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International Standard Serial Number (ISSN): 2319-8141 International Journal of Universal Pharmacy and Bio Sciences 2(3): May-June 2013
INTERNATIONAL JOURNAL OF UNIVERSAL
PHARMACY AND BIO SCIENCES
Pharmaceutical Sciences Research Article……!!!
Received: 23-05-2013; Accepted: 27-05-2013
DEVELOPMENT AND VALIDATION OF A STABILITY-INDICATING
HPLC METHOD FOR ANALYSIS OF LAMOTRIGINE IN BULK DRUG
AND FORMULATIONS
Sanjay Bais*1, Anil Chandewar
2, Alkesh Kakani
3, Indrajeet Singhvi
3 Mrunal Shirsat
3
1Research Scholar, PRIST University, Thanjavur-613001, Tamilnadu.
2P.Wadhwani College of Pharmacy, Yavatmal-445001, Maharashtra.
3Pacific College of Pharmacy, Udaipur-313024, Rajasthan.
KEYWORDS:
Lamotrigine, Stability,
HPLC, Acetonitrile,
Methanol.
For Correspondence:
Sanjay Bais*
Address: Research
Scholar, PRIST
University, Thanjavur-
613001, Tamilnadu.
Email ID:-
m
ABSTRACT
A simple, economic, selective, precise, and stability-indicating HPLC
method has been developed and validated for analysis of Lamotrigine
(LTG), a selective COX-2 inhibitor, both in bulk drug and in
formulation. Reversed-phase chromatography was performed on a C18
column with the mobile phase optimized Buffer (KH2PO4):
Acetonitrile: Methanol (50:25:25 v/v/v). Detection was performed at
225 nm and a sharp peak was obtained for LTG at a retention time of
6.26 ± 0.01 min. Linear regression analysis data for the calibration plot
showed there was a good linear relationship between response and
concentration in the range 16 - 24 µg/ml ; the regression coefficient was
0.9957 and the linear regression equation was y=53978x-63357.. The
detection (LOD) and quantification (LOQ) limits were 0.0832and
0.2522µg mL−1 respectively. The method was validated for accuracy,
precision, reproducibility, specificity, robustness, and detection and
quantification limits, in accordance with ICH guidelines. Statistical
analysis proved the method was precise, reproducible, selective,
specific, and accurate for analysis of LTG. The wide linearity range,
sensitivity, accuracy, short retention time, and simple mobile phase
imply the method is suitable for routine quantification of LTG with high
precision and accuracy.
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1. INTRODUCTION:
Lamotrigine is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder.
For epilepsy it is used to treat partial seizures, primary and secondary tonic-clonic seizures, and
seizures associated with Lennox-Gastaut syndrome. Lamotrigine also acts as a mood stabilizer.
It is the first medication since lithium granted Food and Drug Administration (FDA) approval
for the maintenance treatment of bipolar I. Chemically unrelated to other anticonvulsants,
lamotrigine has relatively few side-effects and does not require blood monitoring. The
mechanism of action of lamotrigine is inhibition of the release of excitatory neurotransmitters
(aspartate and glutamate) and also involvement of the blocking of voltage dependent sodium
channels [1]. The mechanism of action comparable to that of phenytoin carbamazepine, in that
any of these three drugs acts by blocking the voltage-dependent sodium channels thus
prolonging their inactivated state and stabilising the presynaptic membrane. Consequently LTG
acts in particular to prevent the release of excitatory neurotransmitters.
1.1 Chemical structure
Systematic (IUPAC) name: 6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine.
Chemical formula: C9H7Cl2N5 Molecular weight: 256.09 g/mol., soluble in methanol, ethanol,
acetonitrile and water [3].
The principal objective of this study was, therefore, to develop a new, simple, economical,
selective, precise, reproducible, and stability indicating high-performance liquid
chromatographic (HPLC) method with a wide linear range and good sensitivity for assay of
LTG in the bulk drug and in formulations using UV detection. In the method proposed the
mobile phase was used directly for dilution of the formulation after filtration, and then further
used for analysis. Direct use of the mobile phase as diluent for formulations in quantitative
analysis minimizes errors that occur during tedious extraction procedures. The method was
validated in accordance with International Conference on Harmonization (ICH) guidelines
[25].Literature survey reveals that no method with these optimized conditions was reported for
analysis of Lamotrigine from bulk and Tablet dosage form by RPHPLC. Quantitative analysis
of the antiepileptic drugs lamotrigine, oxcarbazepine and its metabolite 10-
monohydroxycarbazepine.
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Separation was performed by elution onto the analytical column (Betasil® C6 5 m, 250mm×4.6
mm) at a flow rate of 1.0 ml/min with potassium dihydrogenphosphate buffer (20 mmol/l,
pH3.0)/acetonitrile (70/30; v/v) as analytical eluent. UV-spectrophotometric detection was set
to 215 nm for all three compounds [4], Lamotrigine and an internal standard were extracted
from plasma using liquid–liquid extraction under alkaline conditions into an organic solvent [5].
Quantitation was performed by measurement of the UV absorbance at a wavelength of 306 nm
by Pela Angelis-Stoforidis [6], Lamotrigine and the internal standard guanabenz were extracted
by Ching-Ling Cheng with 1.2 ml of diethyl ether, after the samples alkalinized with sodium
hydroxide solution (1N). Chromatographic separation was achieved on a silica column with the
mobile phase of acetonitrile–water containing 0.2% phosphoric acid and 0.3% triethylamine
(pH 2.7) (84:16, v/v), at a flow-rate of 1 ml/min. The eluant was detected at 225 nm. The
retention time was about 6 min for lamotrigine and 7 min for guanabenz [7]. The simultaneous
determination of seven antiepileptic drugs (AEDs), including primidone, phenobarbital,
phenytoin, carbamazepine with its two major metabolites carbamazepine-10,11-epoxide and
carbamazepine-10,11-(trans)-dihydrodioland the new AEDs lamotrigine, hydroxycarbazepine
and zonisamide in serum by high performance liquid chromatography (HPLC)-diode array
detector (DAD). After solid-phase extraction, separation is achieved onan Alltima 3C18
analytical column using isocratic elution with a mixture of acetonitrile, methanol and phosphate
buffer at 45 ◦C [8].
Manuela Contin carried out Liquid chromatographic analysis on a Synergi 4_m Hydro-RP,
150mm×4mm I.D. column, using a mixture of potassium dihydrogen phosphate buffer (50 mM,
pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase, at a flow rate of 1.0
mL/min.The UV detector was set at 210 nm (9).HPLC estimation of the antiepileptic drugs
lamotrigine, phenobarbitone, carbamazepine and phenytoin in human serum. After
centrifugation, 10_l of the supernatant was injected onto a NOVA PAK C-18 column and eluted
with a mobile phase consisting of phosphate buffer (10 mM)–methanol–acetonitrile–acetone in
the ratio of 55:22:12:11 (v/v) adjusted to pH 7.0. A UV detector set at 210 nm was employed
for detection [10]. J. Emami carried out HPLC separation on a C18 _-Bondapack column using
a mobile phase of acetonitrile–monobasic potassium phosphate solution (35:65, v/v) containing
orthophosphoric acid to adjust pH to 3.5 at a flow rate of 1.5 ml/min. The UV detector was
operated at 210 nm, and column temperature was adjusted at 40 ◦C [11].
Letizia Antonilli developed HPTLC for quantitative determination of lamotrigine, zonisamide
and levetiracetam in human plasma and compared with HPLC and LC–MS/MS methods.
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Chromatographic separation was achieved on silical gel 60F254 plates using
Ethylacetate:Methanol:Ammonia (91:10:15 v/v/v) as mobile phase. Quantitative analysis was
carried out by densitometry at a wavelength of 312, 240 and 210 nm for LTG, ZNS and LVT,
respectively [12].
Hart A.P analysed lamotrigine by HPLC method for from serum. Serum (0.5 ml) was
alkalinized with borate buffer (pH 9.8). Lamotrigine and the internal standard thiopental were
extracted with 10 ml of chloroform [13]. Liquid chromatography (LC) and ultraviolet
spectrophotometric (UV) methods for lamotrigine determination were validated. The LC
separation was achieved on an ACE RP-18 as stationary phase and 0.3% triethylamine in water
(v/v) pH 4.0 : methanol (62 : 38, v/v) as mobile phase. Detection was achieved with a
photodiode array at 279 nm. [14]. Estimation of Lamotrigine from pharmaceutical formulation
was carried out on a Princeton SPHER C18 (250 mm x 4.6 mm i.d., 5 µ) column with a mobile
phase consisting of acetonitrile: 0.3 % Triethylamine (adjusted to pH 6.5 using orthophosphoric
acid) (25:75 v/v) at a flow rate of 1.0 ml/min. Detection was carried out at 305 nm.
Ondansetron hydrochloride was used as an internal standard. The retention time of Lamotrigine
and Ondansetron hydrochloride was 5.28 and 7.40 min, respectively [15].
Stability indicating reverse phase HPLC method for the determination of lamotrigine on
Hypersil ODS C18 column (250 mm, 4 mm, 5 µm). A mobile phase consisting of methanol:
0.01 mol.L-1 TBAHS (Tetra butyl ammonium hydrogen sulphate) (50:50 % v/v) was used. The
flow rate was 1.0 mL min-1. The separation was performed at room temperature. UV detection
was carried out at 225 nm. The retention time of lamotrigine is found to be 3.383 min. [16].
LC-MS-MS method has been developed and validated for the determination of lamotrigine in
human plasma using multiplexing technique (two HPLC units connected to one MS-
MS). Lamotrigine was extracted from human plasma by solid-phase extraction technique using
Oasis Hydrophilic Lipophilic Balance (HLB) or N-vinylpyrrolidone and divinylbenzene
cartridge [17].
Liquid chromatography tandem mass spectrometry method was developed and validated for
quantification of lamotrigine in human serum. After a simple protein precipitation using
acetonitrile, the analytes were separated on a Shideido 150 mm × 2.0 mm, 5 μm Capcell Pak
C18 MG column using 70% acetonitrile as mobile phase at a flow rate of 200 μl/min.
Lamotrigine was eluted at 1.98 min, ionized using electrospray ionization source, and then
detected by multiple reaction monitoring mode [18]. Liquid chromatography tandem mass
spectrometry method was developed and validated for the quantification of lamotrigine in
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Human plasma by solid-phase extraction and detected in positive ion mode by tandem mass
spectrometry with Electro Spray ionization (ESI) Interface. Chromatographic separation was
performed on a Chromoliths Speed ROD;RP-18ecolumn(50_4.6 mmi.d.) Using acetonitrile:
570.1 mM ammonium formate solution (90:10,v/v) as the mobile phase at a flow rate of
0.500mL/min.[19].
HPLC method was developed for the simultaneous estimation of the antiepileptic drugs
lamotrigine , phenobarbitone , carbamazepine and phenytoin in human serum. The procedure
involves extraction of the AEDs by mixing 200 _l of serum with 200 _l of acetonitrile
containing 10 _g/ml of pentobarbitoneas internal standard (IS). After centrifugation, 10_l of the
supernatant was injected onto a NOVA PAK C-18 column and eluted with a mobile phase
consisting of phosphate buffer (10 mM)–methanol–acetonitrile–acetone in the ratio of
55:22:12:11 (v/v) adjusted to pH 7.0. A UV detector set at 210 nm was employed for detection
[20]. Capillary zone electrophoresis, Samples were deproteinized with acetonitrile containing
an internal standard, acidified with dilute acetic acid and injected into the capillary. The drug
migrated rapidly with the cationic compounds in about 3.5 rain far from any interfering
substances [21].
A HPLC method was developed using a short silica column (30 mm34.6 mm)with an aqueous
methanol mobile phase consisting of methanol–water–NH H PO (94:5.96:0.04) adjusted to a
final 4 2 4 apparent pH of 5.0 and pumped at a flow-rate of 1 ml /min. Ultraviolet detection was
carried out at a wavelength of 280 nm, and serum samples were prepared for HPLC analysis by
extraction into dichloromethane after basification. Lamotrigine was eluted at 0.96 min. [22].
HPLC method for the determination of lamotrigine in plasma is described. The drug was
extracted from 100 ml of plasma with chloroform: isopropanol (95:5% v: v) in the presence of
100 ml of phosphate buffer (10 mM). The extract was evaporated and the residue was
reconstituted with mobile phase and injected onto the HPLC system. The drug and the internal
standard (chloramphenicol) were eluted from a Symmetry C18 stainless steel column at
ambient temperature with a mobile phase consisting of 0.01 M potassium phosphate–
acetonitrile–methanol (70:20:10% v:v:v), adjusted to pH 6.7, at a flow rate of 1.3 ml min_1 and
the detector was monitored at 214 nm.(23) Simultaneous determination of the new generation
antiepileptic drugs lamotrigine oxcarbazepine’s main active metabolite monohydroxy
carbamazepine and felbamate in plasma of patients with epilepsy using HPLC with
spectrophotometric detection. Plasma sample (500_L) pre-treatment was based on simple
deproteinization by acetonitrile. Liquid chromatographic analysis was carried out on a Synergi
4_m Hydro-RP, 150mm×4mm I.D. column, using a mixture of potassium dihydrogen phos-
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-phate buffer (50 mM, pH 4.5) and acetonitrile/methanol (3/1) (65:35, v/v) as the mobile phase,
at a flow rate of 1.0 mL/min. The UV detector was set at 210 nm [24].
2. Experimental
2.1 Instrumentation & Chemicals
LTG was obtained as a gift from Astron Research Limited, Ahmedabad, Gujarat, India HPLC-
grade methanol was purchased from Merck, India High-purity water was prepared using
Millipore purification system. Other chemicals and reagents were of AR grade.
Chromatography Chromatography was performed, under ambient conditions, with A Agilent
1100 series HPLC,UV-Visible detector,Column used was Hypersil BDS C18 (250 mm X 4.6
mm).Samples (20 µL) were injected by means of a Rheodyne injector fitted with a 20-µL loop.
Filter used to filter mobile phase: Nylon 0.45 µm - 47 mm membrane filter, The Mobile Phase
was degassed before use. The detection wavelength selected was 225 nm,The injection volume
was 10 µl.The Temperature was maintained 25 ± 30 C. The detection wavelength selected was
225 nm,The injection volume was 10 µl.The Temperature was maintained 25 ± 30 C.
For degradation study 1 N HCl (86 ml of Concentrated HCl in 1000 ml of tripple distilled
water), 5 N HCl (430 ml of Concentrated HCl in 1000 ml of tripple distilled water),1 N NaOH
(40 gm in 1000 mL of tripple distilled water), 5 N NaOH (200 gm in 1000 ml of tripple distilled
water).,30% H2O2. were used.
2.2 Method Development
A variety of mobile phases were investigated in the development of an HPLC method suitable
for analysis of LTG in the bulk drug and in formulation. The published literature for the
estimation of Lamotrigine anticonvulsant drug and knowledge of the molecule suggest that
reverse phase liquid chromatography (RPLC) is suitable for the analysis of Lamotrigine. In case
of RPLC various columns are available, but as the main aim of the method is to resolve the
interested compound, C18 column (250 mm x 4.6 mm i.d., 5 µm particle size) was preferred
over other columns. Hypersil BDS C18 column was preferred as it has high carbon loading with
very closely packed material to give high resolution over other C18 columns.
Sensitivity and specificity are the most important criterias for this method. It is imperative to
achieve a single sharp peak without interference of impurities or excipients. As per the value of
PKa and solubility of the compound, various compositions of mobile phase were tried. The
compound needed sufficient polar mobile phase to have retention time which is measurable
without interference of any solvent peak. The mobile phase with combination of various polar
solvent like methanol, buffer and acetonitrile were tried and best results were obtained with
mobile phase comprising of all the three solvents mentioned above. To optimize the HPLC
parameters, several mobile phase compositions were tried. Satisfactory peak symmetry for
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Lamotrigine was obtained with mobile phase consisting of buffer (KH2PO4):methanol:
acetonitrile (50:25:25 v/v/v). Quantification was achieved with UV detection at 225 nm based
on peak area.
3. METHOD VALIDATION
3.1 Linearity:
Accurately measured standard stock solution of Lamotrigine (1.6, 1.8, 2.0, 2.2, and 2.4 ml) was
transferred to a series of 100 ml of volumetric flasks and the volume in each flask was adjusted
to 100 ml with methanol. The resulting solution was injected into the chromatography system
and the peak area obtained at retention time 6.26 minutes and flow rate 1.0 ml/min was
measured at 225 nm. Calibration curve from 16 - 24 µg/ml was constructed for Lamotrigine by
plotting peak area versus concentration at 225 nm. Each reading was average of five
determinations.
3.2 Accuracy, as Recovery:-
The accuracy of an analysis is determined by systemic error involved. It is defined as closeness
of agreement between the actual (true) value and analytical value obtained by applying test
method a number of times. Accuracy determines in term of percent recovery. The proposed
method was applied to determine Lamotrigine in tablets. The recovery experiments were carried
out in triplicate by spiking previously analyzed samples of the tablets solutions with three
different concentrations of standards.
3.3 Precision:-
It provides an indication of random error. The precision of an analytical method is usually
expressed as the standard deviation, relative standard deviation or coefficient of variance of a
series of measurements. Precision may be a measure of either the degree of reproducibility or of
repeatability of the analytical method under normal operating conditions.
3.3.1 Method precision (Repeatability)
Method precision experiment was performed by preparing the standard solution of Lamotrigine
(20 µg/ml) and analyzed for six times as per the proposed method. Coefficient of Variation
(C.V) was not more than 2%.
3.3.2 Intermediate precision (Reproducibility)
It expresses within laboratory variations as on different days analysis or equipment within the
laboratory.
3.3.2a Intra-day and inter-day precision:
Variation of results within same day is called Intra-day precision and variation of results
amongst days called Inter-day precision. The Intra-day precision (C.V) was determined for
standard solution of Lamotrigine (16 - 24 µg/ml) for five times on the day.
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3.4 Limit of Detection (LOD)
It is the lowest amount of analyte in a sample that can be detected but not necessarily
quantitated under the stated experimental conditions.
Limit of detection can be calculated using following equation as per ICH guidelines.
LOD = 3.3 × N/S
Where, N is the standard deviation of the peak areas of the drug and S is the slope of the
corresponding calibration curve.
3.5 Limit of Quantification (LOQ)
It is the lowest concentration of analyte in a sample that can be determined with the acceptable
precision and accuracy under stated experimental conditions.
Limit of quantification can be calculated using following equation as per ICH guidelines.
LOQ = 10 × N/S
Where, N is the standard deviation of the peak areas of the drug and S is the slope of the
corresponding calibration curve.
3.6 Analysis of LTG in formulation.
Twenty tablets were weighed and powdered. An accurately weighed quantity of powdered
tablets eq. to 100mg of Lamotrigine was transferred in to 100 ml of volumetric flask. 35 ml of
methanol was added, sonicated for 15 minutes, and was diluted up to mark with methanol. The
solution was filtered through nylon 0.45 µm - 47 mm membrane filter. First few ml of filtrate
was discarded. 2 ml of clear filtrate was diluted to 100 ml with methanol (20 µg/ml).
4.0 FORCED DEGRADATION OF LAMOTRIGINE
In all degradation studies the average peak area of Lamotrigine was obtained after application
of three replicates.
4.1 Acid Degradation
An accurately weighed quantity of powder equivalent to 100 mg Lamotrigine into 100 ml of
volumetric flask was transferred and 70 ml diluent, 5 ml of 5 N HCl was added. The solution
was stored in tight flasks for 6 to 12 hours at 60°C. Before injecting, the solution was cooled at
the room temperature. The above solution was neutralized with 5 N NaOH. The volume was
adjusted with methanol to 100 ml and was filtered through nylon 0.45 µm - 47 mm membrane
filter. API stress sample were prepared similarly and injected into chromatographic system.
4.2 Alkali Degradation
An accurately weighed quantity of powder equivalent to 100 mg Lamotrigine into 100 ml of
volumetric flask was transferred and 70 ml diluent, 5 ml of 1 N NaOH was added. The solution
was stored in tight flasks for 6 to 12 hours at 60°C. Before injecting, the solution was cooled at
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the room temperature. The above solution was neutralized with 1 N HCl. The volume was
adjusted with methanol to 100 ml and was filtered through nylon 0.45 µm - 47 mm membrane
filter. API stress sample were prepared similarly and injected into chromatographic system.
4.3 Oxidative Degradation
An accurately weighed quantity of powder equivalent to 100 mg Lamotrigine was transferred
into 100 ml of volumetric flask and 70 ml diluent, 5 ml of 30% H2O2 was added. The solution
was stored in tight flasks for 6 to 12 hours at 60°C. Before injecting, the solution was cooled to
the room temperature. The volume was adjusted with methanol to 100 ml and was filtered
through nylon 0.45 µm - 47 mm membrane filter. API stress sample were prepared similarly
and injected into chromatographic system.
4.4 Dry Heat Degradation
Twenty tablets was weighed and exposed to heat on a hot air oven at about 105°C for 6 to 12
hours. Before injecting, the sample solution was cooled to the room temperature and was
transferred with weighing quantity of tablets powder equivalent to 100 mg Lamotrigine into
100 ml of volumetric flask. 70 ml diluent was added, sonicated for 15 minutes and the volume
was adjusted with methanol to 100 ml and was filtered through nylon 0.45 µm - 47 mm
membrane filter. API stress sample were prepared similarly and injected into chromatographic
system.
4.0 RESULTS AND DISCUSSION
4.1 Method Development:
The HPLC procedure was optimized with a view to developing a method for stability-indicating
assay. No internal standard was used because no extraction or separation step was involved. Of
several solvents and solvent mixtures investigated mobile phase optimized Buffer (KH2PO4):
Acetonitrile: Methanol (50:25:25 v/v/v) was found to furnish sharp, well-defined peaks with
very good symmetry (1.05) and low tR (6.26 min),Tailing factor:1.30 and theorotical plate
2051. (Fig. 1).
Figure 1: HPLC chromatogram of Lamotrigine with corresponding retention time.
Well-defined peaks and other mobile phases tried either resulted in much lower sensitivity or
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did not give well defined peaks in a short time, and so were not considered.
The final decision on mobile phase composition and flow rate was made on the basis of peak
shape (peak area, asymmetry, tailing factor), baseline drift, time required for analysis, and cost
of solvent, and Buffer (KH2PO4): Acetonitrile: Methanol (50:25:25 v/v/v) was selected as the
optimum mobile phase. Under these conditions the retention time and asymmetry factor were
6.261 ± 0.01 min and 1.05 ± 0.03, respectively
4.2 Validation of the Method
4.2.1 Linearity
The calibration plot of peak area against concentration was linear in the range investigated (16 -
24 µg/ml). Calibration data, with their relative standard deviations, % RSD, standard error, and
95% confidence intervals are listed in Table I. The low values of RSD and standard error show
the method is pre-cise. Statistical calculations were performed at the 5% level of significance.
The linear regression data for the calibration plot are indicative of a good linear relationship
between peak area and concentration over a wide range. The linear regression equation
y=53978x-63357 and the re-gression coefficient was 0.9957. Other linear regression data are
given in Table II. This performance was superior to that of other currently used methods [16].
The correlation coefficient was indicative of high significance. The low values of the standard
deviation, the standard error of slope, and the intercept of the ordinate showed the calibration
plot did not deviate from linearity. There were no significant differences between the slopes of
standard curves constructed on different days.
Figure 2: Calibration curve of Lamotrigine
Table I. Calibration data for LTG with Buffer (KH2PO4): Acetonitrile: Methanol (50:25:25
v/v/v) as mobile phase
Parameters Results
Retention time 6.26 min
Tailing factor 1.30
Asymmetry 1.05
Theoretical plates 2051
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Table II: Optical and regression characteristics for analysis of Lamotrigine by Proposed
method
Parameters Results
Concentration range (µg/ml) 16-24
Limit of Detection (LOD) (µg/ml) 0.0832
Limit of Quantification (LOQ) (µg/ml) 0.2522
Regression equation (y=mx+c)
Slope
Intercept
53978
63357
Correlation coefficient (r2) 0.9957
4.2.2 Accuracy, as Recovery
The recovery of the method, determined by spiking a previously analyzed test solution with
additional drug standard solution, was 97.07- 97.90%. The values of recovery (%), SD listed in
Table III indicate the method is accurate.
Table III: Data of recovery study for Lamotrigine by proposed method
Formulation
Amount of
Drug Taken
(µg/ml)
Amount of
Drug Added
(µg/ml)
Amount of
Drug Found
(µg/ml)
% Recovery ± S.D
(n=5)
Tablet
10 8 17.62 97.90 ± 0.316
10 10 19.86 99.31 ± 0.032
10 12 21.79 99.07 ± 0.100
4.2.3 Precision
Precision was considered at two levels, i.e. repeatability and inter-mediate precision, in
accordance with ICH recommendations. Repeatability of sample injection was determined as
intra-day variation whereas inter-mediate precision was determined by measuring inter-day
variation for triplicate determination of LTG at four different concentrations (16, 18, 20, 22 and
24 μg mL−1
). Results from determination of repeatability and intermediate precision, expressed
as RSD (%), are listed in Table IV, Va & Vb. The low values of RSD indicate the repeatability
of the method.
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Table IV: Method precision data for analysis of Lamotrigine by proposed method
Table Va: Intra-day precision data for analysis of Lamotrigine by proposed Method
Table Vb: Inter-day precision data for analysis of Lamotrigine by proposed method
Lamotrigine
(µg/ml)
Mean ± S.D
(n=5)
% C.V
16 809595 ± 2.8380 0.32
18 909850 ± 2.3915 0.24
20 1007080 ± 4.9001 0.44
22 1112550 ± 6.3314 0.51
24 1245990 ± 6.3146 0.65
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Lamotrigine (20 µg/ml) Area
% Assay
1 1007881 97.70
2 1005819 96.48
3 1007139 97.25
4 1007419 97.68
5 1007072 97.12
6 1007917 97.91
Mean 1007208 97.36
S.D 768.46 0.5254
%C.V 0.076 0.54
Lamotrigine
(µg/ml)
Mean ± S.D
(n=5)
% C.V
16 809557 ± 6.8317 0.77
18 909854 ± 3.1220 0.31
20 1007072 ± 6.2237 0.48
22 1112536 ± 5.9778 0.49
24 1245996 ± 3.9594 0.29
International Standard Serial Number (ISSN): 2319-8141
4.3.4 Specificity
The specificity of the method was determined by exposing a solution of LTG to stress
conditions, i.e. 0.1 M HCl, 0.1 M NaOH, and 3% H2O
2. There was no degradation of LTG in
the presence of 0.1 M HCl or 0.1 M NaOH and no significant change in peak area and retention
time of LTG. In the presence of 3% H2O
2 it was found there was a substantial change in the
peak area of LTG, but not in the retention time. The results from these tests are listed in Table
VI and a chromatogram obtained from.
Table VI: Application of proposed HPLC method to the determination of tablet
Formulations
Labeled/taken
amount (mg)
Amount found
(mg) % Amount found
S.D (n=5)
Tablets
Brand-I
100
98.54
98.54 ± 0.33
Brand-I
100
99.05
99.05 ± 0.09
4.3.5 Acid- and base-induced degradation product:
The chromatograms of the acid degraded samples for Lamotrigine showed additional peak at
retention time of 6.26 min (Figure 4a and 4b). The chromatograms of the base degraded
samples for Lamotrigine showed additional peak at retention time of 6.26 min (Figure 5a and
5b). The concentration of the drug was found to be changing from the initial concentration
indicating that Lamotrigine undergoes degradation under acidic and basic conditions.
4.3.6 Oxidative degradation product:
The sample degraded with 30% v/v hydrogen peroxide (Figure 6a and 6b) showed three
additional peaks at retention times of 2.80, 3.80 and 5.18. The peaks of degraded products were
well resolved from the drug peak.
The samples degraded under dry heat condition (Figure 7a and 7b) showed no additional peaks,
so Lamotrigine is stable under this applied condition.
This indicates that the drug is susceptible to acid–base hydrolysis and oxidation but stable in
dry heat condition. The lower retention time of acid and base degraded product, and oxidized
product indicated that they were more polar than the analyte itself. The results are listed in
Table VII.
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4.3.7 Dry heat degradation product:
The samples degraded under dry heat condition (Figure 7a and 7b) showed no additional peaks,
so Lamotrigine is stable under this applied condition.
This indicates that the drug is susceptible to acid–base hydrolysis and oxidation but stable in
dry heat condition. The lower retention time of acid and base degraded product, and oxidized
product indicated that they were more polar than the analyte itself. The results are listed in
Table VII
Figure 3a Chromatogram of Lamotrigine API as such
Figure 3b Chromatogram of Lamotrigine tablet as such
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Figure 4a Chromatogram of Lamotrigine API in acid degradation condition.
Figure 4b: Chromatogram of Lamotrigine tablet in acid degradation condition.
Figure 5a: Chromatogram of Lamotrigine API in alkali degradation condition
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Figure 5b: Chromatogram of Lamotrigine tablet in alkali degradation condition
Figure 6a: Chromatogram of Lamotrigine API in oxidative degradation condition.
Figure 6b: Chromatogram of Lamotrigine tablet oxidative degradation condition
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Figure 7a: Chromatogram of Lamotrigine API in dry heat degradation condition
Figure 7b: Chromatogram of Lamotrigine tablet in dry heat degradation condition
Table VII Degradation of Lamotrigine
Condition Time (hr) Retention Time
(min)
% Assay
API as such 6 6.26 96.89 %
Tablet as such 6 6.26 96.82 %
Acid Degradation
(5N HCl)
API
Tablet
6
6
6.09
6.09
90.78 %
89.69 %
Base Degradation
(1N NaOH)
API
Tablet
6
6
6.13
6.09
72.53 %
75.15 %
Oxidative Degradation
(30% H2O2)
API
Tablet
6
6
6.06
6.05
10.12 %
9.76 %
Dry Heat Degradation
API
Tablet
6
6
6.25
6.25
96.80 %
96.78 %
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5. CONCLUSION:
From the above observation of the results it was concluded that the develop HPLC method is
suitable for the degradation study of Lamotrigine in bulk and its dosage form. Degradation
study of Lamotrigine was performed under hydrolytic stress conditions (1N NaOH, 5N HCl);
oxidation condition (30% H2O2) and dry heat condition. From the study, it was found that drug
is susceptible for degradation to hydrolytic condition and oxidation but was not affected by dry
heat degradation. The maximum degradation was observed in oxidative condition. Hence, the
Lamotrigine is more susceptible in oxidative degradation. All degradation products were well
separated from the drug. Developed HPLC method was successfully applied to determine drug
in the presence of degraded impurities.
6. ACKNOWLEGEMENT
The authors thankful to Astron Research Limited, Ahmedabad, Gujarat for providing gift
samples for carrying out this research work.
7. REFERENCES:
1. Ben Menachem.E, (2000), New antiepileptic drugs and non-pharmacological treatments,
Current Opinion in Neurology, 13,2, 165–170.
2. Macdonald RL, Kelly KM, (1994), Mechanisms of action of currently prescribed and
newly developed antiepileptic drugs, Epilepsia.; 35 Suppl 4:S41-50.
3. Meldrum BS, (196), Update on the mechanism of action of antiepileptic drugs,
Epilepsia; 37 Suppl 6:S4-11.
4. Christine Greiner, Ekkehard Haen, (2007), Development of a simple column-switching
high-performance liquid chromatography (HPLC) method for rapid and simultaneous
routine serum monitoring of lamotrigine, oxcarbazepine and 10-
monohydroxycarbazepine (MHD), Journal of Chromatography B, 854 , 338–344.
5. Na´dia Rezende Barbosa, Anto nio Fla´vio M´ıdio, (2000), Validated high-performance
liquid chromatographic method for the determination of lamotrigine in human plasma,
Journal of Chromatography B, 741 289–293.
6. Pela Angelis-Stoforidisa, Denis J. Morganb, Terence J. O Briena, Frank J.E. Vajdaa,
(1999), Determination of lamotrigine in human plasma by high-performance liquid
chromatography, Journal of Chromatography B, 727 113–118.
7. 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, 817 199–206.
351 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
8. Vermeij T.A.C, Edelbroek P.M, (2007), Robust isocratic high performance liquid
chromatographic method for simultaneous determination of seven antiepileptic drugs
including lamotrigine, oxcarbazepine and zonisamide in serum after solid-phase
extraction, Journal of Chromatography B, 857 40–46.
9. Manuela Contin, Susan Mohamed, Carmina Candela, Fiorenzo Albani, Roberto Riva,
Agostino Baruzzi, (2004), Simultaneous HPLC–UV analysis of rufinamide, zonisamide,
lamotrigine, oxcarbazepine monohydroxy derivative and felbamate in deproteinized
plasma of patients with epilepsy, Journal of Chromatography B, 878 ,461–465.
10. Patil K.M.,Bodhankar S.L, (2005), Simultaneous determination of lamotrigine,
phenobarbitone, carbamazepine and phenytoin in human serum by high-performance
liquid chromatography, Journal of Pharmaceutical and Biomedical Analysis 39,181-86.
11. Emami J,. Ghassami N, Ahmadi F., (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 40,999–1005.
12. Letizia Antonilli, Valentina Brusadina, Francesca Filipponia, Renzo Guglielmib, Paolo
Nencinia, (2011), Development and validation of an analytical method based on high
performance thin layer chromatography for the simultaneous determination of
lamotrigine, zonisamide and levetiracetam in human plasma, Journal of Pharmaceutical
and Biomedical Analysis 56,763– 770.
13. Hart A.P, Mazarr P S, Blackwell W, Dasgupta A, (1997), A rapid cost-effective high-
performance liquid chromatographic (HPLC) assay of serum lamotrigine after liquid-
liquid extraction and using HPLC conditions routinely used for analysis of barbiturates,
Therapeutic Drug Monitoring, Aug; 19(4): 431-435.
14. Sallustio B.C, Morris R.G, (1997), High-performance liquid chromatography
quantitation of plasma lamotrigine concentrations: application measuring trough
concentrations in patients with epilepsy, Therapeutic Drug Monitoring, 19(6): 688-93.
15. Selvadurai M, (2012), A Simple Development and Estimation of Lamotrigine Tablets by
HPLC, International Journal of ChemTech Research, (.4) 1423-1427.
16. Annapurna M. M, Sharmistha Mohapatraand B.V. V. Ravi Kumar, (2010), Development
and validation of RP-HPLC method for the determination of lamotrigine and its
degradation products in tablets, Journal Pharmacy Education Research 1, (2).
17. Shah H. J, Subbaiah G, Patel D.M, Suhagia B.N, Patel C.N, (2010), Rapid quantification
of lamotrigine in human plasma by two LC systems connected with tandem MS, Jornal
of Chromatographic Science, 48(5): 375-81.
352 Full Text Available On www.ijupbs.com
International Standard Serial Number (ISSN): 2319-8141
18. Lee W, Kim J.H, Kim H.S, Kwon O.H, Lee B.I, Heo K , (2010), Determination of
lamotrigine in human serum by high-performance liquid chromatography–tandem mass
spectrometry, Neurological Science,.31(6): 717-20.
19. Santosh Ghatol, Vatsal Vithlani, Sanjay Gurule, Arshad Khuroo, Tausif Monif, Pankaj
Partani, (2013), Liquid chromatography tandem mass spectrometry method for the
estimation of lamotrigine in human plasma: Application to a pharmacokinetic study,
Journal of Pharmaceutical Analysis;3(2),75–83.
20. Patil K.M, Agarwal A.K, Bhondarkar S.L, (2004), Validated HPTLC method for
estimation of Lamotrigine,Indian journal of Pharmaceutical sciences;66(3); 283-286
21. Shihabi Z.K, Oles K.S., (1996), Serum lamotrigine analysis by capillary electrophoresis,
Journal of Chromatography B, 683, 119 123.
22. Vidal E., Pascual C., Pou L, (1999), Determination of lamotrigine in human serum by
liquid Chromatography, Journal of Chromatography B, 736,295–298.
23. Matar K.M, Nicholls P.J., Bawazir S.A, Al-Hassan M.I., Tekle A. , (1998), A rapid
liquid chromatographic method for the determination of lamotrigine in plasma, Journal
of Pharmaceutical and Biomedical Analysis, 17 525–531.
24. Manuela C, Monica B, Erica C, Carmina C, Fiorenzo A, Roberto Riva, et al, (2005),
Simultaneous liquid chromatographic determination of lamotrigine, oxcarbazepine
monohydroxy derivative and felbamate in plasma of patients with epilepsy, Journal of
Chromatography B, 828 113–117.
25. Validation of Analytical Procedures: Methodology, ICH Harmonized tripartite
Guideline, (1996), 1-8.
353 Full Text Available On www.ijupbs.com