Stability-indicating Simultaneous HPTLC Method for Olanzapine and Fluoxetine in Combined Tablet Dosage Form

Indian Journal of Pharmaceutical Sciences 251 March April 2008 Sciacchitano CJ, Mopper B, Specchio J. IdentiÞ cation and separation of 5. Þ ve cephalosporins by micellar electrokinetic capillary chromatography. J Chromatogr B Biomed Appl 1994; 657:395. Dhaneshwar SR, Kadam SS, Sirisha DV. Development and validation 6. of a HPTLC method for simultaneous estimation of cefuroxime axetil and probenecid. Indian J Pharm Sci 2004; 66:278-80. Pistos C, Kalovidouris M, Kantopoulos G. Application of the ion pair 7. concept to the n-octanol-water partitioning of cefepime and cefpirome. Int J Clin Pharmacol Ther 2004; 42:367-69. REFERENCES

Olanzapine and ß uoxetine (25 mg) each were weighed accurately, dissolved and diluted with methanol to obtain the Þ nal concentration of 100 µg/ml and 1000 µg/ml, respectively.Twenty tablets were weighed accurately and ground to fine powder.Weight equivalent to 25 mg of olanzapine and ß uoxetine were transferred to conical ß ask and mixed with methanol.The solution was sonicated for 15 min.The extracts were Þ ltered through Whatmann Þ lter paper No. 41 and residue was washed thoroughly with methanol.The extracts and washing were pooled and transferred to a 25 ml volumetric flask and volume was made up to 25 ml with methanol.Required dilutions were made to get 100 µg/ml of olanzapine and 1000 µg/ ml of ß uoxetine.
TLC plates were prewashed with methanol.Activation of plates was done in an oven at 50 0 for 5 min.The 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 5×0.45 mm.A deuterium lamp provided the source of radiation.Three µl standard solutions of olanzapine and ß uoxetine were spotted and developed.Photometric measurements were performed at 233 nm in reß ectance mode with Camag TLC scanner 3 using Win CATS software.
Aliquots of 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.The TLC plate was dried, developed and analyzed photometrically as described earlier.The 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 quantiÞ cation, intra-day and inter-day precision and repeatability of measurement as well as repeatability of sample application 13 .Three microlitres of sample solutions of the marketed formulation was spotted on to the same plate followed by development scanning.
Forced degradation or stress studies of drug substance and products play an integral role in the development of pharmaceuticals 9 .The results of degradation studies facilitate the stability indicating method (SIM) development.Literature survey indicates that, stability indicating HPTLC method has not been developed for quantitative determination of olanzapine and ß uoxetine in combined dosage form.The 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 10 .An ideal SIM is one that quantiÞ es the drug and also resolves its degradation product.A very viable alternative for stability indicating analysis of olanzapine and ß uoxetine in combined dosage form is HPTLC.The 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 11,12 .In the present investigation an attempt has been made to develop accurate and precise stability indicating HPTLC method for the simultaneous estimation of olanzapine and ß uoxetine in combined dosage forms 13 .
Olanzapine and fluoxetine standard were procured as a gift sample from Sun Pharmaceuticals Ltd., Baroda.Silica gel 60F 254 TLC plates (10×10 cm, layer thickness 0.2 mm, E. Merck, Mumbai) were used as a stationary phase.All chemicals and reagents used were of analytical grade.Tablets containing 5 mg of olanzapine and 20 mg of ß uoxetine (Olanex-F) were procured from a local pharmacy.A Camag HPTLC system comprising of Camag Linnomate V automatic pharmaceutical formulations.The mobile phase consisting of methanol: toluene (4:2 v/v) gave R f values of 0.63±0.01 and 0.31±0.01for olanzapine and ß uoxetine, respectively (Table 1, (Þ g. 1)).
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.The signal-to-noise ratios of 3 and 10 were considered as LOD and LOQ, respectively.The LOD and LOQ for olanzapine was found to be 30 and 100 ng/spot and for ß uoxetine, 300 and 1000 ng/spot respectively.The intra-day The analysis was repeated in triplicate.The content of the drug was calculated from the peak areas recorded.
Accurately weighed olanzapine (50 mg) and ß uoxetine (500 mg) was transferred in 50 ml of volumetric ß ask and dissolved in methanol (25 ml).Sodium hydroxide, hydrochloric acid solution (25 ml, 1 N) and hydrogen peroxide (25 ml, 3 % v/v) were added.The final solution was transferred in 100 ml of round bottom ß ask and reß uxed at 90±2 0 for 6 h.At time intervals of 0, 30, 60, 90, 120, 180 and 360 minutes, 2.5 ml of the solution was transferred in series of 25 ml of volumetric ß asks and diluted to the mark with mobile phase to stop further degradation.The sample (400 ng/spot) was analyzed employing HPTLC method.For thermal stress, the drug substance in solid state was subjected to dry heat at 60 0 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 and compact spots with significant R f values was desired for quantification of olanzapine and fluoxetine in  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 ß uoxetine showed good degradation in acidic and basic condition, which started to degrade after 30 min and continued up to 180 min.Fluoxetine also 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.Olanzapine and ß uoxetine did not show any degradation in UV light at 254 nm as well as in thermal stress at 60 0 up to 10 days (Table 2, (Þ g. 1).The developed stability indicating HPTLC technique for the simultaneous estimation is simple, precise, speciÞ c, accurate and the statistical analysis proved that method is reproducible and selective for the analysis of olanzapine and ß uoxetine in bulk drug and tablet formulations.
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 Þ ve day over a period of one week.The intra-day and inter-day coefÞ cients of variation for both drugs were found to be in the range of 0.13-0.71% and 0.16-0.61%, respectively.These values indicate that the method is precise.Repeatability 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.The % RSD for peak area values of olanzapine and ß uoxetine were found to be 0.32 and 0.48, respectively.Repeatability of measurement of peak area was determined by spotting 3 µl of olanzapine and ß uoxetine solution on a TLC plate and developing the plate.The 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 0.19 and 0.09, respectively.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.It was observed that the excipients present in the formulation did not interfere with the peaks of olanzapine and ß uoxetine.Recovery studies of the drugs were carried out for the accuracy parameter.These studies were carried out at three levels i.e. multiple level recovery studies.Sample stock solutions from tablet formulation of 100 µg/ml for olanzapine and 1000 µg/ml for ß uoxetine were prepared.Dilutions were made and recovery studies were performed.Percentage Recovery value of olanzapine and fluoxetine were found to be 99.42-100.42±0.75% and 101.33-101.37±0.65 %, respectively while assay value of olanzapine and fluoxetine were found to be 101.5 3±1.06 % and 101.45±0.35%, respectively.The low RSD

Fig 1 :
Fig 1: A typical HPTLC chromatograms of olanzapine and ß uoxetine and their degraded products.(A) Pure drug: peak A and B are of ß uoxetine and olanzapine, respectively.(B) Based induced: peak A is of ß uoxetine, peak C is of ß uoxetine's degraded product, peak D is of olanzapine's degraded product and peak B is of olanzapine.(C) Acid induced: peak A is of ß uoxetine, peak B is of olanzapine and peak D is of olanzapine's degraded product.(D) Hydrogen peroxide induced: peak C is of ß uoxetine's degraded product, peak A is of ß uoxetine and peak B is of olanzapine.