In this work, a sensitive and stability-indicating HPTLC method for the determination of lamotrigine is presented. According to the International Conference on Harmonization guidelines Q1A, lamotrigine was exposed to a variety of stress conditions; these include heating in acidic, basic and neutral media. Its stability towards oxidative stress, humidity, high temperature and direct sunlight was also examined. Separation of the drug from its forced degradation impurities was achieved using TLC silica gel plates and a mobile phase composed of ethyl acetate: methanol: ammonia. The linear regression analysis of the data obtained for the correlation plots showed good linearity over the concentration range of 10–300 ng/spot. The forced degradation studies showed that lamotrigine is susceptible to degradation under acidic, basic, neutral and oxidative conditions, among which alkaline-induced hydrolysis had the highest degradative potential. Alternatively, the drug was stable under heat, humidity, and daylight stress factors. In order to assess the purity and stability of the drug in tablet formulations, the developed method was applied to the analysis of commercial tablets in brand and generic products. The obtained results showed that the degradation of the drug has not occurred in the marketed formulations that were analyzed by the described methodology.
Lamotrigine (LAM) [6-(2,3-dichlorophenyl)-1,2,4-triazine-3,5-diamine] is an antiepileptic drug approved as a monotherapy for partial seizures and for the adjunctive treatment of partial seizures, primary and secondary generalized tonic-clonic seizures, and in patients with Lennox-Gastaut syndrome. It is also approved for use in the maintenance treatment of bipolar disorder to delay the time to occurrence of mood episodes [
The USP describes an HPLC assay with UV detection for the assay of LAM in pure and tablet form [
All reagents and solvents used were of analytical grade. LAM was kindly supplied as a gift sample by Delta Pharm (Cairo, Egypt). 1 mg/mL standard stock solution was prepared in methanol and was further diluted by the same solvent to produce 0.1 and 0.5 mg/mL standard working solutions.
The samples were spotted in the form of bands of 5 mm width with a Camag microlitre syringe under nitrogen stream using a Camag Linomat IV sample applicator (Switzerland). Precoated TLC silica gel aluminum plates 60F254 (20 × 10 cm, 200
Serial volumes of 0.1 mg/mL LAM standard working solution were transferred into a set of 10 mL volumetric flasks, and the volumes were completed to mark with methanol. Each calibration concentration was prepared in triplicates, and 5
Ten tablets were weighed, powdered and thoroughly mixed. An accurately weighed quantity of the powdered tablets equivalent to 50 mg drug according to the label claim was sonicated for 2 × 5 min with 2 × 20 mL methanol portions. The combined extracts were filtered into a 50 mL volumetric flask, and the volume was finally completed to the mark with methanol. Ten mL of this solution was diluted to 100 mL with methanol to produce a 0.1 mg/mL LAM solution. Aliquots of this solution were treated as in Section 2.3. The nominal contents of the tablets were then calculated using the developed regression equation.
The 0.5 mg/mL LAM standard working solution was used in the forced degradation studies to provide evidence of the stability-indicating capacity and the selectivity of the proposed method. With the exception of the photolytic degradation, all forced degradation studies were performed in the dark in order to exclude the possible degradative effect of light.
To 10 mL of the standard working solution, 10 mL of 5 M HCl or 2.5 M NaOH was added separately. These mixtures were refluxed in a thermostatically controlled water bath maintained at 80°C for 2 h. The samples were then cooled to room temperature and quantitatively transferred into 25 mL volumetric flasks. Finally, the volumes were completed to mark with methanol. One
Ten mL of the LAM standard working solution was refluxed with 10 mL water in a thermostatically controlled water bath maintained at 80°C for 72 h. The solution was then treated as in the Section
To 10 mL of the standard working solution, 10 mL of H2O2 (30% v/v) and 20 mL water were added, and the mixture was refluxed in a thermostatically controlled water bath maintained at 80°C for 4 h. The solution was then heated in a boiling water bath for 30 min to remove completely the excess of H2O2. Afterwards, the solution was cooled and quantitatively transferred to a 50 mL volumetric flask and then completed to volume with methanol. Finally, 2
Pure drug powder was spread as a thin layer on a Petri dish and stored in a hot air oven at 100°C for 72 h. A methanolic solution was then prepared using the stressed powder at a concentration of 0.2 mg/mL. One
Pure drug powder was spread as a thin layer on a Petri dish and stored in a humidity chamber (relative humidity = 75%) maintained at 40°C for 72 h. The procedure described in Section
Ten mL of the standard working solution was transferred into a 25 mL volumetric flask. The volume was completed to mark with methanol, and the solution was exposed to direct sunlight for 72 h (12 days, 9 : 00–15 : 00). Finally, 1
In order to optimize the separation of intact drug from its degradation products, drug solutions were spotted and developed in different mobile phases. A system composed of
A typical HPTLC chromatogram of LAM scanned at 310 nm (100 ng/spot,
Six concentrations of the drug over a concentration range of 2–60 ng/
Regression and statistical parameters for the determination of LAM using the proposed HPTLC method.
Parameter | Value |
---|---|
Linearity range (ng/spot) | 10–300 |
|
0.9998 |
|
26.75 |
|
126.31 |
|
32.77 |
|
1.08 |
|
56.27 |
|
0.82 |
The accuracy and the intra- and interday reproducibilities for the determination of the drug among its degradation products were tested using 5 replicates at 3 different concentration levels, namely 30, 150, and 250 ng/spot over 3 consecutive days. The proposed method is deemed accurate as warranted by the low values of percentage relative error (Er%) gathered in Table
Intra- and interday precisions and accuracy of the proposed HPTLC method for the determination of LAM.
Frequency of analysis | Concentration taken |
Concentration found ± SD* (ng/spot) | RSD |
Er |
---|---|---|---|---|
30 | 29.94 ± 0.27 | 0.91 | −0.21 | |
Intraday | 150 | 150.05 ± 1.03 | 0.68 | 0.03 |
250 | 250.26 ± 0.59 | 0.24 | 0.11 | |
| ||||
30 | 30.06 ± 0.15 | 0.50 | 0.19 | |
Interday | 150 | 149.55 ± 1.15 | 0.77 | −0.30 |
250 | 250.32 ± 1.95 | 0.78 | 0.13 |
For the estimation of LOD and LOQ, blank methanol was spotted 6 times following the same parameters described for the construction of the calibration curve, and the noise detected at the same
The peak purity of LAM was assessed by comparing the spectra of the standard and sample solutions at 3 different levels, namely, peak start (S), peak apex (M), and peak end (E) positions of the spot, that is,
A single spot at
Statistical evaluation of the results obtained by the proposed and reference methods for the determination of LAM in tablets.
Pharmaceutical preparation | Proposed method | Reference methodd | ||||||
---|---|---|---|---|---|---|---|---|
Mean ± SDa | RSD% |
|
Mean ± SDa | RSD% |
|
|
| |
Lamictal 100 tablets | 100.08 ± 0.82 | 0.82 | 0.67 | 99.62 ± 0.74 | 0.74 | 0.55 | 0.94 | 1.22 |
Lamictal 50 tablets | 99.58 ± 0.69 | 0.70 | 0.47 | 100.14 ± 0.50 | 0.50 | 0.25 | 1.47 | 1.88 |
Lamictal 25 tablets | 99.68 ± 0.62 | 0.62 | 0.38 | 99.85 ± 0.83 | 0.83 | 0.69 | 1.89 | 1.82 |
Lamotrigine 100 tablets | 99.80 ± 0.58 | 0.58 | 0.34 | 99.85 ± 0.46 | 0.46 | 0.21 | 0.13 | 1.62 |
Lamotrigine 50 tablets | 99.91 ± 0.74 | 0.74 | 0.55 | 100.01 ± 1.15 | 1.15 | 1.32 | 0.17 | 2.40 |
Lamotrigine 25 tablets | 99.63 ± 0.76 | 0.76 | 0.58 | 99.41 ± 0.96 | 0.97 | 0.93 | 0.39 | 1.60 |
Laptorgine 200 tablets | 99.77 ± 0.58 | 0.58 | 0.34 | 99.39 ± 1.04 | 1.05 | 1.08 | 0.73 | 3.20 |
Laptorgine 25 tablets | 99.62 ± 0.82 | 0.82 | 0.67 | 99.45 ± 0.80 | 0.80 | 0.64 | 0.36 | 1.05 |
Larogen 100 tablets | 99.52 ± 1.38 | 1.39 | 1.90 | 99.76 ± 0.85 | 0.85 | 0.73 | 0.34 | 2.60 |
bVariance (
cThe theoretical
d[
The ICH Q1A guidelines provide useful definitions and general comments about forced degradation studies. However, details concerning the scope, timing, and best practice are either general or mainly lacking [
% Recoveries of LAM and
Condition | (%) Recovery |
|
---|---|---|
Acid hydrolysis |
85.25 | 0.01, 0.02, 0.06, 0.1 |
Base hydrolysis |
77.84 | 0.01, 0.1 |
Neutral hydrolysis |
95.91 | — |
Hydrogen peroxide |
87.20 | 0.03, 0.5 |
Dry heat |
99.22 | — |
Humidity |
99.14 | — |
Daylight |
99.92 | — |
HPTLC chromatogram of the base-induced degradation of LAM (200 ng/spot).
HPTLC chromatogram of the acid-induced degradation of LAM (200 ng/spot).
HPTLC chromatogram of the hydrogen peroxide-induced degradation of LAM (200 ng/spot).
According to the ICH guidelines, the safety, quality, and/or efficacy of a drug substance is liable to inadequate storage conditions. Therefore, the need for stability-indicating methods appears indispensable to establish the stability and purity profiles of a drug substance. In this work, LAM was exposed to 7 different stress conditions among which considerable degradation occurred in acidic, basic, and oxidative media. LAM appears more sensitive to alkaline degradation. Whereas heat, humidity, and light had no effect on LAM. The proposed HPTLC method was capable of quantifying low levels of LAM and effectively resolving it from forced degradation impurities. The developed HPTLC assay was also applied to the analysis of tablets, and the ensuing results showed no statistically significance differences from those obtained by a reference method.