Complete Project Edited

CHARACTERIZATION OF L_ASPARAGINASE
A Dissertation submitted to
JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY

HYDERABAD- 500085.
For the partial fulfillment of the degree of
MASTER OF SCIENCE
IN
CHEMISTRY (ANALYTICAL CHEMISTRY)
BY
S.VIKRAM
(16031G2224)
Under the guidance of
T.SPANDANA
HEAD OF THE ANALYTICAL R & D
SAJJALA BIO LABS PVT.LTD
PLOT NO.8, ALEAP INDUSTRIAL ESTATES,
GAJULARAMARAM, MEDCHAL
MALKAJGIRI DIST, TELANGANA
CENTRE FOR CHEMICAL SCIENCES AND TECHNOLOGY

INSTITUTE OF SCIENCE & TECHNOLOGY
(AUTONOMOUS)
HYDERABAD
KUKATPALLY, HYDERABAD – 500085. T.S. (India)
1
(AUTONOMOUS)
HYDERABAD
Certification
Certified that the dissertation work entitled “CHARACTERIZATION OF
L_ASPARAGINASE submitted by S.VIKRAM in partial fulfillment of the
Requirements of Master of Science in Chemistry (Analytical Chemistry) is a bonafied
work and can be placed before the examination board for their consideration.
HEAD OF THE DEPARTMENT
Prof. A. JAYA SHREE
PROJECT SUPERVISOR EXTERNAL EXAMINER
2
REPORT
Title : “CHARACTERIZATION OF L_ASPARAGINASE
Duration : January 10th 2018 TO June 10th 2018
Name : S.vikram
Roll no : 16031G2224
Course : M.Sc. Chemistry (Analytical Chemistry)
Institute : Centre for Chemical Sciences & Technology,

Institute of science and technology (IST),
Jawaharlal Nehru Technological University
(JNTUH), Kukatpally, Hyderabad –500085.
Place of Work : Sajjala Bio Labs Pvt.Ltd
Plot no:8, ALEAP Industrial Estates,
Gajularamaram, Medchal
Malkajgiri Dist, Telangana
3
(AUTONOMOUS)
HYDERABAD
Declaration by the candidate
I, here by certify that the project report entitled “Characterization of

L_ASPARAGINASE” carried out under the guidance and supervision of T.Spandana
Head Of The Analytical R & D Sajjala Bio Labs Pvt.Ltd in partial fulfillment of
the requirements of the award of the degree of Master of Science in Analytical Chemistry
to the faculty of Center for Chemical Sciences and Technology and this is the bonafide
work has not been submitted for the award of any other degree or diploma of this university.
Date: S.VIKRAM
Place: (16031G2224)
4
Acknowledgement
The Satisfaction that accompanies the completion of any task would be

incomplete without the mention of the people who made it possible and whose
constant encouragement and guidance has been a source of inspiration throughout
the course of this project. I take this opportunity to express my gratitude to all
those who have helped us in this project.
I take extreme pleasure to express my deep sense of Gratitude to my project

guide, T.Spanadana Garu Head of the Analytical R&D, Sajjala Bio Labs
Pvt.Ltd ALEAP Industrial Estates Gajularamaram Malkajgiri dist, Hyderabad.
For her inspiring guidance and encouragement throughout the preparation and
progress of the project.
I extend my thanks to Prof. A. Jaya Shree, Prof. B. Sreenivasulu, Dr.
Sadanadam Palle, Dr. Jyothi Vantikommu, Acdaemic Adviser,Mr. B.f.
Mathews for his constant encouragement and guidance, Mr. A. Tejeswara Rao, Mr.
M. Shankaraiah, Mr. P. Naveen, I am greatful to teaching staff, research scholars,
and non-teaching staff, Centre for Chemical Sciences and Technology, I.S.T, J.N.T.
University Hyderabad for their cooperation.
My special thanks to my friends and all my classmates for their help in all
stages of my project work and all those who have directly and indirectly
contributed for the completion of this project.
S.VIKRAM
5
INDEX
S.NO NAME OF THE CHAPTER PAGE.NO.
1. ABSTRACT 7
2 INTRODUCTION 8-30
3 DRUG PROFILE 31-32
4 EQUIPMENT AND CHEMICALS 33
5 METHOD DEVELOPMENT 34-37
6 STANDARD TESTING PROCEDURE 38-45
7 RESULTS AND DISCUSSION 46-55
8 SUMMARY 56
9 BIOGRAPHY 57
6
ABSTRACT
L-Asparginase is an enzyme that acts as an effective anti-tumor agent used for the
remission and improvement of long term survival in patients with acute
lymphoblastic leukemia. In the present study, we have developed simple simple,
sensitive and rapid chromatographic methods for the determination of the purity of
L-Asparaginase in pharmaceutical preparations by using HPLC-PDA technique.
The purity of the L-asparaginase was determined by HPLC-PDA system connected
to UV-detector and reverse phase-C18 column. A low pressure gradient mobile
phase consisting of 0.2% TEA in water (Buffer-A) and acetonitrile and water in the
ratio of 95:5 mixture (Buffer-B) was used for the determination of purity at 225nm.
The observed purity was found to be 98.82%. The HPLC method was also
validated in terms of accuracy, precision, degradation, specificity, linearity and
robustness. The results obtained were within the acceptance criteria.
7
INTRODUCTION
1.1 Analytical Chemistry:
Chemistry is the branch of science that deals with the composition, structure and
properties of the substance and their transformations which they undergo in a
reaction.
Analytical chemistry is a branch of chemistry which analyzes the chemical

composition of natural and artificial materials by using a set of instruments and
methods to separate, identify and quantify.
Pharmaceutical analysis is a branch of analytical chemistry that involves in

separation, identification, quantification and determination of relative substance
present in the compound.
Pharmaceutical analysis plays a crucial role in providing assurance of quality,

safety and efficiency of the new drug.
Method of analysis is routinely developed to validate collaborative study and

optimize the analysis both quantitatively and qualitatively.
QUALITATIVE ANALYSIS:
It is a branch of chemistry that deals with the identification or grouping of
elements based on their chemical and physical properties such as chemical
reactivity, solubility, molecular weight, melting point, radioactivity properties
(emission & absorption) and mass spectra etc.,
More often a substance is complex mixture hence systematic analysis must be
made in order to identify all constituents present in it.
QUANTITATIVE ANALYSIS:
It is a branch of chemistry that deals with the analysis of determination of amount
or concentration of an analyte and expresses in a numerical value in appropriate
units.
Instrumental method of chemical analysis:
8
Instrumental method of chemical analysis deals with all the areas of chemistry and
other areas of pure applied science.
An analytical technique plays an important role in

 Production and evaluation of new drug and its formulation and estimation of
biological fluids.
 Accelerated stability studies of the drug.
 Detection and quantification of impurities and metabolites.
 In-vitro dissolution studies.
 Pharmacokinetics and drug metabolism studies.
 Determination of bioavailability of two or more formulation.
Methods of estimation of drugs are divided into
1. Physical methods.
2. Chemical methods.
3. Physicochemical methods.
A. Physical methods: physical method of analysis involves the studying of
physical properties of the substance such as determination of the solubility
transparency or degree of turbidity, specific gravity, moisture content, melting and
freezing points.
B. Chemical methods: A chemical method of analysis includes the gravimetric

and volumetric procedures which are based on the complex formation, acid base
precipitation and redox reaction.
1.2 TYPES OF CHEMICAL ANALYSIS:
1 .Proximate analysis: The amount of each element in a sample is determined

with no concern with the actual compound present.
2. Partial analysis: Deals with the determination of selected constituent in the
9
sample.
3. Trace constituent analysis: Concerned with the determination of specified

compounds present in minute quantity.
4. Complete analysis: Deals with the determination of each proportion of

component present in the sample.
5. Physiochemical Methods: Physiochemical methods include
A. SPECTROPHOTOMETRIC TECHNIQUES:
 UV-Visible spectrophotometric techniques

 Fluorescence and Phosphorescence techniques
 Atomic spectrophotometric
 Infrared spectrophotometry
 X-ray spectrophotometry
 Nuclear magnetic resonance spectroscopy
 Mass spectroscopy
 Electro spin resonance spectroscopy
B. ELECTROCHEMICAL TECHNIQUES.
 Potentiometry
 Voltametry
 Electrogravimetry
 Conductometry
 Amperometry
C. CHROMATOGRAPHIC TECHNIQUES
10
 High performance liquid chromatography
 Gas chromatography
 High performance thin layer chromatography
 Thin layer chromatography
 GC-MS
 LC-MS
D. MISCELLANEOUS TECHNIQUES
 Thermal analysis
 Kinetic technique
 Electrophoresis
 Micro plate reader
1.3 Introduction to High Performance Liquid Chromatography
Chromatography may be defined as a non destructive procedure for separating

mixture of components into individual components through equilibrium
distribution between two phases."
The technique of chromatography is based on the difference in the rate at which
the components of a mixture move through a porous medium under the influence
of the solvent or gas.
CLASSIFICATION OF CHROMATOGRAPHIC PROCESS

ADSORPTION CHROMATOGRAPHY
 Gas-solid chromatography
 Liquid-column chromatography
 Thin layer chromatography
11
PARTITION CHROMATOGRAPHY
 Gas-liquid chromatography
 Super critical fluid chromatography
 Paper chromatography
ION EXCHANGE CHROMATOGRAPHY
PERMEATION CHROMATOGRAPHY
 Size exclusion chromatography
AFFINITY CHROMATOGRAPHY
 DNA affinity chromatography
ELECTROPHORESIS
 Capillary electrophoresis
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

High performance liquid chromatography (HPLC) has also been reffered to as high
pressure liquid chromatography.
HPLC is a separation technique that involves:
 The injection of a small volume of liquid sample into a tube packed with
particles 3 to 6 micron (µm) in diameter called the stationary phase where
individual components of the sample are moved down the packed tube
(column) with a liquid (mobile phase) forced through the column by high
pressure delivered by a pump.
 These components are separated from one another by the column packing
that involves chemical and/or physical interactions between their molecules
and the packing particles. These separated components are detected at the
exit of this tube (column) by a flow-through device (detector) that measures
their amount. An output from this detector is called as “liquid
chromatogram”.
12
1.3. TYPES OF HPLC TECHNIQUES:
A. BASED ON PRINCIPLE OF SEPARATION

 Adsorption
 Ion exchange
 Size exclusion
 Affinity
 Chiral phase
B. BASED MODES OF CHROMATOGRAPHY
 Normal phase chromatography
 Reverse phase chromatography
C. Based On Elution Technique
 Isocratic elution
 Gradient elution
D. BASED ON SCALE OF OPERATION
 Analytical HPLC
 Preparative HPLC
REVERSE PHASE HPLC:
Reverse phase HPLC (RP-HPLC or RPC) has a non-polar stationary phase and an
aqueous, moderately polar mobile phase. One common stationary phase is silica
which has been treated with RMe2sicl, where R is a straight chain of alkyl group
such as C18H37 or C8H17. With these stationary phases, retention time is longer
for molecules which are less polar, while polar molecules elute more readily.
13
Fig: 1.1
Principe:
The separation mechanism in reversed phase chromatography depends on the
hydrophobic binding interaction between the solute molecule in the mobile phase
and the immobilized hydrophobic ligand, i.e. the stationary phase. The solute
molecules partition (i.e.an equilibrium is established) between the mobile phase
and the stationary phase. The distribution of the solute between the two phases
depends on the binding properties of the medium, the hydrophobicity of the solute
and the composition of the mobile phase.
Chemically bounded octadecyl silane (ODS) and alkaline with 18 carbon atoms is
the most popular stationary phase used in pharmaceutical industry. Organic
solvents water and buffers are used as mobile phase.
HPLC is a method of choice in the field of analytical chemistry since the method is
specific, robust, linear, precise and accurate; LOD is low and also offers
1. Speed (many analysis may be accomplished in 20min)

2. Greater sensitivity ( various detectors can be employed)
3. Improved resolution
4. Reusable column
5. Ideal for substance of low viscosity
6. Easy sample recovery

7. Precise and reproducible
14
1.4 INSTRUMENTATION
Fig:1.2
1. Solvent delivery system:
 The mobile phase is pumped under pressure from one or several reservoirs
and flows through the column at a constant rate.
 With micro particulate packing, there is a high pressure drop across a

chromatographic column.
 Eluting power of the mobile phase is determined by its overall polarity, the
polarity of the stationary phase and the nature of sample components.
 For normal phase separations, eluting power increases with increasing

polarity of the solvent.
 For reverse phase separations, eluting power increases with increasing

solvent polarity.
 Optimum separating conditions can be achieved by making use of mixture

of two solvents.
 The properties of the solvent which need to be considered for a successful

separation are boiling point, viscosity, detector compatibility, flammability
15
and toxicity.
2. PUMP:
The most important component of HPLC in solvent delivery system is the

pump, because its performance directly effects the retention time,
reproducibility and detector sensitivity.
  The role of the pump is to force a liquid (called the mobile phase) through
the liquid chromatography at a specific flow rate, expressed in milliliters per
min (ml/min)
  Normal flow rates in HPLC are in the 1-2ml/min range.
  Typical pumps can reach pressures in the range of 6000-9000psi (400 to
600 bar).
  During the chromatographic experiments, a pump can deliver a constant
mobile phase composition (isocratic) or an increasing mobile phase
composition (gradient).
 Classification of pumps:
 a. Constant pressure
 b. Constant flow pump
 The constant pressure pump is used only for column packing.
 The constant flow pump is the most widely used in all common HPLC
applications.
 A. Constant pressure pumps:
 Advantages:
  Simplicity
  Freedom from pulsations, resulting in smooth baselines
  Cheap and easy to maintain
 Drawbacks:
  Flow rates can’t be changed.
  Flow rates can be changed if the solvent viscosity changes due to either
temperature or composition changes.
 Changes in flow rate can influence both qualitative and quantitative
analysis.
 In qualitative analysis, components identification is primarily based on
matching retention columns. If these changes as a result of flow rates
16
changes, especially during the analysis of complex mixtures, component
location and identification becomes difficult.
 In quantitative analysis, the most common detectors (UV and refractive
index), are concentration sensitive. Changes in the flow rate affect the time
that a chromatographic band passes through the detector cell, which in turn
show up as changes in peak area, which is used for quantitation.
 B. Constant flow pump:
 Constant flow systems are generally of two basic types:
 a. Reciprocating pump
 b. Positive displacement pump
 Reciprocating pump:
 Reciprocating piston pump can maintain a liquid flow for indefinitely long
time. The pumping rate is controlled by piston retracts or by the cam
rotating speed.
 The main draw backs of this pump are a sinusoidal pressure pulsation which
leads to the necessity of the using pulse dampers.
 Dual piston pumps:
 Provide a constant and almost pulse free flow.
 Both the pump chambers are drivers by the same motor through a common
eccentric cam, this common drives allows the one piston to pump while the
other is refilling.
 As a result the two flow profiles, acceleration/deceleration overlap each
other significantly reducing the pulsation downstream of the pump, and this
is visualized below.
 Since the acceleration/deceleration profile is somewhat non linear, the more
efficient types of these pumps use eccentricity shaped cams to obtain the
best overlapping of the pressure curves and to obtain smooth flow.
 Advantages:
  Unlimited solvent reservoir allowing long term unattended use.
  Quick changeover and clean out capability
  Wide flow rate range (0.01 to 10ml/min) is provided without gear change.
 Draw backs:
  Incompletely compensated pulsations might be observable at high
refractive index, detector sensitive’s, especially at flow rates.
17
  Pump reliability depends on the cleanliness of the mobile phase and
continued sealing capability of four check values on each cycle (eg: several
times per minute).
 Recent improvements include:
  A computer designed cam shaft is used to achieve maximum overlap of
pump strokes, resulting in virtually undetectable pulsation or ripple.
  Small volume check valves are used to allow the pumps to function
reliably at flow rates as low as 0.001ml/min.
 Check valves:
 Check valve on the reciprocating pump are the most of the recent HPLC
instruments with the improved dual piston pumps which have three or even
two check valves.
 Working cycle:
  The first piston (low pressure) sucks the liquid from the reservoir.
  The second (high pressure piston) is supplying eluent to the system.
  The first piston refills the second piston very fast, during 1/100 of the
whole pump cycle.
 Two valve design:
 Working cycle:
  The smaller piston dispenses an eluent in the HPLC system and the bigger
piston is sucking the eluent.
  The bigger piston simultaneously refills the smaller chamber and
dispenses eluent into the system.
 Syringe type pumps:
 Syringe type pumps generally consist of a cylinder that holds the mobile
phase which is expelled by a piston. The piston is advanced by a motor
connected through worm gears to provide smooth pulse less flow.
 Advantages:
  High pressure capability (up to 78000psi).
 Constant flow rate
  Infrequent maintenance
  Simple and strong gears.
 Draw backs:
18
  Limited possibility to form gradients
  Limited reservoir capacity
Syringe pumps are now mostly used for SFC (super critical fluid
chromatography) and micro column chromatography.
 Two of the syringe pump can be combined with electric control.
  Gradient – programming operation is possible.
  Continuous elution can be arranged
 III. Mobile phase:
 Mobile phases used for HPLC are typical mixtures of organic solvents and
water or aqueous buffer. Table given below lists the physical properties of
organic solvents commonly used for HPLC, isocratic methods are
preferable more than the gradient methods. Gradient methods will
sometimes be required when the molecule which has to be separated have a
vastly different partition properties. When a gradient elution is used care
must be taken to ensure that all solvents are miscible.
 The following points should also be considered when choosing a mobile

phase:
 1. It is essential to establish that the drug is stable in the mobile phase for at
least the duration of the analysis.
 2. Excessive salt concentration should be avoided. High salt concentration
can results in precipitation, which can damage HPLC equipment.
 3. The mobile phase should have a pH 2.5 to 7.0 for maximize lifetime of
the column.
 4. Reduce cost and toxicity of the mobile phase by using methanol instead
of acetonitrile when possible.
5. Minimize the absorbance of buffers. Since tri flouroacetic acid, acetic acid
or formic acid absorbs at shorter wavelengths, they may prevent detection of
products without chromophores above 220nm.
19
PHYSICAL PROPERTIES OF COMMON HPLC SOLVENTS
SOLVENT MW BP RI UV Density Viscosity Dielectric

cut off constant
g/ml cp
(nm)
Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8
Dioxane 88.1 101 1.420 215 1.034 1.26 2.21
Ethanol 46.1 78 1.359 205 0.789 1.26 24.5
Ethyl 88.1 77 1.372 256 0.901 0.450 6.02

Acetate
Methanol 32.0 65 1.326 205 0.792 0.584 32.7
Dichloro 84.9 40 1.424 233 1.326 0.44 8.93

Dimethane
Iso propanol 60.1 82 1.375 205 0.785 2.39 19.9
n-propanol 60.1 97 1.383 205 0.840 2020 20.3
THF 72.1 66 1.404 210 0.889 0.51 7.58
Water 18.0 100 1.333 170 0.998 1.00 78.5
Table:1.1
20
The constituents of the flexible stage should be degassed and filtered before use.
Two adjustment of the portable stage can be helpful in turn around stage HPLC for
Ionizable compound. One is called particle suspension and the other particle
matching chromatography. In the two methods a cushion is utilized to guarantee
that the pH of the arrangement is steady and generally in any event 1.5 pH units
from a Pka of the medication to guarantee that one type of prevails. On the off
chance that pH is roughly equivalent to Pka , crest expanding can happen. In
particle detachment chromatography, the pH of the watery bit of the portable stage
is changed in accordance with permit the unbiased type of the medication to
prevail. This guarantees the medication is persevering in just a single frame and
result in enhancement of the pinnacle shape and consistency of maintenance time.
In particle matching chromatography the pH of the versatile stage is balanced so

the medication is totally ionized. On the off chance that important to enhance crest
shape or stretch maintenance time, an alkyl sulphonic corrosive salt or massive
anion, for example, triflouro acidic corrosive is added to the particle combine to
cationic medications or an alkyl ammonium salt is added to particle match or
anionic medications.
IV. Dissolvable degassing framework:
The constituents of the versatile stage ought to be degassed and sifted before
utilize. A few strategies are utilized to expel the disintegrated gases in the versatile
stage. They incorporate warming and mixing, vacuum degassing with a suction
apparatus, filtration through 0.45µ channels, vacuum degassing with air
dissolvable film, helium cleansing or mix of these strategies. HPLC frameworks
are additionally given an internet degassing framework, which consistently expels
the broken up gases from the portable stage.
21
V. Test presenting framework
Two means for analyte presentation on the section are infusion into a streaming
stream and a stop stream infusion. These systems can be utilized with a syringe or
an infusion valve. Programmed injector is a miniaturized scale processor controlled
rendition f manual general injector. More often than not up to 100 examples can be
stacked into the auto injector plate. The framework parameters, for example,
stream rate, slope, run time, volume to be infused, and so forth are picked, put
away in memory and successively executed on back to back infusion.
Injector:
•The injector serves to bring the fluid into the stream of the portable stage.
• Typical test volumes are 5 to 20 microlitres (µL).
• Available in programmed injectors and manual injectors.
1. Manual injector:
Client physically stacks test into the example into injector utilizing a syringe. And
after that transforms the handle to infuse test into the streaming versatile stage
which transport the example into the starting (head) of the section, which is at high
weight.
Focal points:
Fast, reproducible, administrator free conveyance of an extensive variety of test

volumes (eg. Upto 60mL up to a few ml) at weight up to 7000psi with under 0.2%
blunder.
22
Disadvantage:
The example circle must be changed to acquire different example volumes

(however this can frequently be accomplished in almost no time).
2. Programmed injector:
• With monetarily accessible programmed testing gadget vast number of tests can
be routinely broke down by LC without an administrator intercession.
• Such hardware is well known for the investigation of routine examples (eg.
Quality control of medications) especially when combined with programmed
information taking care of frameworks. Programmed injectors are vital in
unattended looking (eg. medium-term) for chromatographic parameters, for
example, dissolvable selectivity, stream rate and temperature advancement.
• User loads vials are loaded up with test arrangement into the auto sampler plate
(100 examples) and the auto sampler naturally measures the proper example
volume, infuses the example, at that point flushes the injector to be prepared for
the following example and so on, until the point when all examples vials are
handled. An auto sampler is the programmed rendition for when the client has
numerous examples to break down or when manual infusion isn't handy.
VI. Segment:
Segment is considered as the "heart of the chromatograph". The segment stationary

stage isolates the example parts of enthusiasm utilizing different physical and
substance parameters. The little particles inside the segment are what cause the
high backpressure at ordinary stream rates. The pump must push hard to move the
portable stage through the section and this opposition causes a high weight inside
23
the chromatography.
Sorts of section in HPLC:
• Analytical (inner distance across 1.0-4.6mm; lengths 15-250mm)
• Preparative (i.d.>4.6mm;length 50-250mm)
• Capillary (i.d.0.1-1.0mm; different lengths)
• Nano (i.d.<0.1mm, or some of the time expressed as <100µm)
Materials of development for the tubing:
• Stainless steel (the most prominent; gives high weight capacities)
• PEEK polymer (biocompatible and artificially idle to generally solvents)
• Glass (generally for biomolecules)
• Columns are pressed with little width permeable particles. The most prominent
sizes are 5µm, 3.5µm and 1.8µm.
• Columns are stuffed utilizing high strain to guarantee that they are steady amid
utilize most clients buy pre-pressed sections to use in their fluid chromatographs.
These permeable particles in the section normally have a synthetically fortified
stage on their surface which cooperates with the example segments to isolate them
from each other.
• The procedure of maintenance of the example segments (frequently called

analyte) is controlled by the decision of section pressing and the choice of the
versatile stage to push the analytes through the stuffed segment.
24
BONDED PHASES FOR HPLC AND ABBREVIATIONS
PHASE DESCRIPTION
Si Silica
Classic normal phase material. Suitable for separating polar non-
ionic organic compounds.
C1 TMS SAS (trimethyl silane)

Reversed phase material. Unique selectivity for polar and
multifunctional compounds. Least retentive of all alkyl group
bonded phases for non polar solvents.
C2 RP-2Dimethyl
Reversed phase material, less retentive than C4, C8 or C18. More
retentive than C1.
C3 Propyl
Reversed phase material, used in hydrophobic interaction
chromatography (HIC) of proteins and peptides.
C4 Butyl
Reversed phase material, useful for ion pairing chromatography
offers less retention then C8 and C18 phases for non polar solutes.
C6 Hexyl
Reversed phase material, useful for ion pairing chromatography
offers less retention than C8 and C18 phases.
25
C8 MOS, RP-8, LC8, Octyl
Reversed phase material, similar selectivity to C18 but less
retentive wide applicability (e.g: pharmaceuticals, nucleosides,
steroids).
C18 ODS,RP-18,LC-18,Octadecyl
Classic reversed phase material is most retentive for non polar
solutes are excellent for ion pairing chromatography. It is having
wide applicability for the assay of nucleotides steroids,
pharmaceuticals, vitamins, fatty acids and environmental
compounds.
C6H5 Phenyl
It is a reserved phase material and exhibits unique selectivity. It is
useful analyzing aromatic compounds.
CN CPS, PCN, Cyano, Cyanopropyl, nitryl

It is employed as either a reversed phase or normal phase material.
It is slightly polar and exhibit unique selectivity for polar
compounds in both RP and NP modes. It equilibrates very rapidly
and suitable for gradient separations. It has many pharmaceutical
applications.
NH2 APS, Amino, Amino propyl silyl

Can be employed as reversed phase, normal phase or weak anion
exchange material. Reversed phase useful for separating
carbohydrates. Normal phase is alternative selectivity to silica, not
deactivated by small amounts of water. Ion exchange is weak anion
exchanger when used with buffers separates anions and organic
acids.
26
NO2 Nitro
Normal phase material. Separates aromatic compounds and
compound with double bonds.
OH Diol , glycerol
Can be employed as either a reversed phase or normal phase
material. Reversed phase: used for gel filtration chromatography
(GFC) of proteins and peptides. Normal phase similar selectivity to
silica not deactivated by small amounts of water.
SAX SB, sulphonic acid, strong acid.

Ion exchange material. Strong cation exchangers (acidic) are most
useful for analyzing acidic proteins and peptides.
WAX PEI, DEAF, Polyethylene amine, Diethyl amino ethyl, weak base.
Ion exchange material. Weak anion exchangers are most useful for
analyzing acidic proteins and peptides.
WCX CM, Carboxy methyl, weak acid.

Ion exchange material. Weak cation exchangers (acidic) are most
useful for analyzing basic proteins and peptides.
Table :1.2
VII. Finder:
• The finder can see (identify) the individual atoms that turn out (elute) from the
segment. A finder serves to gauge the measure of those particles with the goal that
the scientific expert can quantitatively dissect the example segments.
27
• The identifier gives a yield to a recorder or PC that outcome in the fluid
chromatogram (i.e. the diagram of the finder reaction)
a. Bulk property indicators: These identifiers depend on differential estimation of a

property, which is normal to both the example and the versatile stage. Models of
such locators are refractive list, conductivity and dielectric consistent indicators.
b.Solute property indicators: solute property reacts to a physical property of solute

which isn't displayed by unadulterated versatile stage. These identifiers measure a
property which is particular to the example either with or without the evacuation of
versatile stage before the discovery. Precedents: incorporate spectrophotometric
(UV or UV noticeable) indicator, fluorescence finder, polorographic identifier,
electrochemical and radioactive locators.
A. Bright (UV) Absorption:
• An bright light pillar is coordinated through a stream cell and a sensor estimates
the light going through the cell.
• If a compound elutes from the segment that ingests this vitality, it will change the
measure of light vitality falling on the sensor.
• The coming about change in this electrical flag is enhanced and coordinated to a
recorder or information framework.
• A UV range is some of the time additionally acquired which help in the

distinguishing may proof of a compound or arrangement of mixes.
B. Mass spectroscopy (MS):
• An MS identifier detects a compound eluting from the HPLC section first by

ionizing it at that point by estimating its mass or dividing the particle into littler
pieces that are one of a kind to the compound.
• The MS finder can once in a while distinguish the compound specifically since its
mass range resembles a finger impression and is very extraordinary to that
compound.
28
C. Refractive Index (RI) recognition:
• The capacity of a compound or dissolvable to avoid light gives an approach to

recognize it.
• The RI is a proportion of atom's capacity to divert light in a streaming versatile

stage in a stream cell with respect to a static portable stage contained in a reference
stream cell.
•The measure of avoidance is relative to fixation.
• The RI identifier is viewed as general finder however it isn't exceptionally

delicate.
D. Fluorescence Detector:
• Compared to UV-Visible identifier fluorescence locators offer a higher

affectability and selectivity that permits measuring and distinguishing mixes and
contaminations in complex grids at to a great degree low fixation levels (follow
level investigation).
• Fluorescence finders sense just those substances that fluorescence.
VIII. Recorders and integrators:
Recorders are utilized to record the reactions acquired from identifiers after
enhancement, if important. They record the gauge and every one of the pinnacles
got, as for the maintenance time for every one of the pinnacles can be gotten from
such chronicles however the territory of the individual pinnacle can't be known.
Integrators are enhanced rendition of recorders with a few information handling
abilities they can record the individual crests with maintenance time, tallness and
width of the pinnacle, crest zone, level of zone and so on.
1.4.1 IMPORTANCE OF HPLC:
A. Quantitative Analysis: The estimation of the measure of a compound in an

example (fixation). There are two fundamental approaches to translate a
chromatogram (i.e, perform evaluation).1. Determination of the peak height of a
chromatographic peak as measured from the baseline.
29
2. Determination of the peak area.
In order to make a quantitative assessment of the compound, a sample with a

known amount of the compound of interest is injected and its peak height or peak
area is measured. In many cases, there is a linear relationship between the height or
area and the amount of sample.
B. Preparation of pure compound:
By collecting the chromatographic peaks at the exit of the detector, and

concentrating the compound (analyte) by removing/ evaporating the solvent a pure
substance can be prepared for later use (e.g. organic synthesis, clinical studies,
toxicology studies etc). This methodology is called preparative chromatography.
C. Trace Analysis:
A trace compound is a compound that is of interest to the analyst but its

concentration is very low, usually less than 1% by weight often parts per
million(ppm).
The determination of trace compounds is very important in pharmaceutical,

biological, toxicology and environmental studies since even a trace substance an
harmful or poisonous in a chromatogram trace substance can be difficult to
separate or detect.
High resolution separations and very sensitive detectors are required.
30
Drug profile:
1.0 Introduction
1.1 L-asparaginase is a relatively wide spread enzyme, found in many

animal tissues, bacteria and plant and in the serum of certain rodents
but not of man. Increasing interest in L-asparaginase stemmed from
its identification as the anti lymphoma factor of Kidd (Broome, 1961).
Although this enzyme has beenisolated from a number of microbial
and vegetal sources, only L-asparaginases derived from E. coli and
Erwinia carotovora have anti-tumor activity, particularly in acute
lymphoblastic leukemia, being beneficial antineoplastic agents when
used in sequential chemotherapy. The fact that not all L-
asparaginase species possess anti-tumor properties seems to be
related to the affinity of the enzyme for the substrate and to factors
affecting the clearance rate from the system.
1.2 L-Asparaginase therapy takes advantage of this fact. l-Asparaginase

performs the opposite reaction: it takes asparagine and pulls off its
amine, releasing aspartate and ammonia. It is generally involved in
balancing the levels of amino acids for the use in protein synthesis.
However, if a large dose of this enzyme is introduced into the blood, it
will circulate and continually break down all asparagine that it finds,
ultimately starving the cells that are rely on the blood-borne supply.
2. Molecular Structure
31
2. About Molecule
2.0 Description: L-Asparaginase from Ecoli
2.1 Molecular Formula: C1377H2208N382O442S17
2.2 Average weight: 31731.9 Da
2.3 Mechanism of Action:

Asparaginase converts asparagine to aspartic acid and ammonia. It facilitates
production of the oxaloacetate which is needed for general cellular metabolism.
Some malignant cells loses the ability to produce asparagine and so the loss of
the exogenous sources of asparagine leads to cell death.
2.4 Toxicity: Complies for no Undue Toxicity
2.5 Storage Conditions:

The material will be stable for 14 days at 2-8°C and for longer period store at -
20°C
32
3. Equipments and Chemicals
Equipment and apparatus used:
1. HPLC instrument used was of SHIMADZU UCT2010 SYSTEM with

auto injector and PDA Dector. Software used is LAB-SOLUTIONS
2. Sonicator (Ultrasonic sonicator)
3. pH meter
4. micro balance(sartorius)
5. Vacume pump
Reagents used:
1.1.1 Triethylamine HPLC Grade (RANKEM)
1.1.2 Acetonitrile HPLC Grade (RANKEM)
1.1.3 Orthophosphoric acid HPLC Grade (RANKEM)
1.1.4 HPLC Grade Water (RANKEM)
33
METHOD DEVELOPMENT
Based on drug solubility and Pka Values following conditions has been
used to develop the method estimation of L_Asperaginase.
Trial 1
Chromatographic conditions
Column : C18, 5µm, 300A°, 4.6x250mm

Flow rate : 0.6 ml per minute
Injection volume : 20µL
Wavelength : 225nm
Column Temperature : 25°C
Sample Temperature : 5°C
Run time : 15 minutes
Mode Isocratic : Channel-A (73%) and Channel-B (27%)
Fig 6.1 Trial chromatogram 1
Observation: peak shape was not good.
34
Trial 2
Column : C18, 5µm, 300A°, 4.6x250mm

Wavelength : 225nm
Observation: peak shape was good but retention time was not
appropriate.
35
Trial 3
Column : C18, 5µm, 300A°, 4.6x250mm

Wavelength : 225nm
Observation: Asparginase eluted with good peak shape and

retention time.
36
Optimized Chromatographic conditions
Column : C18, 5µm, 300A°, 4.6x250mm
Wavelength : 225nm
Fig 6.4 optimized chromatogram 1
Observation: Asparginase eluted with good peak shape and

retention time
37
STANDARD TESTING PROCEDURE (STP)
1.1 Equipment:
HPLC
1.2 Procedure:
1.2.1 Preparation of 10% (v/v) diluted Orthophosphoric acid: Take

5.9ml of Orthophosphoric acid in a 50ml volumetric flask and make
up to volume with water and mix well.
1.2.2 Preparation of Mobile phase A (Buffer): Add 2ml of

Triethylamine in 1000ml of water and mix well adjust the pH to 7.0
with 10% Orthophosphoric acid, filter the mobile phase with 0.45µ
membrane filter and sonicate for 5 minutes.
1.2.3 Preparation of Mobile phase B: Prepare a mixture of Acetonitrile

and water in the ratio of 95:5 (%v/v), mix well and sonicate for 5
minutes.
1.2.4 Preparation of Diluent: Prepare a mixture of Mobile phase A and

Acetonitrile in the ratio of 70:30 (%v/v) and mix well.
1.2.5 Chromatographic Conditions:
Column : C18, 5µm, 300A°, 4.6x250mm

Wavelength : 225nm
Preparation of Solutions:
38
1.2.6 Preparation of Standard Solution: Weigh accurate 50mg of L-
asparaginase working standard in 50ml volumetric flask and dilute
and make up to the mark with diluents.
1.2.7 Preparation of Sample Solution: : Weigh accurate 50mg of L-

asparaginase working standard in 50ml volumetric flask and dilute
and make up to the mark with diluents.
1.2.8 Note: Store the samples and standard samples in 2-8°C. Rinse
and Dry the glassware once with diluents before analysis.
1.2.9 Procedure: Inject Blank, standard solution and sample solution

into the chromatogram.
1.2.10 Inject standard solution for two times and sample solution once
1.3 Acceptance criteria: Not less than 98.00%.
1.0 PARAMETERS TO BE VALIDATED WITH ACCEPTANCE CRITERIA

Validation Parameter Acceptance Criteria
Specificity No significant interference of the Blank should be observed
Degradation studies No other impurities should be developed
Accuracy % Recovery of the L-Asparaginase should be 80.0 to
100.0IU/mg within specified
Linearity Correlation coefficient should not be less than 0.99
Repeatability % RSD for replicate analysis should not be more than 5.0
Precision Intermediate % RSD for replicate analysis between two analysts should
Precision not be more 5.0
Robustness Analytical method should not be affected by small variations
in method parameters.
39
2.0 VALIDATION PROCEDURE
2.1 SPECIFICITY:
2.1.1 Preparation of Diluent: Prepare a mixture of Mobile phase A
(0.2%TEA) and Acetonitrile in the ratio of 70:30 (%v/v) and mix well.
2.1.2 Standard Preparation: Weigh accurately 50mg of L-asparaginase
working standard in 50mL volumetric flask, dilute and make up to the
mark with diluent.
2.1.3 Placebo preparation: Weigh accurately 1.8g of glucose and 0.2g of
NaCL in a 100mL volumetric flask, dilute and makeup with water.
2.1.4 Sample Preparation spiked with placebo: Weigh accurate 50mg of
L-asparaginase sample in 50ml volumetric flask, dilute and make up to
the mark with placebo to obtain a concentration of 1mg/mL.
Note: Store the Samples and Standards in 2-8°C. Rinse and Dry the
glassware once with diluent before analysis.
2.1.5 System suitability: Inject Standard Solution for five times and observe
the %RSD
2.1.6 Procedure: proceed as directed in the STP. Inject Blank (Diluent)

(1inj), Standard Solution(5inj), placebo(1inj) and Sample spiked with
placebo Solution (1inj) to obtain a chromatogram.
2.1.7 Acceptance Criteria:

System suitability: %RSD should be Not More Than 1%
Specificity: No significant interference of Placebo should be observed.
2.2 DEGRADATION STUDY:

2.2.1 Acid Degradation:
Sample Preparation: Weigh about 10mg of sample to be

examined in to 10mL volumetric flask, and add 5mL of 0.1N
Hydrochloric acid and keep for 30min at room temperature and
neutralize with 0.1N sodium hydroxide solution and then make up
to the mark with the diluents.
2.2.2 Alkali Degradation:
Sample Preparation: Weigh about 10mg of sample to be examined in
to 10mL volumetric flask, and add 5mL of 0.1N Hydrochloric acid and
40
keep for 30min at room temperature and neutralize with 0.1N sodium
hydroxide solution and then make up to the mark with the diluent.
2.2.3 Oxidative Degradation:

Sample Preparation: Weigh about 10mg of sample to be examined in
to 10mL volumetric flask, and add 5mL of 0.1N Hydrogen peroxide
and keep for 30min at room temperature and make up to the mark with
the diluent.
2.2.4 Thermal Degradation:
Sample Preparation: Weigh about 10mg of sample to be examined
in to 10mL volumetric flask, dilute and make up till the mark with
diluent and keep at 40oC for 30min in water bath.
2.2.5 Moisture Degradation:
Sample Preparation: Weigh about 10mg of sample to be examined
in to 10mL volumetric flask, dilute and make up to the mark with
diluent and keep at -20oC for 30min in -20 oC freezer. After 30min
takeout the sample and allow to thaw.
2.2.6 Procedure: proceed as directed in the STP, Inject Blank (Diluent)
(1inj), Standard Solution (2inj), Blank of each degraded sample (1inj)
and degraded Samples (2inj) to obtain a chromatogram.
2.2.7 System suitability: Inject Standard Solution for two times and observe
the %RSD
Degradation studies: No other impurities should be developed
2.3 LINEARITY:
2.3.2 Sample Stock Preparation: Weigh about 50mg of sample to be
examined in to 25mL volumetric flask and make up to the mark with
diluent to obtain a concentration of 2.0 mg/mL
2.3.3 Standard Stock Preparation: Weigh about 50mg of L-Asparaginase
Working standard in to 50mL volumetric flask and make up to the
mark with diluent to obtain a concentration of 1.0 mg/mL.
2.3.4 Make serial dilutions from sample stock solutions as mentioned below:
2.3.5 Linearity range: 50-150%
41
2.3.6 50% Nominal: Take the 250µL of sample stock solution and add
750µL of diluents to obtain the concentration between 0.5 mg/mL.
2.3.11 Procedure: Proceed as directed in the STP, Inject Blank (Diluent)
(1inj), Standard Solution (5inj), Sample each concentration (2inj) to
obtain a chromatogram.
Note: For every six injects of sample inject Bracketing standard
the %RSD
2.3.13 Acceptance Criteria :
Linearity: Correlation coefficient should not be less than 0.99
2.4 ACCURACY:
2.4.2 Sample Stock Preparation: Weigh about 50mg of sample to be
examined in to 25mL volumetric flask and make up to the mark with
diluent to obtain a concentration of 2.0 mg/mL
2.4.3 Standard Stock Preparation: Weigh about 50mg of L-Asparaginase
2.4.4 Make serial dilutions from sample stock solutions as mentioned below:
2.4.5 Accuracy range: 50-100%
42
2.4.12 Procedure: Proceed as directed in the STP, Inject Blank (Diluent)
(1inj), Standard Solution (5inj), Sample each concentration (2inj) to
obtain a chromatogram.
the %RSD.
Experimental concentration
% Recovery = ----------------------------------x100
Theoretical concentration

Accuracy: % Recovery of the L-Asparaginase area should be 80.0 to
100% within specified for L-Asparaginase purity.
2.5 PRECISION:
2.5.1 Repeatability:
2.5.1.1 Preparation of Diluent: Prepare a mixture of Mobile phase A
(0.2%TEA) and Acetonitrile in the ratio of 70:30 (%v/v) and mix
well.
2.5.1.2 Standard Preparation: Weigh about 50mg of L-Asparaginase
43
2.5.1.3 Sample Preparation: Weigh about 50mg of sample to be examined
in to 50mL volumetric flask and make up to the mark with diluent to
obtain a concentration of 1.0 mg/mL
2.5.1.4 Procedure: Proceed as directed in the STP, Inject Blank (Diluent)
(1inj), Standard Solution (5inj), Sample 100 nominal concentration
(6inj) to obtain a chromatogram.
2.5.1.5 System suitability: Inject Standard Solution for five times and
observe the %RSD.
2.5.1.6 Acceptance Criteria:
Precision: Relative standard deviation should NMT 1 %.
2.5.2 Intermediate precision:
2.5.2.1 Method precision shall be repeated by different analyst and on
different days.
2.5.2.2 Procedure: Calculate Mean, Standard deviation and Relative standard
deviation
2.5.2.3 Acceptance Criteria:
Intermediate precision: The relative standard deviation of six
preparations should not be more than 1.0% and the difference between
the analyst 1 and analyst 2 should not be more than 2.0%.
2.6 ROBUSTNESS:
2.6.1 Procedure: Prepare standard dilutions and sample dilutions as per STP
and analyze the samples by changing the small variations of
parameters. Inject Blank (Diluent) (1inj), Standard Solution (5inj),
Sample each parameter (2inj) to obtain a chromatogram.
Note: For every six injects of sample inject Bracketing standard.
44
S.No Actual Parameters Change -1 Change – 2
1 Mobile phase A- pH (7.0) 6.0 8.0
Column oven Temperature
2 23°C 27°C
(25°C)
3 Wavelength (225nm) 223nm 227nm
4 Flowrate (0.6mL/min) 0.5mL/min 0.7mL/min
2.6.2 System suitability: Inject Standard Solution for five times and observe the
%RSD.
2.6.3 Acceptance Criteria :
System suitability: %RSD should be Not More Than 1%.
Robustness: Analytical method should not be affected by small variations
in method parameters.
45
RESULTS AND DISCUSSIONS
3.0 VALIDATION PROCEDURE
3.1 SPECIFICITY:
3.1.1 Validation of L-Asparaginase is performed as per ICH guidelines.
3.1.2 Procedure: Blank interference has checked as per the STP procedure
for L-Asparaginase by using HPLC.
3.1.3 Acceptance Criteria: No significant interference of blank should be
observed.
3.1.4 Observation : No significant interference of the Blank is observed
3.2 DEGRADATION STUDY:

3.2.1 Forced degradation experiments were conducted to establish the
stability indicating capability of the method. For each stress
experiment, Drug substance was stressed under conditions shown in
below Table-I and analyzed as per the proposed analytical method
using HPLC.
3.2.2 Table-I
Stress ConditionsStressRT(min
Type Average
Acid 0.1N HCL at RT for 30 NO
) of L- NO
Area
Alkali
degradatio 0.1N NaOH at RT for 30
Mins NO
Asp NO
Oxidative
degradatio
n H O
2 2 at RT for
Mins 30 Mins 0.404 7731544
Thermal
degradatio
n 40C for 30 Mins 0.339 8268177
Moisture
degradatio
n Sample kept at -20C for 0.422 10693202.00
UV
degradatio
n Sample kept under
30 Mins UV 0.413 7777170.00
Agitation
degradatio
n Vortex for
radiation for3030Mins
Mins 0.280 5542350.00
Sample
n RT(min) of L-Asp Area Area%
Standard-1
name 2.65 8988298 99.71
Standard-2 2.66 8987818 99.73
Average NA 8988058 NA
Standard NA 339.41 NA
%RSD
deviation NA 0.00 NA
8.2.3.1 Acceptance Criteria: No other impurities should be developed.
8.2.3.2 Observation: No other impurity peaks were observed.
3.3 ACCURACY:
46
3.3.1 A known amount of analyte, below the normal levels expected in the
sample was prepared and analyzed by the proposed method and the
results are shown in the following Table-II
3.3.2 Table-II
Sample name RT(min) of L- Average Area%
Standard-1 2.64
Asp 12701733
Area 99.76
Standard-2 2.64 12683947 99.76
Standard-3 2.64 12669790 99.76
Standard-4 2.64 12683778 99.75
Standard-5 2.64 12682302 99.76
%RSD
Sample name RT(min) of L- Exp. Avg Theo. Area of
L-Asp_Lyo_50% 2.65
Asp 5406555
Area 6342155
STD
Lyo_60% 2.65 6210852 7610586
Lyo_70% 2.65 7435956 8879017
Lyo_80% 2.65 8311105 10147448
Lyo_90% 2.65 9487139 11415879
Lyo_100% 2.65 10160231 12684310
STANDARD CHROMATOGRAM
47
L-Asp_Lyo_50%, 60%, 70% 80% 90% and 100% CHROMATOGRAM
3.3.3 Procedure: Proceed as directed in the STP
Experimental area
% Recovery = ----------------------------------x100
Theoretical area
3.3.4 Acceptance Criteria: % Recovery of the L-Asparaginase potency
should be 80.0 to 100%. Relative standard deviation of System
Suitability should NMT 1.0 %.
3.3.5 Observation: The observed % Recovery was within specified limit.
%RSD of system suitability was 0.09%.
3.4 LINEARITY:
3.4.1 A known amount of analyte, both above and below the normal levels
expected in the sample was prepared and analyzed by the proposed
method and the results are shown in the following Table-III.
3.4.2 Table-III
Sample name RT(min) of L- Average Area%
Standard-1 2.64
Asp 12701733
Area 99.76
Standard-2 2.64 12683947 99.76
Standard-3 2.64 12669790 99.76
Standard-4 2.64 12683778 99.75
Standard-5 2.64 12682302 99.76
48
%RSD
L-Asp_Lyo_50% 2.64 5455691.00 99.2
L-Asp_Lyo_75% 2.64 7724371.50 99.1
L-Asp_Lyo_100% 2.64 10354015.50 99.1
L-Asp_Lyo_125% 2.64 12485487.00 99.1
L-Asp_Lyo_150% 2.64 14375086.00 99.1
L-Asp_Lyo_50, 75, 100, 125, 150% CHROMATOGRAM
49
3.4.3 Procedure: Proceed as directed in the STP
3.4.4 Acceptance Criteria: Correlation coefficient should not be less than
0.99.
Relative standard deviation of System Suitability should
NMT 1.0 %.
3.4.5 Observation: The observed Correlation coefficient was 0.996. %RSD
of System suitability was 0.09%.
3.5 PRECISION:
3.5.1 Repeatability:
3.5.1.1 Sample Preparation: Weigh about 50mg of sample to be examined
in to 50mL volumetric flask and make up to the mark with diluent to
obtain a concentration of 1.0 mg/mL
3.5.1.2 Note: Inject 6 injections of 100% nominal sample and calculate
the relative standard deviation. The results are shown in the following
Table-IV.
3.5.1.3 Table-IV
Sample name RT(min) of L-Asp Area Area%
Standard-1 2.66 8975236 99.6
Standard-2 2.66 8969758 99.6
Standard-3 2.66 8967836 99.6
Standard-4 2.66 8961586 99.6
Standard-5 2.66 8963249 99.6
Standard
NA 5433.65 NA
deviation
%RSD NA 0.06 NA
50
RT(min) of Area%
Sample name Area
L-Asp
L-Asp_Lyo_1 2.66 12507260.00 99.16
L-Asp_Lyo_2 2.66 12498875.00 99.18
L-Asp_Lyo_3 2.66 12489379.00 99.21
L-Asp_Lyo_4 2.66 12477032.00 99.27
L-Asp_Lyo_5 2.66 12537401.00 99.18
L-Asp_Lyo_6 2.66 12567773.00 99.2
Average NA 12512953.33 NA
Standard NA
NA 33703.38
deviation
%RSD NA 0.27 NA
51
L-Asp_Lyo_1, 2 , 3, 4, 5, 6 CHROMATOGRAM
Procedure: Proceed as directed in the STP

3.5.2 Acceptance Criteria: Relative standard deviation should NMT 1.0 %.
Relative standard deviation of System Suitability should
NMT 1.0 %.
3.5.3 Observation: The observed Relative Standard Deviation was 0.27%.
%RSD of System Suitability was 0.06%.
3.6 INTERMEDIATE PRECISION:

3.6.1 Method precision shall be repeated by different analyst and on
different days.
3.6.2 Procedure: Calculate Mean, Standard deviation and Relative standard
deviation.
3.6.3 Acceptance Criteria: The relative standard deviation of six
preparations should not be more than 1.0%, and the difference between
the analyst 1 and analyst 2 should not be more than 2.0%. The results
are shown in the following Table-V. Relative standard deviation of
System Suitability should be NMT 1.0 %.
52
3.6.4 Observation: The observed Relative Standard Deviation was 0.09%,
and the difference between the analyst 1 and analyst 2 was 0.18%.
%RSD of system suitability was 0.23%.
Table-V
RT(min) of L-
Sample name Area Area%
Asp
Standard-1 2.78 13830037 99.15
Standard-2 2.78 13790000 99.1
Standard-3 2.78 13791605 99.13
Standard-4 2.78 13765319 99.17
Standard-5 2.78 13745604 99.14
Standard deviation NA 31761.45 NA
%RSD NA 0.23 NA
RT(min) of L-
Sample name Area Area%
Asp
L-Asp_Lyo_1 2.78 10626882 99.01
L-Asp_Lyo_2 2.78 10620578.00 99.01
L-Asp_Lyo_3 2.78 10612767.00 99.07
L-Asp_Lyo_4 2.78 10602746.00 99.11
L-Asp_Lyo_5 2.78 10603448.00 99.3
L-Asp_Lyo_6 2.78 10607694.00 99.35
Average NA 10612352.50 NA
Standard deviation NA 9718.16 NA
%RSD NA 0.09 NA
53
L-Asp_Lyo_1, 2, 3, 4, 5, 6 CHROMATOGRAM
3.7 ROBUSTNESS:
3.7.1 Procedure: Prepare sample dilutions as per STP and analyze the
samples by changing the small variations of parameters, as shown in
the Table-VI.
3.7.2 Table-VI
S.No Actual Parameters Change -1 Change – 2
1 Mobile phase A- pH (7.0) 6.0 8.0
Column oven Temperature
2 23°C 27°C
(25°C)
3 Wavelength (225nm) 223nm 227nm
4 Flowrate (0.6mL/min) 0.5mL/min 0.7mL/min
3.7.3 Acceptance Criteria : System suitability should pass in all cases

Observation: Analytical method should not be affected by small
variations in method
54
SUMMARY AND CONCLUSION
L-Asparginase is an enzyme that acts as an effective anti-tumor agent used for the
remission and improvement of long term survival in patients with acute
lymphoblastic leukemia. In the present study, we have developed simple simple,
sensitive and rapid chromatographic methods for the determination of the purity of
L-Asparaginase in pharmaceutical preparations by using HPLC-PDA technique.
The purity of the L-asparaginase was determined by HPLC-PDA system connected
to UV-detector and reverse phase-C18 column. A low pressure gradient mobile
phase consisting of 0.2% TEA in water (Buffer-A) and acetonitrile and water in the
ratio of 95:5 mixture (Buffer-B) was used for the determination of purity at 225nm.
The observed purity was found to be 98.82%. The HPLC method was also
validated in terms of accuracy, precision, degradation, specificity, linearity and
robustness. The results obtained were within the acceptance criteria.
55
BIOGRAPHY
^ Farmer, Peter B.; Walker, John M. (2012). The Molecular Basis of Cancer. Springer Science
& Business Media. p. 279. ISBN 9781468473131. Archived from the original on 2016-12-21.
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CHARACTERIZATION OF L_ASPARAGINASE

JAWAHARLAL NEHRU TECHNOLOGICAL UNIVERSITY

For the partial fulfillment of the degree of

CENTRE FOR CHEMICAL SCIENCES AND TECHNOLOGY

Certified that the dissertation work entitled “CHARACTERIZATION OF

L_ASPARAGINASE submitted by S.VIKRAM in partial fulfillment of the

Requirements of Master of Science in Chemistry (Analytical Chemistry) is a bonafied

HEAD OF THE DEPARTMENT

Prof. A. JAYA SHREE

PROJECT SUPERVISOR EXTERNAL EXAMINER

Title : “CHARACTERIZATION OF L_ASPARAGINASE

Duration : January 10th 2018 TO June 10th 2018

Course : M.Sc. Chemistry (Analytical Chemistry)

Institute : Centre for Chemical Sciences & Technology,

Declaration by the candidate

I, here by certify that the project report entitled “Characterization of

The Satisfaction that accompanies the completion of any task would be

I take extreme pleasure to express my deep sense of Gratitude to my project

S.NO NAME OF THE CHAPTER PAGE.NO.

3 DRUG PROFILE 31-32

4 EQUIPMENT AND CHEMICALS 33

5 METHOD DEVELOPMENT 34-37

6 STANDARD TESTING PROCEDURE 38-45

7 RESULTS AND DISCUSSION 46-55

Analytical chemistry is a branch of chemistry which analyzes the chemical

Pharmaceutical analysis is a branch of analytical chemistry that involves in

Pharmaceutical analysis plays a crucial role in providing assurance of quality,

Method of analysis is routinely developed to validate collaborative study and

An analytical technique plays an important role in

B. Chemical methods: A chemical method of analysis includes the gravimetric

1.2 TYPES OF CHEMICAL ANALYSIS:

1 .Proximate analysis: The amount of each element in a sample is determined

3. Trace constituent analysis: Concerned with the determination of specified

4. Complete analysis: Deals with the determination of each proportion of

5. Physiochemical Methods: Physiochemical methods include

 UV-Visible spectrophotometric techniques

1.3 Introduction to High Performance Liquid Chromatography

Chromatography may be defined as a non destructive procedure for separating

CLASSIFICATION OF CHROMATOGRAPHIC PROCESS

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

HPLC is a separation technique that involves:

A. BASED ON PRINCIPLE OF SEPARATION

REVERSE PHASE HPLC:

1. Speed (many analysis may be accomplished in 20min)

6. Easy sample recovery

 With micro particulate packing, there is a high pressure drop across a

 For normal phase separations, eluting power increases with increasing

 For reverse phase separations, eluting power increases with increasing

 Optimum separating conditions can be achieved by making use of mixture

 The properties of the solvent which need to be considered for a successful

The most important component of HPLC in solvent delivery system is the

 The following points should also be considered when choosing a mobile

SOLVENT MW BP RI UV Density Viscosity Dielectric

Acetonitrile 41.0 82 1.342 190 0.787 0.358 38.8

Dioxane 88.1 101 1.420 215 1.034 1.26 2.21

Ethanol 46.1 78 1.359 205 0.789 1.26 24.5

Ethyl 88.1 77 1.372 256 0.901 0.450 6.02

Methanol 32.0 65 1.326 205 0.792 0.584 32.7

Dichloro 84.9 40 1.424 233 1.326 0.44 8.93

Iso propanol 60.1 82 1.375 205 0.785 2.39 19.9

n-propanol 60.1 97 1.383 205 0.840 2020 20.3

THF 72.1 66 1.404 210 0.889 0.51 7.58

Water 18.0 100 1.333 170 0.998 1.00 78.5