Indo American Journal of Pharmaceutical Research, 2014

ISSN NO: 2231-6876

METHOD DEVELOPMENT AND VALIDATION FOR THE SIMULTANEOUS

ESTIMATION OF OLANZAPINE AND FLUOXETINE HYDROCHLORIDE IN A
PHARMACEUTICAL FORMULATION BY RP-HPLC METHOD
Dondeti Mogili Reddy, Putchakayala Purnachandra Rao and D.Ramachandran*
Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India-522510.
ARTICLE INFO
Article history
Received 08/12/2014
Available online
22/12/2014
Keywords
OLANZAPINE,
FLUOXETINE
HYDROCHLORIDE,
RP-HPLC method,
INERTSIL C18 Column,
Acetonitrile,
Orthophosphoric Acid,
Triethylamine And Validation.

ABSTRACT
An isocratic Simultaneous estimation by RP-HPLC Method were developed and validated for
the quantification of OLANZAPINE and FLUOXETINE HYDROCHLORIDE in tablet
dosage form. Quantification was achieved by using a reversed-phase C18 column (Inertsil
C18 Column , 5, 250 mm 4.6 mm) at ambient temperature with mobile phase consisting of
0.1%v/v Ortho Phosphoric acid in water (pH 3.5 With Triethylamine):Acetonitrile: Methanol
(60:30:10). The flow rate was 1.0 ml/min. Measurements were made at a wavelength of
225nm. The average retention time for OLANZAPINE and FLUOXETINE
HYDROCHLORIDE were found to be 2.19 min and 3.71. The proposed method was
validated for selectivity, precision, linearity and accuracy. The assay methods were found to
be linear from 12-28g/ml for OLANZAPINE and 48-112g/ml for FLUOXETINE
HYDROCHLORIDE. All validation parameters were within the acceptable range. The
developed method was successfully applied to estimate the amount of OLANZAPINE and
FLUOXETINE HYDROCHLORIDE in tablet dosage form.

*Corresponding author

Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical
Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Please cite this article in press as Dondeti Mogili Reddy et al. Method Development and Validation For The Simultaneous
Estimation of Olanzapine And Fluoxetine Hydrochloride In A Pharmaceutical Formulation By RP-HPLC Method. Indo American
Journal of Pharm Research.2014:4(12).

5773

Department of Chemistry,
Acharya Nagarjuna University,
Nagarjuna Nagar, Guntur, Andhra Pradesh, India-522510
mogilireddy@gmail.com,
dittakavirc@gmail.com.
+919848632551,
+919866965335

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INTRODUCTION
OLANZAPINE an atypical antipsychotic agent, is used to treat both negative and positive symptoms of schizophrenia, acute
mania with bipolar disorder, agitation, and psychotic symptoms in dementia. Future uses may include the treatment of obsessivecompulsive disorder and severe behavioral disorders in autism. Structurally and pharmacologically similar to clozapine, olanzapine
binds to alpha(1), dopamine, histamine H1, muscarinic, and serotonin type 2 (5-HT2) receptors.
MOA:Olanzapine's antipsychotic activity is likely due to a combination of antagonism at D2 receptors in the mesolimbic
pathway and 5HT2A receptors in the frontal cortex. Antagonism at D2 receptors relieves positive symptoms while antagonism at
5HT2A receptors relieves negative symptoms of schizophrenia.
Route of Elemination:
It is eliminated extensively by first pass metabolism, with approximately 40% of the dose metabolized before reaching the
systemic circulation. Following a single oral dose of 14C labeled olanzapine, 7% of the dose of olanzapine was recovered in the urine
as unchanged drug, indicating that olanzapine is highly metabolized.
Structure of OLANZAPINE:

FLUOXETINE HYDROCHLORIDE
Fluoxetine hydrochloride is the first agent of the class of antidepressants known as selective serotonin-reuptake inhibitors
(SSRIs). Fluoxetine is a racemic mixture of the R- and S- enantiomers and are of equivalent pharmacologic activity. Despite distinct
structural differences between compounds in this class, SSRIs possess similar pharmacological activity. As with other antidepressant
agents, several weeks of therapy may be required before a clinical effect is seen. SSRIs are potent inhibitors of neuronal serotonin
reuptake. They have little to no effect on norepinephrine or dopamine reuptake and do not antagonize - or -adrenergic, dopamine
D2 or histamine H1 receptors. During acute use, SSRIs block serotonin reuptake and increase serotonin stimulation of somatodendritic
5-HT1A and terminal autoreceptors. Chronic use leads to desensitization of somatodendritic 5-HT1A and terminal autoreceptors. The
overall clinical effect of increased mood and decreased anxiety is thought to be due to adaptive changes in neuronal function that leads
to enhanced serotonergic neurotransmission. Side effects include dry mouth, nausea, dizziness, drowsiness, sexual dysfunction and
headache. Side effects generally occur within the first two weeks of therapy and are usually less severe and frequent than those
observed with tricyclic antidepressants. Fluoxetine may be used to treat major depressive disorder (MDD), moderate to severe bulimia
nervosa, obsessive-compulsive disorder (OCD), premenstrual dysphoric disorder (PMDD), panic disorder with or without
agoraphobia, and in combination with olanzapine for treatment-resistant or bipolar I depression. Fluoxetine is the most anorexic and
stimulating SSRI.

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Mechanism of Action:
Metabolized to norfluoxetine, fluoxetine is a selective serotonin-reuptake inhibitor (SSRI), it blocks the reuptake of serotonin
at the serotonin reuptake pump of the neuronal membrane, enhancing the actions of serotonin on 5HT 1A autoreceptors. SSRIs bind
with significantly less affinity to histamine, acetylcholine, and norepinephrine receptors than tricyclic antidepressant drugs.

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Structure of FLUOXETINE HYDROCHLORIDE:

Fluoxetine, an antidepressant agent belonging to the selective serotonin reuptake inhibitors (SSRIs), is used to treat
depression, bulimia nervosa, premenstrual dysphoric disorder, panic disorder and post-traumatic stress. According to the amines
hypothesis, a functional decrease in the activity of amines, such as serotonin and norepinephrine, would result in depression; a
functional increase of the activity of these amines would result in mood elevation. Fluoxetine's effects are thought to be associated
with the inhibition of 5HT receptor, which leads to an increase of serotonin level. Antagonism of muscarinic, histaminergic, and 1
adrenergic receptors has been hypothesized to be associated with various anticholinergic, sedative, and cardiovascular effects of
classical tricyclic antidepressant (TCA) drugs. Fluoxetine binds to these and other membrane receptors from brain tissue much less
potently in vitro than do the tricyclic drugs.
EXPERIMENTAL:
Equipments:
The chromatographic technique performed on a Shimadzu LC20-AT Liquid chromatography with SPD-20A prominence
UV-visible detector and Spinchrom software, reversed phase C18 column (Inertsil 5, 250 mm 4.6 mm) as stationary phase. Thermo
electron corporation double beam UV-visible spectrophotometer (vision pro-software) ,Ultrasonic cleaner, Shimadzu analytical
balance AY-220,Vaccum micro filtration unit with 0.45 membrane filter was used in the study.
Materials:
Pharmaceutically pure sample of OLANZAPINE and FLUOXETINE HYDROCHLORIDE were obtained as gift samples
from Chandra Labs, Prashanthi nagar, Kukatpally, and Hyderabad, India. The purity of the drug was evaluated by obtaining its
melting point and ultraviolet (UV) and infrared (IR) spectra. No impurities were found. The drug was used without further
purification.
HPLC-grade Acetonitrile Methanol ware from standard reagents pvt ltd. Othophosphoric acid and Triethylamine (AR grade) was from
Merck.
A tablet formulation of OLANZAPINE and FLUOXETINE HYDROCHLORIDE (5mg and 20mg label claims)
Chromatographic conditions
The sample separation was achieved on a C18 (5 , 25 cm 4.6 mm i.d.) INERTSIL column, aided by mobile phase mixture
of 0.1% Orthophosphoric acid (pH:3.5 adjusted with Triethylamine): Methanols : Acetonitrile (60: 10:30), at a flow rate of 1ml/min.
Injection volume is 20 l and detected at 225 nm at ambient temperatures.

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Mobile phase:
Then add 60volumes of buffer and 10 volumes of Methanol and 30volumes of Acetonitrile sonicated for 15 min.

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Preparation of mobile phase:

Buffer Preparation:
Take accurately 1ml of Ortho phosphoric acid in 1000mL Volumetric flask and diluted with 400mL of water then volume
made up to 1000mL with HPLC Grade water then adjusted the pH:3.5 with Triethylamine. Filtered through a 0.45 membrane filter.

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Analysis of formulation
Preparation of standard solution:
A 20mg of standard OLANZAPINE and 80 mg FLUOXETINE HYDROCHLORIDE were weighed and transferred to 100
ml of volumetric flask and dissolved in mobile phase. The flask was shaken and volume was made up to mark with mobile phase to
give a primary stock solution containing 200g/ml OLANZAPINE and 800g/ml of FLUOXETINE HYDROCHLORIDE . From the
above solution 5ml of solution is pipette out into a 50 ml volumetric flask and volume was made up to mark with mobile phase to
give a solution containing 20g/ml OLANZAPINE and 80g/ml of FLUOXETINE HYDROCHLORIDE .
Preparation of sample solution (Tablet Formulation):
For the estimation of the drug in tablet formulation twenty tablets were weighed and their average weight was determined.
The tablets were then finely powdered. Appropriate quantity equivalent to 20mg OLANZAPINE and 80 mg FLUOXETINE
HYDROCHLORIDE ware accurately weighed and The powder was transferred to 100 ml volumetric flask and shaken
vigorously with mobile phase and sonicated for 15 min and volume made up to the mark with mobile phase. The solution was shaken
vigorously and filtered by using whatmann filter no.41. from the above filtered clear solution 5ml of sample pipetted out into a 50 ml
volumetric flask volume made up to the mark with mobile phase to give a solution containing 20g/ml OLANZAPINE and 80g/ml
of FLUOXETINE HYDROCHLORIDE .

After several initial trails with mixtures of methanol, water, ACN and buffer in various combinations and proportions, a trail
with a mobile phase mixture of 0.1% Orthophosphoric acid (pH:3.5 adjusted with Triethylamine): Methanol : Acetonitrile (60:10:30),
brought sharp and well resolved peaks. The chromatogram was shown in Figure-2.

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Figure: 1 U.V Graph of OLANZAPINE and FLUOXETINE HYDROCHLORIDE.

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RESULTS AND DISCUSSIONS

Determination Of Working Wavelength(max):
10 mg of the OLANZAPINE standard drug is taken in a 10 ml volumetric flask and dissolved in methanol and volume made
up to the mark, from this solution 0.1ml is pipetted into 10 ml volumetric flask and made upto the mark with the methanol to give a
concentration of 10 g/ml. The above prepared solution is scanned in uv between 200-400 nm using methanol as blank. The max
was found to be 235nm
10 mg of the FLUOXETINE HYDROCHLORIDE standard drug is taken in a 10 ml volumetric flask and dissolved in
methanol and volume made up to the mark, from this solution 0.1ml is pipette into 10 ml volumetric flask and made upto the mark
with the methanol to give a concentration of 10 g/ml . The above prepared solution is scanned in uv between 200-400 nm using
methanol as blank. The max was found to be 227nm.
The Isosbestic Point of OLANZAPINE and FLUOXETINE HYDROCHLORIDE were found to be 225nm. The U.V Graph
shown in Figure: 1

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Figure:2 Chromatogram of OLANZAPINE and FLUOXETINE HYDROCHLORIDE

METHOD VALIDATION:
Linearity:
Linearity was studied by analyzing five standard solutions covering the range of 12-28 g/ml for OLANZAPINE and 48 to
96g/ml for FLUOXETINE HYDROCHLORIDE of the drug. From the primary stock solution 0.6ml,0.8ml,1.0ml,1.2ml,1.4 ml of
aliquots are pipette into 10 ml volumetric flasks and made up to the mark with the mobile phase to give a concentrations of 12g/mL ,
16g/mL ,20g/mL ,24g/mL and 28 g/mL of OLANZAPINE and 48g/mL , 64g/mL , 80g/mL ,96g/mL and 112 g/mL of
FLUOXETINE HYDROCHLORIDE .
Calibration curve with concentration verses peak areas was plotted by injecting the above prepared solutions and the
obtained data were subjected to regression analysis using the least squares method

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Method precision (repeatability)

The precision of the instrument was checked by repeated injections and measurement of peak areas and retention times of
solutions (n = 6) for, 20 g/ml of OLANZAPINE and 80 g/ml of FLUOXETINE HYDROCHLORIDE without changing the
parameter of the proposed chromatographic method.

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Figure:3 Linearity overlays of OLANZAPINE and FLUOXETINE HYDROCHLORIDE.

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Limit of detection and limit of quantification:

The limit of detection (LOD) and limit of quantification (LOQ) were separately determined based on standard deviation of
the y-intercept and the slope of the calibration curve by using the equations (2) and (3), respectively.
LOD = 3.3 /S . (3)
LOQ =10 /S .. (4)
Where,
= the standard deviation of the response
S = the slope of the calibration curve
The slope S may be estimated from the calibration curve of the analyte.
Accuracy (recovery study):
The accuracy of the method was determined by calculating the recoveries of OLANZAPINE and FLUOXETINE
HYDROCHLORIDE by the standard addition method. Known amounts of standard solutions of OLANZAPINE and FLUOXETINE
HYDROCHLORIDE were added at 10% concentration to pre quantified sample solutions of OLANZAPINE (20, 24, 28g/ml) and
FLUOXETINE HYDROCHLORIDE (80, 96, 112g/ml) (Figure No.5.1 and 5.2). The amount of OLANZAPINE and FLUOXETINE
HYDROCHLORIDE recovered was estimated by using the following formulas.

Robustness:
Robustness is the measure of a method remain unaffected by small, deliberate changes in method parameters like flow rate
and detection wavelength on assay of the analyte of interest. Here the detection wavelength varied 2nm and flow rate was varied 0.2
ml/min. The results were shown in (Table no.4)

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Figure:4 Placebo Chromatogram of OLANZAPINE and FLUOXETINE HYDROCHLORIDE.

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Specificity:
In an assay, demonstration of specificity requires that it can be shown that the procedure is unaffected by the presence of
impurities or excipients. In practice, this can be done by spiking the drug substance or product with appropriate levels of impurities or
excipients and demonstrating that the assay results are unaffected by the presence of these extraneous materials. There should be no
interference of the diluents, placebo at retention time of drug substances.

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Ruggedness:
The ruggedness of the method was studied by analyzing the sample and standard preparations by two analysts. The %RSD
assay values between two analysts was calculated i.e.,(limit <2%). This indicates the method was rugged. The results were shown in
Table no.5.
DISCUSSION
In RP HPLC method, the primary requirement for developing a method for analysis is that the using different solvents and
buffers and columns to get better retention time and theoretical plates, and better cost effective and time saving method than the
previously developed methods. . The Iso bestic Point Of OLANZAPINE and FLUOXETINE HYDROCHLORIDE were found to be
225nm (Figure No: 1) by scanning in UV region. The chromatographic method was optimized with mobile phase consisting of 0.1%
Orthophosphoric acid(pH:3.5): Methanol: Acetonitrile (60:10:30) and C18 Inertsil column. All the validation parameters were
studied at a the wavelength 225nm. Accuracy was determined by calculating the recovery (Table No.3) and the results were in
acceptable range (limit 98-102%). The method was successfully used to determine the amount of OLANZAPINE and FLUOXETINE
HYDROCHLORIDE present in the Tablet. The results obtained were in good agreement with the corresponding labeled amount
(Table No.3). The method was linear in the concentration range of 12 to 28 g/ml for OLANZAPINE and 48 to 112g/ml for
FLUOXETINE HYDROCHLORIDE (Figure no.1 ,Table No.1). Precision was calculated as repeatability (% RSD) for the drug
(Table No.7). Robustness and ruggedness results were in acceptable range (Table No.4 and Table No.5).Summary of all validation
parameters for method is given in Table No.8. By observing the validation parameters, the method was found to be simple, sensitive,
accurate and precise. Hence the method can be employed for the routine analysis OLANZAPINE and FLUOXETINE
HYDROCHLORIDE in tablet dosage form.
Table No: 1
Concentration (g/ml )
12
16
20
24
28

Peak Area
1354.855
1777.900
2129.471
2547.740
2896.682

Table No: 1.1

Concentration (g/ml ) Peak Area
48
3845.286
64
4900.036
80
5782.059
96
6951.370
112
7905.418

Linearity of Fluoxetine

y = 96.33x + 214.5
R = 0.999

3000
2000

10000.000
y = 63.57x + 791.0
8000.000 R = 0.998
6000.000
4000.000

1000

2000.000

0.000
0

10

20

30

50

100

150

Concentration

Concentration

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FigureNo.1&1.1: Linearity (calibration) curve of OLANZAPINE and FLUOXETINE HYDROCHLORIDE.

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Peak Area

4000

Peak Area

Linearity of
Olanzepine

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Table no.2: LOD and LOQ values Calculated from calibration curve:
OLANZAPINE
mcg Area
LOD 0.22 20.88
LOQ 0.66 63.28

FLUOXETINE
mcg Area
1.31 83.54
3.98 253.16

Table No.3: Recovery data.

II.

III.

1
2
3
1
2
3
1
2
3

Amount of
Sample taken (%)
80
80
80
100
100
100
120
120
120

Amount of Standard
Spiked (%)
10%
10%
10%
10%
10%
10%
10%
10%
10%

%Recovery of
OLANZAPINE
99.12%

%Recovery of FLUOXETINE
HYDROCHLORIDE
98.37%

101.36%

101.53%

99.09%

98.75%

Table No.4: Results of Robustness study.

Parameter

Rt of OLANZAPINE

Flow Rate(0.8ml)
1.2ml
1.0ml
Wave Length 225nm
223
227
Parameter
Flow Rate(0.8ml)
1.2ml
1.0ml
Wave Length 225nm
223
227

2.930
1.790
2.223
2.221
2.225
2.225

Tailing
factor
1.440
1.185
1.364
1.820
1.833
1.807

Theoretical
Plates
3494
2986
3144
3044
3045
3001

Rt of FLUOXETINE
HYDROCHLORIDE
4.907
2.980
3.710
3.710
3.711
3.712

Tailing
factor
1.308
1.185
1.219
1.217
1.201
1.200

Theoretical
Plates
5673
4607
5295
5235
5247
5584

Table No.5: Results of Ruggedness.

Analyst-1
Analyst-2
Analyst-1
Analyst-2

OLANZAPINE
FLUOXETINE HYDROCHLORIDE

%Assay
99.98
99.78
100.01
100.34

%RSD
0.08%
0.31%

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I.

S.No

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Table No.6: Assay Results.

OLANZAPINE
1
2098.671
2
2097.800
3
2086.371
4
2108.269
5
2090.360
Average 2096.294
Sample area
1
2060.162
2
2087.965
3
2133.105
4
2091.642
5
2071.128
Average 2088.800
Tablet average weight
200
Standard weight
20
Sample weight
800
Label amount
5
std.purity
99.76
Cal.:
4.97
%Assay 99.42
Standard Area

FLUOXETINE HYDROCHLORIDE
5764.513
5739.332
5780.212
5803.414
5731.065
Average
5763.707
5777.009
5765.820
5809.771
5824.353
5778.627
Average
5791.116
mg
200
mg
80
mg
800
mg
20
99.84
mg
20.07
%
100.33

mg
mg
mg
mg
%
mg
%

Table NO.7: Method Precision (Repeatability).

S.No.
1
2
3
4
5
6
avg
stdev
%RSD

OLANZAPINE
Rt
Area
2.21 2051.034
2.19 2018.514
2.21 2089.940
2.20 2079.924
2.21 2076.366
2.20 2092.363
2.20 2068.024
0.01 28.368
0.38 1.37

FLUOXETINE HYDROCHLORIDE
Rt
Area
3.74
5660.375
3.71
5659.607
3.73
5787.091
3.73
5820.711
3.74
5721.787
3.73
5731.174
3.73
5730.124
0.01
65.360
0.30
1.14

Table No.8: Validation parameters of evaluated method.

S.
No
1.

Parameter

Limit

Value Obtained

ACCURACY(%Recovery)

99.09 to 101.36% (OLANZAPINE)

98.37 to101.53% (FLUOXETINE HYDROCHLORIDE )

2.

Linearity concentrations Range( g/mL)

Regression coefficient (R2 value)

98-102%
In All levels
(80% to 120%)
NLT 0.990

3.

Precision (% RSD)
Method precision
(Repeatability)
(%RSD, n = 6)
Robustness

It should be meet System

suitability criteria

Wavelength Variation
(223nm to 227nm)
Ruggedness
(Intermediate Precision) (%RSD analyst to
analyst variation)

NMT2%

Complies

0.81% For OLANZAPINE and 0.31% for FLUOXETINE

HYDROCHLORIDE

*RSD = Relative standard deviation

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5.

Flow Variation(0.8mL to 1.2mL/min)

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4.

NMT 1%(For Rt)

NMT 2%
(For Area)

12 to 28 g/ml (OLANZAPINE)
R2=0.999
48 to112g/ml(FLUOXETINE
HYDROCHLORIDE )
R2=0.998
%RSD of Rt=0.38% and %RSD of Area 1. 37%
(OLANZAPINE)
%RSD of Rt=0.30% and %RSD of Area 1.14%
(FLUOXETINE HYDROCHLORIDE )

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CONCLUSION
From the above experimental results and parameters it was concluded that, this newly developed method for the simultaneous
estimation of OLANZAPINE and FLUOXETINE HYDROCHLORIDE was found to be simple, precise, accurate and high resolution
and shorter retention time makes this method more acceptable and cost effective and it can be effectively applied for routine analysis
in research institutions, quality control department in meant in industries, approved testing laboratories.
REFERENCES
1. ICH, Q2A validation of analytical procedure: Methodology International Conference on Harmonization, Geneva, October 1994.
2. ICH, Q2B Validation of analytical procedure: Methodology International Conference on Harmonization, Geneva, March 1996.
3. http://www.ich.org/
4. http://www.irjponline.com/admin/php/uploads/1029_pdf.pdf
5. http://www.ncbi.nlm.nih.gov/pubmed/20502563
6. http://en.wikipedia.org/wiki/Olanzapine
7. http://en.wikipedia.org/wiki/Fluoxetine
8. http://www.drugbank.ca/drugs/DB00334
9. http://www.drugbank.ca/drugs/DB00472
10. http://www.ijpsonline.com/article.asp?issn=0250474X;year=2009;volume=71;issue=4;spage=477;epage=480;aulast=Patel
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2865829/

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