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VALIDATED RP - HPLC METHOD FOR THE ESTIMATION OF QUETIAPINE IN FORMULATION

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VALIDATED RP - HPLC METHOD FOR THE ESTIMATION OF QUETIAPINE IN FORMULATION Powered By Docstoc
					                                    M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

                                                                                                ISSN:2249-5347
                                                                                                          IJSID
                      International Journal of Science Innovations and Discoveries                   An International peer
                                                                                                Review Journal for Science


Research Article                                                      Available online through www.ijsidonline.info

    VALIDATED RP - HPLC METHOD FOR THE ESTIMATION OF QUETIAPINE IN FORMULATION
                                M.V. Basaveswara Rao1, A. V. D. Nagendrakumar2*
1*Department  of Chemistry, Krishna University, Machilipatnam-521001, A. P, India
2Department   of Chemistry, GITAM University, Visakhapatnam-530045, A. P, India



   Received: 21.06.2011

   Modified: 15.07.2011                                                         ABSTRACT
   Published: 12.08.2011                          A simple, selective, linear, precise and accurate RP-HPLC
   Keywords: Quetiapine, RP-HPLC,                 method was developed and validated for rapid assay of
   UV detection, 250 nm,
   method development                             Quetiapine in tablet dosage form. Isocratic elution at a flow
   and validation.
                                                  rate of 1.0ml/min was employed on a symmetry C18
   *Corresponding Author                          (250x4.6mm, 5µm in particle size) at ambient temperature.
                                                  The mobile phase consisted of methanol: water: O.P.A
                                                  90:10:01 (V/V/V). The UV detection wavelength was 250 nm
                                                  and 20µl sample was injected. The retention time for
                                                  Quetiapine was 3.64 min. The percentage RSD for precision
                                                  and accuracy of the method was found to be less than 2%. The
                                                  method was validated as per the ICH guidelines. The method
   Address:
                                                  was successfully applied for routine analysis of Quetiapine in
   Name: A.V.D.Nagendra kumar
   Place: Visakhapatnam, AP
   E-mail:
                                                  tablet dosage form.
   Nagendra.git@gmail.com




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                                 M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

                                                INTRODUCTION
       Quetiapine (1-8) is used to treat either schizophrenia or bipolar disorder. In those with bipolar it is
used for depressive episodes, acute manic episodes associated with bipolar I disorder (as either
monotherapy or adjunct therapy to lithium or valproate), and maintenance treatment of bipolar I
disorder (as adjunct therapy to lithium or divalproex). Sometimes it is used as off-label, often as an
augmentation agent, to treat conditions such as obsessive-compulsive disorder, post-traumatic stress
disorder, restless legs syndrome, autism, alcoholism, depression, Tourette syndrome, and has been used
by physicians as asedative for those with sleep disorders or anxiety disorders. Figure-1 represents the
chemical structure of quetiapine.
                                 Figure-1: Chemical structure of Quetiapine




                                         MATERIALS AND METHODS
Chemicals and reagents
       HPLC grade water and methanol were purchased from Merck Specalities Pvt. Ltd.
Instrumentation and analytical conditions
       The analysis of drug was carried out on a PEAK HPLC system equipped with a reverse phase C18
column (250x4.6mm, 5µm in particle size), a LC-P7000 isocratic pump, a 20µl injection loop and a LC-
UV7000 absorbance detector and running on PEAK Chromatographic Software version 1.06. Isocratic
elution with methanol: water: O.P.A 90:09:01 (V/V) (PH-3.4) was used at a flow rate of 1.0ml/min. The
mobile phase was prepared freshly and degassed by sonicating for 5 min before use.
Stock and Working standard solutions
       Accurately weighed and transfer 10mg of Quetiapine working standard into a 10ml volumetric
flask add diluent and sonicate to dissolve it completely and make volume up to the mark with the same
solvent. Further pipette 1ml of the above stock solution into a 10ml volumetric flask and dilute up to the
mark with diluent. Mix well and filter through 0.45µm nylon filter paper and finally 10µg/ml were
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                                 M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

prepared. The calibration curve was plotted with the five concentrations of the 2µg/ml - 10µg/ml
working standard solutions. Calibration solutions were prepared daily and analyzed immediately after
preparation. Chromatographic conditions were represented in table-1.
                                    Table-1: Chromatographic condition
                   Mobile phase                       Methanol:water: O.P.A(90:09:01)
                   PH                                 3.4
                   UV detection                       250 nm
                   Analytical column                  C18
                   Flow rate                          1.0ml/min
                   Temperature                        ambient
                   Injection volume                   20µl
                   Runtime                            8 min
                   Retention time                     3.64 min
Assay of Quetiapine tablets
       Weigh 20 Quetiapine (quetiapine -25mg) tablets and calculate the average weight. Accurately
weigh and transfer the sample equivalent to 10mg of Quetiapine in to a 10ml volumetric flask. Add
diluent and sonicate to dissolve it completely and make volume up to the mark with diluents. Mix well
and filter through 0.45um filter. Further pipette 1ml of the above stock solution into a 10ml volumetric
flask and dilute up to mark with diluents and finally 10µg/ml were prepared. Mix well and filter through
0.45um filter. An aliquot of this solution was injected into HPLC system. Peak area of Quetiapine was
measured for the determination. The results were furnished in Table-2. Figure-2 represents the test
sample chromatogram.
                       Figure-2: Typical chromatogram of Quetiapine Formulation.




                                             Table-2: Assay Results
                    Formulation               Label claim (mg)      % Amount found
                    Quetiapine                25 mg                   93.76%

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Validation procedure
        The objective of the method validation is to demonstrate that the method is suitable for its
intended purpose as it is stated in ICH guidelines. The method was validated for linearity, precision
(repeatability and intermediate precision), accuracy, specificity, stability and system suitability. Standard
plots were constructed with five concentrations in the range of 2µg/ml to 10µg/ml prepared in triplicates
to test linearity. The peak area of Quetiapine was plotted against the concentration to obtain the
calibration graph. The linearity was evaluated by linear regression analysis that was calculated by the
least square regression method. The precision of the assay was studied with respect to both repeatability
and intermediate precision. Repeatability was calculated from six replicate injections of freshly prepared
Quetiapine test solution in the same equipment at a concentration value of 100% (10µg/ml) of the
intended test concentration value on the same day. The experiment was repeated by assaying freshly
prepared solution at the same concentration additionally on two consecutive days to determine
intermediate precision. Peak area of the Quetiapine was determined and precision was reported as
%RSD.
        Method accuracy was tested (% recovery and %RSD of individual measurements) by analyzing
sample of Quetiapine at three different levels in pure solutions using three preparations for each level.
The results were expressed as the percentage of Quetiapine recovered in the samples. Sample solution
short term stability was tested at ambient temperature (25±20C) for three days. In order to confirm the
stability of both standard solutions at 100% level and tablet sample solutions, both solutions protected
from light were re-injected after 24 and 48 hours at ambient temperature and compared with freshly
prepared solutions.
                                             RESULTS AND DISCUSSION
Optimization of the chromatographic conditions
        Proper selection of the stationary phase depends up on the nature of the sample, molecular weight
and solubility. The drug Quetiapine is non-polar. Non-polar compounds preferably analyzed by reverse
phase columns. Among C8 and C18, C18 column was selected. Non-polar compound is very attractive
with reverse phase columns. So the elution of the compound from the column was influenced by polar
mobile phase. Mixture of methanol, water and O.P.A was selected as mobile phase and the effect of
composition of mobile phase on the retention time of Quetiapine was thoroughly investigated. The
concentration of the methanol, water and O.P.A were optimized to give symmetric peak with short run
time (Fig.3).
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                                   M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

                                 Figure-3: Typical chromatogram of Quetiapine




Validation of method
System suitability
The system suitability parameters like capacity factor, asymmetry factor, tailing factor and number of
theoretical plates were also calculated. It was observed that all the values were within the limits
(Table.3). The statistical evaluation of the proposed method was revealed its good linearity,
reproducibility and its validation for different parameters and let us to the conclusion that it could be
used for the rapid and reliable determination of Quetiapine in tablet formulation. The results were
furnished in Table 3.
                                     Table 3: System suitability parameters
                                                Parameters              Values
                        λ max (nm)                                      250
                        Beer’s law limit (µg/ml)                        2-10
                        Correlation coefficient                         0.999
                        Retention time                                  3.64
                        Theoretical plates                              15615
                        Tailing factor                                  1.09
                        Limit of detection ppm                          0.15
                        Limit of quantification ppm                     0.5
                        Slope                                           20448.02
                        Intercept                                       991.76
                        accuracy                                        99.58%
                        R.S.D.                                          0.837
                        % of Quetiapine in formulation                  27.59%




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                                 M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

Linearity
       Five points graphs was constructed covering a concentration range 2-10 ppm (Three independent
determinations were performed at each concentration). Linear relationships between the peak area
signal of Quetiapine the corresponding drug concentration was observed. The standard deviation of the
slope and intercept were low. The statistical analysis of calibration is shown in Table-4.
                                      Table-4: Linearity of Quetiapine

                           S.No. Linearity level          concentration        Area
                             1          I                     2 ppm             37700
                             2         II                     4 ppm            84327.6
                             3         III                    6 ppm            121912.2
                             4         IV                     8 ppm             160396
                             5          V                    10 ppm             204146
Precision
       The validated method was applied for the assay of commercial tablets containing Quetiapine.
Sample was analyzed for five times after extracting the drug as mentioned in assay sample preparation of
the experimental section. The results presented good agreement with the labeled content. Low values of
standard deviation denoted very good repeatability of the measurement. Thus it was showing that the
equipment used for the study was correctl and hence the developed analytical method is highly
repetitive. For the intermediate precision a study carried out by the same analyst working on the same
day on two consecutive days indicated a RSD of 1.64. This indicates good method precision.
Stability
       The stability of Quetiapine in standard and sample solutions were determined by storing the
solutions at ambient temperature (25±20C). The solutions were checked in triplicate after three
successive days of storage and the data were compared with freshly prepared samples. In each case, it
could be noticed that solutions were stable for 48 hrs, as during this time the results did not decrease
below 98%. This denotes that Quetiapine is stable and standard and sample solutions for at least 2 days
at ambient temperature.
Recovery:
       Accuracy of the method was evaluated with std spiking to placebo with 50%, 100% and 150% of
the test concentration level. Results were found to be within the limit and results were tabulated in table-
5



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                                 M.V. Basavewara Rao et al., IJSID 2011, 1 (1), 45-52

                                 Table 5: Recovery studies of Quetiapine
                            % Concentration                   % Recovery

                                         50%                           99.63%
                                        100%                           98.82%
                                        150%                           96.01%
Ruggedness and Robustness:
       Ruggedness and robustnesss of the method was validated with slight changes in the
chromatographic conditions and mobile phase and solutions stability was evaluated and the results
found to be satisfactory.
                                                  CONCLUSION
       A validated RP-HPLC method has been developed for the determination of Quetiapine in tablet
dosage form. The proposed method is simple, rapid, accurate, precise and specific. Its chromatographic
run time of 8 min allows the analysis of a large number of samples in short period of time. Therefore, it is
suitable for the routine analysis of Quetiapine in pharmaceutical dosage form.
                                                  REFERENCES
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   Bolder Ii Study, G. (2006). "Efficacy of Quetiapine Monotherapy in Bipolar I and II
   Depression". Journal of Clinical Psychopharmacology 26 (6): 600.
2. Croissant, B.; Klein, O.; Gehrlein, L.; Kniest, A.; Hermann, D.; Diehl, A.; Mann, K. (2006). "Quetiapine
   in relapse prevention in alcoholics suffering from craving and affective symptoms: a case
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3. Mukaddes, N. M.; Abali, O. (2003). "Quetiapine Treatment of Children and Adolescents with Tourette's
   Disorder". Journal of Child and Adolescent Psychopharmacology 13 (3): 295.
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5. Lieberman, J. A.; Stroup, T. S.; McEvoy, J. P.; Swartz, M. S.; Rosenheck, R. A. Perkins, D. O.; Keefe, R. S.
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   et al. (2005). "Quetiapine and rivastigmine and cognitive decline in Alzheimer's disease: randomised
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7. Validated HPLC–MS/MS method for determination of quetiapine in human plasma, B. Barrett et al,
   Journal of Pharmaceutical and Biomedical Analysis 44 (2007) 498–505
8. A Validated RP-HPLC Method for the Estimation of Quetiapine in Bulk and Pharmaceutical
   Formulations, D. Suneetha et al, E-Journal of Chemistry, 2010, 7(S1), S261-S266




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