Eco-Friendly QbD-Driven RP-HPLC Method for Stability Analysis of Lamotrigine

 

Hindole S. S.1*, Panchabhai V.B.2, Moein S. Attar3, Ram S Sakhare4,

Nandini Hotkar5, Ankita Yadav5

1Assistant Professor and Head Department of Pharmacognosy, Channabasweshwar Pharmacy College (Degree), Latur- 413512, Affiliated to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.

2Professor and Head Department of Pharmaceutical Chemistry, Channabasweshwar Pharmacy College (Degree), Latur- 413512, Affiliated to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.

3Research Scholar, Channabasweshwar Pharmacy College (Degree), Latur- 413512, Affiliated to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.

4Professor and Head, Department Of QA, Channabasweshwar Pharmacy College (Degree), Latur- 413512, Affiliated to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.

5Research Student, Department of Pharmaceutical Chemistry, Channabasweshwar Pharmacy College (Degree), Latur- 413512, Affiliated to Swami Ramanand Teerth Marathwada University, Nanded, Maharashtra, India.

*Corresponding Author E-mail: sunil.pharmalink@gmail.com

 

ABSTRACT:

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the quantification of lamotrigine (LAM) in bulk and tablet dosage forms. The method was systematically optimized using a Quality-by-Design (QbD) framework with Central Composite Design (CCD), evaluating four critical parameters: column type, flow rate, detection wavelength, and mobile phase composition. Optimal chromatographic separation was achieved using an Intersil C8 column (250 mm × 4.6 mm) with a mobile phase of orthophosphoric acid and methanol (55:45v/v), at a flow rate of 1.0 mL/min. The method demonstrated excellent linearity (12.5–75ppm, R˛ = 0.999), precision (%RSD = 0.5), and accuracy (mean recovery = 100.08%), and was validated in accordance with ICH guidelines Q2A and Q2B. Specificity was confirmed through forced degradation studies under acidic, basic, oxidative, thermal, photolytic, and neutral hydrolytic conditions. Maximum degradation was observed under alkaline (5.58%) and acidic (5.43%) stress, with distinct degradant peaks at retention times of 2.827–4.586min. Minimal degradation occurred under neutral (0.64%), thermal (2.30%), photolytic (1.57%), and oxidative (3.48%) conditions, confirming the method’s ability to distinguish LAM from its degradation products. In alignment with green analytical chemistry principles, the method’s environmental impact was assessed using the AGREE metric, yielding a favorable score of 0.82. The validated method is reliable, eco-conscious, and suitable for routine quality control of lamotrigine in pharmaceutical formulations.

 

KEYWORDS: Quality by Design, RP-HPLC, Lamotrigine, Central Composite Design, Estimation, Validation

 

 

INTRODUCTION:

Lamotrigine, a triazine derivative with anticonvulsant properties, possesses a distinct molecular architecture (Figure 1) that underpins its therapeutic efficacy and guides analytical method development. It is approved for the treatment of partial and generalized seizures, including Lennox-Gastaut syndrome, and is also indicated for the maintenance therapy of bipolar I disorder to prevent depressive episodes.1 Its pharmacological action involves the inhibition of voltage-gated sodium channels, thereby stabilizing neuronal membranes and reducing excitatory neurotransmitter release.2

 

 

Figure 1: Molecular structure of lamotrigine, illustrating its triazine-based scaffold and functional groups relevant to its pharmacological activity.

 

Given its narrow therapeutic index and the potential for serious adverse effects such as Stevens-Johnson syndrome, precise and reliable quantification of lamotrigine in pharmaceutical formulations is essential for quality control and regulatory compliance. Several analytical methods have been reported for lamotrigine estimation, including spectrophotometry, capillary electrophoresis, and high-performance liquid chromatography (HPLC).3,4 However, many of these methods lack systematic optimization, involve environmentally hazardous solvents, or fail to demonstrate stability-indicating capability through forced degradation studies.5,6

 

To address these limitations, the present study employs a Quality-by-Design (QbD) approach using Central Composite Design (CCD) to develop a robust reversed-phase HPLC method for lamotrigine quantification. QbD facilitates the identification and control of critical method parameters (CMPs) and ensures method robustness through statistical modeling and design space exploration.6,7 The method was further validated in accordance with International Council for Harmonization (ICH) guidelines Q2A and Q2B, covering parameters such as specificity, linearity, accuracy, precision, and robustness.8

 

In addition to analytical performance, environmental sustainability was considered through the application of green analytical chemistry principles. The method’s greenness was quantitatively assessed using the AGREE metric, which evaluates compliance with the 12 principles of green chemistry and provides a comprehensive score. The developed method achieved an AGREE score of 0.82, indicating high environmental compatibility.5,9

 

Furthermore, the method was evaluated under various forced degradation conditions—acidic, basic, oxidative, thermal, photolytic, and neutral hydrolysis—to confirm its stability-indicating nature. The ability to resolve lamotrigine from its degradation products under stress conditions reinforces its suitability for routine quality control and regulatory submissions.10,11

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Lamotrigine reference standard was obtained from a certified source. Methanol (HPLC grade) and orthophosphoric acid (analytical grade) were procured from Merck (India). Double-distilled water was used throughout the study. All chemicals were of analytical or HPLC grade and used as received.

 

Instrumentation and Chromatographic Conditions:

Chromatographic analysis was performed using a reversed-phase HPLC system equipped with a UV detector. Separation was achieved on an Intersil C8 column (250mm × 4.6mm, 5µm). The mobile phase consisted of orthophosphoric acid and methanol in a 55:45v/v ratio, delivered at a flow rate of 1.0mL/min. Detection was carried out at an optimized wavelength. The injection volume was 20µL, and the column temperature was maintained at ambient conditions. Based on UV-Vis spectral analysis (Figure 2), the detection wavelength for HPLC method development was set at 272nm, corresponding to a secondary absorbance maximum that offers reliable sensitivity for quantitative estimation of lamotrigine.

 

 

Figure 1: UV-Vis spectrum of lamotrigine showing a secondary absorbance maximum at 272nm, selected as the detection wavelength for HPLC method development.

 

Preparation of Standard and Sample Solutions:

A stock solution of lamotrigine (1000µg/mL) was prepared in methanol and serially diluted to obtain working standards in the range of 12.5–75µg/mL. Tablet samples were finely powdered, and an accurately weighed portion equivalent to 100mg of lamotrigine was extracted with methanol, sonicated, filtered, and appropriately diluted for analysis.

 

Method Validation:

The developed method was validated in accordance with ICH guidelines Q2A and Q2B7. Validation parameters included:

·       Linearity: Assessed across the range of 12.5–75 µg/mL.

·       Accuracy: Evaluated through recovery studies at multiple concentration levels.

·       Precision: Determined by intra-day and inter-day repeatability.

·       Specificity: Confirmed by analyzing blank and placebo samples.

·       Robustness and Ruggedness: Assessed by introducing minor variations in chromatographic conditions.

 

Forced Degradation Studies:12,13

To establish the stability-indicating capability of the method, lamotrigine was subjected to stress conditions including acidic, alkaline, oxidative, thermal, photolytic, and neutral hydrolysis, as per ICH Q1A(R2) guidelines(10). Samples were exposed to respective stressors for defined durations, neutralized where necessary, and analyzed using the optimized RP-HPLC method. The extent of degradation and resolution of degradants were evaluated and are discussed in the Results and Discussion section.10,11

 

Greenness Assessment:5,9

The environmental sustainability of the method was evaluated using the AGREE metric, which assesses compliance with the 12 principles of green analytical chemistry. The calculated score reflects the method’s eco-friendly attributes and is presented in the Results and Discussion section.

 

RESULTS AND DISCUSSION:

Quality-by-Design (QbD) Optimization:14–16

A central composite design (CCD) was employed to optimize four critical method parameters—methanol concentration in the mobile phase (40–50% v/v), 0.01 N potassium dihydrogen phosphate buffer strength, flow rate (0.8–1.2 mL/min), and column temperature (28–32 °C)—each at three coded levels (low, mid, high). The CCD matrix comprised 13 experimental runs, enabling estimation of linear, interaction, and quadratic effects on key chromatographic responses: retention time, theoretical plate count, and peak asymmetry. Response surface methodology was applied to construct three-dimensional surface plots, facilitating visualization of how each factor and their interactions influence critical quality attributes. A composite desirability function then integrated retention time, asymmetry, theoretical plates, and peak area to identify the optimal chromatographic conditions (Figure 3).

 

 

Figure 3: (A) 3D response of retention time (B) 3D response of Theoritical Plates  (C) 3D response of Assymetric Factor

 

Chromatographic Optimization and System Suitability:

The RP-HPLC method was optimized to achieve sharp, symmetrical peaks with minimal tailing and consistent retention. Lamotrigine exhibited a well-resolved peak at 3.064 minutes as shown in Figure 4 under the finalized chromatographic conditions using an Intersil C8 column and a mobile phase of orthophosphoric acid:methanol (55:45 v/v). Based on UV-Vis spectral analysis, the detection wavelength was set at 272 nm, corresponding to a secondary absorbance maximum suitable for quantification.

 

System suitability was confirmed through six replicate injections of the standard solution, yielding a %RSD of 1.1% for peak area, which complies with USP acceptance criteria (<2%) and indicates reliable instrument performance.

 

Figure 4: Representative RP-HPLC chromatogram of lamotrigine under optimized conditions

Method Validation:8,17

The method was validated in accordance with ICH Q2(R1) guidelines, covering linearity, accuracy, precision, robustness, and sensitivity.

 

Linearity was demonstrated over the concentration range of 12.5–75µg/mL, with a regression equation of y = 33121x + 14000 and a correlation coefficient (R˛ = 0.9996), indicating excellent proportionality between concentration and response.

 

 

Figure 5: Linearity Curve for LAM

 

Accuracy was assessed via recovery studies at three concentration levels (50%, 100%, and 150%). Mean recoveries ranged from 99.25% to 100.79%, with an overall average of 100.08%, confirming the method’s reliability for quantifying lamotrigine in pharmaceutical matrices.

 

Table 1: Result data for Accuracy study

% Level

Amount Spiked (mg/mL)

Amount recovered (mglmL)

% Recovery

Mean % Recovery

50

25

25

25

25.04

25.08

24.95

100.16

100.33

99.80

100.08

100

50

50

50

49.72

50.06

49.62

99.44

100.12

99.25

150

75

75

75

75.12.

75.47

75.60

100.16

100.63

100.79

 

Precision was evaluated through intra-day and inter-day studies. Method precision yielded a %RSD of 0.7%, while intermediate precision showed 1.1%, both well within acceptable limits. System precision also reflected consistent performance with a %RSD of 1.1% across six injections.

 

Table 1: Precision Data for System Interday and Intraday

Sr. No.

Intraday

Interday

System

Peak Area

Peak Area

Peak Area

1

769847

768486

1643866

2

770123

765603

1627083

3

764964

760150

1654195

4

776262

761367

1676440

5

765486

768540

1631424

6

768460

768193

1652097

Avg.

769190

765390

1647518

SD

4084.8

3769.7

17843.6

% RSD

0.5

0.5

1.1

Robustness was verified by introducing deliberate variations in flow rate, mobile phase composition, and column temperature. The %RSD values under altered conditions ranged from 0.5% to 0.8%, indicating that the method remains unaffected by minor operational changes.

 

Sensitivity parameters were determined using signal-to-noise ratios. The Limit of Detection (LOD) was found to be 0.42µg/mL, and the Limit of Quantification (LOQ) was 1.27µg/mL, confirming the method’s suitability for low-level detection.

 

Greenness Evaluation:

The method’s environmental sustainability was assessed using the AGREE metric, which yielded a score of 0.82. This reflects strong compliance with green analytical chemistry principles, including the use of relatively benign solvents, minimal waste generation, and energy-efficient operation. The score supports the method’s applicability in eco-conscious pharmaceutical quality control settings.5,17

 

 

Figure 6: AGREE Pictogram for the proposed method

 

CONCLUSION:

A robust, precise, and environmentally conscious RP-HPLC method was successfully developed and validated for the quantification of Lamotrigine in pharmaceutical formulations. The method demonstrated excellent linearity over the range of 12.5–75µg/mL, with a correlation coefficient of 0.9996, and high recovery rates across all accuracy levels, confirming its reliability. Precision studies, including intra-day, inter-day, and system precision, yielded %RSD values well within acceptable limits, ensuring reproducibility. Robustness testing further affirmed the method’s resilience to minor variations in chromatographic parameters.

 

The detection wavelength of 272nm, selected based on UV-Vis spectral analysis, provided optimal sensitivity, while the retention time of 3.064 minutes enabled rapid analysis. The method’s greenness, supported by an AGREE score of 0.82, underscores its suitability for routine quality control in compliance with green analytical chemistry principles.

 

 

Overall, the validated method meets regulatory expectations and offers a sustainable, high-performance analytical tool for Lamotrigine estimation in pharmaceutical environments.

 

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Received on 05.12.2025      Revised on 14.01.2026

Accepted on 19.02.2026      Published on 16.04.2026

Available online from April 18, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(2):109-113.

DOI: 10.52711/2231-5675.2026.00016

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