Development And Validation of RP-HPLC Method for The Simultaneous Determination of Olanzapine and Fluoxetine Hydrochloride in Bulk and Pharmaceutical Dosage Forms

Olanzapine¹ is chemically, 2-methyl-4-(4-methyl-1-piperazinyl)-10H-thieno[2,3-b] [1,5] benzodiazepine (Fig. 1a). It is an antipsychotic agent used in the treatment of schizophrenia. Fluoxetine hydrochloride² is chemically, methyl({3-phenyl-3-[4-(trifluoromethyl) phenoxy] propyl}) amine hydrochloride (Fig. 1b). It is a selective serotonin reuptake inhibitor used as an antidepressant agent with non-sedating properties. Literature survey indicated very few methods like HPLC³, Spectroscopy⁴, HPTLC⁵ and Electron spray ionization tandem mass spectrometry⁶ ,  for the simultaneous estimation of these drugs. Hence the combination of these drugs was selected for the simultaneous estimation by RP-HPLC method. The method presently developed has the advantage of being more sensitive to determine both the drugs concurrently by simple and rapid RP-HPLC method for routine analysis.                                                                        

FIGURE (1a).  Structure of olanzapine

FIGURE (1b).  Structure of fluoxetine hydrochloride

1. METHODOLOGY

1. 1.  MATERIALS AND METHODS:

Instrumentation : UV-3000⁺ LABINDIA Double beam with UV win 5 software UV-Visible spectrophotometer with 1cm matched quartz cells was used to determine detection wavelength. WATERSHPLC Aliance 2695, UV- Visible Dual absorbance Detector 2487, with an automated sample injector. The output signal was monitored and integrated using Empower 2 software.

Chemicals and drugs: Acetonitrile, methanol and water are of HPLC grade and ortho phosphoric acid (OPA), pure potassium dihydrogen phosphate is of AR grade were obtained from Merck, Mumbai India. Fluoxetine Hydrochloride and Olanzapine reference standards were obtained as gift samples from Aurobindo Pharmaceuticals Pvt. Ltd., Hyderabad, India. Capsule dosage form containing 25mg of fluoxetine hydrochloride and 12mg of olanzapine (SYMBYAX) was procured from the local market.

Chromatographic method:  A Symmetry XTerra C18 (4.6 x 150mm, 5 mm) column was used for separations. The mobile phase considered was potassium dihydrogen phosphate:acetonitrile in the ratio of 40:60v/v  buffer pH adjusted to 3.0 with orthophosphoric acid. It was pumped at the flow rate of 1ml/min. The mobile phase was passed through 0.45µm membrane filters and degassed before use. The eluent was monitored at 235nm and the injection volume was 20µl.

1. 2. Preparation of solutions:

Preparation of Buffer Solution: 1.36 gm of potassium dihydrogen phosphate was dissolved in 1000mL MilliQ water and pH of this solution was adjusted to 3.0 with ortho phosphoric acid. The solution was mixed well and then filtered through 0.45µ filter paper.

Preparation of Mobile phase: Mobile phase was prepared by mixing pH 3.0 buffer solution and acetonitrile in the ratio 40:60 v/v. Prior to use the mobile phase was filtered through 0.45µ membrane filter after sonication for 8 mins.

Preparation of Standard Stock Solution of Olanzapine: Accurately weighed 100 mg of Olanzapine standard drug was transferred to 100 ml of volumetric flask. Then it was dissolved by adding a little amount of diluent. Mix it well, sonicate  the solution and volume was made up   to 100 ml. This is 1000µg/ml. From this solution 10 ml was transferred to another 100 ml volumetric flask and volume was made up to 100 ml with diluent(100µg/ml).

Preparation of Standard Stock Solution of Fluoxetine Hydrochloride: Accurately weighed 100 mg of Fluoxetine hydrochloride standard drug was transferred to 100 ml of volumetric flask. Then it was dissolved by adding a little amount of diluent. Mix it well, sonicate the solution and volume was made up to 100 ml. This is 1000µg/ml. From this solution 10 ml was transferred to another 100 ml volumetric flask and volume was made up to 100 ml with diluent(100µg/ml).

Preparation of Working Standard Solution: From the above stock solution 1.5ml of OLZ solution and 3ml of FLX solution was transferred to 10 ml of volumetric flask and was made up to with mobile phase. The working standard solution produced contains 15µg/ml of OLZ and 30µg/ml of FLX.

Preparation of Sample Solution: Contents of twenty capsules were weighed accurately and average weight was recorded. 64mg of powder equivalent to 12.5mg of OLX and 25mg of FLX was weighed accurately and transferred to 25ml volumetric flask and dissolved with mobile phase and sonicated for 10mins and filtered through 0.35µ filter paper and volume made up to with mobile phase. The produced sample stock solution is (500µg/ml of OLX and 1000µg/ml of FLX). From this stock solution 0.3ml of solution was transferred to 10ml volumetric flask and made up to volume with mobile phase. The concentration of working solution consists of 15µg/ml of OLX and 30µg/ml of FLX.

1. 3.  Determination of Absorption maxima by uv/visible spectrophotometer:

Both the solutions of olanzapine and fluoxetine hydrochloride were scanned over the range of 190-400nm. Both the drugs showed good response at 235 nm, hence this wavelength was selected for further study. The overlaid uv spectra is shown in the figure 2.

Figure (2). Overlaid UV Spectra of olanzapine and fluoxetine hydrochloride

1. 4.  Optimized Chromatographic conditions:

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Mobile phase: potassium dihydrogen phosphate (pH 3.0): Acetonitrile (40:60) v/v

Flow rate: 1ml/min

Run time (min): 8 min

Detection: 235 nm

Injection volume: 20µl

1. 5.  Procedure: Mixed standard solutions containing fluoxetine hydrochloride and olanzapine in the range of 10µg/ml to 50µg/ml and 5µg/ml to 25µg/ml were prepared and each solution was injected in to the optimized chromatographic system. The chromatograms were recorded and the peak areas were determined for each concentration of the drug solution. Calibration curve of fluoxetine hydrochloride and olanzapine was obtained by plotting the peak areas versus the respective concentrations. The linear correlation coefficient for fluoxetine hydrochloride and olanzapine was found to be 0.999 and 0.999 respectively. A typical chromatogram of fluoxetine hydrochloride and olanzapine was shown in Fig 3.

3.  VALIDATION STUDIES^7,8:
   1.  System suitability studies: The system suitability studies were done for parameters like theoretical plates, tailing factor, repeatability, resolution and the results were given in the table 1.

Table (1).  System suitability parameters

1. 2.  Linearity studies: A series of standard solutions of different concentrations containing 10-50µg/ml of fluoxetine hydrochloride and 5-25µg/ml of olanzapine were prepared and 20µl of each standard was injected. The calibration curves were plotted by taking concentration on x-axis and peak areas on y-axis. The linearity data and calibration curve of the two drugs were given the table 2 and 3 respectively and calibration curves in fig 4 and 5.

Table (2).  Linearity data for Fluoxetine Hcl

Figure (4).  Calibration curve of Fluoxetine Hcl

Table (3). Linearity data for olanzapine

Figure (5). Calibration curve of Olanzapine

1. 3. Precision studies: The system precision and method precision studies were done by using standard and sample solutions respectively. For precision the same injection was repeatedly given and % RSD was calculated. The results were given in the table 4.

Table (4). Precision data for fluoxetine hydrochloride and olanzapine

1. 4.  Accuracy studies: The accuracy studies were done by spiking the standard solutions to 50%, 100%, 150% and the recovery data was given in the table 5.

Table (5). Results of Recovery Studies:

1. 5. LOD and LOQ: The LOD and LOQ were separately determined based on the standard deviation of the response and the slope; the results are presented in table 6.        

Table (6). Results of LOD and LOQ:

1. 6.  Robustness studies: Robustness studies were done by changing the flow rate and the composition of mobile phase.

Table (7). Results of Robustness Studies: