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Validated Simple, Rapid and Accurate HPLC Methods for determination of Metamizole Sodium in Solid and Liquid Dosage Forms Hamide Z. SENYUVA*, Sureyya OZCAN, Burak Veli KABASAKAL TUBITAK-Ankara Test and Analysis Laboratory Abstract: The aim of the study was to develop and validate high performance liquid chromatography (HPLC) assay for the rapid determination of Metamizole Sodium in solid and liquid dosage forms. The experimental procedure involved reversed-phaseHPLC with a Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm), metanolwater volumetric solution (80 : 20, v/v) mobile phase, UV detection at 254 nm for Metamizole Sodium. The flow rate of the, mobile phase was 1 mL/min. The retention time of Metamizole Sodium is ca. 3.1-3.3 min. Formulations components did not give rise to any interfering peaks. Calibration curve was linear over the range 0.5-100 µg/mL for analyte.The metamizole recovery range is at three level (10µg/mL, 15µg/mL and 25µg/mL) 93-100 %. The method was validated with respect to linearity, precision, accuracy, specificity and robustness. Due to its simplicity and accuracy, the assay method is suitable for routine analysis of both solid and liquid formulations. Key words: metamizole, dipyrone, hplc, validation 1-Introduction: Metamizole (synonym: dipyrone) is sodium sulfonate derived from aminopyrine, a pyrazolone non-steroidal anti-inflammatory drug (NSAID). Pharmacologic actions are similar to asprin which is able to inhibit the cyclooxygenase pathaway. this drug produces a relatively high incidence of allergy and intolerance[1]. Metamizole has been determined by titrimetric[2], uv-spectrophotometric [3-4-5], colorometric [6], polarographic [7], TLC, HPTLC[5], gas liquid chromatography (GLC) [8] and HPLC[9] method.Reversed phase HPLC is the seperation method of choice for most pharmaceutial compounds both hydrophilic and hydrophobic, due to the stable, reproducible nature of the HPLC columns, the largely aqueous composition of mobile phase , and relative ease of reproducing the methods in variety of laboratories. A simple, quick, specific and accurate reversed phase HPLC method to assay metamizole in solid and liquid dosage forms.Methot was validated with the parameters of linearity range, precision, recovery and accuracy. 2-Experimental: 2.1- Materials Methanol (HPLC grade J.T. Baker), Ultra pure water obtained from Millipore water purification unit. Certificated Metamizole Sodium obtained from Fine Chemical. 2.2- Instrumentation An Agilent 1100 series High-Performance Liquid Chromatography equipped with DAD, ALS auto sampler and thermostated column module. The HPLC column was a Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm) 2.3- Analytical Method HPLC system was performed by using Zorbax SB C18 column (5 µm particle size, 4.6 ID x 250 mm) column and a mobile phase composed of Methanol:Water (80:20). The injection volume was 100 µL. 25 oC was column temperature. DAD wavelength is 254 nm.All samples were prepared in water. * Corresponding Author: Hamide Z. SENYUVA Fax: +90 312 212 37 49 e-mail: [email protected] Calibration Solvents 100 µg/mL metamizole stock solutions were prepared and diluted with water.Calibration Solvents were prepared freshly at the concentrations of 0.5, 1.0, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0 µg/mL by diluting stock solution with water (r2>0.999). 2.4 Validation of Developed Method Linearity of the method was established by dublicate injections of solutions containing metamizole in the range 0.5-100 µg /ml. Determination of intra-day precision, eight samples of each three different concentrations (5,15,25 µg/ml) were given in one day. For determination of inter-day precision same series of samples were injected during three days.The relative standart deviations and correlation coefficients were calculated for these analyses.Table 1 shows inter-day and intra-day precision.The accuracy of the method was provided by spiking three different concentrations; 10µg/mL,15µg/mL and 25µg/mL of metamizole into placebo. 3- Result and Discussion The simple, quick, specific and accurate method was developed to determine metamizole in solid and liquid dosage forms. 3.1-Linearity Range: Linearity of the method was established by injecting solutions containing 0.5-100 µg ml-1 of metamizole (r2>0.999). 3.2 Detection and Quantification Limit: The detection limit was determined by adding metamizole standart solutions to the placebo which signal/noise ratio was 3. LOD was 0.4 ng/mL for metamizole. LOQ was 1.3 ng/mL at which signal-to-noise ratio was 10. 3.3 Precision: Repeatibility studies were performed by spiking of metamizole at three different levels (5,15,25 µg /ml) in same day dublicate injection (r2 >0.999) . Precision for replicate analyses in placebo ranges from 93 to 100%.Table 1 shows recovery. 3.4- Accuracy: The accuracy of the method was provided by spiking three different concentrations; 10µg/mL, 15µg/mL and 25µg/mL of metamizole into placebo. The placebo contained sodiummetabisulphide, benzyl alcohol and deionized water. .The metamizole recovery range is 93-100 % for each level.Good recoveries were obtained for each concentrations, confirming that the method was accurate.Figure 2 shows the chromatograms of blank and 25 µg/mL spiked sample. Table1.Recovery of spiked Samples Conc.(µg/mL) 10 15 25 Validation Recovery Within-batch (n=8) Mean % s% RSD % 98,6 5,1 5,2 97,4 2,8 2,8 96,4 6,9 7,2 Between Batch (n=3) Mean % s% 96,1 3,1 100,0 2,7 92,8 6,3 RSD % 3,2 2,7 6,8 DAD1 A, Sig=254,16 Ref=off (METVALID\MET00173.D) mAU 400 A 300 blank 200 100 0 2 3 400 B 300 4 3.327 - Ametamizole re a: 20 81 .1 9 0 1 DAD1 A, Sig=254,16 Ref=off (METVALID\MET00176.D) mAU min 25 ug/mL spike 200 100 0 0 1 2 Figure 2. A- Blank Sample 3 4 min B- 25µg/mL Spiked Sample 3.5- Applicability of the method to marketed formulation Seven different samples which three of them are in solid dosage form and four of them are in liquid dosage form, were analyzed by using proposed method for two concentration and each was double injected.Table 2 shows the concentrations of metamizole in marketing drugs. Table2. Resuls of marketing drugs Part No 1 2 3 4 5 6 7 Sample ID Novalgin Ampul Andolor Ampul Adepiron Ampul Novapyrine Ampul Novalgin Tablet Andolor Tablet Devaljine Tablet 1 mg metamizole/2mL 0,99 1,08 1,06 0,99 500 mg metamizole/Tablet 501,17 545.02 460.00 4. Conclusion The method was validated as linearty, precision, accuracy, specificity and was also applied to real marketed samples. The assay can be used to asses stability of the liquid and solid dosage forms. Data obtained for seven products represent two different sample types liquid and solid .The recovery of the drug was essentially quantitative. References 1- Anibarro B., Fontela J.L., MD, ‘Immediate rhinoconjunctivities induced by metamizole and metronidazole.’ Annals of Allergy, Asthma & Immunology Volume 78, April 345346, 1997. 2- Srivastava, M.K. , Ahmad S., Singh D. and Shukla I.C.. Analyst. 110, 735-737 (1985). 3- Erk N. and Onur F., Anal. Lett., 30(6), 1201-1210 (1997). 4- Doğan H.N., Pharmazie, 51(10), 773-774 (1996). 5- Aburjal T., Amro B.I., Aiedeh K., Abuirjeje M. and Al-Khalil S., Pharmazie, 55, 751754 (2000) 6- Hapetle A. M. and Poulet S.A., J. Liq. Chromatogr., 13(2), 357-370 (1990). 7- Belal F., Electroanalysis, 4, 589 (1992). 8- Sioufi A. and Colussi D., J. Chromatogr., 140, 503-507 (1978) 9- Stevens, H.M., Gill, R. Journal of Chromatography, 370 (1986) 39-47