Estimation of Lamotrigine by RP-HPLC Method

A rapid and reproducible reverse phase high performance liquid chromatographic method has been developed for the estimation of lamotrigine in its pure form as well as in pharmaceutical dosage forms. Chromatography was carried out on a Luna C18 column using a mixture of potassium dihydrogen phosphate buffer (pH 7.3) and methanol in a ratio of 60:40 v/v as the mobile phase at a flow rate of 1.0 mL/min. The detection was done at 305 nm. The retention time of the drug was 6.1 min. The method produced linear responses in the concentration range of 10 to 70 μg/mL of lamotrigine. The method was found to be reproducible for analysis of the drug in tablet dosage forms.


Introduction
Lamotrigine [1][2][3][4][5][6] , (6-(2, 3-dichlorophenyl)-1, 2, 4-triazine-3,5-diamine) is an anticonvulsant drug used in the treatment of epilepsy and bipolar disorder.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 medicament since lithium approved by food and drug administration (FDA) for the maintenance treatment of bipolar type I disorder.Chemically unrelated to other anticonvulsants, lamotrigine has relatively few side-effects and does not require blood monitoring.The exact way how lamotrigine works is unknown.

Experimental
A Shimadzu Prominence high pressure liquid chromatographic instrument provided with a Luna C 18 column (150 mm x 4.6 mm, 5 µ), a LC 20 AT quaternary pump, a CTO 20A column oven and an SPD 20A UV-Visible detector was employed in the study.A 20 µL Hamilton injection syringe was used for sample injection.Data acquisition was done by using LC solution software.
Methanol (HPLC grade), potassium dihydrogen phosphate (ExcelaR grade) and sodium hydroxide (ExcelaR grade) of Qualigens, India were used in the study.HPLC grade water prepared using Millipore Milli Q system was used for preparing the mobile phase mixture.
A freshly prepared binary mixture of potassium dihydrogen phosphate buffer (pH 7.3) and methanol in a ratio of 60:40 v/v was used as mobile phase and as diluent for making various working solutions.The mobile phase was filtered through 0.45 µ membrane filter and sonicated before use.The flow rate of mobile phase was maintained at 1 mL/min.The column temperature was maintained at 25±1 0 C. The detection was carried out at 305 nm.

Preparation of stock and working standard solutions
About 100 mg of lamotrigine was weighed accurately and transferred into a 100 mL volumetric flask containing 25 mL of the mobile phase.The solution was sonicated for 20 min and then the volume made up with a further quantity of the mobile phase to get 1 mg/mL solution.An aliquot of this solution was suitably diluted with the mobile phase to get a working standard of 100 µg/mL of lamotrigine.

Linearity and construction of calibration curve
The quantitative determination of the drug was accomplished by an external standard method.The mobile phase was filtered through a 0.45 µ membrane filter before use.The flow rate of the mobile phase was adjusted to 1 mL/min.The column was equilibrated with the mobile phase for at least 30 min prior to the injection of the drug solution.
Linearity of the peak area response was determined by taking measurements at seven concentration points (six replicates at each point).Working dilutions of lamotrigine in the range of 10-70 µg/mL were prepared by taking suitable aliquots of the working standard solutions in different 10 mL volumetric flasks and diluting up to the mark with the mobile phase.Twenty microlitres of the dilutions was injected each time into the column at a flow rate of 1 mL/min.Each dilution was injected six times into the column.The drug in the eluate was monitored at 305 nm and the corresponding chromatograms were obtained.From these chromatograms, the mean peak areas were noted and a plot of concentrations over the peak areas was constructed.The regression of the plot was computed by least square regression method.The linear relationship was found to be of 10-70 µg/mL between the concentration of lamotrigine and peak area.This regression equation was later used to estimate the amount of lamotrigine in pharmaceutical dosage forms.The calibration data and regression parameters are reported in Table 1.

Estimation of lamotrigine in tablet dosage forms
Twenty tablets of Lamitor each containing 25 mg of lamotrigine were weighed and finely powdered in a mortar.From this, the average weight of a tablet is calculated (404.1 mg).An accurately weighed portion from this powder equivalent to 100 mg of lamotrigine was transferred to a 100 mL volumetric flask containing 25 mL of the mobile phase.The contents of the flask were sonicated for about 15 min for complete solubility of the drug.Then the mixture was filtered through a 0.45 µ membrane filter and made up to the volume with the mobile phase.From the above solution a dilution equivalent to 50 µg/mL (a dilution within the linearity range) was prepared using the mobile phase.Twenty microlitres of the above solution was then injected eight times into the column.The mean of the peak areas obtained was calculated and the drug content in the formulation was calculated by using the regression equation obtained for the reference sample.The same procedure is adopted for the estimation of the drug from the tablets of Lamepil containing 25 mg of lamotrigine.

Results and Discussion
The present study was aimed to develop a rapid, precise and accurate HPLC method for the analysis of lamotrigine in pharmaceutical dosage forms.In order to effect analysis of the component peaks under isocratic conditions, mixtures of methanol and potassium dihydorgen phosphate buffer in different combinations were tested as mobile phase on a C 18 stationary phase.A binary mixture of potassium dihydrogen phosphate buffer (pH 7.3) and methanol in a ratio of 60:40v/v was proved to be the most suitable of all the combinations since the chromatographic peak obtained was sharp.The retention time of the drug was found at 6.1 min.A model chromatogram is shown in Figure 1.

Figure 1. A model chromatogram showing the separation of lamotrigine
Each of the samples was injected six times and the same retention times were observed in all cases.The mean peak area of lamotrigine for different concentration setup was calculated and the average value at each concentration is shown in Table 1.A good linear relationship (r=0.9999) was observed between the concentrations of lamotrigine and the corresponding peak areas.The linearity range was found to be 10-70 µg/mL.The regression equation of the calibration curve between concentration of lamotrigine over its peak area was found to be y = 12302 x -45 (where y is the peak area and x is the concentration of lamotrigine).The method was validated in terms of precision, accuracy, robustness, LOD Detector A: 305nm Lamotrigine mV and LOQ.The intra-day and inter-day variations studied at 30, 40 and 50 µg/mL of working drug concentrations showed a low coefficient of variation (Table 2).This reveals that the method is quite precise.The percent recoveries of the drug solutions were studied at three different concentrations levels.The percent individual recovery and the percent RSD values at each level were (100.02-100.05)within the acceptance limits.The results are given in Table 3.The drug content in the tablets was quantified by using the proposed method of analysis.The deliberate changes in the method have not much affected the peak tailing, theoretical plates and the percent assay.This indicates that the present method is robust.The lowest values of LOD and LOQ as obtained by the proposed method indicate the method is sensitive.The working solution of the drug was stable up to 24 hours.The tailing factor, the number theoretical plates and HETP are in the acceptable limits.Therefore, the proposed method can be used for routine quality control and analysis of the drug in bulk samples and in tablet dosage forms.The results of robustness study and system suitability parameters are given in  The mean amount of lamotrigine obtained from two different tablet dosage forms is 99.96 and 100.16% as shown in Table 6 indicates that the method is accurate.The absence of additional peaks in the chromatogram indicates non interference of the commonly used excipients in the tablets and hence the method is specific.

Table 1 .
Calibration of the proposed method

Table 2 .
Precision of the proposed method

Table 4 .
Results of the robustness study

Table 5 .
System suitability parameters

Table 6 .
Recovery of lamotrigine from tablets formulations *Average ± standard deviation of eight determinations