Rousselot D, Bastard JP, Jaudon MC, Delattre J. Consequences of the diabetic status on the oxidant/antioxidant balance. Diabetes Metab 2000;26:163-76. Nourooz-Zadeh J, Rahimi A, Tajaddini-Sarmadi J, Tritschler H, Rosen P, Halliwell B, et al. Relationships between plasma measures of oxidative stress and metabolic control in NIDDM. Diabetologia 1997;40:647-53. Sundaram RK, Bhaskar A, Vijayalingam S, Viswanathan M, Mohan R, Shanmugasundaram KR. Antioxidant status and lipid peroxidation in type II diabetes mellitus with and without complications. Clin Sci 1996;90:255-60. Laight DW, Carrier MJ, Anggard EE. Antioxidants, diabetes and endothelial dysfunction. Cardiovasc Res 2000;47:457-64. Garg MC, Singh KP, Bansal DD. Effect of vitamin E supplementation on antioxidant status of diabetic rats. Med Sci Res 1996;24:325-6. Jennings PE, Jones AF, Florkowski CM, Lunec J, Barnett AH. Increased diene conjugates in diabetic subjects with microangiopathy. Diabetic Med 1987;4:452-6. Mooradian AD. Increased serum conjugated dienes in elderly diabetic patients. J Am Geriatr Soc 1991;39:571-4. Velazquez E, Winocour PH, Kesteven P, Alberti KGM, Laker MF. Relation of lipid peroxide to macrovascular disease in Type 2 diabetes. Diabetic Med 1991;8:752-8. Salah N, Miller NJ, Paganga G, Tijburg L, Bolwell GP. Polyphenolic flavanols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophy 1995;322:339-46. Feillet-Coudray C, Rocke E, Coudray C, Grzelkowska K, Azais-Braesco V, Dardevet D, et al. A Lipid peroxidation and antioxidant status in experimental diabetes. Clin Chim Acta 1999;284:31-43. Drymock W, Warden CJH, Hooper D. In: Pharmacographica Indica. Kegan P, Treneb, editors. London: Trubner and Co; 1890. p. 232. Al Yahya MA. Phytochemical studies of the plants used in traditional medicine of Saudi Arabia. Fitoterapia 1986;57:179-82. Burkill IH. A Dictionary of the Economic Products of the Malay Peninsula Publication Governments of Malaysia and Singapore. Kuala Lumpur: 1966. p. 1153. Eisai PT. In: Medicinal Herb Index in Indonesia. 2nd ed. Indonesia: 1995. p. 77. Kirtikar KR, Basu BD. Indian Medicinal Plants. 2nd ed. Dehradun: International Book Distributors; 1995. p. 371-2. Singh SB, Singh AK, Thakur RS. Chemical constituents of the leaves of Helicteres isora. Indian J Pharm Sci 1984;46:148-9.

22. Qu WH, Li JG, Wang MS. Chemical studies on the Helicteres isora. Zhongguo Yaoke Daxue Xuebao 1991;22:203-6. 23. Bean MF, Antoun M, Abramson D, Chang CJ, Mc Laughlin JL, Cassady JM. Cucurbitacin B and Isocucurbitacin B Cytotoxic components of Helicteres isora. J Nat Prod 1985;48:500-3. 24. Kusumoto IT, Shimada I, Kakiuchi N, Hattori M, Namba T, Supriyatna S. Inhibitory effects of Indonesian plant extracts on Reverse Transcriptase of an RNA Tumour Virus (I). Phytother Res 1992;6:241-4. 25. Otake T, Mori H, Morimoto M, Ueba N, Sutardjo S, Kusumoto IT, et al. Screening of Indonesian plant extracts for anti-human immunodeficiency virus – Type 1 (HIV) activity. Phytother Res 1995;9:6-10. 26. Tezuka Y, Terazono M, Kusumoto TI, Hatanaka Y, Kadota S, Hattori M, et al. Phytoconstituents of some Indonesian plants and their antioxidant activity. Helv Chim Acta 2000;83:2908-19. 27. Kamiya K, Saiki Y, Hama T, Fujimoto Y, Endang H, Umar M. Flavonoid glucuronides from Helicteres isora. Phytochemistry 2001;57:297-301. 28. Hwang BY, Kim HS, Lee JH, Hong YS, Ro JS, Lee KS, et al. Antioxidant benzoylated flavan-3-ol glycoside from Celastrus orbiculatus. J Nat Prod 2001;64:82-4. 29. Miller HE. A simplified method for the evaluation of antioxidant. J Am Oil Chem Soc 1971;18:439-52. 30. Yen GC, Chen HY. Antioxidant activity of various tea extracts in relation to their antimutagenicity. J Agric Food Chem 1995;43:27-32. 31. Kirby MJ, Turner P. The effect of methysergide and other receptor antagonists on fenfluramine-induced glucose uptake into the isolated rat hemidiaphragm. Arch Int Pharmacodyn Ther 1977;225:25-8. 32. Sabu MC, Subburaju T. Effect of Cassia auriculata Linn. on serum glucose level glucose utilization by isolated rat hemidiaphragm. J Ethnopharmacol 2002;80:203-6. 33. Gemmill CL, Hamman LJ. Relation of glucose uptake to macrovascular disease in NIDDM. Johns Hopk Hosp Bull 1941;68:50. 34. Levine R, Goldstein MS. Effect of insulin on glucose uptake by rat hemidiaphragm and its mechanism of action. Recent Prog Horm Res 1955;11:343-80.

Accepted 4 December 2009 Revised 7 September 2009 Received 21 July 2008 Indian J. Pharm. Sci., 2009, 71 (6): 695-699

Validated LC Method for the Estimation of Voriconazole in Bulk and Formulation C. N. PATEL, J. B. DAVE, J. V. PATEL* AND B. PANIGRAHI Department of Pharmaceutical Chemistry, Shri Sarvajanik Pharmacy College, Near Arvind Baug, Mehsana-384 001, India

Patel et al.: Validated LC Method for variconazole Reversed phase high performance liquid chromatographic method was developed and validated for the estimation of voriconazole in bulk and formulation using prominence diode array detector. Selected mobile phase was a combination of water:acetonitrile (35:65 % v/v) and wavelength selected was 256 nm. Retention time of voriconazole *Address for correspondence E-mail: [email protected] November - December 2009

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was 3.95 min. Linearity of the method was found to be 0.1 to 2 µg/ml, with the regression coefficient of 0.999. This method was validated according to ICH guidelines. Quantification was done by calculating area of the peak and the detection limit and quantitation limit ware 0.026 µg/ml and 0.1 µg/ml, respectively. There was no significant difference in the intra day and inter day analysis of voriconazole determined for three different concentrations using this method. Present method can be applied for the determination of voriconazole in quality control of formulation without interference of the excipients. Key words: Reverse phase liquid chromatography, voriconazole, specificity, validation

Voriconazole is designated chemically as (2R,3S)-2(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1-(1H1,2,4-triazol-1-yl)-2-butanol with an empirical formula of C 16H 14F 3N 5O, which is a novel broad spectrum antifungal agent and effective against Aspergillus, candidacies species [1-6]. Assay methods based on high-performance liquid chromatography with UV detection (HPLC–UV) have been developed to determine the concentration of voriconazole in human plasma [7]. Also LC method for quantification and separation of enantiomer of voriconazole is reported[8]. An existing HPLC assay method in human plasma was reported, however this method is based on size exclusion chromatography coupled on-line with a reverse phase HPLC system with column switching and is therefore complex[9,10]. Human pharmacokinetic data for voriconazole have been published[11]. A few methods were reported for the determination of voriconazole in human serum[12,13]. Reports regarding the determination of impurities[14] and separation of stereo isomers[15] also appear in literature. LC-MS has been successfully used to determine other antifungal agents in plasma. The aim of the present work is to develop an accurate, selective, precise and robust RP-HPLC method for the determination of voriconazole and voriconazole in oral suspension powder. The proposed method was validated as per ICH guidelines[16,17] and its updated international convention[18]. A Shimadzu’s HPLC (LC-2010 CHT), SDP-M20A prominence diode array detector and manual injector of 20-µl loop was used. Chromatograms were analyzed using winchrom software provided with the system. Separation was performed on a Kromasil 5 µ 100A CIB, 250×4.6 mm i.d., 5 micron 309811-6 phenomenex column. Mobile phase consisted of water:acetonitrile (35:65 v/v) was used. Flow rate was adjusted to 1.0 ml/min and wavelength was set to 256 nm. Pure voriconazole was obtained as gift sample from 700

Cipla Pharmaceutical Limited (Mumbai, India), acetonitrile and methanol (HPLC grade) were purchased from S. D. Fine Chemicals Ltd., Mumbai, India. Triple distilled water, nylon 0.45 µm-47 mm membrane filter from Gelman laboratory, Mumbai, India and formulation was procured from local pharmacy. Voriconazole standard stock solution (10 µg/ml) was prepared by transferring accurately weighed 10 mg of standard voriconazole to 100 ml volumetric flask and dissolved in methanol. The volume was adjusted up to the mark with methanol. From this solution 5 ml was accurately transferred into a 50 ml volumetric flask and volume was made up to the mark with methanol. Voriconazole suspension powder was accurately weighed. A quantity of powder equivalent to 10 mg of voriconazole was transferred in to a 100 ml of volumetric flask and mixed with methanol (50 ml) and sonicated for 20 min. The solution was filtered through Whatman filter paper No. 41 and the residue was washed thoroughly with methanol. The filtrate and washings were combined in a 100 ml volumetric flask and diluted to the mark with methanol (100 µg/ ml). The solution (1.0 ml) was further diluted to the mark in 10 ml volumetric flask with mobile phase (10 µg/ml). A calibration curve was plotted over a concentration range of 0.1-2 µg/ml for voriconazole. Accurately measured standard stock solution of voriconazole (0.1, 0.3, 0.5, 0.7, 0.9, 1.1, 1.5, 1.7 and 2.0 ml) was transferred to a series of 10 ml corning volumetric flasks and the volume in each flask was adjusted to 10 ml with mobile phase. The resulting solution was injected and the peak area obtained at retention time 3.95 min and flow rate 1.0 ml/min was measured at 256 nm. Calibration curve was constructed for voriconazole by plotting peak area versus concentration at 256 nm. Each reading was average of five determinations.


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Aliquots of prepared sample (10 µg/ml), equivalent to 0.1-2 µg/ml voriconazole, were transferred to series of 10 ml volumetric flasks and procedure was completed as described under construction of calibration curve. The concentrations of voriconazole were determined from the regression equation, and the mean recoveries were calculated. To optimize the HPLC parameters, several mobile phase compositions were tried. Satisfactory peak symmetry was obtained with a mobile phase consisting of water:acetonitrile (35:65 v/v). The quantitation was achieved with prominence diode array detector at 256 nm based on peak area. The retention time was found to be 3.95±0.12 min. The optimized chromatographic conditions are mentioned in Table 1. A representative chromatogram is shown in fig.1. The linearity range obtained for voriconazole was 0.1-2 µg/ml. System suitability tests for HPLC were performed on freshly prepared standard stock solution of voriconazole. Method precision was determined by preparing the standard solution of voriconazole (0.9 µg/ml) and analyzed 6 times as per the proposed method. Percentage relative standard deviation (% RSD) or

coefficient of variation (CV) was not more than 2%. The intra-day precision (CV) was determined for standard solution of voriconazole (0.1-2 μg/ml) for 5 times on the day. The inter-day precision (CV) was determined for standard solution of voriconazole (0.1-2 μg/ml) for 5 days. The proposed method provides acceptable inter-day and intra-day variation for voriconazole at different concentration levels: 0.5, 0.9 and 1.5 μg/ml. The proposed method for extraction and subsequent determination of voriconazole from pharmaceutical formulation after spiking with additional drugs afforded recovery values of 99.06–101.73 %. The developed method was used to quantify voriconazole in its formulation. All determinations were conducted five times. The system suitability and validation parameters are mentioned in Table 2. The proposed validated method was successfully applied to determine voriconazole in bulk powder and in formulation. The estimated result of voriconazole in bulk and formulation are given in Table 3. The percent recoveries obtained was 99.06-101.73, indicates none interference from the common excipients in the formulation. It can be conveniently adopted for routine quality control testing of voriconazole in bulk and in is pharmaceutical dosage forms. TABLE 2: VALIDATION AND SYSTEM SUITABILITY PARAMETERS

Fig. 1: HPLC chromatogram of voriconazole Chromatogram of voriconazole with corresponding retention time at 256 nm.

TABLE 1: OPTIMIZED CHROMATOGRAPHIC CONDITIONS Parameter Column Mobile phase Detection Injection volume Flow rate Run time

Description Kromasil 5 µ 100A CIB, 250×4.6 mm id, 5 micron 309811-6 phenomenex column Water:Acetonitrile::35:65% v/v 256 nm 20 µl 1.0 ml/min 8 min


Parameters

Results

Retention time Tailing factor Resolution factor Theoretical plates Linearity range (µg/ml) Correlation co efficient (r2) Precision (% CV) Intra day (n=5) Inter day (n=5) Accuracy (% Recovery) (n=5) Limit of detection (µg/ml) Limit of quantification (µg/ml) Specificity

3.95 min 0.96 6.42 5643 0.1-2 0.999 0.37-0.63 0.69-01.33 99.08-101.73 0.026 0.1 Specific

TABLE 3: ESTIMATION OF VORICONAZOLE IN FORMULATION Formulations

Labeled/ taken amount (mg)

Bulk powder

10

9.96

99.60±1.23

Formulation

60

60.73

101.21±1.26


Amount found (mg)

% Amount found ±SD (n=5)

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The robustness and specificity of the LC method were established. Changes in the chromatographic system (column, mobile phase, pH, wavelength, and temperature) were used to evaluate the robustness. The retention time observed allowed a rapid determination of the drug, which is important for routine analysis. The use of mobile phase with out buffer which lengthen the column life is the main advantage for the proposed LC method. No interferences from excipients present in pharmaceutical formulation was observed at the detection wavelength. The chromatographic peak of analyte was not attributable to more than one component. At the same concentration, the peak areas of voriconazole standard and sample solutions were identical. All these factors lead to the conclusion that the proposed method is accurate, precise, simple, sensitive, selective, robust and rapid and can be applied successfully for the estimation of voriconazole in bulk and in pharmaceutical formulations.

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Martin MV, Yates J, Hitchcock CA. Comparison of voriconazole (UK109,496) and itraconazole in prevention and treatment of Aspergillus fumigatus endocarditis in guinea pigs. Antimicrob Agents Chemother 1997;41:13-6. Dickinson RP, Bell AS, Hitchcock CA, Narayanaswami S, Ray SJ, Richardson K. A rapid HPLC assay for voriconazole in human plasma. Bioorg Med Chem Lett 1996;16:2031-6. Kappe R. Antifungal activity of the new azole UK-109,496 (voriconazole). Mycoses 1999;42:83. Hegener P, Troke PF, Fätkenheuer G, Diehl V, Ruhnke M. Treatment of fluconazole-resistant candidiasis with voriconazole in patients with AIDS. AIDS 1998;12:2227. Van't Hek LG, Verweij PE, Weemaes CM, van Dalen R, Yntema JB, Meis JF. Successful treatment with voriconazole of invasive aspergillosis in chronic granulomatous disease. Am J Respir Crit Care Med 1998;157:1694-6. O'Neil MJ. The Merck Index, An Encyclopedia of Chemicals, Drugs,

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and Biologicals. 14th ed. Whitehouse Station, NJ; Merck and Co., Inc.: 2006. p. 1728. Perea S, Pennick GJ, Modak A, Fothergill AW, Sutton DA, Sheehan DJ, et al. Comparison of high-performance liquid chromatographic and microbiological methods for determination of voriconazole levels in plasma. Antimicrob Agents Chemother 2000;44:1209. Nagarjuna A, Padmaja K, Mukkanti K, Suryanarayana MV. A validated LC method for separation and quantification of voriconazole and its enantiomer. Chromatographia 2007;66:439-41. Stopher DA, Gage R. Determination of a new antifungal agent, voriconazole, by multidimensional high-performance liquid chromatography with direct plasma injection onto a size-exclusion column. J Chromatogr B Biomed Sci Appl 1997;691:441-8. Gage R, Stopher DA. A rapid HPLC assay for voriconazole in human plasma. J Pharm Biomed Anal 1998;17:1449-53. Purkins L, Wood N, Ghahramani P, Greenhalgh K, Allen MJ, Kleinermans D. Pharmacokinetics and safety of voriconazole following intravenous- to oral-dose escalation regimens. Antimicrob Agents Chemother 2002;46:2546-53. Keevil BG, Newman S, Lockhart S, Howard SJ, Moore CB, Denning DW. Validation of an assay for voriconazole in serum samples using liquid chromatography-tandem mass spectrometry. Ther Drug Monit 2004;26:650-7. 13. Péhourcq F, Jarry C, Bannwarth B. Direct injection HPLC micro method for the determination of voriconazole in plasma using an internal surface reversed-phase column. Biomed Chromatogr 2004;18:719-22. 14. Ferretti R, Gallinella B, Torre F, Zanitti LL. Direct resolution of a new antifungal agent, voriconazole (UK-109,496) and its potential impurities, by use of coupled achiral-chiral high-performance liquid chromatography. Chromatographia 1998;47:649-54. Owens PK, Fell AF, Coleman MW, Berridge JC. Separation of the voriconazole stereoisomers by capillary electrophoresis and liquid chromatography. Enantiomer 1999;4:79-90. ICH, Q2A Validation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva; 1994. ICH, Q2B Validation of Analytical Procedure: Methodology, International Conference on Harmonization, Geneva; 1996. ICH Guideline on Analytical method Validation, International Convention on Quality for the Pharmaceutical Industry. Toronto. Canada; 2002.

Accepted 5 December 2009 Revised 8 September 2009 Received 6 April 2009 Indian J. Pharm. Sci., 2009, 71 (6): 699-702

Spectrophotometric Method for Simultaneous Estimation of Escitalopram Oxalate and Clonazepam in Tablet Dosage Form R. B. KAKDE* AND D. D. SATONE Department of Pharmaceutical Sciences, Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-440 033, India

Kakde and Satone: Spectroscopic Estimation of Escitalopram and Clonazepam *Address for correspondence E-mail: [email protected] 702

