HPLC_PROJECT_K1118688

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FACULTY OF SCIENCE, ENGINEERING
AND COMPUTING
School of Pharmacy and Chemistry
BSc (Hons) DEGREE
IN
Pharmaceutical science
Samali Mukalazi
k1118688
DEVELOPMENT AND VALIDATION OF REVERSE PHRASE
HIGH PREFORMANCE LIQUID CHROMATOGRAPH (HPLC)
FOR THE ANALYSIS OF LAMOTRIGINE.
Date: 27/04/2014
Supervisor: Dr Hall Carl
WARRANTY STATEMENT
This is a student project. Therefore, neither the student nor Kingston University makes any
warranty, express or implied, as to the accuracy of the data or conclusion of the work
performed in the project and will not be held responsible for any consequences arising out of
any inaccuracies or omissions therein.

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Acknowledgment:
I would like to thanks God for giving me the strength to finish this project. Many
thanks to my supervisor Dr Carl Hall for all his guidance and support.
I would also like show my appreciation to the lab technicians of EM 1027 thank you
for being patient with me; I very much appreciate all the help and guidance you have
offered me on this project.

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Abstract
A study was carried out in order to develop and validate a method using reversed
high performance liquid chromatography in order to quantify and determine
lamotrigine in lamotrigine tablet. Lamotrigine was dissolved in HPLC grade
methanol. Separation was performed under isocratic conditions using waters C18
column (150mm x 4.6mm) with a 5 µl particle size) with a flow rate of 1.5ml/min. A
mobile phase of acetonitrile and water (50%:50%) was also used. The UV detector
was operated at 307nm wave length. A well resolved peak of lamotrigine was
obtained at the Stationary phase and the retention time was 1.64.
Validation of the method was carried out according to the international conference of
harmonisation guidelines. The developed method worked well and it was
successfully used for the separation and qualification of lamotrigine. Linearity was
analysed in the concentration range of 7-31µg/mL, generation a regression equation
y=13463x-6202.6 and R2=0.998. Robustness was performed using modified mobile
phase conditions of (55%ACN and 45%water), (45%ACN and 55%water) and
(50%ACN and 50%water) results obtained for the %RSD met the acceptable criterial
of ≤2%. The %RSD obtained for the stability testing of lamotrigine after it was left at
room temperature for 3 day was 6.77% which didn’t met the acceptable criterial of
≤2%, this proved that lamotrigine is not stable when left at room temperature for
3days. % RSD obtained for Precision repeatability and intermediate testing didn’t
exceed ≤2% for the peak and ≤1% for the retention time. The limit detector and limit
quantification were 0.07µg/mL and 0.23µg/mL respectively
Results obtained showed that the method developed was successful and is suitable
for the daily therapeutically drug monitoring.
Key words: lamotrigine, acetonitrile, mobile phase, UV, high performance liquid
chromatography.

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Table of Contents
Acknowledgment ..................................................................................................................2
Abstract ...................................................................................................................................3
Table figures ..........................................................................................................................7
List of table .............................................................................................................................8
List of abbreviation and symbols .........................................................................................9
1.0 Chapter 1 ........................................................................................................................10
1.1 Introduction .....................................................................................................................10
1.2 Lamotrigine and Epilepsy .............................................................................................10
1.2.2 Chemistry of Lamotrigine ...........................................................................................11
1.2.3 Physicochemical properties of lamotrigine .............................................................11
1.2.5 Ultraviolent absorption spectrum ..............................................................................12
1.3 Drug interaction ..............................................................................................................12
1.3.1 Pharmacokinetic and pharmacology properties of Lamotrigine...........................13
1.3.2 Mechanism of action ..................................................................................................15
1.3.3 Clinical use and therapeutic potential of Lamotrigine ...........................................15
1.3.4 Dosage and administration .......................................................................................16
1.4 HPLC –high performance liquid chromatography .....................................................16
1.4.1 HPLC Instrument ........................................................................................................18
1.4.2 Retention and Resolution...........................................................................................19
1.5 Separation techniques: Isocratic, Gradient elution and Derivatizaton ..................20
1.5.1 External and internal standard ..................................................................................20
1.6. Normal phase chromatograph ....................................................................................21
1.7 General method validation steps ................................................................................21
1.8 Proposed chromatographic conditions .......................................................................22
1.8.1 Proposed experimental conditions for HPLC separation......................................23
1.8.2 Description of column of choice ...............................................................................23
1.8.3 Choice of technique HPLC Reverse Phase Chromatograph ..............................23

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1.9 Aims…………………………………………………………………………………..24
1.9.1 Objectives…………………………………………………………………..……24
2.0 Chapter 2 .........................................................................................................................25
2.1 Experiment ......................................................................................................................25
2.1.1 Material and Method ..................................................................................................25
2.1.2 Reagents and Chemicals ..........................................................................................25
2.1.3 Equipment ...................................................................................................................25
2.2 Preparation of lamotrigine solution .............................................................................25
2.2.1 Working standard solution ........................................................................................26
2.2.2 Chromatography analysis separation goals ...........................................................26
3.0 Chapter 3………………………………………………………………………...27
3.1 Discussion and results ..................................................................................................27
3.1.1 UV absorbance of lamotrigine ..................................................................................27
3.2. Selecting the appropriate solvent ………………………………………………27
3.2.1 Selecting the appropriate column ……………………………………………….28
3.2.2 Selecting the appropriate mobile phase ………………………………………...28
4.0 Method validation …………………………………………………………………...30
4.1 International conference on harmonisation (ICH) guidelines .................................30
4.2 Linearity and range ........................................................................................................31
4.2.2 Linearity test results………………………………………………..……31
4.3 Robustness .....................................................................................................................33
4.3.1 Stability ........................................................................................................................36
4.3.2 Stability test results ....................................................................................................36
4.4 Precision of analytical method......................................................................................37
4.4.1 Precision- Repeatability ............................................................................................37
4.4.2 Precision- repeatability test results …………………………………….…37
4.5 Intermediate –Precision……………………………………………………..39

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4.5.1 Intermediate –precision test results…………………………………...….39
4.6 Limit of detector (LOD)………… ……………………………………………40
4.6.1Results of LOD of SD based on the slope of the calibration curve .....................40
4.6.2 Limit of quantification (LOQ) .....................................................................................41
4.6.3 Results of LOQ of SD based on the slope of the calibration curve .....................41
4.7 Analysis of lamotrigine tablet .......................................................................................41
4.7.1 Preparation of Lamotrigine table sample solution..................................................42
4.7.2 Lamotrigine tablet results ..........................................................................................42
5.0 Conclusion ......................................................................................................................44
6.0 Future work .....................................................................................................................47
References ............................................................................................................................48
Appendix ………………………………………………………………………………..….51

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Table figures
Figure 1: chemical structure of lamotrigine……………………………………………12
Figure 2: wavelength of lamotrigine……………………………………………………13
Figure 3: mechanism action of lamotrigine ……………………………………………15
Figure 4: HPLC system. …………………………………………………………………17
Figure 5: Detector wavelength for lamotrigine in methanol …………………….……27
Figure 6: chromatogram of lamotrigine dissolved in acetonitrile. The peak is
unresolved………………………………………………………………………………….28
Figure 7: chromatogram of lamotrigine dissolved in methanol. The peak is
resolved……………………………………………………………………………………..28
Figure 8: chromatogram of the mobile phase of 7O% ACN and 30% water……….29
Figure 9: chromatogram of the mobile phase of 60% ACN and 40% water………..29
Figure 10: chromatogram of the mobile phase of 50% ACN and 50% water………29
Figure 11: Calibration curve generated for analysis of lamotrigine……………..……31
Figure 12: mobile phase 55% ACN and 45% water chromatogram…………………32
Figure 13: mobile phase 45% ACN and 55% water chromatogram………………….33
Figure 14: mobile phase 50% ACN and 50% water chromatogram………………….34
Figure 14: chromatography of lamotrigine tablet ………………………………………40

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List of Table
Table 1: HPLC Instrument ……………………………………………………….…...17
Table 2: Published HPLC methods used for analysis of lamotrigine in dosage form
and biological fluids. ……………………………………………………………………21
Table 3: Proposed experimental conditions for HPLC separation………………..22
Table 4: Optimized chromatography conditions …………………………………….29
Table 5: Linearity test results…………………………………………………….….…30
Table 6: linearity test – comparison of results to acceptance criterial………….….31
Table 7: Robustness under modified conditions - mobile phase 55% ACN and 45%
water……………………………………………………………………………………….32
water………………………………………………………………………………………..33
water………………………………………………………………………………………..34
Table 10: Stability test results day 3…………………………………………………….35
Table 11: Repeatability Test at 7µg/mL…………………………………………………36
Table 12: Repeatability Test at 13µg/mL………………………………………………..36
Table 13: Repeatability Test at 19µg/mL………………………………………….……36
Table 14: Precision test results at 31µg/mL……………………………………………37
Table 15: Precision test results at 31µg/mL……………………………………………37
Table 16: results obtained from linearity…………………………………………….…38
Table 17: Results for the analysis of lamotrigine table at 13µg/mL…………………40

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List of Abbreviations and Symbols
HPLC – High Performance Liquid Chromatography
LOD – Limit of Detection
LOQ –Limit of Quantification
pKa – Acid Dissociation Constant
pH – Negative Logarithm of H+ Concentration
SD - Standard Deviation
RSD – Relative Standard Deviation
V/V – Volume by Volume
ICH – International Conference on Harmonisation Guidelines
UV- Ultra Violent
µL – Microliter
µg/ml – Microgram Per Millilitre
mL – Millilitre
n - Number of Replicates
R2 – Correlation Coefficient
tR – Retention Time
ACN – Acetonitrile
AEDs – Anti-epileptic Drugs
Cmax – Maximum Concentration
IUPAC- international union of pure and applied chemistry

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1.0 Chapter 1
1.1 Introduction
1.2 Lamotrigine and Epilepsy
Lamotrigine (LTG) was for first introduced for the treatment of partial seizures in
1991 in Europe and then in 1994 in the United States. Lamotrigine is known as
antiepileptic drug that belongs to the phenyltriazine class.10, 11
Lamotrigine has the following brand names, Lamictal, Lamictal XR, Lamictal CD and
Lamictal ODT. Lamotrigine is an oral drug which is chemically not related to other
anti-seizure drugs. Lamotrigine was approved by the FDA in 1974. When being
prescribed, Lamotrigine can be prescribed alone or with other anti- seizure drugs
such as carbamazepine for the treatment of partial seizures, tonic-clonic seizures,
including seizures of lennox-gastaut syndrome. The side effects of taking lamotrigine
orally are; dizziness, headache, vomiting, double vision and rash. Patients usually
develop a rash when the lamotrigine dose has been increased. 22
Research from the world health organization, shows that there is an estimated 50
million individuals with epilepsy in the world23. Epilepsy can affect anyone at any age
however the disorder is more increasing in the elderly starting from the age of 65.
Epilepsy is known as a condition which starts from the brain regions resulting in a
range of different seizures. Epilepsy is still not fully understood by researchers
however studies have provided an understanding of epilepsy’s neurobiology in order
to use surgery as a treatment. There is limited knowledge regarding the mechanism
partial seizure disorder, however the partial seizure are much more common in
adults, mostly stemming from focal lesions like stokes, head trauma and tumors.
Circuitry between the thalamus and cerebral cortex are the parts of brain which are
affected by partial seizure. In 1850 bromides were used for the treatment of the
treatment of seizures, there was a theory that epilepsy was caused by an excessive
sex drive. In 1940 phenytoin was also found to be an effective drug for the treatment
of epilepsy, it now known as a first line antiepileptic drug for the partial and second
secondary seizures. Carbamazepine is another drug also used in the treatment of
epilepsy the drug was approved for the treatment of partial seizures in 1974.
Lamotrigine is as an antiepileptic drug it is used to treat epilepsy, partial epilepsy,
Lennox-Gastaut syndrome and bipolar disorder. Epilepsy is described as a brain
disorder; abnormal electrical activity takes place in the brain which results in the
seizures.24, 25

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1.2.1 Chemistry of Lamotrigine
Figure 1: Chemical Structure of Lamotrigine 1
Lamotrigine is as chemically known as 3, 5 diamino-6-(-2, 3-dichlorophenyl)-1,2,4-tri-
azine. The international union of pure and applied chemistry (IUPAC) name for
Lamotrigine is (6-(-2, 3-dichlorophenyl)-1, 2, 4-tri-azine 3, 5 diamino). Lamotrigine
has a molecular weight of is 256.09 and a PKa of 5.7.12
1.2.2 Physicochemical properties of Lamotrigine
Lamotrigine is slightly soluble in water (0.17mg/mL at 25C) and also slightly soluble
in 0.1M HCl (4.1mg/mg at 250c). 12
The melting point of Lamotrigine is 2560C. 12
Lamotrigine is stored at 25 degrees 0C (77 degrees F) and should be stored away
from heat, moisture and light.12

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1.2.3 Ultraviolent Absorption Spectrum
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Figure 2: wavelength of Lamotrigine.14
Lamotrigine is a lipophilic base, at a pH value of 4.5 the UV spectra of lamotrigine
are absorbed at 268 nm however at pH 5.7 of 5.7 there is slightly an increase a
wavelength of 299 nm. Lamotrigine exists as non-ionic form with a wavelength of
308nm at a pH value of 6.8. At the pH of 4.5 Lamotrigine exists as the ionized cation
with a wavelength of 268nm. 14
1.3 Drug Interaction of Lamotrigine
Drug metabolism is not influenced by lamotrigine, it does not interfere with the
efficacy of oral contraceptives agents. Enzyme inhibiting drugs e.g. valproate semi-
sodium, increase lamotrigine plasma concentration and enzyme inducing drug e.g.
carbamazepine, decrease lamotrigine concentration. 13
Lamotrigine is known to be metabolised by glucuronidation. Pharmacokinetic drug
which tend to change lamotrigine blood levels are more expected to do this through
Phase 2 metabolic process. Moreover cytochrome P450 enzyme is not involved in
lamotrigine metabolism.26

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1.3.1 Pharmacokinetic and Pharmacology Properties of Lamotrigine
Lamotrigine undergoes first pass metabolism, bioavailability is 98% for oral
lamotrigine. The absorption of lamotrigine is also not affected by food. Lamotrigine
concentration occurs 1 to 3 hours after a dose. The drug is delivered to all organs,
tissues and the brain, the mean protein binding is between 55 to 68%. Lamotrigine
crosses the placenta and is found in the fetus. In the liver Lamotrigine is broadly
metabolised in the liver, especially along N-glucuronidation. 1.6 to 2.6 L/h is the
clearance and with a mean plasma elimination half-life of 25 to 35 hours. 13
Food does not affect the absorption of lamotrigine. The binding of protein of protein
appears to be quite low at 55% however this doesn’t affect the interaction of
lamotrigine with other drugs. Lamotrigine undergoes phase two conjugation to a 2-N-
glucuronide which is inactive. Lamotrigine can be metabolized outside cytochrome
P450 system of enzymes. Studies have shown that lamotrigine has tendency
towards auto induction in the early stages of treatment. With various dosing and
without drug interactions the elimination half-life results to be ~25 hours.
Pharmacokinetic of lamotrigine can be modified in a lot of ways, it appears that
cirrhosis does not change the Pharmacokinetic. Chronic renal failure has small effect
on the plasma level of lamotrigine however its elimination half-life is prolonged. A
patient’s gender and weather they are obese happens to change lamotrigine
clearance. It was found in studies there is a substantial decrease in blood level of
lamotrigine in pregnancy due to pregnancy evolved and an increase following
delivery. Lamotrigine dosage may be increased if needed during pregnancy in order
to maintain a therapeutic effect. 26
The mechanism of action in epilepsy is still unknown; however lamotrigine doesn’t
have inhibitory effects on voltage sensitive sodium channels. Sodium channel
inhibition results in stabilizing neuronal membranes and modulate release of
excitatory amino acid neurotransmitters. Research was done by ketter and
colleagues investigating multiple mechanisms of action mainly based on
neuroprotective and antigulatmatergic effects as contributors to mood stabilizing
activity. Results that were obtained showed that lamotrigine reduced alpha
aminobutyric acid this therefore suggested that alpha aminobutyric acid mechanism
might also be part involved in the mechanism. Lamotrigine happens to not have an
effect on most neurotransmitters receptors such as dopamine D1 and D2,
muscarinic, serotonin 5-HT2 and N-methyl-D-asparte. Inhibitory effects on dopamine
transporters, 5-H, and norepinephrine are known to be weak. Lamotrigine happens
to be a weak inhibitor of dihydrodroflate reductase however this is useful to in the
mechanism of action.26

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1.3.2 Mechanism Action of Lamotrigine
Lamotrigine is a phenyltriazine compound which acts mainly to inhibit excitatory
amino acid release and stabilises neuronal membranes via blockade of voltage
sensitive sodium channels.35
Lamotrigine blocks hindlimb extension induced by pentyleneterazol and by maximum
electroshock. Lamotrigine also reduces duration of electrically induced after
discharges in different species after focal, cortical and hippocampal stimulation. The
activity in the visually evoked after discharge 35
Lamotrigine blocks low threshold calcium currents. Low threshold calcium channels
are found in high masses of in thalamic neurons, these channels tiger regenerative
bursts which maintain normal and pathologic thalamocortical rhythms and the spike
wave discharges of seizures.
Figure 3: Mechanism Action of Lamotrigine35
The diagram show how lamotrigine blocks influx of sodium ions, by that reducing
excess glamate release and stabilising neuronal membranes. This suggests that
lamotrigine is effective against seizures.35

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1.3.3 Clinical Use and Therapeutic Potential of Lamotrigine
In the UK and Ireland lamotrigine is licenced as an added (with other antiepileptic
drugs) on treatment for patients with partial and secondarily generalised seizures
that are not entirely controlled with other antiepileptic drugs. This drug is used mostly
on children and elderly people however its use in these patients is not recommended
out with control clinical trial. Like most new drugs lamotrigine should not be used in
early pregnancy, unless in the opinion of the patients physician, where the benefits
of the drug outweigh any potential risk to the developing fetus. 35
The dose of lamotrigine depends on exciting treatment this is due to interaction with
other antiepileptic agents. 50mg per a daily is to be given for 2 weeks before the
dose is increased on clinical ground to maintenance amount of 50-100mg twice a
day in adolescents and adults talking sodium valproate alone. 35
The dose for Patients talking other anticonvulsant drugs starts with 50mg twice a
daily and the maintenance dose between 100-200mg twice a daily. Children over 2
years the dose to begin with should be 2mg/kg/day increasing to a maintenance
dose of 5-15mg/kg/day.35
A lower dose should only be used if sodium valproate alone is co-prescribed.
Moreover a higher dose can also be used if seizure continues and the patient is
tolerating the drug well. Abrupt withdrawal of lamotrigine can provoke rebound
seizures, the drug should be tapered over a period of weeks or month when the drug
dose is discontinued. 13
From previous studies it has been shown that lamotrigine is limited in different forms
of epilepsy. Lamotrigine is effective for partial and secondary tonic colonic seizures
and also for primary generalised epilepsies.13
The long elimation half-life and lack of important sedative side effects makes
lamotrigine a promising monotherapy for patients with newly diagnosed epilepsy.13
In the brain, lamotrigine acts by reducing the release of the excitatory amino acid
holding out the possibility of other therapeutic effects. Glutamate induced
neurotoxicity mediates cell injury and death after cerebral anoxia and ischaemia as
well as neuronal loss during severe status epilepticus.in other areas of clinical
medicine e.g. Parkinson’s disease, moteneuron disease and Huntington’s chorea,
lamotrigine is proven effective in these areas.13

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1.3.4 Dosage and Administration
In the United States lamotrigine is approved for the treatment of adults with bipolar 1
disorder in order to delay the time to occurrence of mood episodes in patients
treated for acute mood episodes with standard therapy. The recommended dosage
amount is 25mg/day orally which can be gradually increased to 200mg/day over 6
weeks. Dosages over 200mg/day as monotherapy are not recommended and no
additional efficacy has been demonstrated in clinical trial evaluating dosages up to
400mg/day.12
Patients talking valproate semi sodium, the recommended lamotrigine dosage is
25mg every other day making the target dosage 100mg/day from week 6. When
combined with carbamazepine the recommended dosage is 50m g/day, which can
be increased up to 400mg/ day from week 7. 12
Dosage that is less than 50mg/day can be given once daily. Lamotrigine should be
discontinued at the first sign of any rash unless the rash is not drug related. 12
1.4 HPLC –High Performance Liquid Chromatography:
HPLC –high performance liquid chromatography is an analytical chromatographic
technique which is used separate a mixture of compounds in the area of
biochemistry, analytical chemistry and industrial. HPLC is used mainly to identify,
quantify and to purify each individual components of the mixture. 7,8 Reversed phase
high performance liquid chromatography is a mostly used separation mode. It is
known to provide an excellent retention of compounds which are hydrophobic and
organic.34
Figure 4: HPLC System. 9

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The sample mixture of choice is sent to a stream of mobile phase percolating via the
column to be separated and tested. Columns are available in different types with
sorbents of different particle size and surfaces.34
The mixture moves through the chosen column and interacts with the sorbent. The
sorbent is also known as the mobile phase. The movement speed of each
component in the mixture depends on it chemical nature and the nature of the
chosen column and the composition of the mobile phase. The retention is the time at
which a specific analyte arrives from the column.34
Combinations of water and organic solvents like methanol and acetonitrile are the
mobile phases used the most; however water free mobile phase can also be used.
The mobile phase of aqueous components usually composes acids such as formic,
phosphoric or trifluoroacetic acid or salts which permit the separation of the sample
components. Mobile phase composition can be kept constant or varied during the
chromatographic analysis. For sample components which are not similar in their
affinity for the stationary phase, the constant approach is effective. The composition
of the mobile phase changes from low to high eluting strength, in the varied
approach. Eluting strength of the mobile phase demonstrated by the analyte
retention times, where high eluting strength causes fast elution. Selecting the mobile
phase composition is based on the intensity of interactions between quiet a lot of
sample components and the stationary phase. 34
HPLC is tremendously quick and efficient compared to other chromatographic
techniques, like TLC. HPLC uses a pump instead of gravity, to force a liquid solvent
through a solid adsorbent material. The process takes about 10 to 30 minutes and
gives a high resolution. It is accurate and reproducible because it is largely
automated.34
HPLC can be used in water purification, ligand exchange chromatography, ion
exchange chromatography of proteins and high pH anion exchange chromatography
of carbohydrates and oligosaccharides.34

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1.4.1 HPLC Instrument
Table 1: HPLC Instrument
Instrument Description
Pumps In order for mobile phase to be pushed through the packed
stationary phase high pressure pumps are needed. A pump
pressure between 1000-2000 psi is requires to ensure accuracy
and reproducibility. Maintenance of pumps is required in order to
maintain characteristics of the pumps and to minimize down time.
6
Injectors A single injector or an automated injector system may be used.
The HPLC injector is required to provide sample injection
between 0.1-100Ml of volume with a high reproducibility and
under the pressure of up to 4000 pis. Samples that are solid must
be diluted in the right solvent and the samples that are liquid can
directly be injected for liquid chromatography. 6
Columns Columns come in different lengths, packing materials and bore
sizes. Different separations use different types of column
dimensions. Silica based is the most common used for HPLC
packing material. Octadecyl silica is a popular material with a
C18 coating. Theoretical plates relate chromatographic
separation to theory of distillation and they are also a measure of
column efficiency. The equation below shows how the number of
theoretical plates (n) can be determined. From the equation Tr1
is the total retention time and the band width of the peak is w.6
n = 16 ( tR1 / w)2
LC columns are known to be durable, unless when they are used
with highly acidic, basic eluents or with crude oil samples.
Columns can be re used many times when it’s properly
maintained. It’s very important for a column to be flushed with
mobile phase of high elution strength. Column should be
prevented from drying out when it’s not in use. It is required for
columns to be filtered and a guard column to be utilized.6
Detector The main reason for the use of a detector is to sense the
presence of the compound that is passing through and to also
provide an electronic signal to the data acquisition device.
Fluorescence, ultraviolent and refractive index are the main types
of detectors which can be used in HPLC. When it comes to the
analysis of drugs detectors that respond to absorption of
ultraviolet radiation by the passing through of the solute through
the flow cell inside the detector are most used. The latest
technique known as hyphenated techniques happens to improve
the separating and identifies multiple entities in a mixture. Some
of these techniques are liquid chromatography infrared
spectroscopy and chromatography mass spectrometry. 6

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Mobile Phase
and Reservoir
Separating of components is most affected by the type of
composition of mobile phase. Different HPLC types use different
solvents. The solvent is non-polar for normal phase whereas in
reverse phase the solvent is a mixture of polar organic solvent
and water. Glass Bottles with tubing connecting them to the
pump inlet these are the most used solvent reservoirs.6
Data
Acquisition
Data acquisition is also known as the display system, Data
acquisition for HPLC is a computer. The response to each
component is integrated by the computer and placed into a
chromatograph that is able to read and interpret. Features such
as computer controlled automatic injector, sample fraction
collectors and multi-pump gradient are features that are included
in the data acquisition HPLC system.6
1.4.2 Retention and resolution
Retention or elution volume is the amount of mobile phase needed to pull the sample
through the column. Retention time is how long a component is retained in a column
by the stationary phase relative to the time it residues in the mobile phase. Retention
is also best known as column capacity ratio (k’) that can be used to evaluate the
efficiency of columns. Capacity factor tends to be greater when a component is
retained longer by the column. The following equation shows the column capacity
ratio of a compound (A). VA happens to be the elution volume of compound A
whereas VO is the elution volume of a non-retained compound. Instead of using
retention or elution volumes, retention time (TA and TO) may be used at constant flow
rate. 6
K’ = TA-TO/TO = VA-VO/VO
Resolution enables the column to separate peaks on the chromatography.
Resolution is the ratio of the distance between two peak maxima to the mean value
of the peak width at base line, TB representing the retention time of component B and
TA the retention time of component A. the peak width of component is the
represented by WB. Components appear to be fully separated when R is equal TO or
more than 1 however resulting in components overlapping.6
R = (TB-TA)2 /WA + WB

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1.5 Separation Techniques: Isocratic, Gradient Elution and Derivatizaton
Isocratic method is a very simple technique, in this method the composition of the
mobile phase remains constant. Gradient elution techniques are known to be
methods of pumping mobile phase through a column, in this method this method
during separation process the composition changes. 6
Derivatizaton can be used to enhance the selectivity and selectivity of detection
when present detectors are not good enough for the inderivatized compounds. A
drug is unnecessary to achieve adequate chromatography in HPLC Derivatizaton. 6
1.5.2 External and Internal Standard
This method depends on the reproducibility of the injection volume. For this method
to be carried out the standard solutions of unknown concentrations of the needed
compound are prepared with one standard which is the same in concentration to the
unknown. Fixed amount of sample is injected. The peak area or height is plotted
versus the concentration of each compound and it is to be linear, going through the
origin. The formula below is used to determine the unknown concentration.6
Conc. unknow n = (Area unknow n / Area know n) conc. unknow n
Accurate and precise results are more yielded by the internal standard method.
Equal amount of an internal standard is added to both samples and standard
solutions in this method. In order to have the same retention time and the same
derivatize to the analyte, the chosen internal standard has to be chemically similar.
The internal standard sample must be stable and must not interrupt with the sample
components. In order for the extraction efficiency to be evaluated the internal
standard must be added before the preparation of the sample. Quantification is
carried out by the use of the area of the component or peak height to the internal
standard. 6
Conc. unknow n = (AreainternalStd.in know n / Area internalStd.in unknow n) x = (Area unknow n / Area know n)
x (conc. know n)

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1.6 General Method Validation Steps
There are five steps in the method validation process System qualification, sampling,
preparation, and analysis and data evaluation.27
The system qualification process allows analysts to ensure that the instrument is
suitable for the proposed analysis and also if the material are well suitable for use in
analytical determinations. Analysts must possess training and qualifications. 27
Sampling gives assurance that the sample chosen represents the material as a
whole for the purpose of meaningful statistical inferences. The substantial body is
found within the category of statistical literature moreover the time and relative costs
involved within each strategy must be evaluated in advance. 27
In the analytical laboratory the sample preparation represents 60 to 80 percent of the
work activity and operating costs. For sample preparation the literature is always well
documented. Selection of the analytical concentration, the sample size, instrument
technique and sample matrix, depending on the analyst they have to be
remembered.27
Analysis steps deals more with the instruments used to obtain quantitative or
qualitative information from sample with the recommended uncertainty level. The
choice of the specific analysis is based on the chemical properties of the analytical
species, cost, the speed, the concentration of the analytic the sample and the matrix
of the sample. Analysis can be visualised or even on a system with three
interconnected basic element such as input, converted, output.27
The data evaluation step helps to summarise and gain more information about a
specific data set by using mathematical and statistical approaches in order to draw a
conclusion about the input and outputs. This step is important in the validation
process. 27
1.7 Normal Phase Chromatograph
Normal phase liquid chromatography is a technique that is known to use columns
that are packed with a polar stationary phase together with non-polar mobile phases
in order to separate the components of mixtures. In the Normal phase liquid
chromatography each solute moves at in the according to its polarity. Solutes which
are less polar tend to move much faster and are therefore detected first in the
column however solutes that are more polar result in slow movement.20

22
1.8 Proposed Chromatographic Conditions
Different techniques are used in the separation and quantitative analysis of
lamotrigine in different formulations. A range of analytical methods published for the
analysis, either alone or in combination with other antiepileptic drug are displayed in
table 2.
Table 2: Published HPLC Methods Used For Analysis Of Lamotrigine in
Dosage Form And Biological Fluids.
Method Column Mobile phase Retentio
n time
Detectio
n
Flow rate
mL/min
M. Mathrusri
Annapurna,
Sharmistha
Mohapatra and B.V.
V. Ravi Kumar15
C18 Methanol: 0.01
mol.L-1 TBAHS
(Tetra butyl
ammonium
hydrogen sulphate)
(50:50 % v/v)
3.383
min
225 nm 1.0
mL/min
J. Emami17 C18 Acetonitrile–
monobasic
potassium
phosphate solution
(35:65, v/v)
containing
orthophosphoric
acid to adjust pH to
3.5
10 210 nm 1.5
mL/min
T. Vijaya Bhaskara
Reddy, G. Ramu, A.
Biksham Babu, and
C. Rambabu16
C8 Acetonitrile and
potassium
dihydrogen
phosphate buffer of
pH = 7. 0 in the ratio
60 : 40 v/v
2.797 min 215 nm 0.7
mL/min
Ching-Ling Chenga,
Chen-Hsi Chou17
C18 Acetonitrile–water
containing 20 mM
phosphate buffer
(pH 7) (35/65, v/v)
4.5 min 290 nm 1 mL/min

23
The HPLC parameters and UV detector were obtained for the analysis of
lamotrigine, according to analytical methods shown in the literature table 1 and with
the use of equipment that was available in the laboratory.
Table 3: describes the chromatographic conditions that were used to obtain the
optimised conditions required for method validation.
1.8.1 Proposed Experimental Conditions for HPLC Separation
Table 3: Proposed Experimental Conditions for HPLC Separation
Separation variables Initial choice
Column C18 waters column (3.9mm x 150mm).
Mobile phase Acetonitrile and water
Flow rate 1.0 mL/min
Sample size 5µl
Detection wave length 307nm
Temperature Room temperature
1.8.2 Description of Column of Choice
C18 column are mostly used in science and chemical analysis to analyze chemical
mixtures. The C18 substance is used as the mobile phase for C18 column. When it
comes to HPLC, C18 column are known to be commonly used they happen to be
available in different sizes containing different particles and pores sizes, different
degrees of hydrophobicity and thy are also capable of separating acidic or basic
mixtures . In a C18 columns there are 18 carbon atoms. When selecting the column
it is important to identify the characteristics of the selected compound this helps in
choose a well suitable C18 column. The column that was selected for the
development and validation of reverse phrase HPLC for the analysis of lamotrigine
was a C18 waters column (3.9mm x 150mm).19
1.8.3 Choice of Technique HPLC Reverse Phase Chromatograph
Reverse phase chromatography is a technique that is used to separate hydrophobic
molecules. A solute molecule binds to a molecule that is immobilized hydrophobic in
a polar solvent. C18 and C4 columns are used mostly in reverse phase. Reverse
phase chromatography uses a polar mobile phase therefore hydrophobic molecules
in the polar mobile phase result in absorbing the hydrophobic stationary phase, in
the mobile phase hydrophilic molecules pass through the column and therefore elute
first. 21

24
1.9 Aim
The main aim of the study was to develop and validate a simple and fast reverse
phase high performance liquid chromatography method for quantification and
determination of lamotrigine.
1.9.1 Objectives
The aim was achieved by developing a reverse phase HPLC method for the analysis
of lamotrigine and by also investigating validity, precision of method, accuracy and
data collection. The whole study was also evaluated.

25
Chapter 2
2.0 Experiment
2.1 Material and Method
2.1.1 Reagents and Chemicals
 Lamotrigine powder ≥98% from sigma Aldrich
 HPLC grade Acetonitrile from sigma Aldrich
 HPLC grade Methanol from sigma Aldrich
 Distilled water from Kingston university laboratory
 Lamotrigine tablet from GlaxoSmithKline
2.1.2 Equipment
 C18 waters column (3.9mm x 150mm)
 Varian 920 LC for manual injection
 Schimadzu HPLC LC-2010A HT with auto sampler
 Ultra bath sonic
 Four digit analytical weighing balance
2.2 Preparation of Lamotrigine Solution
Lamotrigine Stock Solution
By using a weighing balance (0.01g) 10mg of lamotrigine was accurately weighed
out and then transferred into a 25ml volumetric flask. About 10 mL of methanol was
added to the sample in the volumetric flask and then placed in an ultrasonic bath for
3mintues for it to dissolve and made up to the volume with methanol. This was stock
solution with a concentration of 0.4mg/ml.

26
2.2.1 Working Standard Solution
From the stock solution further dilution was done by using a micropipette a volume of
175 µl (concentration of 7µg/mL) was pipetted into a 10ml volumetric flask and this
was made up with methanol, the solution was then placed in an ultrasonic bath for
3mintues for it to dissolve. After 3 minutes the solution was ready to be injected into
the Varian HPLC system using an HPLC syringe. In the Varian HPLC system the
solution joined the mobile phase and travelled through the stationary phase.
Other concentrations of 13 µg/mL, 19 µg/mL, 25 µg/mL, and 31 µg/mL were also
prepared from the stock solution in 10ml volumetric flasks and made up to 10mL with
methanol in order to investigate linearity.
2.2.2 Chromatography analysis separation goals
The goals of HPLC separation
 A run time of less than 5 minutes per injection.
 Lamotrigine peak must be well resolved from the sample components.
 Separation of lamotrigine must have a narrow peak for large signal to noise
ratio.
 During each run the operating pressure must be stable and not too high this is
because column tends to plug less and auto samplers, pumps and sample
valves operate better at low pressure.

27
3.0 Chapter 3
3.1 Discussion and Results
Method Development
3.1.1 UV Absorbance of Lamotrigine
Selecting the wavelength was carried out using the UV spectroscopy by scanning
lamotrigine in methanol in the range of 200 – 800 nm. Figure 5 shows the
absorbance of lamotrigine at 210 nm and 307 nm. The detector could have been set
at either wavelength however at 307 nm the intensity is higher. At the wavelength
210nm organic compound and methanol is best detected therefore this could result
in peaks of organic compound and methanol being detected therefore making it
difficult for the lamotrigine peak to be identify. Therefore 307nm was the chosen
wavelength.
Figure 5: UV spectrum of Lamotrigine in Methanol
3.1.2 Selecting the Appropriate Solvent
When selecting the solvent for the analysis of lamotrigine solubility of the analyte in
the solvent was investigated in order to find which solvent lamotrigine was more
soluble in. lamotrigine is slightly soluble in water and hydrochloric acid and
completely soluble in methanol. Acetonitrile was also experimented with to see how

28
soluble lamotrigine is in in acetonitrile this is shown in Figure 6. Methanol was also
used to prove if lamotrigine was really soluble in it, this is also shown in figure 7.
It was found that lamotrigine is more soluble in methanol than in acetonitrile this is
shown in figure 7.
Figure 6: Chromatogram of Lamotrigine Dissolved in Acetonitrile. The peak is
unresolved.
Figure 7: Chromatogram of Lamotrigine Dissolved in Methanol. The peak is
resolved.
3.1.3 Selecting an Appropriate Column
There was limited column sizes provided in the laboratory therefore there was not
much choice to select from as most column that were provided were C18 columns.
C18 waters column 3.9mm X 150mm was the selected column for the analysis of
lamotrigine. The chosen column was used throughout the experiment and it was not
changed.
3.1.4 Selecting an Appropriate Mobile Phase
The manual injector was used for the method development of lamotrigine to begin
with. When developing the method a suitable mobile phase was searched by
experimenting with different mobile phase in order find which mobile phase gives a
well resolved peak. A mobile phase of 7O% ACN and 30% water (figure 8) which

29
had a retention time of 1.67 the mobile phase was then changed to 6O% ACN and
40% (figure 9) the retention time obtained was 1.68. Both peaks obtained for the
mobile phase of 7O% ACN and 30% water and 6O% ACN and 40% water were not
well resolved therefore a mobile phase of 50% ACN and 50% water (figure 10) was
used the retention time obtained was 1.64, the peak was well resolved as shown in
figure 10 this becomes the chosen mobile phase for the validating the method.
Figure 8: Chromatogram of the Mobile Phase of 7O% ACN and 30% Water.
Figure 9: Chromatogram of the Mobile Phase of 60% ACN and 40% Water.

30
Figure 10: Chromatogram of the Mobile Phase of 50% ACN and 50% Water.
Table 4: Optimized Chromatography Conditions
Separation variables Initial choice
HPLC system Schimadzu
Column C18 waters column (3.9mm x 150mm).
Mobile phase 50%Acetonitrile and 50% water
Flow rate 1.0 mL/min
Sample size 10µL
Detection wavelength 307nm
Temperature Room temperature
4.0 Method Validation
Method validation is carried out in order to obtain consistent, reliability and accurate
data. The results obtained can be used to judge the quality, reliability and
consistency.
4.1 International Conference on Harmonisation (ICH) Guidelines
ICH guidelines have been established in the EU, United States and Japan. ICH
guidelines are mostly used in the pharmaceutical industry. The main aim of ICH is to
propose ways of achieving greater harmonisation in the interpretation and
application of the technical guidelines and requirements for product registration for
the reduction need to duplicate the testing achieved during research and

31
development of the new medicines. Validation of the analytical method includes
robustness, repeatability and intermediate precision, detection limit and
quantification limit, linearity and range. Method validation is included in the ICH
guidelines; validation is carried out on the analytical method to ensure that the
method is applicable for its required purpose.28, 29
4.2 Linearity and Range
Linearity is the ability of the method to produce test results that corresponding to
analyte concentration within a provided range. Units from the test results from the
methods are the same units used in expressing the range. A minimum of five
concentration levels are required according to the ICH guidelines. A coefficient of
>0.999 is mostly recommended as the acceptable data for the regression line in
linearity. The range is usually expressed in the same units as the test results
obtained by the method. RSD percentage when calculated it should give a value that
isn’t greater than 2%. Linearity is analysed by visual analysis of a plotted analyte
concentration and by repeating the measurements of the five concentrations. 30This
is displayed in table 5.
4.2.1 Linearity Test Results
Table 5: Linearity Test Results (n=3)
Concentration
µg/mL
Peak
Area
Peak Area
mean
Response
factor
Standard
deviation
%RSD
7 91942 91661.6 13094.15 448.82 0.48
91899
91144
13 158752 163685 12589.07 9694.55 0.5
157449
174854
19 239832 246998 12999.89 12504.65 5.06
239725
261437
25 339200 336405 13456.2 4561.09 1.35
331142
338874
31 410223 409177.3 13199.26 914.93 0.22
408785
408524

32
Response factor = Peak Area/Concentration.
Mean of response factors = 13067.714
SD = 317.26
%RSD = 2.42 %
Figure 11: Calibration curve generated for analysis of lamotrigine.
Table 6: Linearity Test – Comparison of Results to Acceptance Criteria
Parameters Acceptable criteria of
linearity 30
Results of linearity
Regression y = 13463x - 6202.6
Correlation Coefficient R² = ≥0.999 R² = 0.998
y-intercept ≤2.0% -6202.6
Residual standard
Deviation
≤2.0% 2.42%
Linearity Range 80-120% 7-31%
Results show that the linearity test meets the acceptable criterial.
y = 13463x - 6202.6
R² = 0.998
0
50000
100000
150000
200000
250000
300000
350000
400000
450000
0 5 10 15 20 25 30 35
PeakAreaMv
concentration µg/mL
calibration Curve of Lamotrigine Powder

33
4.3 Robustness
Robustness is preformed when a method is being validated. Robustness of an
analytical method is a measure of its ability to remain unaffected with a change of
conditions. Robustness of a method can be investigated by changing method
parameters such as different HPLC columns, mobile phase, flow rate etc. ICH
recommends for robustness to be investigated in the early stages of the method
development. 30, 32
The % RSD for the modified conditions obtained should not be more than 2% as this
is the recommended acceptable criteria
Robustness was analysed using a mobile phase of 55% ACN and 45% water and
then modified to a mobile phase of 45% ACN and 55% water and 50%ACN and 50%
water. The flow rate was kept at 1mL/ minute and the wavelength remained at
307nm.
Figure 12: Mobile phase 55% ACN and 45% water chromatogram
Table 7: Robustness under modified conditions - mobile phase 55% ACN and
45% water (n=3)
Concentration µg/mL Retention time (min) Peak Area
25 1.953 361037
1.926 360056
1.921 360035
Mean 1.933 360376
SD 0.017 572.5
RSD% 0.87 0.15
min
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
5
10
15
20
25
Detector A 307nm

34
There is an extra peak display in figure 13 at the retention time 1.697 this could have
appeared due to impurities in the sample, the methanol used to make up the solution
could have been contaminated resulting in the impurities eluting or the glass ware
used might have been dirty causing impurities in the sample.
55% water (n=3).
Concentration µg/mL Peak Area Retention time (min)
25 353067 2.211
351987 2.220
348744 2.203
Mean 351266 2.211
SD 2249.8 0.008
RSD % 0.64 0.36
min
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0
5
10
15
Detector A 307nm

35
50% water (n=3).
Concentration µg/mL Peak Area Retention time (min)
25 321580 1.729
323514 1.723
325311 1.726
Mean 323468.3 1.726
SD 1865.9 0.003
RSD% 0.57 0.17
All three peaks of the different mobile phase compositions, 45% ACN and 55%
water, 55% ACN and 45% water and 50% ACN and 50% water show a well resolved
peak of lamotrigine. Results obtained from the robustness method meet the
acceptable criteria with a % RSD of no more than 2% therefore the results indicate
that the HPLC method developed is robust

36
4.4 Stability
The objective of stability testing is to show evidence of how the quality of the drug
substance varies with time under the effects of a variety of environment factors like
humidity and light in order to establish the drug shelf life and storage conditions. 33
% RSD meets the acceptable criterial if the % RSD value is ≤ 2% in the sample
response, relative to freshly made standards
The stability test was assessed by storing the same concentrations 7-31 µg/mL
which were used for the analysis of linearity, the samples were left for 3 days left for
three days at room temperature for analysing.
4.4.1 Stability Test Results
Table 10: Stability Test Results Day 3 (n=3)
Concentration
µg/mL
Peak Area Peak
Area
Mean
Response
Factor
Standard
Deviation
%RSD
7 94571 95371.6 13624.52 861.94 0.009
96284
95360
13 148898 152506.3 11731.25 6376.67 0.041
148752
159869
19 231298 236837.6 12465.14 4817.22 0.020
239172
240043
25 303029 310280.6 12411.22 7000.25 0.022
310814
316999
31 412523 426554 13759.81 12151.21 0.028
433587
433552
Mean of response factors = 12798.388
SD = 866.972
%RSD = 6.77%

37
The %RSD was 6.77% therefore the results did not meet the acceptable criteria, the
peaks were distorted and the retention time was not consistent. The height of the
peaks decreased compare to normal. From the results it is clear that lamotrigine is
not stable after three days when left at room temperature.
4.5 Precision of Analytical Method
Precision of an analytical method shows the closeness of agreement between a
series of measurements of results collected from the sample with the same
conditions. Precision is considered at the following three levels, repeatability;
intermediate precision and reproducible.30
4.5.1 Precision- Repeatability
The ICH guidelines recommended a minimum of 9 determinations conditions for
repeatability. Repeatability precision is defined as the results of the procedure
operating over a short time interval under the same conditions.
The acceptable criterial – for each concentration level the % RSD of the lamotrigine
peak area in the three replicate injections should not exceed more than 2%. The
retention time of the lamotrigine peak should also not exceed more than 1%.
For the study of lamotrigine repeatability will be investigated using three different
concentrations of lamotrigine concentrations. 30, 31
4.5.2 Repeatability Testing Results
Table 11: Repeatability Test at 7µg/mL (n=3)
Concentration µg/mL Retention time (min) Peak area
7 1.729 92158
1.726 89667
1.723 92096
Mean 1.726 91307
SD 0.003 1420.6
%RSD 0.17 1.55

38
The retention time of the lamotrigine peak did not exceed more than 1% therefore
the % RSD obtained results met acceptable criterial. The peak area of the three
concentrations also had a % RSD of not more than 2 % this indicates that the results
obtained for both the retention time and the peak area show adequate repeatability.
13 1.744 174894
1.729 175799
1.732 172295
Mean 1.735 174329.3
SD 0.007 1818.9
%RSD 0.40 1.04
19 1.748 263136
1.741 263174
1.755 264988
Mean 1.748 263766
SD 0.007 1058.45
%RSD 0.40 0.004

39
4.5.3 Intermediate –Precision
Intermediate precision is defined as results which are obtained within the laboratory
variations because of different events like equipment, different day, analysts etc.
The main purpose of Intermediate precision is to ensure that the same results can be
obtained even when the method developing stage has been complete. Just like for
the Repeatability precision three different concentrations of lamotrigine
concentrations will be used for the study.30
The acceptable criterial – The % RSD should not exceed more than 2 % for the 3
samples prepared for 100% lamotrigine active ingredient analysed on different days.
4.5.6 Intermediate –Precision Test Result
Precision Day 1
Table 14: Precision Test Results at 31µg/mL (n=3).
Precision Day 2
Table 15: Precision Test Results at 31µg/mL (n=3).
The results obtained for both day one and two demonstrate that the % RSD was
below the value of 2% therefore results obtained were within the acceptable range.
Concentration µg/Ml Retention time(min) Peak Area
31 1.758 402539
1.769 399189
1.780 394150
Mean 1.769 398626
SD 0.011 4222.7
%RSD 0.62 1.0
31 1.760 407950
1.787 408783
1.774 409326
Mean 1.773 408686.3
SD 0.013 693.1
%RSD 0.73 0.16

40
4.6 Limit of Detection (LOD)
The lowest concentration of an analyte in a sample which can be detected is defined
as the limit of detection (LOD). The aim of the limit test is to investigate if an analyte
is below or above a certain value. LOD can be calculated based on the SD of the
response and calibration curve.30
4.6.1 Results of LOD of SD based on the slope of the calibration curve
The linearity table 5 was used to calculated LOD
Table 16: Results Obtained From Linearity
Mean of response factor 13067.714
SD 317.26
% RSD 2.42%
Slope of calibration curve 13463
LOD calculations:
LOD = 3 X (SD/Slope of calibration curve)
LOD = 3 X (317.26/13463) =0.07 µg/mL
4.6.2 Limit of Quantification (LOQ)
LOQ is known as the lowest concentration of an analyte in a sample which can be
determined with the required accuracy and precision under chosen operational
working conditions of the method. The calculation for the LOQ is also based on the
SD of the response and calibration curve.30

41
4.6.3 Results of LOQ of SD based on the slope of the calibration curve
The linearity table 5 was used to calculated LOQ
LOQ Calculation
LOQ = 10 X (SD/Slope of calibration curve)
LOQ = 10 X (317.26/13463) = 0.23µg/mL
LOD and LOQ results were calculated from the linearity table 5
LOD = 3 X (317.26/13463) =0.07µg/mL
LOQ = 10 X (317.26/13463) = 0.23µg/mL
Results demonstrate that stability for linearity was very sensitive.
4.7 Analysis of Lamotrigine Tablet
4.7.1 Preparation of Lamotrigine table sample solution
The developed method was used to analyse lamotrigine tablet by crushing the
lamotrigine table which weighed 50 mg (0.05 g). The 50mg of the lamotrigine powder
was then transferred into a 250 mL volumetric flask, methanol was then added to the
sample and placed in the ultrasonic bath for three minutes in order for the sample to
fully dissolve. This was then the stock solution of 50mg/250 mL (0.2 mg/mL).
Further dilution was carried out by pipetting 325µl using a micro pipette from the
stock solution, this was then transferred to a 10 mL volumetric flask, methanol was
added to this to making a concentration of 13 µg/mL of the working standard
solution. Using a pipette the prepared solution of 13µg/mL was then transferred to a
2 mL vial for analysis in the automatic HPLC machine.

42
4.7.2 Lamotrigine tablet results
A well resolved peak of lamotrigine tablet was obtain at a retention time of 1.671 with
a peak area of 3245 this is clear prove that the method developed for the analysis of
lamotrigine was accurate.
Figure 15: Chromatography of lamotrigine tablet
Table 17: Results for the analysis of lamotrigine table at 13µg/mL (n=3)
A well resolve peak of the lamotrigine table was obtained and the % RSD for the
results at the concentration 13 µg/mL didn’t exceed more 2 % which indicates that
the results met the acceptable criterial and the method developed worked for the for
the analysis of lamotrigine table.
13 1.673 3176
1.671 3245
1.672 3264
Mean 1.672 3228.3
SD 0.001 46.306
%RSD 0.05 1.43
min
0.0 0.5 1.0 1.5 2.0 2.5 3.0
0.0
0.1
0.2
0.3
Detector A 307nm

43
The main objective for this study was to develop and validate a simple and fast
reverse phase high performance liquid chromatography method for quantification
and determination of lamotrigine. There were a lot of challenges faced in validating
the method. Some of the main errors faced were poor integration this was due the
wrong base line adjustment however this was amended by running the base line for
30 minutes for each time the mobile phase was changed. During some of the runs
separation appeared to be incomplete and the peaks over lapped with other
impurities that were detected during the run, this could have been due to non-
optimized selectivity and insufficient separation power.
No mobile phase is chemically clean and majority of mobile phase used are known
to be sensitive to light and oxygen this can therefore cause impurities in the mobile
phase, in the analysing it was ensure that the mobile phase used was always close d
with the tube lid through the experiment to avoid impurities.
The column temperature is very important in liquid chromatography as it affects the
separation power, if separation is not carried out accurately in the column this may
result in an error on the shape of the peak. Problems issued by the column were
solved by purging the column with isopropanol in order to remove all impurities. In
order to minimise errors and impurities, before each run the pump was primed to
remove residual air bubbles and the LC column was equilibrated using the mobile
phase for 30 minutes.
In weighing out the appropriate amount of lamotrigine using the weighing balance,
the weighing balance was very sensitive therefore when weighing out the required
amount of lamotrigine; it was ensure that there was nothing surrounding the
weighing balance to avoid errors in the measurements.
Reverse phase columns have a hydrophobic stationary phase which is known to
work well for retention of most organic analyte. Water was used as a mobile phase
with a less polar solvent which was acetonitrile.
It is very important to use the right flow rate, flow rate effects HPLC system pressure
and analysis time therefore it is required for the right flow rate to be used. A very
High flow rate is likely to affect the quality of the chromatography not providing the
analyte enough time to interact with the stationary phase. A very low flow rate is also
likely to result in a long wait for the peak to appear at the detector.

44
5.0 Conclusion
The main goals for this study were to obtain a well resolved lamotrigine peak from
the sample components and to also develop a method that worked for the analysis of
lamotrigine tablet.
A C18 waters column (3.9mm X 150mm) was the chosen column used for the
separation under isocratic elution with acetonitrile 50% and 50% water. A sharp peak
with a short retention 1.64 was obtained.
In order to detect the wavelength lamotrigine was scanned in methanol using UV
spectroscopy in the range of 200- 800nm, results are shown in figure 5. Form
research the wavelength was operated at 225nm for the analysis of lamotrigine.36 In
the study the method was modified by operation the wave length at 307nm and a
well resolved peak of lamotrigine was obtained with the use of this wavelength
The method was validated using the appropriate conditions, flow rate 1mL/min,
mobile phase ACN 50% and water 50%, detector wave length 307nm; however the
conditions were modified to test the robustness for validation of the method.
Results which were obtained from method validation were within the acceptable
criterial of not exceeding more than 2% for the %RSD of the peak area and not more
than 1% for the retention time for the %RSD for the retention time. Stability testing is
the only test which didn’t meet the acceptable criterial; this is because lamotrigine is
not stable when it’s left at room temperature for 3 days.
Linearity of the calibration curve was investigated by plotting the average peak area
against the concentration. The regression equation was obtained. The results that
were obtained from the slope, intercept and correlation efficient were discovered to
be 13463, -6202.6, and 0.998. From the results there is clear evidence that there is a
good correlation between the peak area and the concentration of lamotrigine. The
linearity results were within the acceptable criterial.
Research shows 0.999 for the correlation coefficient for lamotrigine and the
concentrations that were used for the linearity were between 5-25 µg/mL.36
Comparing the result obtain form the study to the results from the research, it is

45
shown clearly that results that were obtained from the study match up with the
results in the research however only the concentration were modified. When the
concentration from the research was experimented with the results did not give a
good correlation between the peak area and the concentration of lamotrigine
therefore the concentrations between 7-31 µg/mL were used.
Robustness was investigated by modifying the mobile phase percentage of
acetonitrile and water to 45%:55, 55%:45 and 50%:50%. The flow rate and detector
wavelength were not modified. In table 7, 8 and 9 displays the results obtained. The
peak in figure 13 displayed an extra peak which was not expected in the results, the
extra peak could have been due to impurities in the sample or impurities from the
glass ware which could have been contaminated with the working sample. The
results of the %RSD didn’t exceed 2% therefore the results obtain met the
acceptable criterial, indicating that the method which was developed was robust.
Stability of lamotrigine was analysed by storing the same lamotrigine samples which
were used for linearity testing. The samples were stored for 3 days at room
temperature. After 3 days the samples were then analysed however the results
obtained did not meet the acceptable criterial. The %RSD results obtained exceeded
2%; this showed that lamotrigine was not stable after 3 days when stored at room
temperature.
Precision repeatability test was evaluated by analysing 3 independent of the
concentration 7, 13 and 19 µg/mL. The results met the acceptable criterial for both
the retention time and peak area this is shown in table 6 and 7. This proves that the
method developed was precise.
Intermediate precision was carried out by analysing the peak area for the
concentration 31 µg/mL. The results obtained for day 1 and 2 both met the
acceptable criterial for the %RSD of no more than 2% showing that the method
developed was precise.
LOD and LOQ were determined by using the calibration curve and the formula for
the standard deviation of the response. The LOD and LOQ of lamotrigine were found
to be 0.07µg/mL and 0.23µg/mL respectively. The LOD and LOQ results indicate
that the method is sensitive and can therefore be used to detect and qualify
lamotrigine over an expanded rang of concentrations.

46
From research the LOD and LOQ of lamotrigine were found to 0.008 µg/mL and
0.029 µg/mL.36 Comparing the results to the results obtained in the study, there is
not much of a big difference with the LOQ results however there seem to be a
difference in the LOD results compared to the results from the study indicating that
lowest concentration of an analyte in a sample which can be determined is could be
less than what was obtained from the study.
The method was used to evaluate the lamotrigine tablet. The results from the
analysis met the acceptable criterial. The peak obtained was a well resolved peak
with a short retention time; this also proved that the method developed was
successful.
From research the method that was chosen to being with was by Ching-Ling
Chenga, Chen-Hsi Chou.17 The method contained a mobile phase of 35%
acetonitrile-water and 65% phosphate buffer, wavelength of 290nm and the flow rate
at 1min/mL. The expected retention time was 4.5 min. when this method was
experimented with the results obtained didn’t match the results from the search. The
peak was not well resolved and the retention time was more than 4.5 minutes. The
method was therefore modified to 50% acetonitrile and 50% water, the wavelength
was operated at 307nm and the flow rate was kept the same. A well resolved peak
was obtained from this method with a short retention time hence why this method
was used to validate the method.
When ordering the lamotrigine powder for the analysis, the order didn’t arrive on time
for hence why the evaluation of lamotrigine was not complete because this resulted
in a limited amount of time to carry out the analysis of lamotrigine. The HPLC
machines were also not working on some of the days this also delayed progress of
the study.
Overall the method developed proved to be working for both the lamotrigine powder
and lamotrigine tablet. The method also proved to be robust and obtained a wide
range of linearity. The mobile phase was provided in the laboratory and it was very
easy to prepare. Taking all those factors in consideration the method developed is
surely suitable for the determination and quantification of lamotrigine in
pharmaceutical formulation

47
6.0 Future work
In order to fully validate the method using reversed high performance liquid
chromatography to quantify and determine lamotrigine in lamotrigine tablet,
further work is require as validation of lamotrigine was not complete. Some of the
steps included in validating the method were not carried out, like precision testing
which consists of three levels however only two of those levels were carried out,
leaving out the precision reproducible test. The stability test could also be
expanded by analysing the drug over seven days. The lamotrigine tablet could
also be investigated further by carrying out the validation steps on the tablet.
Lamotrigine could also be analysed in a mixture with other anti-epileptic drugs
and compared. Impurities in the chromatograms could also be investigated and
prevented. The robustness testing could also be expanded further by
investigating and modifying other condition like the wavelength, flowrate and
temperature. It would have been interesting to validate the method using normal
phrase high performance liquid chromatography and compare the results with the
reverse phase.

48
References
1. Ualberta.ca, (2015). Transbuccal delivery of lamotrigine across porcine buccal
mucosa: in vitro determination of routes of buccal transport, pp54-62.
2. Messenheimer, J. (1995). Lamotrigine. CNS Drug Reviews, 1(2) Vol.1,
pp.190-203.
3. Cheney, M., Shan, N., Healey, E., Hanna, M., Wojtas, L., Zaworotko, M.,
Sava, V., Song, S. and Sanchez-Ramos, J. (2010). Effects of Crystal Form on
Solubility and Pharmacokinetics: A Crystal Engineering Case Study of
Lamotrigine. Crystal Growth & Design, 10(1) Vol.10, pp.394-405.
4. Westercamp, N. and Sharma, A. (2012). Case Report. The Primary Care
Companion For CNS Disorders Vol.14, pp.1110-1278.
5. Drugbank.ca, (2015). DrugBank: Lamotrigine.
6. Tom, kupie. (2014).quality control analytical methods high performance liquid
chromatograph. Analytical research laboratories, pp. 223-227
7. Fekete, S., Kohler, I., Rudaz, S. and Guillarme, D. (2014). Importance of
instrumentation for fast liquid chromatography in pharmaceutical analysis.
Journal of Pharmaceutical and Biomedical Analysis, 87, pp.105-119.
8. Hassan, B. (2015). HPLC Uses and Importance in the Pharmaceutical
Analysis and Industrial Field. Pharmaceutica Analytica Acta, pp. 11800.
9. Czaplicki, S. (2013). Chromatography in Bioactivity Analysis of Compounds.
Column Chromatography, pp.101-121.
10.Fumisuke, mastuo. (1999). Lamotrigine. Epilesia Vol. 40, pp. 30-36
11.Vincenzo, P. Francesca, B. Cesare, B. and Maria, A. (2005) Analysis of
lamotrigine and its metabolites in human plasma and urine by micellar
electrokinetic capillary Chromatography. Electrophoresis, Vol.26, pp. 935-942.
12.Messenheimer, J. (1995). Lamotrigine. CNS Drug Reviews, 1(2) Vol.1,
pp.190-203.
13.Goldsmith, D., Wagstaff, A., Ibbotson, T. and Perry, C. (2003). Lamotrigine.
Drugs, 63(19), pp.2029-2050.
14.Ching-Ling Chenga, Chen-Hsi Choub,∗, Oliver Yoa-Pu Hu. (2005).
Determination of lamotrigine in small volumes of plasma by high-performance
liquid chromatography. Journal of Chromatography B Vol.817, pp. 199–206.
15.M. Mathrusri Annapurna*, Sharmistha Mohapatra and B.V. V. Ravi Kumar.
(2010). Development and validation of RP-HPLC method for the
determination oflamotrigine and its degradation products in tablets. J Pharm
Educ Res Vol. 1, pp.83-87.
16.J. Emami∗, N. Ghassami, F. Ahmadi. (2006).Development and validation of a
new HPLC method for determination of lamotrigine and related compounds in
tablet formulations. Journal of Pharmaceutical and Biomedical Analysis,
pp.999–1005.
17.Reddy, T., Ramu, G., Biksham Babu, A. and Rambabu, C. (2013).
Development and Validation of HLPC Method for the Estimation of

49
Lamotrigine in Bulk and Pharmaceutical Formulations. Journal of Chemistry
Vol.2013, pp.1-4.
18.Ching-Ling Chenga, Chen-Hsi Choub. (2005).Determination of tadalafil in
small volumes of plasma by high-performance liquid chromatography with UV
detection. Journal of Chromatography B Vol.822, pp. 278–284.
19.Labcompare.com, (2015). C18 Column / C18 HPLC Columns.
20.Cooper, W. (2006). Normal-Phase Liquid Chromatography. Applications,
Theory and Instrumentation, pp.33-47.
21.J. Emami∗, N. Ghassami, F. Ahmadi. (2006). Development and validation of a
new HPLC method for determination of lamotrigine and related compounds in
tablet formulations. Journal of Pharmaceutical and Biomedical Analysis, pp.
999–1005.
22.Omudhome Ogbru, P. (2015). lamotrigine, Lamictal: Drug Facts, Side Effects
and Dosing.
23.Mike May. (2014). Epilepsy. Nature Outlook Vol.511, pp. 1.
24.Chang, Bernard S, Lowenstein, Daniel H. (2003). Epilepsy.The New England
Journal of Medicine, pp.1257-66.
25.Emedicine.medscape.com, (2015). Epilepsy and Seizures.
26.Ben Johns, w. (2015). CNS Spectrums: Lamotrigine in Psychiatry:
Pharmacology and Therapeutics, pp. 224-232.
27.Pedro Araujo. (2009). Key aspects of analytical method validation and
linearity evaluation. Journal of Chromatography B Vol.877, pp.2224–2234
28.Vadscorner.com, (2015). ICH - GCP Guidelines for Clinical Trials.
29.Monika Bakshi, Saranjit Singh. (2002). Development of validated stability-
indicating assay methods—critical review. Journal of Pharmaceutical and
Biomedical Analysis Vol.28, pp. 1011–1040
30.Ghulam A. Shabir. (2003). Validation of high-performance liquid
chromatography methods for pharmaceutical analysis: Understanding the
differences and similarities between validation requirements of the US Food
and Drug Administration, the US Pharmacopeia and the International
Conference on Harmonization. Journal of Chromatography A Vol.987, pp. 57–
66
31.Isabel Taverniers, Marc De Loose, Erik Van Bockstaele. (2004).Trends in
quality in the analytical laboratory. II. Analytical method validation and quality
assurance. TrAC Trends in Analytical Chemistry Vol.23, pp. 535–552.
32.Sepscience.com, (2015). Implementing Robustness Testing for HPLC
Methods: Part 1 - Separation Science: Premier Learning for Analytical
Chemists.
33.U.S. Department of Health and Human Services Food and Drug
Administration Center for Veterinary Medicine: Guidance for Industry:
Specifications: Test Procedures and Acceptance Criteria for New
Biotechnological/Biological Veterinary Medicinal Products (VICH GL40).
(2006). Biotechnology Law Report, 25(4), pp.447-460.

50
34. Azom.com, (2015). High Performance Liquid Chromatography – Methods,
Benefits and Applications.
35. Brodie, Martin J. (1992), Lamotrigine. ProQuest Science Journals, Vol.339,
pp. 1397-1400.
36. Vidya Sagar. K*, Prsanth Naidu. Y, Suresh. S, Anusha. Ch, Somnath De and
Aneela. S.(2011), A new simultaneous RP-HPLC method for development
and validation of Lamotrigine tablets, Journal of Chemical and Pharmaceutical
Research, 3(3):651-658

52
COSHH Assessment Form
COSHH Risk Assessment No:
Assessor:
SAMALI MUKALAZI
Site: Room No:
PRPM1028
Date:21/10/2014
Describe the activity or work process.
(Include how long and how often this is
carried out and the quantity of substance
used).
HPLC METHODS VALIDATION OF LAMOTRIGINE
FROM LAMICTA.
Location of process being carried out PRPM1028 Qty in Use:
Identify the persons at risk:

Academ
ic

Technic
al

Student
s
☐ Other
Hazards associated with compounds/substances used and measures to be adopted to prevent
exposure and to control the risk (Notes 1 and 2)
Substance Hazard & Risk Measure to control risk
(All substances to be included) R - Risk Phrases S – Safety Phrases
Lamotrigine
Toxic if swallowed. May
cause drowsiness or
dizziness. Exposure to
atmospheric concentrations
in excess of the
occupational exposure limit
may cause symptoms
including: dizziness,
headache, blurred or
double vision, lack of
coordination, sleepiness,
nausea, vomiting, and rash
Wear protective clothing. Avoid
contact with skin and by
inhalation. Avoid breathing dust.
Methanol Flammable. Toxic by
inhalation, in contact with
skin and if swallowed.
Toxic: danger of very
serious
Irreversible effects through
Wear protective clothing. Avoid
contact with skin and by
inhalation. Avoid breathing dust.
Keep away from heat, sparks,
open flames, hot surfaces. No
smoking
phosphate Slightly hazardous in case
of skin contact (irritant), of
eye contact (irritant), of
Do not ingest. Do not
breathe dust. Wear
suitable protective clothing.

53
ingestion, of Inhalation. In case of insufficient
ventilation, wear suitable
respiratory equipment. If
ingested, seek medical
advice immediately and
show the container or the
label.
Hazard Pictograms
 Very toxic/toxic 
Skin & eye
irritant
Environmental

Flammable, self-
reactive,
pyrophoric

Gases under
pressure
Oxidising

Mutagen,
carcinogen,
reproductive

Corrosive,
serious skin &
eye damage
Explosive
Hazard Type
       ☐
Gas Vapour Mist Fume Dust Liquid Solid Other
If other please state:
Route of Exposure
    ☐
Inhalation Skin Eyes Ingestion Other
Workplace Exposure Limits (WELs) (Note 3) Please state n/a where not applicable
Short-term (15 min) ppm Long-term (8 Hr)
Substance ppm Mg m³ Substance ppm Mg/m³
methanol 60 102 methanol 40 68
Is health surveillance or monitoring required? ☒ ☐Yes No

54
Personal Protective Equipment (State type and standard)
☐ ☐
Dust Mask Visor
☐ 
Wear splash resistant safety goggles
Respirator Goggles

Wear appropriate chemical
resistant gloves. 
Wear Lab coat
Gloves Overalls

Covered shoes
☐
Footwear Other
First Aid Measures – Including immediate treatment
Eye Contact: Remove any contact lenses. In case of contact, immediately flush eyes with plenty of
water for at least 15minutes. Cold water may be used. Get medical attention if irritation occurs.
Skin Contact: Wash with soap and water. Cover the irritated skin with an emollient. Get medical
attention if irritation develops. Cold water may be used.
Inhalation: If inhaled, remove to fresh air. If not breathing, give artificial respiration. If breathing is
difficult, give oxygen.
Ingestion: Do NOT induce vomiting unless directed to do so by medical personnel. Never give
anything by mouth to an unconscious person. Loosen tight clothing such as a collar, tie, belt or
waistband. Get medical attention if symptoms appear.
Accidental Release Measures/spillage
Shut off all sources of ignition.
Inform others to keep at a safe distance.
Wear appropriate protective clothing.
Ensure supply of fresh air in enclosed rooms.
Absorb on an inert absorbent, transfer to a suitable container and arrange disposal via special waste
route.
Wash site of spillage thoroughly with water and detergent.
For large spillages liquids should be contained with sand or earth and both liquids and solids
transferred to salvage containers.
Any residues should be treated as for small spillages.

55
Storage
Methanol: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.
Containers which are opened must be carefully resealed and kept upright to prevent leakage.
Lamotrigine: Tablets should be stored at 15to 25 degrees in a dry place and protected from light.
Phosphate: Keep container tightly closed. Keep container in a cool, well-ventilated area.
Disposal of Substances & Contaminated Containers
 Hazardous Waste ☐ Skip ☐ Return to supplier ☐ Other
Is exposure adequately controlled? ☐ Yes  No
Risk rating following control measures
 High ☐ Medium ☐ Low
Additional safety information

56
Appendix 2: Project proposal

57
Project Proposal: DEVELOPMENT AND VALIDATION OF REVERSE
PHRASE HIGH PREFORMANCE LIQUID CHROMATOGRAPH (HPLC)
FOR THE ANALYSIS OF LAMOTRIGINE.
Background information:
Lamotrigine is used to treat epilepsy. People with epilepsy are prone to having periods of
uncontrolled electrical activity in the brain. These periods of uncontrolled electrical activity
may lead to seizures. There are many different types of seizure. Lamotrigine helps to control
electrical activity in the brain. This reduces the chances of having seizures. Lamotrigine is
also used to treat depressive episodes in people who have bipolar disorder. [3]
Aims
The main aim of the study was to develop and validate a simple and fast reverse phase high
performance liquid chromatography method for quantification and determination of
lamotrigine.
Objectives
The aim was achieved by developing a reverse phase HPLC method for the analysis of
lamotrigine and by also investigating validity, precision of method, accuracy and data
collection. The whole study was also evaluated.
Methodology
Step 1:
Order lamotrigine powder and lamotrigine tablet.
Select mobile phase and buffer solutions.
Step2:
Pure lamotrigine will be used as the stock sample in validating the method. After the method
has been developed lamotrigine table with then be analysed to ensure that the method is
working.
Step3:
Select the appropriate column size to be used in order to determine and obtain accurate
results.
Choose an appropriate mobile phase which gives a well resolved peak of lamotrigine.
Chemical structure

58
[1]
LAMICTAL (lamotrigine).
Chemical name is 3,5-diamino-6-(2,3-dichlorophenyl)-as-triazine,
Molecular formula is C9H7N5Cl2,
Molecular weight is 256.09.
Lamotrigine is a white to pale cream-colored powder and has a pKa of 5.7. Lamotrigine
slightly soluble in water and slightly soluble in 0.1 M HC1 [2]
Material and Method
Reagents and Chemicals
• Lamotrigine powder ≥98% from sigma Aldrich
• HPLC grade Acetonitrile from sigma Aldrich
• HPLC grade Methanol from sigma Aldrich
• Distilled water from Kingston university laboratory
• Lamotrigine tablet from GlaxoSmithKline
Equipment
• C18 waters column (3.9mm x 150mm)
• Varian 920 LC for manual injection
• Schimadzu HPLC LC-2010A HT with auto sampler
Table 2: Published HPLC Methods Used For Analysis Of Lamotrigine in Dosage Form
And Biological Fluids.
Method Column Mobile phase Retention
time
Detection Flow rate
mL/min

59
M. Mathrusri
Annapurna,
Sharmistha
Mohapatra and B.V.
V. Ravi Kumar15
C18 Methanol: 0.01 mol.L-
1 TBAHS (Tetra butyl
ammonium hydrogen
sulphate) (50:50 %
v/v)
3.383 min 225 nm 1.0
mL/min
J. Emami16
C18 Acetonitrile–
monobasic potassium
phosphate solution
(35:65, v/v) containing
orthophosphoric acid
to adjust pH to 3.5
10 210 nm 1.5
mL/min
T. Vijaya Bhaskara
Reddy, G. Ramu, A.
Biksham Babu, and
C. Rambabu17
C8 Acetonitrile and
potassium dihydrogen
phosphate buffer of
pH = 7. 0 in the ratio
60 : 40 v/v
2.797 min 215 nm 0.7
mL/min
Ching-Ling Chenga,
Chen-Hsi Chou18
C18 Acetonitrile–water
containing 20 mM
phosphate buffer (pH
7) (35/65, v/v)
4.5 min 290 nm 1 mL/min
Expected results:
• A run time of less than 5 minutes per injection.
• Lamotrigine peak must be well resolved from the sample components.
• Separation of lamotrigine must have a narrow peak for large signal to noise ratio.
• During each run the operating pressure must be stable and not too high this is
because column tends to plug less and auto samplers, pumps and sample valves operate
better at low pressure.
Time plan
Oct Nov Dec Jan Feb May
Research
about the
project,
collect
articles
Research
about the
project,
collect
articles
Submit
project
Lab
work
Lab
work
Write up
project

60
How the project will be recycled or stored after completion.
After preforming the HPLC, it will be ensure that all products left over and used are poured
into the waste bottle.
References
1) Pubchem.ncbi.nlm.nih.gov, (2014). lamotrigine - PubChem.
2) RxList, (2015). Lamictal (Lamotrigine) Drug Information: Description, User Reviews,
Drug Side Effects, Interactions - Prescribing Information at RxList.
3) Nhs.uk, (2014). lamotrigine - Epilepsy medicines and drugs - NHS Choices.
4) M. Mathrusri Annapurna*, Sharmistha Mohapatra and B.V. V. Ravi Kumar. (2010).
Development and validation of RP-HPLC method for the determination oflamotrigine
and its degradation products in tablets. J Pharm Educ Res Vol. 1, pp.83-87.
5) J. Emami∗, N. Ghassami, F. Ahmadi. (2006).Development and validation of a new
HPLC method for determination of lamotrigine and related compounds in tablet
formulations. Journal of Pharmaceutical and Biomedical Analysis, pp.999–1005.
6) Reddy, T., Ramu, G., Biksham Babu, A. and Rambabu, C. (2013). Development and
Validation of HLPC Method for the Estimation of Lamotrigine in Bulk and
Pharmaceutical Formulations. Journal of Chemistry, 2013, pp.1-4.
7) Ching-Ling Chenga, Chen-Hsi Choub. (2005).Determination of tadalafil in small
volumes of plasma by high-performance liquid chromatography with UV detection.
Journal of Chromatography B, pp. 278–284.

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