ETORICOXIB AND PREGABALIN OF METHOD DEVLOPMENT IN RPHPLC BY UPEXA BAVADIYA

GUIDED BY
DHIRENDRA KUMAR TARAI
PREPARED BY:
UPEKSHA J. BAVADIYA
Department of Pharmaceutical Quality Assurance
GUJARAT TECHNOLOGICAL UNIVERSITY
Chandkheda, Ahmadabad – 382424 - Gujarat
“Development and Validation for Simultaneous
Estimation of Etoricoxib and Pregabalin in Bulk
and Tablet Dosage Form by RP-HPLC”

CONTENT
• Introduction
• Drug Profile
• Review of Literature
• Rational
• Aim and Objectives
• Material and Equipment
• Identification of API
• Method development and
validation
• Summary and conclusion
• Comment sheet
• References
• Paper Publication
2

INTRODUCTION OF DRUG (ETORICOXIB)(1)
4
 Etoricoxib, sold under the trade name Arcoxia, is a selective COX-2
inhibitor from McOLSON Research Laboratories. Currently it is
approved in more than 80 countries worldwide but not in the US, where
the Food and Drug Administration (FDA) has required additional safety
and efficacy data for Etoricoxib before it will issue approval
 Etoricoxib is indicated for the treatment of rheumatoid arthritis,
psoriatic arthritis, osteoarthritis, ankylosing spondylitis, chronic low
back pain, acute pain, and gout. Approved indications differ by country.
In the U.K., it is also "used for the short term treatment of moderate
pain after dental surgery" of adults.
 MOA : Like any other COX-2 selective inhibitor Etoricoxib selectively
inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2), preventing
production of prostaglandins (PGs) from arachidonic acid.

INTRODUCTION OF DRUG (PREGABALIN)(2)
 Marketed under the brand name Lyrica among others, is a medication used to
treat epilepsy, neuropathic pain, fibromyalgia, restless leg syndrome, and
generalized anxiety disorder Its use in epilepsy is as an add-on therapy for
partial seizures. When used before surgery, it reduces pain but results in
greater sedation and visual disturbances It is taken by mouth
 Common side effects include headache, dizziness, sleepiness, confusion,
trouble with memory, poor coordination, dry mouth, problem with vision,
and weight gain. Serious side effects may include angioedema, drug misuse,
and an increased suicide risk.
 MOA : Act as a ligand of the alpha2-delta subunit of calcium channels.
Decreased calcium entry into nerve endings. less glutamate released from
nerve endings .than relief of neuropathic pain.
5

Introduction to Dosage form
Brand Name Contents Manufacturer Formulation
Etoshine NP
Etoricoxib
+
Pregabalin
Sun Pharma
laboratory pvt. Ltd
Tablet
6

TECHNIQUES OF METHOD DEVLOPMENT
7

INTRODUCTION OF ANALYTICAL METHOD (3)
 Analytical chemistry is divided into two branches:
 Qualitative: A qualitative analysis provides information about the identity of
atomic or molecular species or functional groups in sample.
 Quantitative: A quantitative analysis provides numerical information as to the
relative amount of one or more of these components.
 Analytical methods development and validation play important roles in the
discovery, development and manufacture of pharmaceuticals with the objectives. To
qualify and quantify the active pharmaceutical ingredients in bulk as well as
dosage form To establish impurity profile and limit of allowable impurities in
dosage form.
8

9
INTRODUCTION OF HPLC METHOD (4)
 HPLC is an analytical technique widely used for identification, separation, detection
and quantification of various drugs and its related degradents.
 High performance liquid chromatography (HPLC), is a separation technique based
on a solid stationary phase and a liquid mobile phase. Most of the drugs in multi-
component dosage forms can be analyzed by HPLC method because of the several
advantages like rapidity, specificity, accuracy, precision and ease of automation in
this method. HPLC method eliminates tedious extraction and isolation procedures.
 Principle of separation:
 The principle of separation in normal phase mode and reverse phase mode is
adsorption. When mixtures of components are introduced in to a HPLC column,
they travel according to their relative affinities towards the stationary phase. The
component which has more affinity towards the adsorbent travels slower. The
component which has less affinity towards the stationary phase travels faster. Since
no two components have the same affinity towards the stationary phase, the
components are separated.

CONT…..
 Different modes of separation in HPLC:
1) Normal phase mode.
2) Reverse phase ion pair chromatography.
3) Reversed phase mode.
4) Size exclusion chromatography.
10

Method development by RP-HPLC (5)
11
 Reversed-phase chromatography is the mainly used in chromatographic
mode, it is used to separate neutral molecules in solution based on their
hydrophobicity.
 As the name suggested that, reversed-phase chromatography is the
reverse of normal-phase chromatography in the intelligence that it involves
the employ of a polar mobile phase and a non-polar stationary phase.
 It ensures that a decrease in the polarity of the mobile phase results in a
decreases in solute retention.

12
HPLC Techniques
Normal phase
Mode
Stationary phase : Polar
Mobile phase : Non polar
Reverse phase Mode
Stationary phase : Non
polar
Mobile phase : Polar

13
Fig. A schematic diagram of HPLC equipment.

VALIDATION OF ANALYTICAL METHOD (14)
14
Validation
Validation is a process of establishing documented evidence, which provides a high
degree of assurance that a specific activity will consistently produce a desired result
or product meeting its predetermined specifications and quality characteristics.
Method Validation
Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use.
Results from method validation can be used to judge the quality, reliability and
consistency of analytical results.

Validation of Analytical method
15

2. Drug profile of Etoricoxib (17)
INTRODUCTION
CAS Number 202409-33-4
Description
Etoricoxib is a synthetic, nonsteroidal anti-inflammatory drug
(NSAID) with antipyretic, analgesic, and potential antineoplastic
properties. Etoricoxib specifically binds to and inhibits the
enzyme cyclooxygenase-2 (COX-2), resulting in inhibition of the
conversion of arachidonic acid into prostaglandins.
Structure
Chemical
Formula
C18H15ClN2O2S
17

Mol. Weight 358.84 gm/mol
IUPAC Name
5-chloro-2-(6-methylpyridin-3-yl)-3-(4-methylsulfonyl) phenyl)
pyridine.
Categories
 COX-2 Inhibitors
 Anti-Inflammatory Agents
Solubility Slightly Soluble in water, freely soluble in methanol
Mechanism
Of Action
Like any other COX-2 selective inhibitor Etoricoxib selectively
inhibits isoform 2 of cyclo-oxigenase enzyme (COX-2),
preventing production of prostaglandins (PGs) from arachidonic
acid.
LogP 3.7
pKa 4.96
Melting point 134-135°C
18

2. Drug profile of Pregabalin (15-16)
INTRODUCTION
CAS Number 148553-50-8
Description
Pregabalin is structurally similar to gamma-
aminobutyric acid (GABA) - an inhibitory
neurotransmitter. It may be used to manage
neuropathic pain, postherpetic neuralgia, and
fibromyalgia among other conditions
Structure
Chemical
Formula
C8H17NO2
19

Mol. Weight 259.22 gm/mol
IUPAC Name 3-isobutyl GABA, (S)-3-isobutyl-γ-aminobutyric acid
Categories
 Anticonvulsants
 Analgesics
Solubility Freely Soluble in water
Mechanism
Of Action
Act as a ligand of the alpha2-delta subunit of calcium channels.
Decreased calcium entry into nerve endings. less glutamate
released from nerve endings .than relief of neuropathic pain.
LogP -1.3
pKa 4.2
Melting point 176 - 178ºC
20

3. Review of Literature
 Official
Drugs Method Year Abstract Summary Ref. No
Etoricoxib Monograph IP
2010
Column: stainless steel column 30 cm x 3.9 mm,
packed with phenyl silica gel
Mobile phase : mixture of 26 volumes of
acetonitrile and 74 volumes of a 0.272 per cent
w/v solution of sodium acetate,
Flow rate: 1.0 ml/min
Wavelength: 256 nm
18
Pregabalin Monograph IP
2010
Column: stainless steel column 25 cm x 4.6 mm
packed with octadecylsilane bonded to porous
silica (5 μm)
Mobile phase : mixture of62 volumes of a buffer
solution prepared by diluting about 7.18 ml
of triethylamine to 1000 ml water, adjusted to pH
3.0 with orthophosphoric acid and 38 volumes
of acetonitrile,
Wavelength: 340 nm
19
22

 Non - official
Drugs Method Authors/year
/article /journal
Abstract Summary Ref. No
Etoricoxib RP-HPLC Bhattacharya I
Bhattacharya SP .
Asian J. Research
Chem
2009
297-299
Column: C18 column
Mobile Phase: phosphate buffer ( pH-7.8)
and methanol (90:10 v/v)
Wavelength: 235 nm., Flow rate: 1 ml/min
Retention times: 3.428 min
Linearity range: Etoricoxib - 10-200 μg/ml
20
Etoricoxib HPLC Haque M ,
Shaema N.
Am. J. Pharm Tech
Res.
2012
275 – 283
Column: C18 column (250 mm × 4.6 mm.
5μm particle size)
Mobile Phase: Ammonium Acetate
21
23

Drugs Method
s
authors/ year
/articals
Abstract Summary Ref.
No
Etoricoxib RP-
HPLC
Venugopal S,
Tripathi UM,
Devanna N,
E-Journal of
Chemistry
2011
S119-S126
Column : Zorbax SB CN (250 × 4.6 mm , 5µm)
Mobile phase : buffer : methanol
50:50
Flow rate : 0.8 ml/min
Wavelength : 235 nm
22
Pregabalin RP-
HPLC
Kasawar GR,
Faroogui MN .
Indian Journal of
Pharmaceutical
Sciences. 2010
517-519
Column: Hypersil BDS(C8, 150×4.6 mm,5 μm)
column
Mobile Phase: phosphate buffer pH 6.9 and
acetonitrile in the ratio of 95:05 Flow rate: 1 ml/min
23
Pregabalin RP-
HPLC
Prathima SSP.
INDO AMERICAN
JOURNAL OF
PHARMACEUTICAL
SCIENCES
2015
1038-1047
Column: AGILENT ZORBAX SB-ODS C18(250×4.6
mm)5 μm
Mobile Phase: dipotassium hydrogen phosphate:
methanol in the ratio of 60:40% v/v
Wavelength: 247 nm. ,Flow rate: 1 ml/minRetention
times: 2.00 min
Linearity range: 50 μg/ml to 150 μg/ml
LOQ: 0.53 𝜇g/ml LOD: 1.61 𝜇g/ml
24
24

/article /journal
No
Etoricoxib
(ETR) and
Thiocolchico
side (THC)
RP-
HPLC
Kumar US ,
Natraj D,
Khan A
INTERNATIONAL
JOURNAL OF
RESEARCH IN
PHARMACY AND
CHEMISTRY
2011
649-65
Column: C18 stainless steel column (InertSil
ODS-3, 250 mm x 4.6 mm ID, particle size 5μm)
Mobile Phase: phosphate buffer (PH6, adjusted
with ortho phosphoric acid) and methanol
(30:70 v/v)
Wavelength: 255 nm. , Flow rate: 1.2 ml/min
Retention times: Etoricoxib - 2.506 min
Thiocolchicoside - 4.600 min
Linearity range: Etoricoxib - 40-80 μg/ml
Thiocolchicoside 2-6 μg/ml
25
Paracetamol
(PCM) and
Etoricoxib
(ETO)
HPLC Baheti KG
Shaikh S
International
Journal of
PharmTech
Research vol -3
2011
1719-1727
Mobile Phase: phosphate buffer : acetonitrile
(60:40v/v)
Wavelength: 242 nm.
Retention times: PCM – 1.51 min
ETO – 4.31 min
Linearity range: PCM - 5-30 μg/ml
ETO 1-6 μg/ml
26
25

/article /journal
NSAIDs
and their
commonly
prescribed
combinatio
n drugs
RP-HPLC Gananadhamu S,
Patel PN .
Chromatography
Research
International
2013
1-13
Column: Kromasil C18 (250 × 4.6 mm, 5 𝜇m)
Mobile Phase: phosphate buffer pH 3.25 and
acetonitrile with gradient elution
Wavelength: 230 nm.
LOQ: 0.64 to 3.24 𝜇g/Ml
LOD: 0.04 to 0.97 𝜇g/mL
27
Etoricoxib
and
Paracetam
ol
RP-HPLC Rao KP,
Damanu G .
Journal of Advanced
Studies in
Agricultural,
Biological and
Environmental
Sciences
2014
2394-2606
Column: Hypersil BDS C18 (250 x 4.6 mm
i.d., 5μm particle size)Mobile Phase: 0.05 M
sodium dihydrogen phosphate buffer
(adjusted pH 3.2 with o-phosphoric acid):
acetonitrile (35:65 v/v)
Wavelength: 235 nm.
Flow rate: 1 ml/min
concentration range:
Etoricoxib - 1000 to 3000μg/ml 40-80 μg/ml
Paracetamol - 1200 to 3600μg/ml
28
26

Drugs Method Authors/
year/ artical
Thiocolchic
oside and
Etoricoxib
RP-HPLC Padmavati k,
Rao MS.
World Journal
of
Pharmaceutic
al Sciences.
2016
76-79
Column: Hypersil BDS C18 (250 x 4.6 mm i.d.,
5μm particle size)Mobile Phase: phosphate
buffer(pH-3.4) and acetonitrile in the ratio of
35:65 v/v
Retention times: Etoricoxib – 6.92 min
Thiocolchicoside – 2.83 min
29
Pregabalin
And
Methalcob
alamine
RP-HPLC Bhatt KK,
Patelia EM,
Mori A,
Journal of
Analytical &
Bioanalytical
Techniques
2018
1-4
Column : C18 ( 250× 4.6 mm) 5µm
Mobile phase : Methanol : water 40:60
Wavelength : 218 nm
Retension time : 6.4
PH :6.5
30
27

Drugs Method Authors / year
/ artical
No
Epalrestat
and
Pregabalin
RP-
HPLC
Parmeshweri
SA ,Anunam G.
International
Journal of
Pharmaceutica
l Sciences and
Research
2018
1844-1850
Column: C18 column 250 × 4.6 mm
Mobile Phase: 0.1% ortho phosphoric acid buffer and
acetonitrile ( ratio of 45: 55)
Wavelength: 244 nm. , Flow rate: 1 ml/min
Detector: PDA
Linearity range: Epalrestat - 37.5 – 225 μg/ml
Pregabalin - 18.75 - 112.5 μg/ml
Retention times: Epalrestat – 2.407 min
Pregabalin – 3.272 min
31
Nortriptyli
ne and
Pregabalin
RP-
HPLC
Potkuri H, Rao
SB.
Journal of the
Chilean
Chemical
Society.62
2017
62
Column: C18 column BDS (250mm x 4.6 mm, 5m)
Mobile Phase: Perchloric acid (0.1%) and acetonitrile
in the ratio of 55:45
Wavelength: 210 nm. , Flow rate: 1 ml/min
concentration range: nortriptyline – 37.5 - 22.5
μg/ml
Pregabalin - 37.5 – 22.5 μg/ml
Retention times: nortriptyline – 2.407 min
32
28

Drugs Method Authors
/year/art.
Epalrestat
and
Pregabalin
RP-HPLC Goday S,
Ragaman A.,
Asian
journal of
pharmaceuti
cal and
clinical
reaserch,
vol -112018
319-324
Precision : epalrestat - 0.2% Pregabalin – 0.3%
LOQ : Epalrestat - 0.65 μg/ml
Pregabalin – 0.25 μg/ml
LOD : Epalrestat - 0.21 μg/ml
Linearity range: Epalrestat - 30–180 ppm
Pregabalin - 15–90 ppm
33
Pregabalin
and
Celecoxib
RP-HPLC Swapna G
Merugu M,
World
Journal of
Pharmaceuti
cal Research,
2017
1354-1360
Column: Hypersil BDS(150 mm x 4.6 mm, 5m)
Mobile Phase: potassium di hydrogen orthophosphate
buffer of pH 6.5 and acetonitrile in the ratio of (70:30)
Wavelength: 238 nm.
Flow rate: 1 ml/min
Linearity range: Celecoxib - 100 μg/ml -750 μg/ml
Pregabalin-37.5μg/ml-281.25 μg/ml
34
29

Drugs Method Author/year
/ artical
No
Pregabalin
and
Aeclofenac
stability
RP-HPLC
Suchitta TJ
Gurupadoyy
a B M .
Acta
scientific
Pharmaceuti
cal sciences.
2018
20-26
Wavelength: Aeclofenac - 276 nm., Pregabalin - 406 nm
concentration range: Aeclofenac – 10 - 50μg/ml μg/ml
Pregabalin - 50 - 500μg/ml μg/ml
LOQ: Aeclofenac - 2μg/ml , Pregabalin - 4μg/ml
LOD: Aeclofenac – 0.50 μg/ml ,Pregabalin – 0.6 μg/ml
35
Epalrestat
and
Pregabalin
stability-
indicating
RP-HPLC
Prasad R ,
Madhuharik
a MY.
International
journal of
pharmacy
2017
157-164
Column: C18 (150x4.6mm, 5μ) column
Mobile Phase: Ammonium acetate buffer (pH 10): ACN
70:30 % v/v , Flow rate: 1 ml/min
LOQ : Epalrestat - 0.57 μg/ml Pregabalin – 1.51
μg/ml , LOD : Epalrestat - 0.19 μg/ml Pregabalin –
0.50 μg/ml
Linearity range: Epalrestat - 37.5 – 225 μg/ml
Pregabalin - 18.75 - 112.5 μg/ml
36
30

Drugs Method Authors /year/
articals/
journal
No
Etoricoxib
and
Drotaveri-ne
RP- HPLC Syal PK, Sahoo
M, Ingale KD,
Ingale SS,
Choudhari VP
And Kuchekar
BS,
Scholars
Research Library
2010
93-102
Column: kramosil C18 ( 250 × 4.6 mm,
5µm)
Mobile phase : methanol: buffer
51:49
Flow rate : 0.9 ml/min
Wavelength : 244 nm
37
Pregabalin
And
Nor-
triptiline
RP- HPLC Mewada NA,
Patel BR, Patel
JG, Vegad KL and
Patel VS,
International
Journal for
Pharmaceutical
Research
Scholars
2017
1-7
Column : BDS Hypersil C18 column
(250×4.6 mm ,5 µm)
Mobile Phase : buffer : methanol
70:30
Flow rate : 1 ml /min
Wavelength : 210nm
38
31

Drugs Method Authors /year/
articals/ journal
Epalrestat
and
Pregabalin
RP-HPLC Sivagami I,
Kavyalalitha S,
INTERNATIONAL
RESEARCH
JOURNAL OF
PHARMACY
2018
9
Column: C8 (250 x 4.6 mm, 5μm) Column.
Mobile Phase: Potassium Dihydrogen
Phosphate Buffer: Acetonitrile taken in the
ratio 45:55
Flow rate: 1 ml/min, Wavelength: 274 nm
LOQ : Epalrestat - 0.73 μg/ml
LOD : Epalrestat - 0.24 μg/ml
39
32

34
 The drug selected for the present study is Etoricoxib and Pregabalin
in tablet from combination dosage form.
 A review of literature reveals that only few chromatographic and
spectrophotometric methods have been reported for the estimation
of Etoricoxib and Pregabalin separately but not combined in
simultaneous method and RP-HPLC method.

5. AIM AND OBJECTIVES
36
 Aim
 My prime aim is to develop a simple, rapid, efficient, accurate, reproducible,
less time consuming and reliable new validated RP-HPLC chromatographic
method of analysis for Etoricoxib and Pregabalin in pharmaceutical dosage
form.
 Objectives
 Development of rapid, sensitive and accurate RP-HPLC method for the
simultaneous estimation of Etoricoxib and Pregabalin
 To estimate Etoricoxib and Pregabalin simultaneously from tablet dosage
form by developed method and validation of proposed method.

6. MATERIAL AND EQUIPMENT
38
Equipment Manufacturer
UV Visible Spectrophotometer Shimadzu 1800
HPLC CYBERLAB LC100
FTIR Shimadzu
Ultrasonicator HINTRON CD 4820
Measuring cylinders 50, 100, 1000 ml
pH meter LABTRONICS LT-14
ISO:2008
Volumetric flask (graduated) 10, 25, 50, 100 ml
Pipettes 1, 2, 5,10 ml
Beakers 250, 500, 1000 ml
 Equipment used in method :

39
 Materials used in method :
S.NO. NAME MANUFACTURER
1. Etoricoxib SUN PHARMA PVT.LID
2. Pregabalin SUN PHARMA PVT.LID
3. Potassium dihydrogen
phosphate
Merck
4. Sodium perchlorate Merck
5. Perchloric acid Merck
6. Ortho phosphoric acid Merck
7. Methanol Merck
8. Acetontrile Merck
9. Water Merck
10. 0.45 µm Nylon filter Axivia
11. 0.45µm PVDF filter Rankem

40
 Melting point of Etoricoxib and Pregabalin have been determined by
using open capillary method.
Sr.
No.
APIs
Melting point (˚C)
Reported Measured
1 Pregabalin 176 - 178°C 176-177 ˚C
2 Etoricoxib 134-135 °C 134-135˚C
7. Melting Point Determination of Etoricoxib and Pregabalin

41
 IR Spectra of Etoricoxib:
8. Identification by FTIR Spectroscopy

42
Sr. No. Functional Group Frequency
1 N-H 3058
2 C-H 2917
3 C=C 1599
4 S=O 1297
5 C-N 1143
6 C-Cl 840
7 C-H 772
 IR Spectral interpretation of Etoricoxib :
Structure of etoricoxib

43
 IR Spectra of Pregabalin :

44
Sr. No. Functional Group Practical Peaks
1 N-H 2964
2 C-H 2666
3 C=O 2206
4 C-N 1333
5 C-O 1278
6 OH 1469
 IR Spectral interpretation of Pregabalin
Structure of Pregabalin

45
The solubility of Etoricoxib and Pregabalin practically determined separately by
taking 100 mg of both the drugs in 100 ml volumetric flasks, adding required
quantity of solvent at room temperature and shaken for few minutes. Solubility data
for each study was observed and recorded.
Description Terms Relative Quantities of solvent for 1prts of solute
Very Soluble Less than 1 part
Freely Soluble From 1 to 10 parts
Soluble From 10 to 30 parts
Sparingly Soluble From 30 to 100 parts
Slightly Soluble From 100 to 1000 parts
Very Slightly Soluble From 1000 to 10000 parts
Practically Insoluble More than 10000 parts
 Solubility table as per IP 2010 specification
9. Solubility Study

46
 Solubility data of Etoricoxib and Pregabalin
Solvent Etoricoxib Pregabalin
Water
Soluble Freely Soluble
0.1 N HCl soluble soluble
0.1 N NaOH Very soluble Soluble
Methanol Freely soluble Freely soluble

47
 Selection of Wavelength:
• 60µg/ml solution of Etoricoxib and 75µg/ml solution Pregabalin was
prepared using methanol as solvent.
• The above mentioned solutions were scanned individually from 190 to 400
nm in UV-Visible spectrophotometer.
• The optimal response for the overlain spectrum of Etoricoxib and
Pregabalin was obtained at 234 nm.
• Hence the complete method was processed at the wavelength of 234 nm.
• Spectrums are shown in Fig.
10. DEVELOPMENT AND OPTIMIZATION OF RP-HPLC METHOD

48
UV spectrum of Etoricoxib and Pregabalin for detection of wave.

49
 Preparation of Phosphate buffer
Weighed 2.950 grams of potassium dihydrogen phosphate and 540mg of dipotassium
hydrogen ortho phosphate into a 1000 ml beaker, dissolved and diluted to 1000 ml
with HPLC water, the pH was found to be 6.26.
 Preparation of mobile phase
Mixed the above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%) and
degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 µ under vacuum
filtration.
 Diluent preparation
Mixed the above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%) and
degassed in ultrasonic water bath for 5 minutes. Filtered through 0.45 µ under vacuum
filtration.
Preparation of solution

50
 Standard Solution Preparation :
 Accurately weighed and transferred 60 mg each of Etoricoxib & 75 mg of Pregabalin
working standard into a 10ml clean dry volumetric flask added about 7ml of Diluent
and sonicated to dissolve it completely and made volume up to the mark with the
same solvent (Stock solution).
 Further pipetted 1ml of Etoricoxib & 1 ml of Pregabalin the above stock solution
into a 10ml volumetric flask and diluted up to the mark with diluents.
 The solution contains 60µg/ ml and 75 µg/ ml concentration of Etoricoxib and of
Pregabalin respectively
 Sample Solution Preparation :
 Accurately weighed and transferred equivalent to 60mg of Etoricoxib/ 75 mg of
Pregabalin sample into a 10ml clean dry volumetric flask added about 7ml of
Diluent and sonicated to dissolve it completely and made volume up to the mark
with the same solvent (Stock solution).
 Further pipetted 1.0ml of Etoricoxib & 1.0 ml of Pregabalin of the above stock
solution into a 10ml volumetric flask and diluted up to the mark with Diluent.
 The solution contains 60µg/ ml and 75 µg/ ml concentration of Etoricoxib and of
Pregabalin respectively.

51
Sr
no
Mobile phase Ratio Remarks
1 Phosphate buffer : methanol 45:55 Retension time are very
low
2 Phosphate buffer : methanol 50:50 Peack are not clear
3 Phosphate buffer : methanol 40:60 No clear peak observed
4 Phosphate buffer : methanol 30:70 Peak shape are not clear
5 Phosphate buffer: ACN 50:50 No clear peak observed
6 Phosphate buffer : ACN 25 :75 Clear peak observed

52
 TRAIL-1
 Preparation of Mobile phase
 The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol
in the ratio of 45:55. The mobile phase is then sonicated using Ultra-Sonicator to
remove the impurities and dissolved gases, as they may lead to unwanted peaks in
the chromatogram.
 Mobile phase: Phosphate buffer : methanol (45:55)
 Chromatographic Parameters
Equipment
High performance liquid chromatography
equipped with UV detector
Column
C18 column
Flow rate 1.0ml/min
Wavelength 234nm
Injection volume 20 µl
Column oven Ambient
Run time 9min

53
Chromatogram-1
 Conclusion
 The retention time was very low for Pregabalin which has been tried for many
concentrations.

54
 TRAIL-2
 Preparation of Mobile phase :
 The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and
methanol in the ratio of 50:50. The mobile phase is then sonicated using Ultra-
Sonicator to remove the impurities and dissolved gases, as they may lead to
unwanted peaks in the chromatogram.
 Mobile phase: Phosphate buffer: methanol (50:50)
 Chromatographic Parameters
Equipment
Column
C18 column
Flow rate 1.0ml/min
Wavelength 234nm
Column oven Ambient
Run time 6min

55
 Conclusion
 The plate count was found to be less and the retention time was found to be
less for Etoricoxib with an less tailing which was found unsatisfactory.
Chromatogram-2

56
 TRAIL-3
methanol in the ratio of 40:60. The mobile phase is then sonicated using Ultra-
 Chromatographic Parameters:
Equipment
Column
C18 column
Flow rate 0.9 ml/min
Wavelength 234nm
Column oven Ambient
Run time 9min

57
Chromatogram-3
 Conclusion
 The small hump was seen in chromatogram with less tailing and plate count which
was carried for many times hence found unsatisfactory.

58
 TRAIL-4
 The mobile phase is prepared by mixing Phosphate buffer (pH-6.26) and methanol in
the ratio of 30:70. The mobile phase is then sonicated using Ultra-Sonicator to
remove the impurities and dissolved gases, as they may lead to unwanted peaks in
the chromatogram.
 Chromatographic Parameters:
Equipment
Column
C18 column
Flow rate 1.0ml/min
Wavelength 234nm
Column oven Ambient
Run time 6min

59
 Conclusion
 The peak shape was not clear for Pregabalin resolution was found to be less with
less plate count hence tried by changing the concentration which gave rise to the
optimized method.
Chromatogram-4

60
 TRAIL : 5
 Preparation of mobile phase :
Acetonitrile in the ratio of 30:70. The mobile phase is then sonicated using Ultra-
 Mobile phase: Phosphate buffer: ACN (50:50)
 Chromatographic Parameters :
Equipment
Column
C18 column
Flow rate 1.0ml/min
Wavelength 234nm
Column oven Ambient
Run time 6min

61
Chromatography 5
 Conclusion
 The peak shape was not clear for Pregabalin resolution was found to be less
with less plate count hence tried by changing the concentration which gave rise.

62
 TRAIL : 6
 Preparation of mobile phase :
 Mixture of above buffer 250 ml (25%) and 750 ml of Acetonitrile HPLC (75%)
are mixed and degassed in ultrasonic water bath for 5 minutes. Filtered
through 0.45 µ under vacuum filtration. The mobile phase is sonicated using
Ultra-Sonicator for 5min to remove the impurities and dissolved gases, as they
may lead to unwanted peaks in the chromatogram.
 Mobile phase: phosphate buffer: Acetonitrile (25:75).
 Chromatographic Parameters :
Equipment
Column
C18
Flow rate 0.8 ml/min
Wavelength 234nm
Injection volume 20µl
Column oven Ambient
Run time 8 min
pH 6.26

64
 Conclusion :
 The retention time, shape, resolution were found to be good when compared
to other methods in trails, hence this method was finalized for the
estimation of Pregabalin and Etoricoxib.
 A simple and sensitive reverse phase HPLC method has been developed
for simultaneous analysis of Pregabalin and Etoricoxib in combined dosage
form.
 The method utilizes sample preparation followed by separation on a
Cosmosil C18. Analytes were monitored by UV detection at 234 nm using an
isocratic mode with phosphate buffer (pH-6.26): Acetonitrile in the ratio
25:75 as mobile phase.
 The flow rate was set at 0.8 ml/min and effluent was monitored at 234nm.
 The retention time was 2.475min and 6.528 min for Pregabalin and
Etoricoxib respectively.

65
Analyze the placebo, Etoricoxib and Pregabalin separately. A solution of placebo was
spiked with the Etoricoxib and Pregabalin at its working concentration. The solution
was analyzed as per the RP-HPLC method described.
(i) Placebo solution:
Weigh accurately about 135 mg of powdered placebo into a clean 100 ml volumetric
flask. Add 25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and
make up the volume with the water and filter (Concentration: 4.30 mg/ml of placebo).
(ii) Standard solution (Etoricoxib):
Weigh accurately about 60 mg of Etoricoxib working standard into a clean 100 ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate for
5 minute, and make up the volume with the water (Concentration: 1.0 mg/ml of
Etoricoxib).
(iii) Standard solution (Pregabalin):
Weigh accurately about 75 mg of Pregabalin working standard into a clean 100ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution. Sonicate
for 5 minute, and make up the volume with the water (Concentration: 0.10 mg/ml of
Pregabalin).
Preparation of Solution

66
(iv) Standard solution (Etoricoxib + Pregabalin):
Weigh accurately about 60 mg of Etoricoxib working standard and 75 mg of
Pregabalin working standard into a clean 100 ml volumetric flask. Dissolve in
25 ml of water and 0.2 ml of ammonia solution. Sonicate for 5 minute, and
make up the volume with the water (Concentration: 1.0 mg/ml of Etoricoxib
and 0.10 mg/ml of Pregabalin).
(v) Standard + placebo solution:
Weigh accurately about 135 mg of placebo, 60 mg of Etoricoxib working
standard and 75 mg of Pregabalin working standard into a clean 100 ml
volumetric flask. Dissolve in 25 ml of water and 0.2 ml of ammonia solution.
Sonicate for 5 minute, and make up the volume with the water (Concentration:
4.30 mg/ml of Placebo, 1.0 mg/ml of Etoricoxib and 0.10 mg/ml of Pregabalin).
 Acceptance criteria:
The placebo chromatogram should not show any peak at the retention time of
Etoricoxib and Pregabalin.

67
 Method Validation
1. Specificity:
Placebo solution was prepared separately at a concentration of 4.3 mg/ml with
matrix blend. Etoricoxib was prepared at its working concentration and analyzed as
per the method. Pregabalin was prepared at its working concentration and
analyzed as per the method. A typical chromatogram for specificity is shown in
Figure.
Summarizes the retention time and relative retention time values for placebopeaks,
Figure 7.1: Chromatogram of blank

68
Figure 7.2: Chromatogram of etoricoxib
Figure 7.3: Chromatogram of Pregabalin

69
Figure 7.4: Chromatogram of standard
Figure 7.5: Chromatogram of standard + placebo

70
Peak name Retention time
(minutes)
Blank peak 3.43
Etoricoxib 4.53
Pregabalin 9.79
Table 7.1: Summary of retention time and relative retention time values for
placebo peaks, Etoricoxib and Pregabalin.
 No peak was observed at the retention time of Etoricoxib and Pregabalin in
the chromatogram of placebo.

71
2. Linearity and Range:
(a) Linearity of Etoricoxib
The linearity of the RP-HPLC method was demonstrated for Etoricoxib ranging
from 40 µg/ml to 80 µg/ml. Five solutions at the concentrations within
the mentioned range were prepared and analyzed as per the method. The
linearity results obtained are shown in Figure shows the line of best fit for peak
area versus concentration of etoricoxib .
(b) Linearity of Pregabalin
The linearity of the RP-HPLC method was demonstrated for Pregabalin
solutions ranging from 25 µg/ml to 125 µg/ml. solutions at the concentrations
within the mentioned range were prepared and analyzed as per the method.
The linearity results obtained are in Figure shows the line of best fit for peak
area versus concentration of Pregabalin. A typicshownal chromatogram for
linearity is shown in

72
Figure 7.6: Overlay chromatogram of different concentration

73
Sr no Conc (µg/ml) Peak area
1 40 3202601
2 50 4162205
3 60 5122685
4 70 5859779
5 80 7028796
Correlation coefficient 0.9976
Table 7.4: Linearity of Etoricoxib
0
3202601
4162205
5122685
5859779
7028796
y = 86815x - 111409
R² = 0.9976
-1000000
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
0 10 20 30 40 50 60 70 80 90
Area
Con.
Figure 7.13: Linearity graph for Etoricoxib

74
Sr No Conc (µg/ml) Peak area
1 25 289460
2 50 501587
3 75 728768
4 100 929599
5 125 1154724
Correlation coefficient 0.9997
Table 7.5: Linearity of Pregabalin
0
289460
501587
728768
929599
1154724
y = 9075.7x + 43460
R² = 0.9997
0
200000
400000
600000
800000
1000000
1200000
1400000
0 20 40 60 80 100 120 140
Area
Con.
Figure7.6: Linearity graph for Pregabalin

75
3. Accuracy:
• The accuracy of the method was determined by using three solutions containing
placebo spiked with Etoricoxib and Pregabalin at approximately 80%, 100% and
120% of its working strength. Each level was analyzed as per the method.
Level Working strength Theoretical
Conc ( µg/ml)
Measured
Conc (µg/ml)
%Recovery
80%
60 48 48.1 101.43
60 48 48.2 101.09
60 48 48.0 101.44
100%
60 60 59.2 98.83
60 60 59.01 98.35
60 60 59.2 98.80
120%
60 72 72.1 101.75
60 72 72.0 101.71
60 72 72.1 101.38
Table 7.6: Accuracy of Etoricoxib
 The percentage recovery values were in the range of 98.35%- 101.75% which
is within the acceptance criteria.

76
Level Working
strength
Theoretical
Conc (µg/ml)
Measured
Conc (µg/ml)
% Recovery
80%
75 60 60.1 100.84
75 60 60.2 100.18
75 60 60.1 100.98
100%
75 75 75.0 100.70
75 75 75.0 100.01
75 75 74.6 99.58
120%
75 90 89.1 99.09
75 90 88.7 98.59
75 90 89.4 99.44
 The percentage recovery values were in the range of 98.59% - 100.98%
which is within the acceptance criteria.
Table 7.7: Accuracy of Pregabalin

77
4. Precision:
1. Repeatability
The data for repeatability of Peak Area measurement for Etoricoxib (60μg/ml) and
Pregabalin (75μg/ml) based on six measurements of same solution of Etoricoxib
(60μg/ml) and Pregabalin (75μg/ml). The % RSD for Etoricoxib and Pregabalin was
found to be
2. Intraday precision
Standard solution containing (50, 60, 70μg/ml) of Etoricoxib and (50, 75, 100μg/ml)
of Pregabalin were analyzed three times on the same day and % R.S.D was calculated.
3. Interday precision
Standard solution containing (50, 60, 70μg/ml) of Etoricoxib and (50, 75,
100μg/ml) of Pregabalin were analyzed three times on the different day and % R.S.D
was calculated.

78
Precision of Etoricoxib
Conc. (µg/ml) Mean peak area
(mAu*sec) ±SD (n=3)
% RSD
Intraday precision
50 417566±1.78 0.789
60 532568±1.98 1.124
70 586578±2.45 0.362
Interday precision
50 425566±3.89 1.305
60 543568±2.67 0.950
70 596578±3.78 0.572
Repeatability
% RSD
60 4985023±2.65 0.24

79
Precision of Pregabalin
% RSD
Intraday precision
50 501564±2.69 0.547
75 728546±3.69 0.754
100 928512±2.65 0.369
Interday precision
50 565864±2.09 0.863
75 723546±3.87 0.968
100 935612±3.59 0.652
Repeatability
% RSD
75 740081±4.23 0.60

80
• The LOD and LOQ of the developed method were determined by analyzing
progressively low concentration of the standard solution using the developed
methods.
• The LOD is the concentration of the analyte that gives a measurable response (signal
to noise ratio 3.3).
• The LOQ is the lowest concentration of the analyte, which gives a response that can be
accurately quantified (signal to noise ratio of 10).
5. LOD and LOQ
• Calibration curve was repeated for five times and the standard deviation of the
intercept was calculated. Then LOD and LOQ were calculated as follows,
Where, SD = Standard deviation of intercepts of 6 calibration curves
Slope = Mean slope of the 5 calibration curves

81
Etoricoxib Pregabalin
LOD = 3.3 x (SD / Slope)
=3.3 x (86815/111409)
=2.5715 μg/ml
LOD = 3.3 x (SD / Slope)
=3.3 x (9075.7/43460)
=0.6891μg/ml
Etoricoxib Pregabalin
LOQ = 10 x (SD / Slope)
=10 x (86815/111409)
=7.7924 μg/ml
LOQ = 10 x (SD / Slope
= 10 x (9075.7/43460)
= 2.088 μg /ml
Limit of Quantitation
Limit of Detection

82
5. Robustness:
The following table (Table 7.11) shows the parameters of the method that were altered to
test the robustness of the method. The robustness of the method is to be determined by
analyzing the standard solution six times with varying RP-HPLC conditions as described
below. A typical chromatogram for robustness is shown in Figure.
Parameter Actual Low High
Flow rate 1.00 ml/min 0.90 ml/min 1.10 ml/min
Mobile phase ratio 75 : 25 78 : 22 73 :27
Buffer pH 2.20 2.10 2.30
Column oven temperature 30°C 28°C 32°C
Table 7.11: Parameters altered for robustness test

83
6.1 Chromatographic conditions (Actual):
Column : C18, 250 X 4.6 mm, 5 μm or equivalent
Flow rate : 1.0 ml / min
Detection wavelength : 234 nm
Injection volume : 20 ml
Oven temperature : 30°C
Mobile phase : phosphate buffer: Acetonitrile (25:75).
Figure 7.20: Chromatogram of robustness-
standard (Actual)
sample (Actual)

84
6.2 Chromatographic conditions (Flow-Low):
Mobile phase : phosphate buffer: Acetonitrile (78 : 22).
standard (Flow low)
sample (Flow low)

85
6.3 Chromatographic conditions (Flow-High):
Mobile phase : phosphate buffer: Acetonitrile (73 :27)
standard (Flow high)
sample (Flow high)

86
Flow
Actual Low High
ET PRE ET PRE ET PRE
RT (min) 4.30-
4.53
9.09-
9.27
4.22-
4.23
8.12-
8.16
4.57-
4.59
10.16-
10.43
Area 4966028-
4991449
745914-
779360
4836134-
4872558
764132-
771434
4940974-
5020412
782565-
791915
Tailing
factor 1.19 1.05 1.21 1.03 1.21 1.01
Theoretical
plates 8586 14487 8604 13715 8586 14487
Average
(Area) 4978738 762637 4850959 772652 4967201 786025
%RSD
(Area) 0.27 0.65 0.28 0.73 0.59 0.43
Table 7.12: Summary of robustness results of standard (Flow)

87 Table 7.13: Summary of robustness results of sample (Flow)
Flow
Actual Low High
ET PRE ET PRE ET PRE
RT (min) 4.31-
4.40
9.09-
9.27
5.14-
5.22
11.29-
11.38
4.13-
4.15
8.99-
9.05
Area 5003737-
4966028
735393-
745914
5775729-
5640003
873982-
890362
4585191-
4469026
715423-
720801
Tailing
factor 1.23 1.05 1.19 1.02 1.20 1.03
Theoretical
plates 8638 14392 10171 16407 8501 14020
Average
(Area) 4985023 740081 5705806 885693 4519688 718206
%RSD
(Area) 0.24 0.60 0.98 0.73 1.14 0.38

88
 DISCUSSION
• The working condition for the RP-HPLC method were established for Pregabalin and
Etoricoxib then applied on pharmaceutical dosage form.
• Mobile phase system were prepared and used to provide ratio of Phosphate
Buffer: ACN (75:25) gave a better resolution and sensitivity. The retention time of
Pregabalin and Etoricoxib were found to be 9.79 and 4.53 respectively. The
asymmetry factor or tailing factor of Pregabalin and Etoricoxib is 1.05 and 1.23
respectively which indicates symmetrical nature of peak.
• The number of theoretical plates of Pregabalin and Etoricoxib was 14392 and 8638
respectively which indicates efficient performance of column. The retention time of
drugs were found to be within the limits of 0-10 minutes. These parameters
represent the specificity of method.

89
• From the linearity studies, specified concentration range was determined. It was
observed that Pregabalin and Etoricoxib were in the range of 50% - 150% each for
the target concentration. The linearity range is 40 – 80 µg/ml for Etoricoxib and 25 –
125 µg/ml for Pregabalin were found to obey linearity with correlation coefficient of
0.9976 for Etoricoxib and 0.9997 for Pregabalin. The validation for the proposed
method was verified by system precision and method precision. There was %RSD of
system precision for Pregabalin and Etoricoxib was 0.60 and 0.24 respectively. The
method precision was conducted and the % label claim was obtained as 103.71% for
Etoricoxib and 105.16% for Pregabalin. The % recovery value of 98.35% - 101.75%
for Etoricoxib and 98.58% - 100.98% for Pregabalin indicates non-interferences of
excipient in the formulation.
• The validation of the proposed method was verified by recovery studies. The
robustness studies were performed by changing the flow-rate, pH, column
temperature and mobile phase concentration. The ruggedness studies were
performed. All parameters including flow-rate, temperature, detector, wavelength and
sensitivity were maintained constant throughout the procedure. The analytical
method validation was carried as per the ICH guidelines.

90
 SUMMARY REPORT:
S.No Parameters Observation Acceptance Criteria
1 Specificity
Placebo Interference
No peak was observed at the
retention time of Etoricoxib
and Pregabalin in the
chromatogram of placebo
The placebo chromatogram
should not show any peak at
the retention time of
2
Linearity & Range
(a) Etoricoxib 0.9976
Correlation coefficient: > 0.995
(b) Pregabalin 0.9997
3
Accuracy
(a) Etoricoxib 98.35 – 101.75 %
98.0 – 102.0%
(b) Pregabalin 98.58 – 100.98 %
4
Precision (ET)
Intraday Precision
(%RSD, n=3)
0.75
% RSD: < 2.0
Interday Precision
(% RSD, n=3)
0.94
Repeatability (% RSD, n=6) 0.24

91
Sr. No Parameters Observation Acceptance criteria
Precision (PRE)
Intraday Precision (%RSD, n=3)
0.55
% RSD: < 2.0
Interday Precision(% RSD, n=3)
0.82
Repeatability (% RSD, n=6)
0.60
5
LOD
(a) Etoricoxib 2.57 >2 or 3
(b) Pregabalin 0.68
6
LOQ
(a) Etoricoxib 7.79 <10
(b) Pregabalin 2.08
7
Robustness
(a) Etoricoxib
1.19 – 1.23 Tailing factor: <2.0
8501 – 10171 Theoretical plates: > 2000
0.06 – 1.14 % RSD: < 2.0
(b) Pregabalin
1.00 – 1.05 Tailing factor: < 2.0
13715 –16403 Theoretical plates: > 2000
0.32 – 0.73 % RSD: < 2.0

92
 CONCLUSION
• C18 column as stationary phase and phosphate buffer: Acetonitrile (25: 75) as a
mobile phase and 234 nm is selected for the detection in this RP-HPLC.
• The system precision shows RSD value obtained was below 1 which indicate precision
and retention time for Etoricoxib and Pregabalin were 4.53 and 9.79 respectively.
• Quantitative estimation of Etoricoxib and Pregabalin gives accuracy which between
98.35% – 101.75% for Etoricoxib and 98.58% - 100.98% for Pregabalin.
• The percentage purity of Etoricoxib and Pregabalin lies in between 103.71% and
105.16% respectively.
• The proposed RP-HPLC method was simple, precise, rapid & accurate and involves
easy sample preparation. The linearity, reproducibility and recovery data confirms no
major interferences in the assay determination. So, this method can be used for
routine quality control analysis of this drug.

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