METHOD DEVELOPMENT AND VALIDATION OF QUETIAPINE FUMARATE BY RP - HPLC METHOD

About Author:
Sharath Kumar Pallikonda*¹, Srikanth Subburu², Shanker Reddy Soma², Chandra Shekar Reddy^3
1,2Vathsalya College Of Pharmacy,
Bhongir, Dist: Andhra Pradesh, India - 508 116

Abstract
A simple, sensitive, rapid, robust and reproducible method for the determination of Quetiapine fumarate in bulk and pharmaceutical formulation (Tablets) was developed using reverse phase high performance liquid chromatographic method (RP-HPLC). The RP-HPLC analysis was performed isocratically on XTERRA C18 (4.6X150mm), analytical column using a mobile phase consisting of ortho phosphorus buffer and acetonitirle in the Ratio of 60:40v/v, with a flow rate of 0.6ml/min. The analyte was monitored with UV detector at 290nm. The developed method Quetiapine fumarate elutes at a run time of 10 min. The proposed method is having linearity in the concentration range from 40 to 80 μg/mL of Quetiapine fumarate. The present method was validated with respect to system suitability, linearity, precision, limit of detection (LOD) and limit of quantification (LOQ), accuracy (recovery), ruggedness, and robustness. The proposed method can be readily utilized for bulk drug and pharmaceutical formulations.

REFERENCE ID: PHARMATUTOR-ART-1141

Introduction
Quetiapine Fumarate is chemically 2-[2-(4-dibenzo[b,f][1,4]thiazepin-11-yl-1-piperazinyl) ethoxy] ethanol hemifumarate. It is anatypical antipsychotic approved for the treatment of schizophrenia, acute episodes of bipolar disorder (manic, mixed or depressive), and as n augmentor for the maintenance treatment of depression and bipolar disorder.The literature survey (AshishBaldi et al., 2010) reveals that there is some HPLC methods have been reported. The aim of the present study was to develop and validate a simple, isocratic RP-HPLC (Remington, 2007; Skoog,  2004; Chatwal GR, 2004) method for the determination of Quetiapine fumarate in tablets. The developed method was validated using ICH guidelines for validation (ICH, 1995). Today, RP-HPLC is the most popular analytical technique for separating complex mixtures in the chemical, pharmaceutical and biotechnological industry. RP-HPLC is the opposite of normal-phase chromatography, with a nonpolar stationary phase and a polar, largely aqueous mobile phase. The most common stationary phases used are octadecyldimethyl (C18) phases with silica as the solid support. Silica has a small pH range (3 to 8) where mixtures can be separated without degradation of the column performance. Above pH 8, silica supports dissolve and destroy the column. Below Ph 3, the silicon-carbon bond is cleaved, and the column is destroyed. The separation is achieved by analytes having different interactions with the stationary phase. In RP-HPLC, solutes are separated using their hydrophobicity. A more hydrophobic solute will be retained on the column longer than a less hydrophobic one. Also, polar solutes will interact with the silica surface to cause peak tailing. The mobile phase is one of the two components involved in the separation process. Water is generally one of the components of a binary mixture in RP-HPLC. Water is considered to be the weak component of the mobile phase and does not interact with the hydrophobic stationary phase chains. The RP-HPLC method reported in this study was validated in accordance with the International Conference on Harmonization (ICH) guideline (ICH, 1997) and best practice (Shabir GA et al., 2007; Shabir GA et al., 2003; USFDA, 2000). Specificity, linearity, precision (repeatability and intermediate precision), accuracy, robustness, limit of detection and limit of quantitation were evaluated.

OBJECTIVE
Literature reveals different methods for their analysis in their formulations. But our present objective is to develop a new, simple, precise & accurate method for its analysis in formulation after a detailed study a new RP-HPLC method was decided to be developed and validated. Applying developed method to marketed formulation.

METERIALS, INSTRUMENTS AND METHODS

Chromatographic conditions

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METHOD DEVELOPMENT:    (Lloyd R Snyder et al.,2007; Synder KL et al., 1983)

Solubility:
According to literature, Quetapine is freely soluble in methanol and Water. And it was checked for different dilutions of methanol for solubility of Quetapine. Finally Methanol was chosen as solvent for present work.

Selection of wavelength: (λ_max)
An ideal wavelength is one that uses good response for the drugs to be detected. Quetapine in diluents the spectra was scanned on UV Visible spectrophotometer meter in the range of 200nm to 400nm against diluents as blank.UV-Spectrum is done manually performed. The maximum absorbance for Quetapine is observed at 290nm.

Selection of initial chromatographic conditions:
Appropriate selection of chromatographic method depends upon the characteristic nature of the sample (ionic or ionisable or neutral), its molecular weight and solubility. The nature of Quetapine is polar. Hence reverse phase chromatography is used. The reverse phase HPLC was selected for initial chromatographic condition because of its simplicity and suitability

Mobile phase preparation:
Mix a mixture of above buffer 600 mL (60%) and 400mL of Acetonitrile HPLC (40%) and degas in ultrasonic water bath for 5 minutes. Filter through 0.45 μ filter under vacuum filtration

Diluents: HPLC Water as diluents.

Standard preparation: (stock preparation)
Accurately weigh and transfer 10mg of Quetapine Working standard into a 10 mL volumetric flask add about 7 mL of Diluent and sonicate to dissolve it completely and make volume up to the mark with the same solvent. Further pipette 0.6 ml of the above stock solution into a 10ml volumetric flask and dilute up to the mark with diluents. Mix well and filter through 0.45μm filter

Stock solution: QUETIAPINE-1000PPM

Preparation of Phosphate buffer:
Weigh 7.0 grams of Potassium di hydrogen phosphate into a 1000ml beaker, dissolve and diluted to 1000ml with HPLC water. Adjusted the pH to 3 with ortho phosphoric acid.

RESULTS AND DISCUSSIONS
HPLC separation of Quetiapine fumarate was carried out on an Xterra C18 column by an isocratic elution with ortho phosphoric buffer- acetonitrile (60:40 v/v). The flow rate was constant at 0.6 ml/min and the column temperature was at room temperature (24±1°). The UV wavelength was set at 290 nm. No interference from diluents, impurities, or excipients present in the pharmaceutical formulation was observed at this detection wavelength. Before each run LC column was equilibrated with the mobile phase for about 15 min. A sharp, symmetrical peak was obtained for Quetiapine fumarate when analyzed under these conditions.

This retention time enable rapid determination of the drug, which is important for routine quality control analysis. System suitability test was established from eight replicate injections of a solution containing Quetiapine fumarate 20μl. The percent relative standard deviation (RSD) of the peak area was calculated. The peak tailing for drug was measured. A useful and practical measurement of peak shape, the peak tailing and theoretical plate count was determined. Column plate number was determined using the formula, N = 5.54(t R /w h) 2, where w h is the bandwidth at 50% of peak height. The proposed method met these requirements within the United States Pharmacopoeia (USP) accepted limits (Tailing factor < 1.5, Theoretical plates > 2000). The stability of Quetiapine fumarate in solution was investigated in the method development phase. Eight solutions containing 20 μl of Quetiapine fumarate were tested. The solutions were stable during the investigated time and the RSD was < 1.0% for retention time (min), peak area and height. The solutions were shown to be stable with no significant change in Quetiapine fumarate concentration over this period.

METHOD VALIDATION: (Yuri Kazakevin et al., 2007;Bently et al., 1985; David Harvey et al., 2000; Sethi PD,2006)

Linearity
Appropriate amounts of Quetiapine fumarate stock solutions were diluted with mobile phase to given concentration of 40, 50, 60, 70 and 80 μg/ml. Each solution was injected calibration plot was prepared. Linearity was evaluated by linear least-squares regression analysis. Good linearity was observed over the concentration range evaluated (40-80 μg/ml) as shown in the linearity curve in figure 1. The correlation coefficientwas found 1.0

Precision
The precision of the method was investigated with respect to repeatability and intermediate precision.The repeatability (intra-day precision) of the method was evaluated by assaying five replicate injections of the Quetiapine fumarate at 100% of test concentration (60 μg/ml) on the same day. The %RSD of the retention time (min) and peak area were calculated. Intermediate precision (inter-day precision) was demonstrated by evaluating the relative peak area percent data the LC system at three different concentration levels (50%, 100%, and 150%) that cover the assay method range (40-80 μg/ml). The %RSD of the system was calculated from the individual relative percent peak area mean values at the 50%, 100%, and 150% of the test concentration. The intraday (n= 5) and inter-day (n= 3) %RSD are given in table. All the data are within the acceptance criteria of 2%.

Accuracy
Accuracy of the method was evaluated by fortifying a Quetiapine fumarate sample solution (with respect to the target assay concentration)  by Injecting the standard solution, Accuracy 50%, Accuracy 100% and Accuracy 150% solutions. Calculate the amount found and amount added for Quetiapine  and calculate the individual recovery and mean recovery values Percent recoveries were calculated form differences between the peak areas obtained for fortified and unfortified solutions. Good recoveries were obtained within the acceptance criteria (98.0-102.0%). No significant differences were observed between amounts of Quetiapine fumarate added and the amounts found.

LOD & LOQ
The limit of detection (LOD) and limit of quantitation (LOQ) tests for the procedure were evaluated by serial dilutions of Quetiapine fumarate stock solutions in order to obtain signal-to-noise ratios. LOD Average Baseline Noise obtained from Blank : 51μV .Signal Obtained from LOD solution (0.12% of target assay concentration) : 149 μV ,S/N = 149/51 = 2.92. LOQ Average Baseline Noise obtained from Blank : 51μV Signal Obtained from LOQ solution (0.4% of target assay concentration) : 512μV S/N = 512/51 = 10.03

Robustness
Robustness of the method was evaluated by the analysis of Quetiapine fumarate under different experimental conditions such as changes in the organic composition of the mobile phase and flow rate. The percentage of methanol in the mobile phase was varied ±10%, the flow rate was varied ±0.1 ml/min. Their effects on the USP plate count, USP tailing at 10%, recovery and repeatability were studied. Deliberate variation of the method conditions had no significant effect on assay data or on chromatographic performance, indicating the robustness of method and its suitability for routine use and transfer to other laboratories.

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DISCUSSION
A RP-HPLC method with UV detection for the assay of Quetiapine fumarate was developed and validated. The results showed that the method is very selective, no significant interfering peak was detected; accurate, with the percentage recoveries > 99; and reproducible, with the %RSD < 1%. The method was sensitive; a little as 0.072μg/ml could be detected with the LOQ of 0.24μg/ml. The method involves use of a simple methanol with the HPLC water and minimum sample preparation, encouraging its application in quality control for analysis of Quetiapine fumarate in bulk samples, raw materials and final dosage forms.

Acknowledgements
Authro are grateful to GNV Chandra Shekar, Pharma Train, Kukatpally, Hyderabad for authentication of drug and methodology.

REFERENCES
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