HPLC GC analysis

1. “Quantitative Analysis of Lamotrigine using HPLC & GC analysis
and the impact on light on peak value”
Authors – Birla, Himanshu
Guide
- Professor John Nicholson J.W.Nicholson@greenwich.ac.uk
2008
1

2. CHAPTER 1
INTRODUCTION
1.1 OBJECTIVE
2

3. 1. The objective of the experiment was to partially validate a high performance
liquid chromatography technique for the quantitative analysis of lamotrigine.
2. The drug utilized for the study was lamotrigine taken from Sigma-Aldrich in
powder form. The generic lamotrigine came with an amber bottle in 10mg
which was then analysed for quantitative analysis.
1.2 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
3

4. Liquid chromatography is a separation method which is widely used in the chemical,
pharmaceutical and biotechnology industry and is of great importance. The principle
of liquid chromatography includes the injection of the sample into the system which
contains a particulate stationary phase as a column and a moving mobile phase in
pushed through the system and hence column. The separation of substances takes
place due to differences in the rates of movement through the column and interaction
with the stationary phase and movement through the column is facilitated with the
help of mobile phase. Chromatography is a general term applied to the separation
techniques involving the separation partitioning of the sample in presence of a mobile
phase which is generally a liquid or gas and a stationary phase which may be a solid
or a liquid. [1]
Chromatography is used widely to separate the mixtures and isolate them. It is
generally divided into two types, namely Preparative chromatography and Analytical
chromatography. Preparative chromatography is used to separate components of the
mixture for further use whereas analytical chromatography is done on smaller amount
of materials and helps in finding out the proportion of the constituents in the
analytical sample [1]. The aim of separation is to obtain the qualitative and
quantitative information about the sample. The all important aim of validation of a
sample is used to obtain the Quantification of the substance which was observed in
this experiment. [1, 2]
1.2.1 HISTORY OF CHROMATOGRAPHY
The term “chromatography” was first coined by Tswett after finding a band of colours
on the column while separating pigments of green leaves [2]. The thin layer
chromatography and gas chromatography came into existence in 1950’s and after that
various gel exclusion techniques came in 1960’s. In 1960’s the Journal of Gas
Chromatography changed its name to Journal of Chromatography Science and this
cerated the interest in liquid chromatography and hence start of a new modern liquid
chromatography era. Gas chromatography was a well known and established
technique at that time but gas chromatography had a limitation [2]. It was unable to
Handle thermally unstable and non-volatile compounds and this limitation drove the
interest in favour of liquid chromatography. [2]
4

5. Liquid chromatography also helped in selective interaction of two chromatographic
phases, easy sample recovery and room temperature conditions for the technique to
work. Liquid chromatography showed the limitation of inability to show
quantification of compounds and resolution between similar compounds. At this stage
pressure liquid chromatography showed good separations with the decrease in the
separation time with pressurised flow of mobile phase through the column and hence
reducing the purification times of compounds. The increase in the pressure to move
the mobile phase in liquid chromatography laid the foundation of high performance
liquid chromatography (HPLC). [2]
High performance liquid chromatography was developed in 1970’s and it was further
improved by the use of packed columns and better resolution detectors and now it is
the most common techniques used in the analysis and separation of compounds. Even
in late 1970’s another technique called reversed phase chromatography helped to
improve separation between very similar compounds. [3].
1.2.2 REVERSED PHASE CHROMATOGRAPHY: - [2]
Reversed Phase Chromatography is a technique slightly different from the normal
chromatography. The difference in reversed phase chromatography is the stationary
phase. In the reversed phase chromatography the stationary phase is non-polar and
hence it is more advantageous as compared to normal chromatography. As the
stationery phase is non-polar, water can be used as mobile phase and the strength of
the mobile phase can be increased with the help of organic solvents. The aqueous
mobile phase provides transparent mobile phases at low cost and they have high
compatibility with biological samples and electrochemical detectors. The interactions
of the solute with the stationary phase are weak, which provides mass transfer with
less possibility of irreversible adsorption and provides rapid equilibrium with solvent
changes and hence helpful in gradient elution methods.
HPLC gained importance because of its reliable technique that is high pressure
application on the mobile phase to push its movement through the densely packed
5

6. column. This technique hence enables faster separations and increased resolution as
compared to liquid chromatography. Until 1980’s HPLC was commonly used to
separate the chemical compounds but later with further improvements in the
technique the quantification, purification and identification of the compounds also
started to immerge with the use of HPLC. The development of computerised methods
also added in improved results and reproducibility and hence helping the HPLC as the
most common and widely used technique for the analysis and separation of
compounds.[3]
1.2.3 THEORY OF OPERATION [2]
Chromatography is a separation technique which employs of two phases, one
stationery phase and one mobile phase. The mixture is injected into the system with
the help of the injection through an injector and is carried by the mobile phase into
contact with the stationary phase. Separation takes place as the components of the
mixture move and partition differently between the stationery and mobile phases. The
theory of operation basically works on two parameters which include: - [2]
1) Thermodynamic considerations
2) Kinetic considerations
Thermodynamic considerations involve the basic interaction between the mobile
phases containing the compound with that of the stationary phase. Hence
thermodynamic considerations define the retention times of the sample. [2]
The time required for the sample to elute from the system is equal to the time the
sample spends in the moving mobile phase plus the time spent in the stationary phase.
If there is no interaction takes place with the stationary phase then the retention times
becomes the hold up time or dead time and it is the time required for the mobile phase
molecules to cross the column. [2]
Kinetic considerations in an HPLC experiment refers to the moving speed of the
mobile phase and hence kinetics of the system is responsible for peak maxima, peak
6

7. width and band broadening and hence thermodynamic and kinetic factors of an HPLC
system defines the peak and hence separation of the compound. [2]
1.2.4 INSTRUMENTATION OF HPLC
[3]
The HPLC technique is carried out with the help of different experimental parts, but
the most important part is the column as the column is responsible for separation and
hence the analysis of the sample. The instrumentation of HPLC contains following
parts which are discussed in details later. The instrumentation includes:1) Column Efficiency
2) Mobile Phase
3) Stationary Phase
4) Injectors
5) Pumps
6) Columns and Column packing
7) Detectors
1.2.4.1 Column Efficiency
Column efficiency basically defines the capability of the mobile phase to distinguish
and separate the analytical compounds. The column efficiency basically depends on
the number of theoretical plates and the efficiency of the column to separate; it
defines column’s packing and kinetics of the mobile phase through the column. The
change in the column efficiency can be seen with the help of peak shapes [3]. If the
peaks show “Tailing” then it implies that there is some problem in the column and
hence the change in the packing is causing the peak shape to change. The column
efficiency is calculated by the following equation:-
N = A (TR/W2)
Where,
“N” is the number of theoretical plates.
“A” is the constant dependent upon the height where peak width measured.
7

8. “TR” is the retention time.
“W” is the peak width.
[2]
[Fig.1 taken from ref. no. 3]
Mobile Phase:The mobile phase is another important part
of the HPLC. The mobile phase is a
continuous solvent which runs across the
HPLC system throughout the experiment.
The mobile phase is basically a carrier for
the compound which is subjected to
analysis. The mobile phase carries the sample into the column where the separation
takes place. The interaction of the mobile phase with the sample and the column
collectively defines the extent of separation and movement of the sample through the
system [2]. Methanol is a common mobile phase used in the HPLC, though other
polar organic compounds, such as acetonitrile, are also widely used. The interaction
of the sample with that of the mobile phase determines its movement and separation.
If the sample is having a high affinity with that of the solvent then the sample will
elute from the system faster and the retention time of the sample at the column and the
system will decrease and the elution will be fast hence it may lead to less separation
and vice-versa is also true. The mobile phase is subjected to a number of alterations to
get the best suited mobile phase for the sample in order to get maximum separation.
[2, 3]
1.2.4.2 Stationary Phase:
The stationary phase is the base at which the separation takes place. The solid support
containing the column forms the stationary phase and mobile phase continuously runs
over the stationery phase during the experiment. The sample comes into the system
from the injector and hence joins the mobile phase from the injector and runs over the
stationary phase (column) where the separation takes place. When the sample solution
moves to the stationary phase along with the mobile phase then the interactions
between the sample solution and the column helps the separation. The amount of time
8

9. sample solution spends at the stationary phase or the column interface before finally
eluting out is called the retention time of the sample. Samples having higher
interaction towards the mobile phase as compared to the column will elute faster. [3]
The samples having good interaction towards the column will elute out from the
system slower hence the samples having higher affinity towards the column or
stationary phase show greater retention times as compared to the samples which have
higher affinity to the mobile phase. There are different stationary phases used
according to the need of the experiment and the sample and detailed discussions is not
included in this part of introduction. The different columns include: - [3]
1) Solid-Liquid columns (Adsorption)
2) Liquid-Liquid
3) Size Exclusion
4) Normal Phase
5) Reversed Phase
6) Ion Exchange
7) Affinity
1.2.4.3. Injector:The injector is the source from where the sample enters the HPLC system and hence
meets the mobile phase and move towards the column. The injection technique plays
a very important role in the experiment, as the injection technique for sample injection
is responsible for total peak width and hence the injection of the sample should be
done with care. The injection of the sample in the HPLC system is a bit difficult
because of the high pressure on the mobile phase exerted by the pumps to push the
mobile phase through the column. The difficult injection technique is responsible for
improper precision and reproducibility and is termed as “syringe error”. The problem
of syringe error is now been sorted out with the help of auto samplers. Until now the
most common method of sample injection is through the Rotary Valve injectors.
These injectors contain ports which allow the sample to be injected into the external
9

10. loop. The activation of ports helps the sample to get incorporated into the mobile
phase flow and the sample is delivered into the system. Since the introduction of the
auto samplers the precision and reproducibility has been greatly improved. The most
common injection loops volumes are from 5 to 20uL. [3]
1.2.4.4 Pumps:The pumps play an important role in the HPLC system as they are responsible for the
constant flow of mobile phase through the system. Pumps are responsible for the
constant flow and pressure of the mobile phase and hence help in getting proper
precision during the experiment. There are different types of pumps applied in the
HPLC. Some commonly used pumps include: - [3]
Reciprocating Piston Pumps- These pumps consists of a motor driven piston which
moves back and forth in a hydraulic chamber. These pumps help in moving the
mobile phase from the reservoir and then push the mobile phase into the system. The
flow rate can be changed by changing the settings of such pumps. [3]
Syringe Type Pumps- These pumps are responsible for small columns as they deliver
a definite amount of volume and then they need to get refilled. These pumps generally
have the volume ranging from 250-500uL. The pump operates with the help of a
motor and delivers mobile phase at constant rate. [3]
Constant Pressure Pump- These types of pumps provide a constant pressure to
mobile phase but the mobile phase is pushed through the system with pressure from
gas cylinder. A low pressure gas source is needed for this pump. These pumps provide
continues mobile phase flow rates. [3]
1.2.4.5 Detectors: - [3]
The detectors are the instruments which take the response from the HPLC system and
show them as a peak in the Chromatogram. Detectors work at the elution of the
sample and define the separation or sample change as a peak in the chromatogram.
Detectors work just after the stationary phase and detect the sample as soon as they
elute out from the stationary phase. The bandwidth and the peak areas can be adjusted
with the help of these detectors as they can be subjected to a change easily and are
10

11. easily adjustable. The most common detector used in the HPLC is the UV
spectrometer.
The UV spectrometer works on the principle of absorbance and shows that with the
increase in the concentration of the sample the peak area increases. There are many
detectors used in the HPLC instrumentation and some of them are listed below: - [3]
1) UV Detectors
2) Fluorescent Detectors
3) Electrochemical Detectors
4) Mass Spectroscopy Detectors
5) NMR Detectors
6) Light Scattering Detectors
7) Near Infrared Detectors.
1.3 VALIDATION
When a method has been developed it is important to validate it to confirm that it is
suitable for its intended use. The validation of the system tells how good the methods
are and how good they are specifically for the intended application [1]. Validation is a
technique which establishes the documented evidence that produces high degree of
assurance about the product or technique and its accuracy and precision. Validation of
an analytical technique is a procedure which is carried out to obtain justified evidence
of the ability of the technique to provide accurate and précised results [4]. In the
pharmaceutical industry validation and validation methods have a great importance.
All analytical techniques used for the development of pharmaceutical products and for
the determination of their quality, validation are done [4].
Partial validation is the process in which the validation is carried out on the
analytical sample and system but in partial validation all the components of validation
are not included. Partial validation is carried out on the following components: -
11

12. 1) Accuracy
2) Precision
3) Repeatability
4) Reproducibility
5) Linearity and Range
6) Robustness
7) Limit of Detection
8) Limit of Quantification
9) Recovery. …………………………………….[1]
1.3.1 Accuracy and Precision: The precision of an analytical technique is the closeness of the series of individual
measurements of a sample when the analytical procedure is applied repeatedly to
multiple experiments of the same sample. The precision in a technique is calculated
by the coefficient of variation between the techniques and hence defines the standard
deviation from the results found earlier. This is also called as relative standard
deviation (RSD) and is further divided into repeatability (Intra-day precision) and
(Inter-day precision) and reproducibility (between-laboratory precision). [1]
1.3.2 Linearity and Range:The linearity of an analytical method is the ability of the technique to produce the
results which are directly proportional to the concentration of the sample. The results
are then transformed into a mathematical expression and then plotted in a graph mode
to give the linearity in the results. Linearity is calculated by the equation of
regression. The results of the experiment are plotted on the graph. Generally 6-8
points are taken on the graph to find out the linearity of the results. The linearity of
the sample defines that the change in the sample is directly proportional to the results
obtained that is with the increase of the sample concentration the peak area in the
12

13. result increase and hence defines the peak area increases proportionally with the
increase in sample concentration. [1]
1.3.3 Limit of Detection is defined as the concentration of the sample that results in a
peak height three times the noise when injected into the chromatographic system.
LOD is the lowest quantity of the substance which can be distinguished from the
absence of that substance. It is estimated from the mean of blank and the standard
deviation of the blank. [1]
1.3.4 Limit of Quantification is defined as the lowest concentration of the sample
that can be quantified with acceptable accuracy and precision.
1.3.5. Recovery of the sample is the desirable result of sample preparation and it is an
important part of extraction experiments. The absolute recovery is the ratio of
response measured for a spiked sample treated according to the whole analytical
procedure and with the same quantity the sample substance and directly injected into
the HPLC system. The recovery is referred as the ratio of the responses between
extracted spiked samples and extracted pure samples. The relative recovery can be
used to calculate the absolute recovery and hence can be useful in the detection of
sample losses during the process. [1]
1.3.6 Robustness is the test which is used to examine the effects that can change the
results of the experiment. In robustness the experimental parameters are altered to a
small extent and then the results are taken. Robustness is the technique which is not
approved by FDA guidelines and is not considered most appropriate for validation.
The parameters of the experiment are subjected to a small change to find out the
changes in the result. The parameters which can be altered include pH of the buffer,
mobile phase, and temperature and detection wavelength. [1]
13

14. INSRTUMENTATION OF HPLC
14

15. Fig 2………………….. [7].
1.4 LAMOTRIGINE :- (LAMICTAL)
15

16. Lamotrigine is a new antiepileptic agent drug which is chemically not similar to other
anticonvulsants and generally used as an add-on therapy in patients with epilepsy.
Lamotrigine is effective against partial and secondary generalised tonic clonic
seizures as monotherapy or as an add-on. [5].
Lamotrigine or Lemictal is an anticonvulsant drug used in the treatment of
epilepsy and bipolar disorder. Lamotrigine is commonly used in epilepsy to treat
partial seizure, primary and secondary tonic clonic and Lennox Gas taut
syndrome. Lamotrigine is found to work in the treatment of mood disorders as
well, which includes rapid cycling and mixed bipolar state. The main advantages
of lamotrigine over other antiepileptica agents like valproate is that it has fewer
side effects and does not require blood monitoring. [6].
The CNS and nerves are made up of many nerve cells called neurons which
communicate among themselves through electrical signals. These signals are carefully
regulated for the brain and nerves to function properly. Sometimes there is an
abnormality in the brain leads to rapid and repetitive electrical signals firing at the
nerve endings, the brain becomes over-stimulated and normal function is disturbed.
This results in fits or seizures. [7]
1.4.1 MECHANISM OF ACTION: Lamotrigine is a sodium channel blocker which prevents epileptic fits by preventing
excessive electrical activity in the brain. Lamotrigine prevents sodium from entering
nerve cells and stops the conduction of the electrical signals and block use dependesnt
sodium channels. Sodium entry into the nerve cells is necessary for the electrical
signal to be passed on to other nerve cells. Lamotrigine prevents the sodium entry and
hence helps stabilise the electrical activity in the brain. [7]
Lamotrigine prevents the conduction of electrical impulse as well as it prevents the
release of a neurotransmitter called glutamate from the nerve cells in the brain.
Neurotransmitters are chemicals that are stored in nerve cells and are involved in
conducting messages between the nerve cells. Glutamate is a neurotransmitter that
acts as a natural 'nerve-exciting' agent. It is released when electrical signals
16

17. conduction takes place in the nerve cells and due to its nature it excites more nerve
cells. It is believed that glutamate plays a key role in causing epileptic seizures. [3]
Reducing the release of glutamate from the nerve cells in the brain also helps in
stabilising the electrical conductance in the brain and hence helps in preventing
epilepsy. [7]
Lamotrigine is also used as a mood stabiliser for treating people with the psychiatric
illness, bipolar affective disorder. Lamotrigine proved to be effective in people with
bipolar disorder who have not responded to the traditional mood stabilisers (lithium,
carbamazepine, valproate). Lamotrigine is used for treating episodes of high or low
mood and for helping to prevent episodes of ill health in these people. It is not fully
understood how lamotrigine works in this illness, but is thought to be to do with the
reduction of glutamate in the brain. [6]
1.4.2 Properties of Lamotrigine: - [6]
Chemical Formula
C9 H7 Cl2 N5
6-(2, 3-dichlorophenyl)-1, 2, 4-triazine-3, 5-diamine
Figure 1 (Taken from google image)
Mol. Mass- 256.091 g/mol
Pharmacokinetic data
Bioavailability- 98%
Protein binding -55%
17

18. Metabolism -Hepatic
Half life -24-34 hours (healthy adults)
Excretion- Renal
Storage- 2-8o C [8]
Insoluble in water [8]
1.4.3 Market Preparations of Lamotrigine: - Lamictal and Lamictin from GSK is
the main market preparation of lamotrigine. Lamotrigine is available as an oral
medication intended for oral use and it is available in tablet form with the dosage of
25 mg, 100mg, 150mg and 200mg. The tablet of the lamotrigine is given in the figure:
- [6]
Tablet of Lamotrigine
Fig.2 (taken from Wikipedia)
1.4.4 INDICATIONS: - [6]
Labelled indications: - Epilepsy and Bipolar Disorders I
1. Lennox-Gastaut syndrome
2. Partial Seizures
3. Secondarily Generalised Tonic Clonic seizures
4. Bipolar Mania I
5. Mixed and Rapid Cycling Mania
Off-Labelled Indications:1. Peripheral Neuropathy
2. Trigeminal Neuralgia
3. Neuropathy Pain
18

19. 4. Migraine
CHAPTER 2
MATERIALS AND METHODS
FOR
QUANTITATIVE ANALYSIS OF LAMOTRIGINE
19

20. 2.1 Materials
Chemical Name
Supplier
Orthophosphoric acid
Fisher scientific UK Ltd.
HPLC grade methanol
Fisher scientific UK Ltd.
Lamotrigine powder
Sigma Aldrich UK Ltd.
Acetonitrile
Fisher scientific UK Ltd.
De-ionized water
Made in the HPLC lab of University of
Greenwich
2.2 Equipment
1. HPLC pump: (I) Model :- 6000 A solvent delivery system manufactured by
WATER ASSOCIATES, serial no. 18105.
(II) AGILANT 1200 Systems 1200 Series isocratic pump
41310 A Serial No. DE 62956516
2.
Detector :-
(I) PHILIPS Pye Unicham PU 4025 UV Detector.
(II) AGILANT Systems 1200 Series VWD 41314 B.
Serial No. DE 71359377
3. Integrator: - Model number SP4270, Manufactured by: Spectra Physics.
4. Column: -
C 18 Bondapack column from WATER ASSOCIATES
Length 25 cm , Serial No. 27324.
20

21. 5. Injector: -
RHEODYNE 7725i 25uL injector made in USA.
6. Syringe: -
50 uL Syringe manufactured by; - SGE Ltd. Australia.
7. Membrane Filters: - Manufactured by Fisher UK limited.
8. Weighing Balance: - Model AC 88, Manufactured by: Metter instrument.
2.3 Chromatographic conditions:1. The Flow rate was 1ml/min.
2. UV Detection was done at 210nm.
3. A Rheodyne 20-µl loop injector was used.
4. C 18 Bondapack column from WATER ASSOCIATES 25 cm long.
5. 50 µL Syringe for injection.
2.4 Mobile Phase:1. The Mobile phase was prepared using Acetonitrile and Water ( 49.9:49.9 v/v)
with 0.2 % Orthophosphoric acid.
2. The Mobile phase was prepared and the Degassed using membrane filters
of 0.45 µm thickness under vaccum.
2.5 Chromatographic system and conditions:HPLC method was performed using an Agilant systems HPLC pump with a
Rheodyne 7725 I injector. The absorbance was taken with AGILANT
Systems 1200 Series VWD 41314 B. The separation was done on C-18
µBondapack column. The mobile phase was prepared with acetonitrile and
21

22. water and the Flow rate was kept at 1ml/min. The experiment was done at
room temperature and 20 µl of samples was injected into the HPLC system.
2.6 Standard Stock Solution of Lamotrigine:The Lamotrigine powder was taken from Sigma-Aldrich ltd in 10 mg amber
coloured bottle. The lamotrigine standard solution was prepared with help of
methanol. Accurate amount of lamotrigine was weighed and transferred to a
10 ml volumetric flask and made it up to 10 ml. This gave lamotrigine
standard solution as 10mg of lamotrigine in 10 ml methanol.
2.7 Working Standard solution:The working standard solution contained 25µg/ml of lamotrigine. 250 uL of
the standard solution was taken and transferred to 100ml volumetric flask and
the solution was made up to 100ml with methanol.
.
2.7 Procedure:Preparation of Lamotrigine Stock solution
10 mg of Lamotrigine was taken and dissolved in Methanol and made upto 10 ml in
volumetric flask. Then further dilutions to get desired test solutions were done from
this stock solution using the calculations:-
22

23. The equation used for making the dilutions for test solutions:
C1V1= C2V2
Where,
C1 = Concentration of the Stock solution
V1= Unknown volume to be calculated
C2 = Concentration of dilution to be prepared
V2= Volume of the volumetric flask in which the dilution is made
2.8 DILUTIONS MADE FOR THE EXPERIMENT:Dilutions made from working standard solution:Lamotrigine
0.5 ml
1.0 ml
1.5 ml
2.0 ml
2.5 ml
Methanol (vol. make up )
100 ml
100 ml
100 ml
100 ml
100 ml
Conc. in PPM
5
10
15
20
25
These different dilutions were injected in the HPLC system to get the results.
CHAPTER 3
PARAMETERS FOR PARTIAL VALIDATION
3.1-Determination of Standard Deviation:S. No.
Concentration
Peak Area (AUC)
1
10 µg/ml
2.70
2
10 µg/ml
2.68
23

24. 3
10 µg/ml
2.74
4
10 µg/ml
2.74
5
10 µg/ml
2.75
6
10 µg/ml
2.73
The Mean of the above readings comes to be:Mean = 2.73
Median = 2.74
The Standard Deviation of the above readings was calculated from the
site http://www.physics.csbsju.edu/stats/descriptive2.html.
The Standard Deviation of the above readings came as:Standard Deviation (S.D) = 0.02495
From the Standard Deviation we can calculate the Relative Standard
Deviation.
Relative Standard Deviation (RSD) = (SD/Mean) x 100
i.e. RSD = (0.02495/ 2.73) x 100
= 0.91%
3.2 Linearity:Linearity of the test sample was calculated by plotting the graph of different
concentrations against their respective peak areas. The graph when plotted showed a
straight line with the Regression Coefficient r2 value 0.9994. From the graph, the
value of slope was taken for further calculation of LOD and LOQ.
Readings for the Concentration and respective AUCs of different concentrations
Concentration
Mean Peak area (AUC)
24

25. 1
5
10
15
20
25
400
1204
2293
3543
4649
5746
Chart Title
7000
y = 225.52x + 115.91
R2 = 0.9994
6000
5000
4000
peak area AUC
3000
Linear (peak area AUC)
2000
1000
0
0
10
20
30
From the above graph we get the equation:-
y= 225.52x + 115.91
Hence we get, Slope=S= 225.52
Table for Peak areas of different concentrations with their Standard Deviation:Concentration
Peak
Peak
S.No
µg/ml
Area (1) Area (2)
Peak
Area (3)
Mean
S.D
25

26. 1
1 µg/ml
362
409
430
400
0.34
2
5 µg/ml
1213
1198
1201
1204
0.79
3
10 µg/ml
2280
2298
2301
2293
0.114
4
15 µg/ml
3547
3539
3544
3543
0.040
5
20 µg/ml
4658
4619
4671
4649
0.27
6
25 µg/ml
5790
5772
5675
5746
0.61
Total S.D = 2.164
From the above table we get the Total Standard Deviation S.D = 2.164
3.3 Limit of Detection (LOD) = 3 x S.D/s
Where s = slope = 225.52
Hence LOD = 3 x 2.164/225.52
= 0.028 µg/ml
3.4 Limit of Quantification (LOQ) = 10 x S.D/s
Hence LOQ = 10 x 2.164/225.52
= 0.096 µg/ml.
The limit of detection and limit of quantification showed the results that the
concentration of the sample was very less and hence the system was very sensitive to
even smaller amounts of samples as well.
3.4 Quantification:Readings were taken from equal injections of a test solution and standard solution and
compared and then the content of lamotrigine in solution was calculated by
Cs (At/As)
At
As
26

27. 3695
5545
3620
5571
3672
5574
Where Cs is the concentration of lamotrigine in the working standard solution which
is 25 µg/ml. Hence
Quantified value of the test solution = 25(3662/5563)
= 16.45µg/ml
3.6 Precision and Repeatability:The Precision of HPLC method was done by Repeatability which includes Intra Day
and Inter Day precision. The results were taken by injecting the concentrations of 5,
10 and 20 and the peak areas were taken for precision by calculating the standard
deviation.
3.6.1 INTRA-DAY PRECISION ----- TABLE 1. Date 13/12/2007
Concentration
S.No.
(PPM)
Peak Area
AUC
1173
S.D
R.S.D
27

28. 1.
5
1169
0.050
0.42
0.076
0.32
0.226
0.48
1179
2306
2.
10
2311
2321
4661
3.
20
4674
4705
TOTAL RSD = 1.22
3.6.2 INTRA-DAY PRECISION ------TABLE 2
Concentration
S.No.
1.
Peak Area
(PPM)
AUC
1174
S.D
R.S.D
5
1179
.00251
0.021
0.151
0.64
0.66
1.40
1177
2337
2.
10
2366
2344
4723
3.
20
4624
4749
3.6.3 INTER-DAY PRECISION AND REPEATEBILITY
Concentration
S.No.
1.
Date 14/12/2007
Peak Area
(PPM)
AUC
1127
S.D
R.S.D
5
1131
.00208
0.018
0.214
0.91
0.110
0.22
1128
2349
2.
10
2359
2318
4853
3.
20
4869
4874
TOTAL RSD = 1.14
28

29. The results obtained for Repeatability and Precision taking the interday and intra
Day results which shows that all the readings show RSD below 2.0%.
The results were taken from inter and intraday precision and repeatability and the
RSD for the results were taken and compared. All the results showed the RSD under
the limits of acceptance and the method came out to be linear and precise.
CHAPTER 4
RESULTS AND CONCLUSION
4.1 RESULT AND CONCLUSION:The Partial Validation of Quantitative Analysis of Lamotrigine was done using HPLC
and the parameters taken to partially validate the technique include Linearity,
Quantification, Specificity, Precision and Repeatability. The results obtained in the
technique along with their use in validation are detailed below:4.2- LINEARITY: - The linearity was evaluated with the help of linear regression
analysis and linearity gave the values of LOQ and LOD.
29

30. 4.3- Regression coefficient r2 was 0.9994 which shows great degree of linearity.
4.4- LOQ and LOD: - From the linearity taken the LOD and LOQ were calculated
with the help of slope from the regression equation. The calculated
LOQ = 0.096 µg/ml
LOD = 0.028 µg/ml#
4.5- PRECISION AND REPEATIBILITY:The precision and repeatability was taken by Intra Day and Inter Day analysis and the
results were taken to calculate the Relative Standard Deviation (RSD). The RSD for
both Intra day and Inter day came under the desired value of 2 % and hence the results
comply within the limits.
RSD for Inter day came to be 1.14 %
RSD for Intra day came to be 1.22 %
4.6 -CONCLUSION:The HPLC validation technique for the Quantitative analysis of Lamotrigine gives
results under the limits of acceptance and the method used is linear and precise.
CHAPTER 5
DISCUSSION
5.1 DISSCUSION:The method used in this experiment for the HPLC of lamotrigine was slightly
different from the method taken as reference. The changes in the mobile phase were
done and a simpler mobile phase was used containing acetonirile and water. The
method showed linearity with regression coefficient 0.9994 and precision with RSD
less than 2% for both Inter and Intra day precision. According to the USP the value of
RSD (which is the measure of the spread of data in comparison to the mean of the
data should not be more than 2%) [9] And hence the results came under the limits of
acceptance and the method was found to be linear and precise.
30

31. REFERENCES
1. Johan
Lind
Holm,
Development
methods,Comprehensive
and
Validation
of
HPLC
Summaries of Uppsala Dissertations from
Faculty of Science and Technology 995, Acta Universities Upsaleinsis, pp
87 Sweden 2004.
2. Thomas H Stout, Handbook of Pharmaceutical Analysis, chapter 3.
Published in
3. A
Acid Free paper 2002, United States of America.
Guide
to
HPLC,
http://www.pharm.uky.edu/ASRG/HPLC/hplcmytry.html
4. N.A Epshtein, Validation of HPLC Techniques for Pharmaceutical Analysis,
Pharmaceutical Chemistry Journal vol. 38, no.4, 2004.
31

32. 5. J. Emami, N Ghassami, Development and Validation of HPLC method for
Determination of Lamotrigine, Journal of Pharmaceutical and Biomedical
Analysis 40 (2006) 999-1005.
6. Wikipedia, Lamotrigine last modified 03:27, 13 December 2007.
7. netdoctor.co.uk/medicines/100001450.html last updated 15/3/2007.
8. Sigma-Aldrich
sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/L37911000014
50.html last updated 15/3/2007.
9. J.C Miller, Statistics for Analytical chemistry, III rd edition. 1993.
32

33. 33