College of Pharmaceutical Education and Research, College Campus, Modasa-383 315, India 2 L.College of Pharmacy, Navrangpura, Ahmedabad-380 009, India Date of Submission 21-Jul-2006 Date of Decision 25-Oct-2007 Date of Acceptance 21-Apr-2008 CORRESPONDENCE ADDRESS: C R Shah Department of Quality Assurance, Shri B.Shah College of Pharmaceutical Education and Research, College Campus, Modasa-383 315 India SOURCE OF SUPPORT: None, CONFLICT OF INTEREST: None Abstract A rapid, selective and stability-indicating high performance thin layer chromatographic method was developed and validated for the simultaneous estimation of olanzapine and fluoxetine inbined tablet dosage form.And fluoxetine were chromatographed on silica gel 60 F 254 TLC plate using methanol:toluene (4:2 v/v) as the mobile phase and spectrodensitometric scanning-integration was performed at a wavelength of 233 nm using a Camag TLC Scanner III.System was found to givepact spots for both olanzapine (R f value of 0.And fluoxetine (R f value of 0.The polynomial regression data for the calibration plots showed good linear relationship with r 2 =0.The concentration range of 100-800 ng/spot for olanzapine and 1000-8000 ng/spot for fluoxetine with r 2 =0.The method was validated in terms of linearity, accuracy, precision, recovery and specificity.Limit of detection and the limit of quantification for the olanzapine were found to be 30 and 100 ng/spot, respectively and for fluoxetine 300 and 1000 ng/spot, respectively.And fluoxetine were degraded under acidic, basic and oxidation degradation conditions which showed all the peaks of degraded product were well resolved from the active pharmaceutical ingredient.Drugs were not further degraded after thermal and photochemical degradation.Method was found to be reproducible and selective for the simultaneous estimation of olanzapine and fluoxetine.The method could effectively separate the drugs from their degradation products, it can be employed as a stability-indicating method.KEYWORDS: Stability indicating method, forced degradation, olanzapine, fluoxetine, HPTLC, simultaneous estimation HOW TO CITE THIS ARTICLE: Shah CR, Suhagia BN, Shah NJ, Patel DR, Patel NM.HPTLC method for olanzapine and fluoxetine inbined tablet dosage form.J Pharm Sci 2008;70:251-5 HOW TO CITE THIS URL: Shah CR, Suhagia BN, Shah NJ, Patel DR, Patel NM.HPTLC method for olanzapine and fluoxetine inbined tablet dosage form.J Pharm Sci ;70:251-5.Olanzapine is an antipsychotic agent, chemically a thienobenzodiazepine described as a 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno ,3-b] ,benzodiazepine and fluoxetine is an antidepressant agent, selective serotonin reuptake inhibitor (SSRI), chemically described as a (±)-N-methyl-3-phenyl-3-propylamine .Inbination with fluoxetine is used in treatment-resistant depression (TRD).A slightly smaller increase of serotonin than fluoxetine alone .Survey reveals that few spectrophotometric, GC and HPLC methods are reported for the estimation of olanzapine and fluoxetine alone in formulations and biological samples such as urine and plasma ,,, .Or stress studies of drug substance and products play an integral role in the development of pharmaceuticals .Results of degradation studies facilitate the stability indicating method (SIM) development.Survey indicates that, stability indicating HPTLC method has not been developed for quantitative determination of olanzapine and fluoxetine inbined dosage form.Current ICH guidelines requires that the analysis of stability samples should be done by using stability indicating assay methods developed and validated after stress testing on drug under variety of conditions, including hydrolysis (at various pH's), oxidation, photolysis and thermal degradation .Ideal SIM is one that quantifies the drug and also resolves its degradation product.Very viable alternative for stability indicating analysis of olanzapine and fluoxetine inbined dosage form is HPTLC.Advantages of HPTLC is that several samples can be run simultaneously by using a small quantity of mobile phase unlike HPLC, thus lowering analysis time and cost per analysis , .Present investigation an attempt has been made to develop accurate and precise stability indicating HPTLC method for the simultaneous estimation of olanzapine and fluoxetine inbined dosage forms .Standard were procured as a gift sample from Sun Pharmaceuticals Ltd.Silica gel 60F 254 TLC plates (10Ч10 cm, layer thickness 0.E.Mumbai) were used as a stationary phase.Chemicals and reagents used were of analytical grade.5 mg of olanzapine and 20 mg of fluoxetine (Olanex-F) were procured from a local pharmacy.Camag HPTLC systemprising of Camag Linnomate V automatic sample applicator, Hamilton syringe (100 µl), Camag TLC Scanner 3, Camag WinCATS software, Camag Twin-trough chamber (10Ч10 cm) and ultrasonicator were used during study.Fluoxetine (25 mg) each were weighed accurately, dissolved and diluted with methanol to obtain the final concentration of 100 µg/ml and 1000 µg/ml, respectively.Tablets were weighed accurately and ground to fine powder.Equivalent to 25 mg of olanzapine and fluoxetine were transferred to conical flask and mixed with methanol.Solution was sonicated for 15 min.Extracts were filtered through Whatmann filter paper No.And residue was washed thoroughly with methanol.And washing were pooled and transferred to a 25 ml volumetric flask and volume was made up to 25 ml with methanol.Dilutions were made to get 100 µg/ml of olanzapine and 1000 µg/ml of fluoxetine.Were prewashed with methanol.Of plates was done in an oven at 50 o for 5 min.Chromatographic conditions maintained were precoated silica gel 60F 254 aluminum sheets (10x10 cm) as stationary phase, methanol: toluene (4:2 v/v) as mobile phase, chamber and plate saturation time of 30 min, migration distance allowed was 72 mm, wavelength scanning was done at 233 nm keeping the slit dimension at 5Ч0.A deuterium lamp provided the source of radiation.µl standard solutions of olanzapine and fluoxetine were spotted and developed.Measurements were performed at 233 nm in reflectance mode with Camag TLC scanner 3 using Win CATS software.1-8 µl of standard solution of olanzapine (100 µg/ml) and 1-8 µl of fluoxetine (1000 µg/ml) were applied on the TLC plate.Plate was dried, developed and analyzed photometrically as described earlier.Standard calibration curve was generated using regression analysis with Microsoft excel.The developed method was validated in terms of linearity, accuracy, limit of detection, limit of quantification, intra-day and inter-day precision and repeatability of measurement as well as repeatability of sample application .Of sample solutions of the marketed formulation was spotted on to the same plate followed by development scanning.Was repeated in triplicate.Content of the drug was calculated from the peak areas recorded.Accurately weighed olanzapine (50 mg) and fluoxetine (500 mg) was transferred in 50 ml of volumetric flask and dissolved in methanol (25 ml).Hydroxide, hydrochloric acid solution (25 ml, 1 N) and hydrogen peroxide (25 ml, 3 % v/v) were added.Final solution was transferred in 100 ml of round bottom flask and refluxed at 90±2 o for 6 h.Time intervals of 0, 30, 60, 90, 120, 180 and 360 minutes, 2.The solution was transferred in series of 25 ml of volumetric flasks and diluted to the mark with mobile phase to stop further degradation.Sample (400 ng/spot) was analyzed employing HPTLC method.Thermal stress, the drug substance in solid state was subjected to dry heat at 60 o for 10 days and for photo degradation, the drug substance in solid state was exposed to UV at 254 nm for 10 days.A solvent system that would give dense andpact spots with significant R f values was desired for quantification of olanzapine and fluoxetine in pharmaceutical formulations.Mobile phase consisting of methanol: toluene (4:2 v/v) gave R f values of 0.And 0.For olanzapine and fluoxetine, respectively .The linear regression data (n=5) showed a good linear relationship over a concentration range of 100-800 ng/spot and 1000-8000 ng/spot for olanzapine and fluoxetine, respectively.Signal-to-noise ratios of 3 and 10 were considered as LOD and LOQ, respectively.LOD and LOQ for olanzapine was found to be 30 and 100 ng/spot and for fluoxetine, 300 and 1000 ng/spot respectively.Intra-day precision was determined by analyzing standard solutions in the concentration range of 200 ng/spot to 500 ng/spot for olanzapine and 2000 ng/spot to 5000 ng/spot for fluoxetine for three times on the same day while inter-day precision was determined by analyzing corresponding standards daily for five day over a period of one week.Intra-day and inter-day coefficients of variation for both drugs were found to be in the range of 0.% and 0.%, respectively.Values indicate that the method is precise.Of sample application was assessed by spotting 3 µl of drug solution seven times on a TLC plate followed by development of plate and recording the peak area for 5 spots.% RSD for peak area values of olanzapine and fluoxetine were found to be 0.0.Repeatability of measurement of peak area was determined by spotting 3 µl of olanzapine and fluoxetine solution on a TLC plate and developing the plate.Separated spot was scanned seven times without changing the position of the plate and % RSD for measurement of peak area of olanzapine and fluoxetine were found to be 0.0.To confirm the specificity of the proposed method, the solution of the formulation was spotted on the TLC plate, which was than developed and scanned.Was observed that the excipients present in the formulation did not interfere with the peaks of olanzapine and fluoxetine.Studies of the drugs were carried out for the accuracy parameter.Studies were carried out at three levels i.Level recovery studies.Stock solutions from tablet formulation of 100 µg/ml for olanzapine and 1000 µg/ml for fluoxetine were prepared.Were made and recovery studies were performed.Recovery value of olanzapine and fluoxetine were found to be 99.% and 101.Respectively while assay value of olanzapine and fluoxetine were found to be 101.% and 101.%, respectively.Low RSD value indicated the suitability of the method for routine analysis of olanzapine and fluoxetine in pharmaceutical dosage forms.Degradation studies (for 0-360 min) indicate that olanzapine and fluoxetine showed good degradation in acidic and basic condition, which started to degrade after 30 min and continued up to 180 min.Showed good degradation in oxidation condition, started to degrade after 30 min and continued to degrade up to 360 min, while olanzapine did not show any degradation up to 360 min.Fluoxetine did not show any degradation in UV light at 254 nm as well as in thermal stress at 60 o up to 10 days .Developed stability indicating HPTLC technique for the simultaneous estimation is simple, precise, specific, accurate and the statistical analysis proved that method is reproducible and selective for the analysis of olanzapine and fluoxetine in bulk drug and tablet formulations.Acknowledgements The authors are thankful to Sun Pharmaceuticals Ltd.For the gift sample of olanzapine and fluoxetine.Are thankful to Principal and management for providing necessary facilities and encouragement.References Manickam A, Donna A, William CW, Stephen RM.By high performance liquid chromatography with electrochemical detection.Drug Monit 1997;19:307-13.Labat L, Deveaux M, Dallet P, Dubost JP.Of new antidepressants and their metabolites by micellar electrokinetic capillary chromatography.Chromatogr B Biomed Sci Appl 2002;773:17-23.Zhang W, Kenneth WP, David TW, Potts BD, Bao J, Tollefson GD, et al.Of olanzapine and other antipsychotic agents inbination with fluoxetine on norepinephrine and dopamine release in rat prefrontal cortex.2000;23:250-62.Raggi MA, Casamenti G, Mandrioli R, Lzzo G, Kenndler E.Olanzapine in tablets by HPLC, CZE, derivative spectrometry and linear voltammetry.Pharm Biomed Anal 2000;23:973-81.Bugamelli F, Volterra V, Raggi MA, Casamenti G, Mandrioli R.Fluoxetine and norfluoxetine in human plasma by high-pressure liquid chromatography with fluorescence detection.Biomed Anal 1998;18:193-9.Bao J, Potts BD.Of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection.Chromatogr B Biomed Sci Appl 2001;752 : 61-7.Dasoondi B, Shah P, Gandhi C, Bhat KM.Of HPLC assays method for the estimation of olanzapine in human plasma and its application to pharmacokinetics.J Pharm Sci 2003;40:350-4.Khan GJ, Trivedi C, Soni K, Khan IJ, Saraf MN.Method development, validation andparative bioavailability studies of fluoxetine extended release tablets in healthy human volunteers.2005;42:580-4.Reynolds DW, Facchine KL, Mullaney FJ, Alsante KM, Hatajik TD, Michel MG.Guidance and best practices for conducting forced degradation studies.2002;15:48-56.ICH.Stability testing of new drug substances and products.On Harmonization, Geneva: 1993 October.Sethi PD.Performance thin layer chromatography: Quantitative analysis of pharmaceutical formulation.Ed.Delhi: CBS Publishers; 1996.3-62.Fried B, Sharma J.Layer chromatography: Techniques and application.Ed.York: Marcel Dekker; 1994.11-22.ICH.Read more Beyond insulin replacement: addressing the additional needs of the diabetes patient
Get more Effects of aspirin, nimesulide, and sc-560 on vasopressin-induced contraction of human gastroepiploic artery and saphenous vein *.
