CONTENTS

Introduction
Aim And Objective
Review and Literature
Materials and Methods
Results And Discussions
Summary and Conclusion
References
 INTRODUCTION
 High Performance Liquid Chromatography (HPLC) is a process of separating
components in a liquid mixture. A liquid sample is injected into a stream of solvent
(Mobile phase) following through a column packed with a separation medium
(Stationary phase). Sample components separate from one another by a process of
differential migration as they flow through the column.
 As bands emerge from the column, flow carries them to one or more detectors which
deliver a voltage response as a function of time. This is called a Chromatogram. For
each peak, the time at which it emerges identifies the sample constituent with respect to a
standard. The peak’s area represents the quantity.
 Principle
 High Performance Liquid Chromatography (HPLC) is a process of separating
components in a liquid mixture. A liquid sample is injected into a stream of solvent
(Mobile phase) following through a column packed with a separation medium
(Stationary phase). Sample components separate from one another by a process of
differential migration as they flow through the column

 This is passed with slow speed and especially at packing granules were small enough to
efficient separation, then delivery under gravity decreases even up to few drops per minute.
The obvious way to increase the flow rate and get efficient separation to force the liquid by
positive displacement pump or by gas pressure. HPLC is a large resolution and highspeed
liquid chromatography. It is used for speedy resolution of complex mixture.
 INSTRUMENTATION
 Mobile Phase reservoir and Solvent treatment system
 Pumps
 Pre-columns
 Sample injectors
 Liquid chromatographic columns
 Detectors
1. U V – Visible detector
2. Refractive index detector
3. Fluorescence detector
4. Electro chemical detector
5. Mass detector
 AIM & OBJECTIVES OF THE
STUDY
AIM
Simultaneous Estimation Of Clonazepam And Escitalopram In Pure And Formulation
By Using RP-HPLC
OBJECTIVES
The main objectives of the present research work are;

 To perform method development for the drug,

 To determine the drug content in given pharmaceutical dosage form,
 To validate analytical methods as per ICH (Q2) guidelines,
 To demonstrate that it suitable for its intended pur
 REVIEW OF LITERATURE

Prawez Alam et al., (2022) The study aimed to develop a new reverse-phase high-performance liquid
chromatography (RP-HPLC) method with diode array detection (DAD) detection for simultaneous
estimation of escitalopram (EST) and clonazepam (CZP) in tablet dosage forms with a quality by design
(QbD) approach. The chromatographic conditions were optimized by Box-Behnken design (BBD) and
developed method was validated for the linearity, system suitability, accuracy, precision, robustness,
sensitivity, and solution stability according to International Council for Harmonization (ICH) guidelines.
EST and CZP standard drugs peaks were separated at retention times of 2.668 and 5.046 min by C-18
column with dimension of 4.6 × 100 mm length and particle size packing 2.5 µm. The mobile phase was
methanol: 0.1% orthophosphoric acid (OPA) (25:75, v/v), with a flow rate of 0.7 mL/min at temperature
of 26 °C. The sample volume injected was 20 µL and peaks were detected at 239 nm. Using the standard
calibration curve, the % assay of marketed tablet was founded 98.89 and 98.76 for EST and CZP,
respectively. The proposed RP-HPLC method was able to detect EST and CZP in the presence of their
degradation products, indicating the stability-indicating property of the developed RP-HPLC method.
The validation parameter’s results in terms of linearity, system suitability, accuracy, precision, robustness,
sensitivity, and solution stability were in an acceptable range as per the ICH guidelines. The newly
developed RP-HPLC method with QbD application is simple, accurate, time-saving, and economic. 38
 JAGDISH V. MANWAR ET AL., (2021) RP-HPLC METHOD WAS
DEVELOPED FOR SIMULTANEOUS DETERMINATION OF
ESCITALOPRAM OXALATE (ESC) AND FLUPENTIXOL HCL (FLU)
IN TABLET DOSAGE FORM. MOBILE PHASE CONSISTING OF
MIXTURE OF ACETONITRILE AND POTASSIUM PHOSPHATE
BUFFER (PH 7.0 WITH 0.1% TRIETHYLAMINE) IN THE RATIO 60:
40 AT FLOW RATE OF 1ML/MIN USING C18 GRACE (250MMX
4.6MM) COLUMN AT 231 NM. THE RETENTION TIME OF ESC
WITH FLU WAS FOUND TO BE 2.96 MIN AND 6.98 MIN,
RESPECTIVELY. THE LINEARITY RANGE FOR ESC WITH FLU
OBSERVED WAS 5-25 ΜG/ML AND 10-50 ΜG/ML, RESPECTIVELY.
METHOD WAS VALIDATED AS PER ICH GUIDELINES.
VALIDATION PARAMETERS STUDIED WERE LINEARITY AND
RANGE, RECOVERY STUDY, PRECISION, LOD, LOQ AND
ROBUSTNESS. STATISTICAL DATA OBTAINED WAS FOUND TO
SATISFACTORILY.39
 Drug Profile:
Drug : Escitalopram
Synonym : Escitalopram
Escitalopramum
Drug category : Anticholinergic Agents
Chemical name/ Nomenclature / IUPAC Name : (1S)-1-[3-(dimethyl amino) propyl]-1-(4-
fluorophenyl)-1,3-dihydro-2-benzofuran-5-carbonitrile.

Molecular Formula : C22H23FN2O5

Molecular Weight : 414.433 g/mol

Official Pharmacopoeia : USP

PHYSICOCHEMICAL PROPERTIES:
Description (Physical State): Escitalopram oxalate occurs as a fine, white to
slightly-yellow powder.
Solubility : Escitalopram was found to be freely soluble in
methanol and dimethyl sulfoxide (DMSO), soluble in isotonic saline solution,
sparingly soluble in water and ethanol, slightly soluble in ethyl acetate, and
insoluble in heptane.
Dosage : 10mg

Melting point : 152 to 153°C

pKa (strongest basic) : 9.78

Log P : 3.5

PHARMACOKINETIC PROPERTIES:

Bioavailability : 80%
Half-life : 27 to 32 hrs

Absorption : The absolute bioavailability of citalopram is about 80%

relative to an intravenous dose.

Volume of Distribution : 12 L/kg

Protein binding : 56 %

Metabolism : Mainly hepatic. Escitalopram undergoes N-

demethylation to S-demethyl citalopram (S-DCT) and S-didemethyl citalopram (S-
DDCT). CYP3A4 and CYP2C19 are the enzymes responsible for this N-demethylation
reaction.

Time of peak action : 3 to 6hr

 MATERIALS AND METHODS

INSTRUMENTS USED
CHEMICALS USED:
Table-: Instruments used
Table-: Chemicals used
S.No Instruments And Glass
Model
. wares S.No Chemical Brand names
WATERS Alliance 2695 separation
1 Escitalopram (Pure) Sura labs
1 HPLC module, Software: Empower 2, 996
PDA detector. 2 Clonazepam (Pure) Sura labs
2 pH meter Lab India
Water and Methanol for LICHROSOLV
3 Weighing machine Sartorius 3
HPLC (MERCK)
4 Volumetric flasks Borosil
4 Acetonitrile for HPLC Merck
5 Pipettes and Burettes Borosil
6 Beakers Borosil
7 Digital ultra sonicator Labman
Preparation of standard solution:

Accurately weigh and transfer 10 mg of Escitalopram and Clonazepam working standard into a
10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and
removal of air completely and make volume up to the mark with the same Methanol.

Further pipette 0.6ml of Escitalopram and 1ml of Clonazepam from the above stock solutions into
a 10ml volumetric flask and dilute up to the mark with Methanol.

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms,
note the conditions of proper peak elution for performing validation parameters as per ICH
guidelines.
Mobile Phase Optimization:

Initially the mobile phase tried was Methanol: Water and ACN: Water with
varying proportions. Finally, the mobile phase was optimized to Methanol: 0.1%
Orthophosphoric acid in proportion 64:36 v/v respectively.

Optimization of Column:
The method was performed with various C18columns like Symmetry, X
terra and ODS column. Symmetry ODS C18 (4.6mm×150mm) 5µm Particle Size
was found to be ideal as it gave good peak shape and resolution at 1ml/min flow
 OPTIMIZED CHROMATOGRAPHIC CONDITIONS:

Instrument used : Waters Alliance 2695 HPLC with PDA Detector 996
model.

Temperature : 38ºC

Column : Symmetry ODS C18 (4.6mm×150mm) 5µm Particle

Size

Mobile phase : Methanol: 0.1% Orthophosphoric acid

(64:36% v/v)

Flow rate : 1ml/min

Wavelength : 224nm

Injection volume : 20µl

 .
PREPARATION OF DRUG SOLUTIONS FOR LINEARITY:

Accurately weigh and transfer 10 mg of Escitalopram and Clonazepam working standard into a 10ml of
clean dry volumetric flasks add about 7ml of Diluents and sonicate to dissolve it completely and make
volume up to the mark with the same solvent. (Stock solution)

Preparation of Level – I (20ppm of Escitalopram and 60ppm of Clonazepam):

Pipette out 0.2ml of Escitalopram and 0.6ml of Clonazepam in to a 10ml volumetric flask and make the
volume upto mark by using diluent and sonicate for air entrapment.

Preparation of Level – II (40ppm of Escitalopram and 80ppm of Clonazepam):

Pipette out 0.4ml of Escitalopram and 0.8ml of Clonazepam in to a 10ml volumetric flask and make the
volume upto mark by using diluent and sonicate for air entrapment.
Preparation of Level – III (60ppm of Escitalopram and 100ppm of Clonazepam):

Pipette out 0.6ml of Escitalopram and 1ml of Clonazepam in to a 10ml volumetric flask and make
the volume upto mark by using diluent and sonicate for air entrapment.

Preparation of Level – IV (80ppm of Escitalopram and 120ppm of Clonazepam):

Pipette out 0.8ml of Escitalopram and 1.2ml of Clonazepam in to a 10ml volumetric flask and
make the volume upto mark by using diluent and sonicate for air entrapment.

Preparation of Level – V (100ppm of Escitalopram and 140ppm of Clonazepam):

Pipette out 1ml of Escitalopram and 1.4ml of Clonazepam in to a 10ml volumetric flask and make
the volume upto mark by using diluent and sonicate for air entrapment.
 PRECISION

REPEATABILITY

Preparation of Escitalopram and Clonazepam Product Solution for Precision:

Accurately weigh and transfer 10 mg of Escitalopram and Clonazepam working standard into a 10ml of
clean dry volumetric flasks add about 7ml of Diluents and sonicate to dissolve it completely and make
volume up to the mark with the same solvent. (Stock solution)

Further pipette out 0.6ml of Escitalopram and 1ml of Clonazepam from the above stock solutions into a
10ml volumetric flask and dilute up to the mark with Diluent.

The standard solution was injected for five times and measured the area for all five injections in HPLC. The
%RSD for the area of five replicate injections was found to be within the specified limits.
INTERMEDIATE PRECISION:

To evaluate the intermediate precision (also known as Ruggedness) of the method, Precision was
performed on different days by maintaining same conditions.

Procedure:

DAY 1:

The standard solution was injected for Six times and measured the area for all Six injections in HPLC. The
%RSD for the area of Six replicate injections was found to be within the specified limits.

DAY 2:

The standard solution was injected for Six times and measured the area for all Six injections in HPLC. The
%RSD for the area of Six replicate injections was found to be within the specified limits.
ACCURACY:

For preparation of 50% Standard stock solution:

Accurately weigh and transfer 10mg of Escitalopram and Clonazepam working standard into a 10ml of clean
dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make volume up
to the mark with the same solvent. (Stock solution)

Further pipette out 0.3ml of Escitalopram and 0.5ml of Clonazepam from the above stock solutions into a
10ml volumetric flask and dilute up to the mark with Diluent.

For preparation of 100% Standard stock solution:

Accurately weigh and transfer 10 mg of Escitalopram and Clonazepam working standard into a 10ml of
clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it completely and make
volume up to the mark with the same solvent. (Stock solution)

Further pipette out 0.6ml of Escitalopram and 1ml of Clonazepam from the above stock solutions into a
10ml volumetric flask and dilute up to the mark with Diluent.
FOR PREPARATION OF 150% STANDARD STOCK SOLUTION:
ACCURATELY WEIGH AND TRANSFER 10 MG OF ESCITALOPRAM
AND CLONAZEPAM WORKING STANDARD INTO A 10ML OF CLEAN
DRY VOLUMETRIC FLASKS ADD ABOUT 7ML OF DILUENTS AND
SONICATE TO DISSOLVE IT COMPLETELY AND MAKE VOLUME UP
TO THE MARK WITH THE SAME SOLVENT. (STOCK SOLUTION)
FURTHER PIPETTE OUT 0.9ML OF ESCITALOPRAM AND 1.5ML OF
CLONAZEPAM FROM THE ABOVE STOCK SOLUTIONS INTO A 10ML
VOLUMETRIC FLASK AND DILUTE UP TO THE MARK WITH
DILUENT.
ROBUSTNESS:

The analysis was performed in different conditions to find the variability of test results. The
following conditions are checked for variation of results.

For preparation of Standard solution:

Accurately weigh and transfer 10 mg of Escitalopram and Clonazepam working standard into
a 10ml of clean dry volumetric flasks add about 7mL of Diluents and sonicate to dissolve it
completely and make volume up to the mark with the same solvent. (Stock solution)

Further pipette out 0.6ml of Escitalopram and 1ml of Clonazepam from the above stock
solutions into a 10ml volumetric flask and dilute up to the mark with Diluent.
Effect of Variation of flow conditions:

THE SAMPLE WAS ANALYZED AT 0.9 ML/MIN AND 1.1 ML/MIN INSTEAD
OF 1ML/MIN, REMAINING CONDITIONS ARE SAME. 20ΜL OF THE ABOVE
SAMPLE WAS INJECTED AND CHROMATOGRAMS WERE RECORDED.
EFFECT OF VARIATION OF MOBILE PHASE ORGANIC COMPOSITION:
THE SAMPLE WAS ANALYZED BY VARIATION OF MOBILE PHASE I.E.
METHANOL: 0.1% ORTHOPHOSPHORIC ACID (64:36% V/V) WAS TAKEN IN
THE RATIO AND 69:31, 59:41 INSTEAD OF 64:36 REMAINING CONDITIONS
ARE SAME. 20ΜL OF THE ABOVE SAMPLE WAS INJECTED AND
CHROMATOGRAMS WERE RECORDED.
RESULTS AND DISCUSSION
Trial 1:
Mobile phase : Methanol + Water (40:60%v/v)
Column : Thermo BDS Hypersil C18 column having 250 x
4.6mm 5μ,
Flow rate : 0.5ml/min
Wavelength : 224 nm
Column temp : Ambient
Injection Volume : 10 µl
Run time : 6minutes

USP USP USP

S. Peak Heigh
Rt Area Resol Taili Plate
No Name t
ution ng count

Escitalo 2.47 64874 5879

1 1.24 1254
pram 6 5 4

Figure-: Chromatogram for

Trail 2:
Mobile phase : Acetonitrile: Methanol (40% -60%v/v)
Column : Zorbax C18 (4.6mm×250mm) 5µmparticle size
Flow rate : 0.8 ml/min
Wavelength : 224 nm USP USP USP
S. Peak Hei
Column temp : Ambient Rt Area Resolu Tailin plate
No. Name ght
Sample Temp : Ambient tion g count
Injection Volume : 10 µl Escitalop 2.4 5365 598 1.13 4265
Run time : 9 minutes 1
ram 85 21 98

Clonaze 4.0 8658 879 1.26 3412

2 2.35
pam 30 452 84
Table: Peak Results for Trail 2

Table: Peak Results for Trail 2

Trail 3:
S. USP USP USP
Mobile phase : Acetonitrile: 0.1% Peak Hei
N Rt Area Resol Taili plate
Orthophosphoric acid (70:30% v/v) Name ght
o ution ng count

Column : Develosil C18 Clonazep 1.5 1852 523

1 1.26 2653
(4.6mm×250mm, 5µm) am 73 456 65
Flow rate : 1.0 ml/min 2
Escitalop 4.6 1258 254
4.35 0.92 4536
Wavelength : 224 nm ram 11 75 6
Column temp : Ambient
Sample Temp : Ambient Figure-: Chromatogram for Trail 3
Injection Volume : 0.9µl/min
Run time : 10 minutes

Table: Peak Results for

Trail 3
Trail 4:
Mobile phase : Methanol: 0.1% Orthophosphoric acid (50:50% v/v)
Column : Symmetry ODS C18 (4.6mm×150mm) 5µm Particle Size
Flow rate : 1.0 ml/min
Wavelength : 224 nm
Column temp : 360C
Sample Temp : Ambient
Injection Volume : 1.0µl/min
Run time : 10 minutes

USP USP USP

S. Peak Hei
Rt Area Resol Taili plate
No Name ght
ution ng count
426 32
Clonaze 6.0 1365.
1 598 56 0.96
pam 25 6
5 85
45
Escitalo 7.9 536
2 68 2.51 1.08 3856.5
pram 77 25
5
Figure-: Chromatogram for Table: Peak Results for Trail 4
Trail 4
SUMMARY AND CONCLUSION

OPTIMIZED CHROMATOGRAPHIC CONDITIONS:

INSTRUMENT USED : WATERS ALLIANCE 2695 HPLC WITH PDA DETECTOR
996 MODEL.
TEMPERATURE : 38ºC
COLUMN : SYMMETRY ODS C18 (4.6MM×150MM) 5ΜM PARTICLE SIZE
MOBILE PHASE : METHANOL: 0.1% ORTHOPHOSPHORIC ACID (64:36%
V/V)
FLOW RATE : 1ML/MIN
WAVELENGTH : 224NM
INJECTION VOLUME : 20ΜL
RUN TIME : 7.0MINUTES
 BIBLIOGRAPHY

1. Settle FA, In: Handbook of Instrumental Techniques for Analytical Chemistry. 1st ed,
Singapore, Pearson Education Inc.2004.

2. Willard HH and Dean AJ. Instrumental Methods of Analysis. CBS Publishers and
distributors, 7th ed, 1986, 513-515.

3. Connors AK. In: A Text Book of Pharmaceutical Analysis. A Wiley Interscience

Publication, 3rd ed, 2005, 373-400.

4. Ahuja S. In: High Pressure Liquid Chromatography of Comprehensive Analytical

Chemistry. Elsevier Publications. 2006.

5. Principles and Methods. In: Amesham Biosciences of Reversed Phase Chromatography.

THANK YOU