INTRODUCTION:

The metabolic condition known as type 2 diabetes mellitus (T2DM) is characterized by insulin resistance and malfunction of β-cells, and it is a chronic and progressive disease¹. The complex and multi-faceted pathophysiology of type 2 diabetes has led to the development of multiple oral antidiabetic drugs (OADs), each of which targets a different mechanism. One group of OADs that has been developded so far is the thiazolidinedione (TZD) family, which mainly targets insulin resistance². Insulin sensitivity is enhanced by thiazolidinediones because they promote the activation of peroxisome proliferator-activated receptor γ (PPARγ)³. A recently developed TZD, lobeglitazone sulfate (LGZ) aims to meet the requirement for a safe and efficient TZD. The chemical name for it is 5-[4-(2-{[6-(4-Methoxy-phenoxy)-pyrimidin-4-yl]-methyl-amino}-ethoxy)-benzyl]-thiazolidine-2,4-dione hydrosulfuric acid^4,5.

Multiple studies have demonstrated that lobeglitazone is a new type of TZD that is highly effective and has a positive safety profile. Compared to pioglitazone, lobeglitazone has similar effectiveness in controlling blood sugar levels but requires a lower dose because it has a stronger bond with PPARγ. Since lobeglitazone is primarily broken down by the liver and is excreted minimally through the kidneys, it is expected to be safe for use in patients with kidney problems without needing a dosage adjustment. Additionally, there is a possibility that lobeglitazone has a lower risk of causing bladder cancer compared to other TZDs. Clinical trials conducted so far have also reported positive findings regarding the safety of LGZ⁴. The drug's molecular structure is shown in Figure 1.

Figure 1. Chemical structure of LGZ.

The LOBG® tablet, containing 0.5 mg of LGZ, was introduced in the Indian market by Glenmark Pharmaceuticals Limited (Mumbai, India) in late 2022 for the management of type 2 diabetes in adults. Because it increases the reactivity of human cells to insulin, lobeglitazone acts as an antidiabetic drug. Lobeglitazone was approved for the management of type 2 diabetes in Korea in July 2013, after being created in Seoul, Korea, by Chong Kun Dang Pharmaceutical Corporation. After a randomized, double-blind Phase 3 clinical trial with adults aged 18 and above who had type 2 diabetes, Glenmark was given the green light to produce and sell lobeglitazone by the Drug Controller General of India, an Indian drug regulatory body. The study claims that lobeglitazone displayed better and faster glycemic control in the trials^6,7. To measure the amounts of LGZ in biological samples or medication formulations, several analytical methods have been described in the literature, such as HPLC⁸ and LC-MS^9,10. HPTLC methods were not available for the quantification of LGZ in bulk drugs or pharmaceutical formulations. An HPTLC methodology for the analysis of LGZ in tablet formulations was therefore developed and validated in this work. The objective of this study is to create a unique HPTLC method for evaluating the tablet formulation of the innovative medicine LGZ that is easy to use, quick to respond, accurate, and exact.

The validation process adhered to the techniques outlined in the ICH Q2(R1) standards¹¹ to verify the consistency of the recently created HPTLC approach. The HPLC and LC-MS/MS techniques have specific sample preparation steps and are intricate, expensive, and time-consuming. On the other hand, HPTLC has a number of benefits, such as reducing analysis time and costs by analyzing more samples concurrently with a smaller mobile phase. HPTLC permits immediate qualitative evaluations through visual examination of separated chemical bands, in contrast to LC-MS, which depends on detectors for detection and quantification. Unlike the HPLC and LC-MS/MS procedures, the HPTLC method does not require any specific sample preparation due to its simplicity. The use of potentially harmful organic solvents in developing new methods is also decreased^12,13. In addition, a table (Table 4) has been supplied for comparison, showing how the method described in this study was compared to previously reported methods.

MATERIALS AND METHODS:

Chemicals and Reagents:

Dalton Pharma Chem of Vadodara, Gujarat, India, kindly provided us with a free sample of LGZ (98.5% w/w) to use as a reference standard during our inquiry. The marketed formulation was procured from a local pharmacy in Mumbai, Maharashtra, India, and was manufactured by Glenmark Pharmaceuticals Ltd. The solvents and reagents, including acetonitrile, ammonium acetate, toluene, and triethylamine, were supplied by Loba Chemie Pvt. Ltd., Mumbai, Maharashtra, India.

Chromatography and analytical conditions:

The TLC plates covered with silica gel 60F254 on an aluminum sheet of 10 cm × 10 cm × 0.2 mm manufactured by Merck KGaA, Darmstadt, Germany, were used for the densitometric analysis. The sample was applied using a CAMAG Linomat 5 automated sampler manufactured by CAMAG in Muttenz, Switzerland, together with a 100 μL Hamilton micro-syringe. A 10 × 10 cm CAMAG glass twin trough was used as the development chamber. By utilizing the absorbance mode at a wavelength of 248 nm, densitometric scanning was carried out using the CAMAG TLC Scanner 4. The detection wavelength of LGZ and the overlapping spectra are displayed in Figure 2. In order to evaluate the results, the system was linked to the Server HPTLC software, version 3.0.20196.1, developed by CAMAG of Muttenz, Switzerland. The study was carried out in a controlled environment with a relative humidity of 40% and a temperature of 25±2°C. A deuterium lamp (filter: K320) was used to generate radiation. The Linomat 5sample applicator was used to constantly apply 150 nL/s of test and reference solutions to the TLC plate. The bands, each 6 mm in length, were spaced 10 mm apart on the TLC plate, with 15 mm between each edge and 10 mm from the bottom edge. The TLC plates were left to air dry for 5 minutes after spotting before being moved to the development chamber. A mixture of toluene: ammonium acetate (1 M in methanol): acetonitrile and triethyl amine 4:2.5:1.5:0.2 v/v/v/v was used as the solvent system. For 20 minutes at room temperature, with a development distance of 80 mm, the TLC plates were developed in a linear-ascending fashion in a pre-saturated developing chamber. After the plates were allowed to air dry, densitometric scanning was performed at a speed of 100 mm/s using a slit measurement of 6 × 0.45 mm. A planar chromatography program called Server HPTLC, version 3.0.20196.1, developed by CAMAG of Muttenz, Switzerland, was used to analyze the TLC densitogram. Subsequently, calculations were performed using an Excel sheet from Microsoft Corporation, USA. The weighing process was carried out using an Adventurer Pro AVG264C electronic balance (Ohaus Corporation, Pine Brook, NJ, USA).

Figure 2. Overlain spectra of LGZ showing detection wavelength of 248 nm.

Preparation of Solutions:

Preparation of Standard Solution:

To prepare the reference stock solution, 1 mg of LGZ was measured and placed in a 10 mL volumetric flask. Methanol was added to bring the volume to 10 mL, creating a standard stock solution with a concentration of 100 μg/mL of LGZ.

Preparation of Sample Solution:

To prepare the sample solution, 1 mg of the powdered tablet formulation (LOBG® 0.5 tablet, Glenmark Pharmaceuticals Ltd., India) was measured and added to 10m of methanol in a volumetric bottle. To produce the volume 10 mL, sonicating the liquid for 10 minutes was followed by the addition of methanol. Whatman filter paper no. 41 was then used to filter the solution. In order to estimate LGZ, filtered liquid with a concentration of 100 µg/mL was spotted directly.

Validation of Chromatographic Method:

By consulting the ICH recommendations and other published methods, the established methodology was found to be legitimate^11,14-48.

Linearity and Range:

To test the method's linearity, several volumes of the working standard (LGZ: 100, 250, 500, 1000, 1500 ng/band) were applied to TLC plates using Linomat 5, with volumes ranging from 1 to 15 µL. A total of six replicates were examined. Following plate development, chromatograms were obtained, and a calibration curve was constructed by plotting the mean peak area versus concentration (ng/band). Regression analysis was performed using the least squares method to determine the intercept, slope, and correlation coefficient.

Precision:

Three distinct metrics were used to evaluate precision: repeatability, intra-day precision, and inter-day precision. Six separate applications of the test solution, each with a concentration of 500 ng/band for LGZ, were performed on a pre-coated TLC plate to evaluate repeatability. A pre-coated TLC plate was used with test solutions at three distinct concentration levels (250, 500, and 1000 ng/band for LGZ) to ensure intra and inter-day precision. Experiments were conducted over the period of three days to assess inter-day precision, whereas samples were checked on the same day to assess intra-day precision. The analysis was conducted in triplicate, and the results were expressed as the percentage relative standard deviation of the analyte peak area, demonstrating precision.

Accuracy:

The established approach was tested for practicality and reliability by recovery analysis utilizing the customary standard addition procedure. Reference LGZ at 80,100, and 120 percent concentrations were added to pre-analyzed test solutions with concentrations of 100, 200, and 500 ng/band for LGZ. We estimated the % recoveries after re-analyzing the solutions using the projected methodology. By calculating the fraction of reference LGZ recovered from the formulation using the following formula, we were able to assess the accuracy of the predicted approach.

(Quantity of anlyte found after adding standard drug

- Quantity of anlyte found before addition of standard drug)

% Recovery = ------------------------------------- × 100

Quantity of standard analyte added

LOD and LOQ:

The sensitivity of the suggested technique was evaluated by calculating the LOD and LOQ for LGZ using the equation indicated below, in accordance with ICH strategies.

LOD = 3.3 x 𝜎/ S

LOQ = 10 x 𝜎/ S

Where 𝜎 = The SD of the response, S = The slope of the linear graph.

Robustness:

The ability of an analytical method to withstand deliberate and small changes to its parameters is what we mean when we talk about its robustness. When the critical values of a process are well within the acceptable range, it can be said that it is suitable for routine laboratory use. Modifications were done to parameters including toluene content volume (4±0.5 mL), development distance (8±0.5cm), chamber saturation period (20±5 min), wavelength (248±2 nm), and temperature (25±2°C) in order to assess the approach's robustness. To evaluate robustness, the effect of these variations on Rf values and peak areas was determined.

Specificity:

The specificity of the intended method was evaluated by analyzing the peak purity of the drug's densitogram. The peak purity of LGZ was assessed by comparing the spectra produced from the chromatogram at different wavelengths of the test and standard analyte at the beginning, middle, and end of the peak.

Solution Stability:

The stability of the solution was assessed by comparing it with newly generated solutions and examining any changes in the Rf value, peak area, tailing factor, and UV spectral pattern. The efficacy of the solutions was regularly assessed throughout storage at ambient temperature (25±2°C) and under refrigerated circumstances (6°C).

System suitability tests (SST):

A pre-coated TLC plate was used to apply the LGZ solution (1000 ng/band) in order to undertake the system suitability investigation. To show that the system as a whole performed satisfactorily, we computed test parameters such tailing factor, peak area, and retardation factor (Rf).

Analysis of sample solution (tablet formulation):

The steps for making the test solution were covered in the section on materials and procedures earlier. To estimate LGZ on the TLC plate, the sample solution (LGZ: 100 µg/mL) was applied six times, with 5 µL of the solution used each time. After that, the TLC plate was developed under the ideal chromatographic circumstances. The TLC plate was scanned after development, and the peak area was measured. The drug concentrations were estimated using regression equations based on the measured peak area.

Comparison of proposed method with reported methods:

In order to prove that the proposed approach is unique, we compared them to previously published research papers^[8-10] in terms of technique used, range, sensitivity, specificity, system precision, and applicability (Table 7).

RESULTS AND DISCUSSION:

Method development:

When it comes to quantifying drug substances and pharmaceutical preparations, including those derived from nature, HPTLC is the way to go because of its adaptability in method development, ease of sample preparation, affordability, and capacity to examine multiple analytes simultaneously and rapidly. A technique for quantitatively determining the tablet formulation of the anti-diabetic medication LGZ was developed in this work by utilizing thin layer chromatography. A band application was employed to introduce both the sample and reference solutions. The slit dimension was maintained at 6.0 × 0.45 mm, and optimal chamber saturation was achieved after 20 minutes. The wavelength of 248 nm was selected to ensure an appropriate balance between absorbance and sensitivity. The mobile phase composition that produced the best chromatographic result and the most consistent and acceptable Rf value was determined after extensive experimentation with many solvent combinations. More than that, we tried various solvent combinations including triethyl amine, chloroform, methanol, acetonitrile, ammonium acetate, toluene, ethyl acetate, and glacial acetic acid. Lastly, the optimal mobile phase showed a clear, symmetrical peak for the analyte and consisted of toluene: ammonium acetate (1 M in methanol): acetonitrile and triethyl amine 4:2.5:1.5:0.2 v/v/v/v. The exemplary densitogram of LGZ, with an Rf value of 0.721 ± 0.009, is shown in Figure 3. A pre-coated TLC plate of 10 cm × 10 cm × 0.2 mm was used to administer LGZ reference and sample solutions. The bands, each 6 mm long, were spaced 10 mm apart, situated 15 mm from either side, and 10 mm from the plate's lowest boundary.

Figure 3. Standard densitogram of LGZ (1000 ng/band).

Method Validation:

In line with ICH rules, the validity of the proposed method was evaluated. The outcomes for different validation parameters are covered in the section that follows.

Linearity and Range:

The HPTLC method that was developed showed linearity for LGZ in the range of 100-1500 ng/band. The analyte concentrations and the matching peak areas from the LGZ densitograms at each level were plotted to construct the calibration graphs. The linearity of the approach was confirmed by the good linear correlation (r2) of 0.9991 for LGZ in the calibration curve. Figure 4 displays the three-dimensional linearity densitograms, while Table 1 contains the data from the linear regression.

Table 1. A concise overview of the data acquired through linear regression, along with the validation of the proposed methodology.

Parameters	LGZ
Detection Wavelength (nm)	248
Linearity range (ng/band)	100-1500
Correlation coefficient	0.9991
Regression equation	y = 0.0896x + 4.9231
LOD (ng/band)	17.31
LOQ (ng/band)	52.46
Specificity	No interferences

n = 6 (No. of determinations).

Precision:

In accordance with the ICH suggested limitations (˂2), the results of the precision experiments were reported as %RSD. Table 2 shows that the results of suggested procedure had very little variation both within and between days, and it was very repeatable.

Accuracy:

Using standard addition methodology to determine the analyte retrieval, the accuracy of the predicted approach was evaluated. According to Table 3, the suggested methodology was accurate for LGZ, since the recovery rates of the repeated inquiry ranged from 96% to 104%.

LOD and LOQ:

To calculate LOD and LOQ, we used the formula given The limits of detection (LOD) and quantification (LOQ) were calculated using the formula specified in the ICH Q2(R1) guideline. The LOD and LOQ for LGZ were found to be 17.31 ng/band and 52.46 ng/band, respectively. As presented in Table 1, the obtained values indicate the high sensitivity of the proposed method.

Figure 4. Three-dimensional overlain densitogram of LGZ (100-1500 ng/band).

Table 2. Outcome of precision study by the proposed high-performance thin-layer chromatographic methodology.

Concentration (ng/band)	Peak area ±SD	% RSD
Repeatability (n=6)*
500	51.56 ± 0.24	0.46
Intra-day precision (n=3)*
250	29.02 ±0.25	0.85
500	51.15 ± 0.35	0.68
1000	94.64 ± 0.77	0.81
Inter-day precision (n=3)*
250	29.20 ± 0.39	1.33
500	52.43 ± 0.89	1.70
1000	95.69 ± 1.28	1.34

*n=Number of estimations.

Robustness:

Minor and intentional modifications to the chromatographic conditions that may occur during regular analysis did not significantly alter the peak area and Rf value of LGZ, suggesting that the technique remained unchanged. The findings confirm the robustness of the method, with the corresponding data presented in Table 4.

Table 4. Outcomes of Robustness study.

Modification	LGZ
Modification	R_f value*	Peak area*
Toluene content (4 ± 0.5 mL)	0.711 ± 0.010	51.07 ± 0.68
% RSD	1.41	1.34
Development distance (8 ± 0.5 cm)	0.714 ± 0.010	50.89 ± 0.59
% RSD	1.44	1.15
Chamber saturation time (20 ± 5 min)	0.713 ± 0.005	51.11 ± 0.56
% RSD	0.66	1.10
Wavelength (248 ± 2 nm)	0.706 ± 0.005	51.19 ± 0.30
% RSD	0.65	0.59
Temperature (25 ± 2°C)	0.717± 0.005	50.90 ± 0.33
% RSD	0.66	0.64

*mean ± SD, (n=3) number of determinations.

Specificity:

We compared the spectra of the test and standard samples taken at the beginning, peak, and end of the spots to determine the peak purity of the LGZ. To confirm the specificity of the suggested method, Figure 5 displays a peak purity spectrum comparing the test and standard analytes. The spectra obtained at the beginning (s), peak (m), and end (e) of every peak showed a high connection. This examination confirms the peaks are pure, since it shows that neither the drug's standard solution nor the sample solution had any impurities or excipients that would have altered them.

Figure 5. Comparison of test and standard analytes using peak purity spectra to affirm the specificity of the proposed approach.

Stability of the Solution:

The solution exhibited no significant change in its Rf value, tailing factor, or UV spectral pattern during two days at ambient temperature (25 ± 2°C) and ten days while kept in the refrigerator (6°C), as indicated by the results of the stability test. After 10 days in the fridge and two days at ambient temperature, the peak area did, however, diminish.

System suitability tests:

Calculations of SST parameters, including retardation factor (Rf), selectivity, and tailing factor, were done to verify that the whole system performed satisfactorily during the trial. According to the ICH criteria, the values that were collected and displayed in Table 5 fall within the permitted ranges. This proves the system worked as expected during the experiment.

Table 5. SST parameters of the suggested HPTLC method.

Parameters	LGZ (Mean ± SD)	% RSD	Acceptance Criteria
Reproducibility of R_fvalue	0.752 ± 0.005	0.67	%RSD <2
Reproducibility of Peak area	94.57 ± 0.43	0.72	%RSD <2
Tailing factor	0.955	-	<2

Determination of LGZ present in tablet:

Results ranging from 98 to 101% were obtained after the defined approach for the investigation of LGZ was carried out. Six further determinations verified this degree of precision, yielding a dataset that could be trusted statistically. Therefore, as shown in Table 6, the outlined methodology may be used with confidence to evaluate LGZ in tablets.

Table 6. Outcomes of tablet formulation investigation employing approach.

Drug	Labeled amount in tablet (mg)	Amount Found (mg)	Labeled amount Found (%)*	RSD (%)
LGZ	0.5	0.497 ± 0.006	99.37 ± 1.26	1.26

*Mean ± SD (n = 6).

Comparison of proposed method with reported methods:

The range, sensitivity, specificity, system precision, and applicability of the proposed approach were compared to those of previously published research articles (Table 7). According to the comparative results, the suggested strategy is very comparable in terms of a number of outcomes. Table 7 shows that compared to the reported methods, the proposed one stands out due to its sensitivity, system precision, and relative ease of use. The presented approach has been validated according to ICH standards and offers several benefits, such as being easy to use, having a wide concentration range, high sensitivity, and a low reference and sample preparation barrier. To evaluate LGZ in pharmaceutical formulations, the suggested technique was determined to be competitive with the described methods and able to compensate for their deficiencies; so, it can be used in conjunction with the reported methods or as a replacement for them.

CONCLUSION:

In some cases, HPTLC is favored over HPLC because of the many advantages it offers over other analytical processes. Due to its reduced solvent usage and capacity to perform speedy and simultaneous analysis of several samples, it is believed to be more cost-effective and environmentally friendly. The novel anti-diabetic medicine LGZ was evaluated in tablet form using a simple, sensitive, accurate, and robust HPTLC method that we devised in this work. The specificity, sensitivity, linearity, accuracy, precision, and robustness standards set out by the ICH were satisfied by the suggested method. Additional evidence of the approach's great responsiveness was the noticeably decreased LOD and LOQ values. In addition, the results of the recovery process were highly consistent with what was stated on the label. All things considered, the analytical performance of the suggested approach was satisfactory, proving that it is a good fit for the quality control department's routine examination of LGZ in bulk medicine and pharmaceutical formulations.

LIST OF ABBREVIATIONS:

CDSCO: central drug standard control organization; ICH: International Conference on Harmonization; HPTLC: high-performance thin layer chromatography; LGZ: lobeglitazone sulfate; OADs: oral antidiabetic agents; PPAR: peroxisome proliferator-activated receptor γ (PPARγ); SST: system suitability tests; T2DM: type 2 diabetes mellitus; TZD: thiazolidinedione.

CONFLICT OF INTEREST:

The authors declare no financial or any other competing interests related to this work.

ACKNOWLEDGEMENTS:

The authors express their sincere gratitude to Sumandeep Vidyapeeth Deemed to be University, situated in Piparia, Waghodia, Vadodara-391760, Gujarat, India, for providing the necessary resources to conduct this research.

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Received on 03.08.2024 Revised on 07.12.2024

Accepted on 04.03.2025 Published on 01.10.2025

Available online from October 04, 2025

Research J. Pharmacy and Technology. 2025;18(10):4695-4703.

DOI: 10.52711/0974-360X.2025.00675

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Methods compared	Drug	Name of Method	Range	Sensitivity	Specificity	System suitability	Application
Proposed method	LGZ	HPTLC	100-1500 ng/band	LOD: 17.31 ng/band LOQ: 52.46 ng/band	Specific	Performed	Marketed formulation
Gulhane et al., 2023 ^[8]	LGZ	HPLC	3.12-50 µg/mL	LOD: 1.05 µg/mL LOQ: 3.04 µg/mL	Specific	Performed	Marketed formulation, pharmacokinetic study
Kim et al., 2012 ^[9]	LGZ	LC-MS/MS	Plasma: 0.5-1000 ng/mL	LLOQ: 0.5 ng/mL	Not mentioned	Not mentioned	Pharmacokinetic study
Kim et al., 2012 ^[9]	LGZ	LC-MS/MS	Urine: 0.2-250 ng/mL	LLOQ: 0.2 ng/mL	Not mentioned	Not mentioned	Pharmacokinetic study
Lee et al., 2009 ^[10]	LGZ	LC-MS/MS	Rat plasma: 50-10000 ng/mL	LLOQ: 50 ng/mL	Specific	Not mentioned	Pharmacokinetic study

Drug	Recovery Level (%)	Conc. of pre-analyzed sample solution (ng/spot)	Amount of standard drug spiked	Recovery (%)*	RSD (%)
LGZ	80	100	80	99.09 ± 1.66	1.67
	80	200	160	99.32 ± 1.40	1.40
	80	500	400	101.92 ±1.51	1.48
	100	100	100	97.74 ± 1.31	1.34
	100	200	200	100.07 ± 1.90	1.89
	100	500	500	98.15 ± 0.77	0.79
	120	100	120	99.37 ± 1.55	1.56
	120	200	240	98.03 ± 1.48	1.51
	120	500	600	97.82 ± 1.40	1.43