International Standard Serial Number (ISSN): 2319-8141 .... RPA1301002003.pdf · International...

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:-

[email protected]

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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International Standard Serial Number (ISSN): 2319-8141

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.




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


http://www.ncbi.nlm.nih.gov/pubmed?term=Hart%20AP%5BAuthor%5D&cauthor=true&cauthor_uid=9263385



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-




-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




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.




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




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




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




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.




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


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



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.




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




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




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




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 %




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.

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