A Systematic Design Of Experiment-Driven High Performance Thin Layer Chromatography Approach for the Quantification of Glimepiride and Linagliptin in Synthetic Blends

INTRODUCTION

Type 2 diabetes mellitus is referred as increasing blood sugar level which requires combination therapies for improvement of blood glucose regulations. where Linagliptin (a DPP-4 inhibitor) and Glimepiride (a sulfonylurea) act synergistically (Beckett, 2007; Indian Pharmacopoeia Commission, 2022; Mohan, 2019; Ojo, 2023; Guedes, 2013; Neumiller, 2012).

To ensure this drug combination is safe and effective, we need a dependable method to check its quality and stability. Although techniques like HPLC and spectroscopy have been used to test each drug separately, there isn’t a well-established method for testing both at the same time in a lab-prepared mixture.

High-Performance Thin-Layer Chromatography (HPTLC) could be a great option because it’s simple, fast, cost-effective, and allows testing of multiple samples at once (Neumiller, 2012; International Council for Harmonization, 2023; Ks, 2024; Falguniben, 2024).

Therefore, this study focuses on the development and validation of a simple, rapid, and accurate HPTLC method for the simultaneous determination of GLP and LNG in a synthetic mixture. Using Design of Experiments principles to test the robustness of High-Performance Thin-Layer Chromatography (HPTLC) techniques guarantees a methodical, scientific approach to method optimization. Finding important method variables and evaluating their effect on method performance within a specified design space are made easier with the aid of Design of Experiment (DoE). This tactic increases method reliability while reducing variability.

MATERIALS AND METHODS

Drug Components and Reagents

Glimepiride and linagliptin working standards were generously given as a free sample by Glenmark Pharmaceuticals Ltd. in Ankleshwar, India. The study's solvents and chemicals were all acquired from Merck Specialities Pvt. Ltd., in India.

Glimepiride 2 mg and Linagliptin 5 mg in combined tablet dosage form is approved by Central Drugs Standard Control Organisation (CDSCO) Indian regulatory authority. Hence, to ensure the accurate dose incorporated in tablet dosage form, this novel approach has been discovered.

HPTLC Systems

The chromatographic analysis was conducted using CAMAG instrumentation from Switzerland. The auto-sampler employed was the CAMAG-LINOMATE 5, equipped with a Hamilton syringe specifically designed for precision sampling. The Twin Trough chamber (20 × 20 cm) from CAMAG was utilized as the developing chamber, ensuring controlled and uniform plate development. The detection of analytes was performed using the CAMAG-TLC Scanner 4, which offers high sensitivity and accuracy. Data acquisition and analysis were carried out using Vision CATS software, version 13, which is optimized for advanced TLC method development and result interpretation.

Preparation of Standard Solution (Glimepiride 20 µg/mL, Linagliptin 50 µg/mL)

Transferred 1 mL of Glimepiride solution (200 µg/mL) and 1 mL of Linagliptin solution (500 µg/mL) were transferred into a 10 mL volumetric flask. The volume was made up to the mark with methanol to obtain a working standard solution containing 20 µg/mL of glimepiride and 50 µg/mL of linagliptin.

Preparation of Synthetic Mixture as per approved by Central drug standard control organisation

A synthetic mixture of Glimepiride and Linagliptin was prepared by accurately weighing 2 mg of Glimepiride and 5 mg of Linagliptin along with 77 mg of Microcrystalline Cellulose, 20 mg of Starch, 10 mg of Hydroxypropyl Cellulose, 4 mg of Talc, and 2 mg of Magnesium Stearate. All components were transferred to a clean, dry glass mortar and triturated uniformly using a pestle to obtain a homogeneous powder blend. The prepared mixture was employed for subsequent analytical evaluation and method validation.

Method Validation

System Suitability

System suitability testing proves the method's suitability. A standard solution of Glimepiride and Linagliptin was spotted five replicates at 2000 ng/band and 5000 ng/band Respectively. The retardation factor and peak area were measured using specified chromatographic conditions.

Specificity

The ability of an analytical technique to precisely quantify a target analyte in the presence of other elements that are probably present in the sample matrix is known as specificity, as the name suggests. The experiment utilized excipients such as cross carmellose sodium, talc, magnesium stearate, microcrystalline cellulose, starch, and hydroxypropyl cellulose to prepare the synthetic mixture. There are no interferences of any excipients used in preparation of Synthetic mixture (placebo).

Linearity and Range

At six concentration levels within the ranges of 2000-10000 ng/band (for Glimepiride) and 5000-25000 ng/band (for Linagliptin), the method's linearity was assessed through regression analysis. Peak area was plotted against concentration (n=5) to create the calibration curves. Results are shown in Table 5.

Precision

Precision was evaluated through repeatability, intra, and interday measurements. Repeatability involved six measurements of the same concentration Glimepiride (2000 ng/band) and Linagliptin (5000 ng/band) within a day, computing mean standard deviation and % RSD.

To determine intra-day precision, sample solutions of Glimepiride (2000, 4000, 6000 ng/band) and Linagliptin (5000, 10000, 15000 ng/band) were analysed at three levels concentrations of the calibration curve on the same day (n=3).

The inter-day precision was assessed by examining sample solutions of Glimepiride (2000, 4000, and 6000 ng/band) and Linagliptin (5000, 10000, and 15000 ng/band) at three concentration levels across three days (n=3). Mean values and relative standard deviation (% RSD) were calculated based on the peak areas obtained.

Accuracy

To determine the method's accuracy, the % recovery of Glimepiride and Linagliptin were calculated. Glimepiride and Linagliptin were added in known quantities at 80%, 100%, and 120% to a pre-quantified physical mixture. The solutions were spotted to the TLC plate, and development took place. The peak area was assessed, and the quantities of Glimepiride and Linagliptin were determined.

DL and QL

Detection and quantification were determined through the calibration curve method. The standard deviation of the y-intercepts of regression lines is used as the standard deviation. It was calculated as per ICH guidelines as described below:

"σ" stands for the standard deviation of regression y-intercepts, while "S" is the slope of a calibration curve.

Robustness Study

To evaluate the robustness of the developed HPTLC method, a Fractional Factorial Design (FFD) of type 2^4-1 was employed. (Software: Design Expert version 13 was used for both experimental design and optimization analysis). This approach enabled the systematic assessment of four critical method parameters with a limited number of experiments. The selected factors were solvent front distance (A), detection wavelength (B), volume of ethyl acetate in the mobile phase (C), and chamber saturation time (D). Each parameter was varied at two levels high and low to simulate minor, deliberate changes from the optimized method conditions. All experiments were carried out in randomized order to minimize the impact of uncontrolled variables. The synthetic mixture of Glimepiride and Linagliptin was spotted onto HPTLC plates and analysed under the specified conditions for each experimental run. The Retention Factor (R_f) values for both drugs were recorded and assessed for variability to determine the method's robustness.

Analysis of Glimepiride and Linagliptin in Synthetic Mixture

A synthetic mixture equivalent to 2 mg of Glimepiride and 5 mg of Linagliptin (650 mg total) was transferred to a 100 mL volumetric flask, dissolved in methanol, and ultrasonicated for 20 min. The solution was filtered through a 0.45 µm Whatman filter paper and diluted to obtain 2 µg/mL of Glimepiride and 5 µg/mL of Linagliptin. Aliquots were applied to HPTLC plates, developed using the optimized mobile phase, and scanned at 259 nm. Quantification was performed using the respective calibration curves.

RESULTS

The developed method demonstrated excellent linearity over the tested concentration ranges of 2000-10000 ng/band for Glimepiride and 5000-250000 ng/band for Linagliptin, with correlation coefficients of 0.9981 and 0.9995, respectively. The Retention factor (R_f) values were found to be 0.655 for Glimepiride and 0.210 for Linagliptin.

The limits of detection were 229.129 ng/band for Glimepiride and 763.76 ng/band for Linagliptin, while the limits of quantification were 95.262 ng/band and 288.67 ng/band, respectively, indicating high sensitivity of the method.

Accuracy studies showed recovery values between 99.30% and 100.30%, whereas precision studies yielded %RSD values below 2%, confirming the reliability of the method. Robustness evaluation revealed that small variations in chromatographic conditions had no significant effect on retention factors, indicating the robustness of the method.

DISCUSSION

Type 2 diabetes mellitus is characterized by increased blood glucose levels and often requires combination therapy for effective glycemic control. Glimepiride and Linagliptin are commonly used for blood sugar regulation. A combined tablet dosage form containing Glimepiride 2 mg and Linagliptin 5 mg has been approved by the Central Drugs Standard Control Organisation (CDSCO). Therefore, ensuring accurate drug content in such formulations is essential, which led to the development of this method (Figure 1).

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Among the different combinations evaluated, the mobile phase consisting of Methanol: Toluene: Ethyl Acetate: Chloroform (2:3:0.5:4.5, v/v/v/v) provided optimal separation (Figure 2). This composition produced well-defined bands with consistent R_f values and minimal tailing, and was selected for further HPTLC analysis (Table 1).

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System suitability was evaluated by performing five replicate analyses at concentrations of 2000 ng/band for Glimepiride and 5000 ng/band for Linagliptin under fixed chromatographic conditions. The method demonstrated good specificity, with no interference observed at the respective R_f values of the analytes, as shown in Figures 2 (A, B). The calibration curves showed strong linear relationships, with correlation coefficients (R²) of 0.9981 for Glimepiride and 0.9995 for Linagliptin, as presented in Figures 3, 4, and Table 2.

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Precision studies, including repeatability, intraday, and interday assessments, showed %RSD values below 2% for all standards. The results of intraday precision are shown in Table 3, while interday precision results are presented in Table 4. Accuracy studies demonstrated recovery values within the range of 98-102%, with %RSD values below 2%, complying with ICH guidelines (Table 5). The method was also capable of detecting and quantifying small amounts of both drugs with high precision (Table 6).

Robustness studies were carried out using Design Expert version 13, and the factorial model for the R_f values of both drugs is presented in Table 7. Model validation using ANOVA is shown in Table 8. The adequate precision values were above 4, indicating a satisfactory signal-to-noise ratio. The p-values obtained were greater than 0.05, indicating that the models were statistically non-significant. This confirms that small deliberate variations in experimental conditions do not significantly affect the method performance.

Perturbation plots were used to evaluate the influence of solvent front (A), wavelength (B), volume of ethyl acetate (C), and chamber saturation time (D) on the R_f values. For Glimepiride, all factors showed minimal influence, with slight downward trends observed for A and B, while C and D remained nearly constant, as shown in Figure 4. For Linagliptin, factors A, C, and D showed slight decreasing trends, whereas factor B remained almost unchanged, as shown in Figure 5. The absence of sharp variations confirmed that these parameters do not significantly affect the chromatographic response.

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Three-dimensional response surface plots further supported these findings. For Glimepiride, interactions between solvent front and wavelength (Figure 6), solvent front and ethyl acetate volume (Figure 7), and solvent front and chamber saturation time (Figure 8) showed nearly flat or slightly varying surfaces. For Linagliptin, a slight decrease in R_f was observed with increasing wavelength (Figure 9), while interactions with ethyl acetate volume (Figure 10) and chamber saturation time (Figure 11) remained nearly uniform. These results indicate that the method is stable and robust, with minimal influence from the studied variables.

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The quantification of the synthetic mixture confirmed the applicability of the developed method, as presented in Table 9.

CONCLUSION

The current novel method is found to be linear (R²>0.998), highly sensitive (Low LOD and LOQ concentrations), highly accurate (recovery is equivalent to 99-100%), highly precise (%RSD <2%), robust (against minor variations). Its successful application to synthetic mixtures further highlights its practical utility, making it an ideal tool for pharmaceutical quality control and regulatory compliance.