J4#04-07-flu–

International Journal of Generic Drugs
ANALYTICAL METHOD
AM-V-0206-0300
P R O C E D U R E S
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination Method is suitable for:ý In-process controlþ Product Releaseþ Stability indicating analysis (Suitability - US/EU Product) CAUTIONFLUOXETINE HYDROCHLORIDE IS A HAZARDOUS CHEMICAL AND SHOULD BE HANDLED ONLY UNDER CONDITIONS SUITABLE FOR HAZARDOUS WORK.
IT IS HIGHLY PRESSURE SENSITIVE AND ADEQUATE PRECAUTIONS SHOULD BE TAKEN TO AVOID ANYMECHANICAL FORCE (SUCH AS GRINDING, CRUSHING, ETC.) ON THE POWDER.
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International Journal of Generic Drugs
ANALYTICAL METHOD
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination TABLE OF CONTENTS
INTRODUCTION.
PRECISION .
System Repeatability .
Method Repeatability .
Intermediate Precision .
LINEARITY .
RANGE .
ACCURACY.
Accuracy of Standard Injections .
Accuracy of the Drug Product .
VALIDATION OF FLUOXETINE HCl AT LOW CONCENTRATION .
Linearity at Low Concentrations.
Accuracy of Fluoxetine HCl at Low Concentration.
System Repeatability .
Quantitation Limit.
Detection Limit .
VALIDATION FOR META-FLUOXETINE HCl (POSSIBLE IMPURITIES) .
Meta-Fluoxetine HCl linearity at 0.05% - 1.0%.
Detection Limit for Fluoxetine HCl.
Quantitation Limit for Meta Fluoxetine HCl .
Accuracy for Meta-Fluoxetine HCl .
Method Repeatability for Meta-Fluoxetine HCl.
Intermediate Precision for Meta-Fluoxetine HCl.
SPECIFICITY - STABILITY INDICATING EVALUATION OF THE METHOD .
FORCED DEGRADATION OF FINISHED PRODUCT AND STANDARD .
1. Unstressed analysis.
2. Acid Hydrolysis stressed analysis .
3. Base hydrolysis stressed analysis .
4. Oxidation stressed analysis .
5. Sunlight stressed analysis .
6. Heat of solution stressed analysis .
7. Heat of powder stressed analysis .
System Suitability stressed analysis .
Placebo.
STABILITY OF STANDARD AND SAMPLE SOLUTIONS .
Standard Solution .
Sample Solutions.
ROBUSTNESS .
Extraction.
Factorial Design .
CONCLUSION .
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International Journal of Generic Drugs
ANALYTICAL METHOD
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination BACKGROUND
Therapeutically, Fluoxetine hydrochloride is a classified as a selective serotonin-reuptakeinhibitor. Effectively used for the treatment of various depressions. Fluoxetine hydrochloridehas been shown to have comparable efficacy to tricyclic antidepressants but with feweranticholinergic side effects. The patent expiry becomes effective in 2001 (US).
INTRODUCTION
Fluoxetine capsules were prepared in two dosage strengths: 10mg and 20mg dosagestrengths with the same capsule weight. The formulas are essentially similar andgeometrically equivalent with the same ingredients and proportions. Minor changes in non-active proportions account for the change in active ingredient amounts from the 10 and 20mg strength.
The following validation, for the method SI-IAG-206-02 , includes assay and determinationof Meta-Fluoxetine by HPLC, is based on the analytical method validation SI-IAG-209-06.
Currently the method is the in-house method performed for Stability Studies. The Validationwas performed on the 20mg dosage samples, IAG-21-001 and IAG-21-002.
In the forced degradation studies, the two placebo samples were also used.
PRECISION
SYSTEM REPEATABILITY
Five replicate injections of the standard solution at the concentration of0.4242mg/mL as described in method SI-IAG-206-02 were made and the relativestandard deviation (RSD) of the peak areas was calculated.
PEAK AREA
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination PRECISION - Method Repeatability
The full HPLC method as described in SI-IAG-206-02 was carried-out on the finished
product IAG-21-001 for the 20mg dosage form. The method repeated six times and the
relative standard deviation (RSD) was calculated.
of labeled amount
(%) Average
PRECISION - Intermediate Precision
The full method as described in SI-IAG-206-02 was carried-out on the finished product IAG-
21-001 for the 20mg dosage form. The method was repeated six times by a second analyst
on a different day using a different HPLC instrument. The average assay and the relative
standard deviation (RSD) were calculated.
of labeled amount
Average (%)
The difference between the average results of method repeatability and the intermediateprecision is 1.7%.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination LINEARITY
Standard solutions were prepared at 50% to 200% of the nominal concentration required bythe assay procedure. Linear regression analysis demonstrated acceptability of the methodfor quantitative analysis over the concentration range required. Y-Intercept was found to beinsignificant.
STD. CONC.
PEAK AREA
Correlation coefficient (RSQ)
Y - Intercept
response at 100%
Different concentrations of the sample (IAG-21-001) for the 20mg dosage form wereprepared, covering between 50% - 200% of the nominal weight of the sample.
Conc. (%)
Conc. (mg/mL)
Peak Area
of labeled amount
(%) Average
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination The results demonstrate linearity as well over the specified range.
Correlation coefficient (RSQ)0.99981 Slope ACCURACY
ACCURACY OF STANDARD INJECTIONS
Five (5) replicate injections of the working standard solution at concentration of0.4242mg/mL, as described in method SI-IAG-206-02 were made.
INJECTION
PEAK AREA
ACCURACY
The percent deviation from the true value wasdetermined from the linear regression line ED. N0: 04
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination ACCURACY OF THE DRUG PRODUCT
Admixtures of non-actives (placebo, batch IAG-21-001 ) with Fluoxetine HCl wereprepared at the same proportion as in a capsule (70%-180% of the nominalconcentration).
Three preparations were made for each concentration and the recovery wascalculated.
Fluoxetine
Accuracy
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination VALIDATION OF FLUOXETINE HCl
AT LOW CONCENTRATION
LINEARITY AT LOW CONCENTRATIONS
Standard solution of Fluoxetine were prepared at approximately 0.02%-1.0% of theworking concentration required by the method SI-IAG-206-02. Linear regressionanalysis demonstrated acceptability of the method for quantitative analysis over thisrange.
ACCURACY OF FLUOXETINE HCl AT LOW CONCENTRATION
The peak areas of the standard solution at the working concentration weremeasured and the percent deviation from the true value, as determined from thelinear regression was calculated.
µg/100mL
ACCURACY
(%) Average
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination System Repeatability
Six replicate injections of standard solution at 0.02% and 0.05% of working concentration asdescribed in method SI-IAG-206-02 were made and the relative standard deviation wascalculated.
FLUOXETINE HCl AREA
Quantitation Limit - QL
The quantitation limit ( QL) was established by determining the minimum level atwhich the analyte was quantified. The quantitation limit for Fluoxetine HCl is 0.02%of the working standard concentration with resulting RSD (for six injections) of 5.0%.
Detection Limit - DL
The detection limit (DL) was established by determining the minimum level at whichthe analyte was reliably detected. The detection limit of Fluoxetine HCl is about0.01% of the working standard concentration.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination VALIDATION FOR META-FLUOXETINE HCl
Meta-Fluoxetine HCl linearity at 0.05% - 1.0% Relative response factor for Meta-Fluoxetine HCl was determined as slope ofFluoxetine HCl divided by the slope of Meta-Fluoxetine HCl from the linearity graphs(analysed at the same time).
Detection Limit (DL) for Fluoxetine HCl
The detection limit (DL) was established by determining the minimum level at whichthe analyte was reliably detected.
Detection limit for Meta Fluoxetine HCl is about 0.02%.
Quantitation Limit (QL) for Meta-Fluoxetine HCl
The QL is determined by the analysis of samples with known concentration of Meta-Fluoxetine HCl and by establishing the minimum level at which the Meta-FluoxetineHCl can be quantified with acceptable accuracy and precision.
Six individual preparations of standard and placebo spiked with Meta-Fluoxetine HClsolution to give solution with 0.05% of Meta Fluoxetine HCl, were injected into theHPLC and the recovery was calculated.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination META-FLUOXETINE HCl
[RECOVERY IN SPIKED SAMPLES].
Known Conc.
Area in Spiked
Found Conc.
Recovery
(µg/100ml)
(µg/100mL)
(%) AVERAGE
The recovery result of 6 samples is between 80%-120%.
QL for Meta Fluoxetine HCl is 0.05%.
Accuracy for Meta Fluoxetine HCl
Determination of Accuracy for Meta-Fluoxetine HCl impurity was assessed usingtriplicate samples (of the drug product) spiked with known quantities of MetaFluoxetine HCl impurity at three concentrations levels (namely 80%, 100% and120% of the specified limit - 0.05%).
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International Journal of Generic Drugs
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination META-FLUOXETINE HCl
[RECOVERY IN SPIKED SAMPLES]
Known Conc.
Area in spiked
Found Conc.
Recovery
(µg/100mL)
(µg/100mL)
RECOVERY DATA DETERMINED IN SPIKED SAMPLES ED. N0: 04
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination REPEATABILITY
Method Repeatability - Meta Fluoxetine HCl
The full method (as described in SI-IAG-206-02) was carried out on the finisheddrug product representing lot number IAG-21-001-(1). The HPLC method repeatedserially, six times and the relative standard deviation (RSD) was calculated.
IAG-21-001 20mg CAPSULES - FLUOXETINE
% Meta-Fluoxetine1 in
Fluoxetine
Spiked Solution
AVERAGE (%)
All results are less than QL (0.05%) therefore spiked samples with 0.05% Meta FluoxetineHCl were injected.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination Intermediate Precision - Meta-Fluoxetine HCl
The full method as described in SI-IAG-206-02 was applied on the finished productIAG-21-001-(1) .
It was repeated six times, with a different analyst on a different day using a differentHPLC instrument.
The difference between the average results obtained by the method repeatabilityand the intermediate precision was less than 30.0%, (11.4% for Meta-Fluoxetine HClas is and 28.5% for spiked solution).
IAG-21-001 20mg - CAPSULES FLUOXETINE
Percentage
% Meta-fluoxetine1
Meta-fluoxetine
in spiked solution
(%) AVERAGE
All results obtained were well below the QL (0.05%) thus spiked samples slightly greaterthan 0.05% Meta-Fluoxetine HCl were injected. The RSD at the QL of the spiked solutionwas 14.5% ED. N0: 04
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination SPECIFICITY - STABILITY INDICATING EVALUATION
Demonstration of the Stability Indicating parameters of the HPLC assay method [SI-
IAG-206-02] for Fluoxetine 10 & 20mg capsules, a suitable photo-diode array
detector was incorporated utilizing a commercial chromatography software managing
system2, and applied to analyze a range of stressed samples of the finished drug
product.
GLOSSARY of PEAK PURITY RESULT NOTATION (as reported2):
Purity Angle
is a measure of spectral non-homogeneity across a peak, i.e. the weighed average of all spectral contrast angles calculated by comparing all spectra in theintegrated peak against the peak apex spectrum.
Purity Threshold
is the sum of noise angle3 and solvent angle4. It is the limit of detection of shape differences between two spectra.
Match Angle
is a comparison of the spectrum at the peak apex against a library Match Threshold
is the sum of the match noise angle3 and match solvent angle4.
3Noise Angle
is a measure of spectral non-homogeneity caused by system noise.
4Solvent Angle
is a measure of spectral non-homogeneity caused by solvent OVERVIEW
The assay of the main peak in each stressed solution is calculated according to the
assay method SI-IAG-206-02, against the Standard Solution, injected on the same
day.
If the Purity Angle is smaller than the Purity Threshold and the Match Angle is
smaller than the Match Threshold, no significant differences between spectra can be
detected. As a result no spectroscopic evidence for co-elution is evident and the
peak is considered to be pure.
The stressed condition study indicated that the Fluoxetine peak is free from any
appreciable degradation interference under the stressed conditions tested.
Observed degradation products peaks were well separated from the main peak.
1® PDA-996 Waters™ ; 2[Millennium 2010] ED. N0: 04
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination FORCED DEGRADATION OF FINISHED PRODUCT & STANDARD
1. UNSTRESSED SAMPLE
1.1. Sample IAG-21-001 (2) (20mg/capsule) was prepared as stated in SI-IAG-206-02 and
injected into the HPLC system. The calculated assay is 98.5%.
SAMPLE - UNSTRESSED
1.2. Standard solution was prepared as stated in method SI-IAG-206-02 and injected into
the HPLC system. The calculated assay is 100.0%.
2. ACID HYDROLYSIS
2.1. Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI-
IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weighed into a 50mL
volumetric flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL
of conc. HCl was added to this solution The solution was allowed to stand for 18 hours, then
adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected
into the HPLC system after filtration.
Fluoxetine peak intensity did NOT decrease. Assay result obtained - 98.8%.
SAMPLE- ACID HYDROLYSIS
2.2. Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine
HCl were weighed into a 50mL volumetric flask. 20mL Diluent were added. 2mL of conc.
HCl were added to this solution. The solution was allowed to stand for 18 hours, then
adjusted to about pH = 5.5 with NaOH 10N, made up to volume with Diluent and injected
into the HPLC system.
Fluoxetine peak intensity did NOT decrease. Assay result obtained - 97.2%.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination STANDARD - ACID HYDROLYSIS
3. BASE HYDROLYSIS
3.1. Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-
IAG-206-02 : An amount equivalent to 20mg Fluoxetine was weight into a 50mL volumetric
flask. 20mL Diluent was added and the solution sonicated for 10 minutes. 1mL of 5N
NaOH was added to this solution. The solution was allowed to stand for 18 hours, then
adjusted to about pH = 5.5 with 5N HCl, made up to volume with Diluent and injected into
the HPLC system.
Fluoxetine peak intensity did NOT decrease. Assay result obtained - 99.3%.
SAMPLE - BASE HYDROLYSIS
3.2. Standard stock solution was prepared as per method SI-IAG-206-02 : About 22mg
Fluoxetine HCl was weighed into a 50mL volumetric flask. 20mL Diluent was added. 2mL
of 5N NaOH was added to this solution. The solution was allowed to stand for 18 hours,
then adjusted to about pH=5.5 with 5N HCl, made up to volume with Diluent and injected
into the HPLC system.
Fluoxetine peak intensity did NOT decrease - 99.5%.
STANDARD - BASE HYDROLYSIS
4. OXIDATION
4.1. Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as per method SI-
IAG-206-02. An equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask.
20mL Diluent added and the solution sonicated for 10 minutes.
1.0mL of 30% H2O2 was added to the solution and allowed to stand for 5 hours, then madeup to volume with Diluent, filtered and injected into HPLC system.
Fluoxetine peak intensity decreased to 95.2%.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination SAMPLE - OXIDATION
4.2. Standard solution was prepared as in method SI-IAG-206-02 : about 22mg Fluoxetine
HCl were weighed into a 50mL volumetric flask and 25mL Diluent were added. 2mL of
30% H2O2 were added to this solution which was standing for 5 hours, made up to volume
with Diluent and injected into the HPLC system.
Fluoxetine peak intensity decreased to 95.8%.
STANDARD - OXIDATION
5. SUNLIGHT
Sample solution of IAG-21-001 (2) (20mg/capsule) was prepared as in method SI- IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1hour. The BST wasset to 35°C and the ACT was 45°C. The vials were placed in a horizontal position (4mmvials, National + Septum were used). A Dark control solution was tested. A 2%w/v quininesolution was used as the reference absorbance solution.
Fluoxetine peak decreased to 91.2% and the dark control solution showed assay of 97.0%.
The difference in the absorbance in the quinine solution is 0.4227AU.
Additional peak was observed at RRT of 1.5 (2.7%).
The total percent of Fluoxetine peak with the degradation peak is about 93.9%.
SAMPLE - SUNLIGHT
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination SUNLIGHT (Cont.)
Working standard solution was prepared as in method SI-IAG-206-02 . The solution was exposed to 500w/hr. cell sunlight for 1.5 hour. The BST was set to 35°C and the ACTwas 42°C. The vials were placed in a horizontal position (4mm vials, National + Septumwere used). A Dark control solution was tested. A 2%w/v quinine solution was used as thereference absorbance solution.
Fluoxetine peak was decreased to 95.2% and the dark control solution showed assay of99.5%.
The difference in the absorbance in the quinine solution is 0.4227AU.
Additional peak were observed at RRT of 1.5 (2.3).
The total percent of Fluoxetine peak with the degradation peak is about 97.5%.
STANDARD - SUNLIGHT
6. HEAT OF SOLUTION
6.1. Sample solution of IAG-21-001-(2) (20 mg/capsule) was prepared as in method SI-
IAG-206-02 . Equivalent to 20mg Fluoxetine was weighed into a 50mL volumetric flask.
20mL Diluent was added and the solution was sonicated for 10 minutes and made up to
volume with Diluent. 4mL solution was transferred into a suitable crucible, heated at 105°C
in an oven for 2 hours. The sample was cooled to ambient temperature, filtered and
injected into the HPLC system.
Fluoxetine peak was decreased to 93.3%.
SAMPLE - HEAT OF SOLUTION [105oC]
6.2. Standard Working Solution (WS) was prepared under method SI-IAG-206-02 . 4mL
of the working solution was transferred into a suitable crucible, placed in an oven at 105°C
for 2 hours, cooled to ambient temperature and injected into the HPLC system.
Fluoxetine peak intensity did not decrease - 100.5%.
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HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination STANDARD - HEAT OF SOLUTION
7. HEAT OF POWDER
Equivalent to 20mg Fluoxetine were weighed from powder of IAG-21-001-(2)(20mg/capsule) into a suitable test tube with cover and heated to 100°C for 4 hours. Thepowder after cooled to room temperature was transferred into a 50mL volumetric flask with20mL Diluent, sonicated for 10 minutes, made up to volume with Diluent and injected intothe HPLC system after filtration. - Fluoxetine peak intensity decreased to 86.6%.
SAMPLE - HEAT OF POWDER
About 45mg Fluoxetine HCl were weighed into a suitable test tube with cover and heated to100°C for 4 hours. The powder after cooled to room temperature was transferred into a100mL volumetric flask with Diluent, made up to volume with Diluent and injected into theHPLC system.
Fluoxetine peak intensity did not decrease significantly - 95.9%.
STANDARD - HEAT OF POWDER
Fluoxetine peak:
Purity Angle
Match Angle
SYSTEM SUITABILITY
A System Suitability Test was performed as described in the method SI-IAG-206-02 .
The resolution calculated (according to USP) between Meta Fluoxetine peak and Fluoxetinepeak should be not less than 2.0.
The Tailing Factor [TF] for Fluoxetine peak should be not more than 2.0.
Typical retention time of Fluoxetine peak is about 9.5 - 10.0 minutes.
ED. N0: 04
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ISSN 0793 694X US/Canada
ISSN 0793 7784 Euro
ISSN 0793 7822 Pacific Rim
International Journal of Generic Drugs
ANALYTICAL METHOD
AM-V-0206-0300
P R O C E D U R E S
of Pages: 24
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination A mixture of non-actives (placebo, batch IAG-21-001-1 for 10mg dosage and IAG-21-001-3for 20mg dosage) was prepared and subjected to analysis. No interfering peaks wereobserved.
STABILITY OF STANDARD & SAMPLE SOLUTIONS
STANDARD SOLUTION TEST
A Standard Solution (1) was initially prepared, held under refrigerated conditions [4 o - 8o C]
for up to 30 days and tested against two freshly prepared Standard Solutions (2) and (3).
Standard No.
Weight (mg)
Preparation
Response
Comparison between Standards (%)
Standard
CONCLUSION
Under refrigerated conditions 4 o - 8o C the Standard Solution remains stable for up to 30days storage ED. N0: 04
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ISSN 0793 694X US/Canada
ISSN 0793 7784 Euro
ISSN 0793 7822 Pacific Rim
International Journal of Generic Drugs
ANALYTICAL METHOD
AM-V-0206-0300
P R O C E D U R E S
of Pages: 24
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination SAMPLE SOLUTIONS
Two sample solutions from batch IAG-21-001 (20mg/capsule) were prepared and analyzedas in method. The sample solution was refrigerated for 48 hours and re-analyzed.
Preparation Date
Analysis Date
Average (%)
ROBUSTNESS
EXTRACTION EFFICIENCY PROFILE
Demonstration of the method's extraction efficiency was achieved using triplicate samplesolutions as described in the HPLC method via the selection of the three extraction times - 5minutes apart - as shown in the table.
EXTRACTION EFFICIENCY
Sonication
Time (min.)
Percentage
Percentage
The results demonstrated that an extraction time of 10 minutes by sonication is suitable toensure a complete extraction of the active material.
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ISSN 0793 694X US/Canada
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International Journal of Generic Drugs
ANALYTICAL METHOD
AM-V-0206-0300
P R O C E D U R E S
of Pages: 24
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination ROBUSTNESS - (Cont.)
SPECIFICATION - FACTORIAL DESIGN
The HPLC condition as performed in the analytical method SI-IAG-206-02 are as follows: : Shandon, Hypersil BDS C-8, 5µ, 150×4.6mm
Mobile phase
: Solution A: solution B (55 : 45)
: Triethylamine buffer*: acetonitrile: tetrahydrofuran (75:15:10)
: Triethylamine buffer*: acetonitrile: tetrahydrofuran (65:20:15)
Flow rate
: 1.4 - 1.6mL/min
Temperature
: 30°C (±2°C)
Injection volume
Detector
: UV detector λ = 227nm, Flow Cell = 10mm.
: SOLUTION B
The factorial design variations were performed on the above set of specifications.
Changes were made to the following parameters, using the system suitability
parameters.
n percentage eluent composition
n flow rate parameters
n column temperature
n column [tpe/make]
Incident
Flow Rate
HPLC COLUMN TYPE
(mL/min.)
Solution A
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ISSN 0793 694X US/Canada
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ISSN 0793 7822 Pacific Rim
International Journal of Generic Drugs
ANALYTICAL METHOD
AM-V-0206-0300
P R O C E D U R E S
of Pages: 24
HPLC ASSAY with DETERMINATION OF META-FLUOXETINE HCl.
ANALYTICAL METHOD VALIDATION
10 and 20mg Fluoxetine Capsules HPLC Determination FACTORIAL DESIGN (CONT.)
Resolution1
Retention
Solution
(mL/min)
1The resolution is calculated between the Fluoxetine and Meta-Fluoxetine peaks
Conclusion of results from Factorial Design - based on the obtained results, theanalytical procedures are determined to be robust.
CONCLUSION
HPLC Method SI-IAG-206-02 for assay evaluation was demonstrated to be bothaccurate and precise for performing the stability indicating assay and thedetermination of Meta-Fluoxetine HCl analysis for product release and stabilitystudies and profiling of Fluoxetine Capsules 10 and 20mg.
1. Millennium 2010 chromatography manager, PDA software user's guide, manual No. 038993 revision 2, chap. 9, January 1993.
2. FLUOXETINE CAPSULES (Fluoxetine HCl equivalent to 10mg or 20mg Fluoxetine per capsule ASSAY AND DETERMINATION OF META- FLUOXETINE HCl-ANALYTICAL METHOD VALIDATION REPORT IAG-SI-V-01- EU IAG-206-02 /10/20-013.
3. USP/NF XXIV Rockville Maryland USA4. Martindale Pharmaceutical UK5. DAB FRG and Eu. Pharm.
6. IAG-SI -221X8-03 FLUOXETINE CAPSULES 03-97-03 Containing: Fluoxetine HCl equivalent to 10mg or 20mg 7. Int. J of Drug R&D Vol. 01 No 06 Analytical Overview of FLUOXETINE8. Stark, P. And Hardison, C.D. (1985). J. Clin. Psychiatry, 46, (3, Sec. 2), 53) ED. N0: 04
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ISSN 0793 7822 Pacific Rim

Source: http://www.iagim.org/pdf/AOM-09.pdf