Analytical Quality by Design-Based RP-HPLC Method for Simultaneous Estimation of Lobeglitazone sulfate and Glimeperide in Pharmaceutical Formulations

Laxman A. Kawale¹, Mayuri V. Gophane², Surabhi H. Patil³, Vandana S. Nade⁴

¹Assistant Professor (Pharmaceutical Chemistry), MVP Samaj’s College of Pharmacy, Nashik.

²M. Pharm Student (Pharmaceutical Quality Assurance), MVP Samaj’s College of Pharmacy, Nashik.

³Assistant Professor (Pharmaceutics), MVP Samaj’s College of Pharmacy, Nashik.

⁴Assistant Professor (Pharmacology), MVP Samaj’s College of Pharmacy, Nashik.

*Corresponding Author E-mail: kawalela@rediffmail.com, mayurigophane5@gmail.com, shpatil@mvpcpn.edu.in, vsnade@mvpcpn.edu.in

ABSTRACT:

A simple, precise, accurate, and robust High-Performance Liquid Chromatographic (HPLC) method has been developed for simultaneous estimation of Lobeglitazone sulfate and Glimeperide both in bulk and tablet dosage form. Fishbone diagram was used to identify variables that can affect quality of analytical method. Analytical quality by design (AQbD) approach was implemented for method development process. Box-Behnken Design (BBD) was used to optimise chromatographic conditions. Three factors were selected to design the matrix- percent organic ratio, mobile phase pH and flow rate. Analysis of variance (ANOVA) was applied to confirm if selected factors were significant. The analysis was done on Phenomenex C₁₈(250mm, 4.6mm, 5μm) column, using mobile phase as Acetonitrile: Water [% Orthophosphoric acid (OPA)] (80:20% v/v), (pH: 2.5), flow rate of 1ml/min with 243nm detecting wavelength. ICHQ2 (R2) guidelines were followed for method validation. The method was found to be linear in the range of 5-25μg/ml for Lobeglitazone sulfate and 10-50μg/ml for Glimeperide with correlation coefficients of 0.999. Percent recovery for both drugs was found to be in the range of 98-102%. To estimate precision and repeatability Percent Relative Standard Deviation (%RSD) was calculated. It was found to be below 2% confirming that the proposed method showed good accuracy, robustness.

KEYWORDS: RP-HPLC, Analytical Quality by Design, Lobeglitazone sulfate, Glimeperide, Box-Behnken Design.

1. INTRODUCTION:

Type 2 Diabetes Mellitus (T2DM) is a chronic, progressive metabolic disorder, characterised by insulin resistance and β-cell dysfunction. It has long-term effects on the body.

Lobeglitazone sulfate (LOBG) belongs to the thiazolidinedione class of antidiabetic medications and its IUPAC name is (5-4- (2- {[6- (4- Methoxyphenoxy)- 4- pyrimidinylamino} ethoxy) benzyl]-1, 3-thiazolidine-2, 4- dione sulfate). It has molecular weight of 578.6 g/mol. It primarily function as an insulin sensitizer by binding and activating Peroxisome Proliferator-Activated Receptors (PPAR)-gamma within fat cells. PPAR is a transcription factor that plays a role in regulating metabolism. It promotes the binding of insulin to fat cells, lobeglitazone has shown to reduce blood sugar levels, lower haemoglobin A1C levels, and improved lipid and liver profiles^1-3 .

Glimeperide (GLM) is a sulfonylurea antidiabetic agent. Chemically, it is 1-[[p-[2-(3-ethyl-4-methyl-2-oxo-3-pyrroline-1-carboxamido) ethyl] phenyl] sulfonyl]-3-(trans-4-methylcyclohexyl) urea. It has molecular mass of about 490.617g/mol. It is a Second -generation sulfonylurea derivative commonly used in the treatment of non-insulin-dependent Type 2 Diabetes Mellitus. Glimeperide is a second-generation Sulfonylurea agent. The mechanism of action of glimeperide in lowering blood glucose is dependent on stimulating the release of insulin from functioning pancreatic Beta cells and increasing sensitivity of peripheral tissues to insulin. Glimeperide likely binds to ATP-sensitive potassium channel receptors on the pancreatic cell surface, reducing potassium conductance and causing depolarization of the membrane. Membrane depolarization stimulates calcium ion influx through voltage-sensitive calcium channels. This increase in intracellular calcium ion concentration induces secretion of insulin^1-3 .

Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) is a widely used analytical technique in pharmaceutical analysis for separating, identifying, and quantifying compounds in complex mixtures. Its versatility, sensitivity, and ability to handle a wide range of molecules, including polar and non-polar compounds, make it a preferred method in drug development and quality control.

In RP-HPLC, the stationary phase is typically hydrophobic (non-polar), and the mobile phase is relatively polar, leading to the separation of compounds based on their polarity and interactions with the stationary phase^4,5,28-32 . The pharmaceutical industry has increasingly prioritized product quality, safety, and efficacy. Advances in product quality have been achieved through the adoption of scientific tools such as QbD. The principles of Quality by Design in analytical method development is becoming increasingly popular to attain high robustness and improved method performance^6-13. According to ICH, QbD is defined as “a systematic approach to development that starts with predefined objectives and starts focuses on understanding the product and process, along with process control, grounded in sound science and quality risk management.” In a similar vein, AQbD extends these principles to analytical processes, ensuring that analytical methods are thoroughly understood, robust, and suitable for their intended purpose throughout the product lifecycle^14-17 .

The Box-Behnken Design (BBD) was employed to optimise chromatographic settings of HPLC method in this investigation. BBD is a special type of three-level fractionate factorial design, which allows modelling 1st and 2nd order response surfaces. These designs are more cost-effective than three-level full factorial designs, particularly for large number of input factors ^18-19 . The review of literature indicated that several analytical techniques have been used for determination of Lobeglitazone sulfate and Glimeperide separately. Therefore, there was need for the creation of a single approach for simultaneous determination of both the drugs. The prime focus of this research was to provide a robust analytical method using AQbD concept. According to ICH criteria, the proposed approach was optimized and validated^20-26 .

2. EXPERIMENTAL:

2.1 Chemicals and reagents:

The standard API drug LOBG and GLM was obtained from Glenmark Pharmaceuticals, Sinnar, Maharashtra. HPLC grade organic phase Acetonitrile was obtained from Alpha Chemika, Nashik, Maharashtra. Analytical grade Orthophosphoric acid was procured from Molychem, Nashik, Maharashtra. LOBG-1 (LOBG 0.5 mg and GLM 1mg) tablets were acquired from the local pharmacy.

2.2 Instrumentation and Optimized HPLC condition:

Method development and analysis was performed using isocratic separation on HPLC Waters 1525, waters scientific with solvent delivery pump, autosampler injector and a UV/visible detector. Separation was achieved using Phenomenex (C₁₈,250mm,4.6 mml). The solution’s pH adjustment was carried out using pHCal model pH meter. The solvents were degassed using an ultrasonicator. The optimum mobile phase comprised of acetonitrile and water (pH adjusted with orthophosphoric acid) in the proportion of 80:20v/v; flow rate at 1ml/min; injection volume 20µl and detection wavelength at 243nm.

2.3 Software:

The data was collected and processed using the waters- Empower software. Design expert trailed version 13 software was used in experimental design for optimization of various chromatographic parameters as well as to create design space.

2.4 Preparations:

2.4.1 Preparation of standard solution:

Accurately weighed about 5 mg of Lobeglitazone sulfate and 10 mg of Glimepiride and dissolved in 10mL acetonitrile and sonicated for 10 minutes. Standard solution having 500 µg/ml and 1000µg/ml concentrations respectively was obtained.

2.4.2 Preparation of Sample Solution (Test Solution):

For analysis of the marketed formulation 20 tablets (LOBG-GI) having combination of Lobeglitazone and Glimepiride were weighed and crushed to a fine powder. Accurately weighed and dissolved a quantity of sample equivalent to 5 mg Lobeglitazone and 10 mg Glimepiride in 10 ml Acetonitrile. The obtained solution was sonicated and filtered through 0.45 µm membrane filter. From this solution, 0.1 mL was pipette out and diluted to 10mL with Acetonitrile to give 10 µg/ml test solution of Lobeglitazone and 20 µg/ml of test solution of Glimepiride.

2.4.2 System suitability:

The standard solutions comprising LOBG 15 µg/ml and GLM 30 µg/ml were injected in six replicate injections to determine the system appropriateness parameters. All %RSD were calculated for resolution, retention time (Rt), and theoretical plates.

2.5 Method Validation

The developed method for estimation of Lobeglitazone sulfate and Glimeperide was validated as per ICH Q2(R2) guidelines. Different parameters assessed were accuracy, precision, linearity, limit of detection (LOD), limit of quantification (LOQ), specificity, and robustness¹⁶.

3. RESULT AND DISCUSSION:

3.1 Method development as per experimental design.

The important chromatographic factors were selected, based on preliminary trials, prior knowledge from the literature, and by using the Ishikawa diagram. The factors were selected for method development, including % organic ratio in mobile phase, mobile phase pH, and flow rate. BBD was employed to assess the effects of three independent variable on the three defined key response or dependent variable. A 2³ factorial design indicated that there were two levels and three factors were involved. The two levels were low (-1) and high (+1), whereas factors were (X1) % organic phase used in mobile phase (70% and 90%), (X2) flow rate of mobile phase (0.8 and 1.2 mL/min), and (X3) pH of mobile phase (2 and 3). The retention time of LOBG (Y1) and retention time of GLM (Y2), employed as responses in experimental design. 17 experiments were constructed in Box -Behnken design using the conditions and observed responses given in (Table 1)^{18,25-27,33,37,38} .

3.2 Risk Assessment:

Conducted risk assessment study to identify potential sources of variability and risks associated with the method. Different risk management tools given in ICH Q9 guideline. In this work Ishikawa diagram was used for risk assessment given in (Fig 1) ¹⁵ .

Figure.1 Ishikawa fishbone diagram

Table 1 Layout of 17 experimental trials by BBD

Std	Run	Factor 1 A= Mobile phase (% ACN) (X1)	Factor 2 B= pH of Mobile phase (X2)	Factor 3 C= Flow rate (mL/min) (X3)	Response 1 Retention time of LOBG(Y1)	Response 2 Retention time of GLM(Y2)
1	5	70	2	1	3.082	4.545
2	1	90	2	1	3.532	4.377
3	6	70	3	1	4.515	5.879
4	13	90	3	1	3.254	3.517
5	15	70	2.5	0.8	5.115	6.815
6	2	90	2.5	0.8	4.153	4.668
7	8	70	2.5	1.2	3.415	4.55
8	4	90	2.5	1.2	3.42	4.539
9	17	80	2	0.8	3.845	5.671
10	9	80	3	0.8	4.89	6.018
11	12	80	2	1.2	2.574	3.797
12	14	80	3	1.2	2.794	3.082
13	7	80	2.5	1	3.581	4.218
14	10	80	2.5	1	3.598	4.251
15	3	80	2.5	1	3.592	4.281
16	16	80	2.5	1	3.595	4.286
17	11	80	2.5	1	3.587	4.431

Table 2 ANOVA table for retention time of Lobeglitazone Sulfate

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	7.10	9	0.7894	48.72	<0.0001	Significant
A-Mobile Phase (%ACN)	0.3907	1	0.3907	24.12	0.0017
B-pH of Mobile phase	0.7321	1	0.7321	45.18	0.0003
C-Flow rate	4.21	1	4.21	259.53	<0.0001

Analysis of variance (ANOVA) for the retention time of Lobeglitazone Sulfate response 1:

The Analysis of Variance (ANOVA) was performed to identify the significant and insignificant factors. ANOVA results help identify which factors have a statistically significant impact on the response variable(s). Factors with significant effects are considered critical and need to be controlled or optimized for method robustness. The term significant represent that selected factor having critical impact on responses.it depends on p value, if p value is less than 0.05 then considered as a model is significant. The results of ANOVA for the response 1 are as presented in table 2:

The Model F-value of 48.72 implies the model is significant. There is only a 0.01% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant.

Figure no.2 of Contour plots and 3D surface plots shows that as concentration (conc.) of organic phase i.e. % ACN and Flow rate increases, RT of Lobeglitazone sulfate decreases and as Mobile phase pH increases, RT of Lobeglitazone sulfate increases.

Response Surface plots for LOBG:

The Model F-value of 12.64 implies the model is significant. There is only a 0.15% chance that an F-value this large could occur due to noise. P-values less than 0.0500 indicate model terms are significant.

Figure 2. Contour plot (a-c), and 3-D response plot (d-f) for retention time (RT) of Lobeglitazone sulfate.

Table 3 ANOVA table for retention time of Glimeperide

Source	Sum of Squares	df	Mean Square	F-value	p-value
Model	13.63	9	1.51	12.64	<0.0001	significant
A-Mobile Phase (%ACN)	2.75	1	2.75	22.92	0.0017
B-pH of Mobile phase	0.0014	1	0.0014	0.0117	0.0003
C-Flow rate	6.49	1	6.49	54.12	<0.0001

Figure 3. contour plot (g-i), and 3-D response plot (j-l) for retention time (RT) of Glimeperide.

Response surface plot for GLM

Figure no.3 of Contour plots and 3D surface plots shows that as conc. of organic phase i.e. % ACN and Flow rate increases, RT of Glimepiride decreases and as Mobile phase pH increases, RT of Glimeperide increases.

Method Operable Design Region Design Space:

The Graphical optimization done with the help of Design Expert software provided design space as shown in following Figure 4. A design space plot is a graphical representation of the region in which we get optimal conditions or certain criteria which imputed in goal.

Figure 4 Method Operable Design Region Design Space

Optimized Method:

Figure 5 shows Optimized Method with chromatogram

Figure.5 Chromatogram of Optimized Condition

Figure 6 overlay Chromatogram of linearity

The Mobile phase composition such as acetonitrile as to (OPA) Water 80:20, flow rate (1 mL/min), and pH 2.5 which resulted in retention time of LOBG (Y1) 3.592, retention time of GLM (Y2) 4.281.

3.3. System suitability:

The outcomes of the system suitability test are represented in Table 4. The % RSD obtained for all parameters was less than 2.

Table 4. System suitability results for the developed HPLC method.

Parameters	Average ±SD	%RSD
Retention time (RT1)	0.023	0.639
Retention time (RT2)	0.044	1.044
Tailing Factor of LOBG	0.0014	0.898
Tailing Factor of GLM	0.004	0.349
Plate count of LOBG	52.65	0.127
Plate count of GLM	60.49	1.0

The system suitability parameters were computed, and the values were obtained within acceptable limits.

3.4. Method validation:

3.4.1. Linearity, LOD, and LOQ:

A significant linear relationship was obtained among the concentration and peak areas of LOBG and GLM over the ranges 5-25 μg/ml and 10-30 μg/ml, respectively, under optimized chromatographic conditions with the correlation coefficient of R2 = 0.9991 for LOBG and R2 = 0.999 for GLM. The analytical outcomes for linearity with slope and intercept are depicted in Table 4. The values of LOD and LOQ were computed using the Standard Deviation (SD) of response and regression line slope, which indicates the high sensitivity of the proposed method. The values for LOD and LOQ were obtained to be 0.0266, 0.0806 for LOBG and 0.04094,0.124 for GLM, respectively.

Figure 6 shows overlay chromatogram for Linearity.

Table 5. The results of Linearity parameters for LOBG and GLM.

Parameters	LOBG	GLM
Concentration (µg/ml)	5-25 µg/ml	10-30 µg/ml
Regression equation	249796x -1756.9	379014x + 1179.7
Correlation coefficient (R^2)	0.9991	0.999
LOD (µg/ml)	0.0266	0.04094
LOQ (µg/ml)	0.0806	0.124

Table 6. Precision results for LOBG and GLM.

LOBG					GL M
Conc.	Intraday		Interday		Conc.	Intraday		Interday
	SD	%RSD	SD	%RSD		SD	%RSD	SD	%RSD
10	0.044	1.23	0.14	0	10	0.0081	0.221	0.0169	0
15	0.024	0.669	0.047	1.1	15	0.0081	0.223	0.0047	0.1
20	0.0081	0.224	0.038	0.9	20	0.0081	0.224	0.0163	0.38

3.4.2. Precision and accuracy:

All the precision studies were conducted, and their outcomes are described in Table 6. The percentage RSD was not more than 2 %, indicating reliable precision. The accuracy results of the developed method show a good range of percent recovery, representing good accuracy for the recommended method. The outcomes of accuracy are depicted in Table 7.

Table 7. Accuracy results for LOBG and GLM.

Level of addition	%mean recovery	SD	%RSD
LOBG   80%	99.55	1.919	1.92
100%	99.96	0.733	0.73
120%	99.94	0.68	0.68
GLM     80%	99.97	0.227	0.227
100%	99.996	0.37	0.37
120%	99.993	0.291	0.291

4. CONCLUSION:

An extensive literature survey has revealed lack of Reverse Phase High Performance Liquid Chromatographic (RP HPLC) method available to simultaneously estimate of Lobeglitazone sulfate and Glimeperide in pharmaceutical dosage tablet forms with use an experimental design approach. The current work involves the systematic development of simple, precise, rapid, accurate and robust RP-HPLC method for simultaneous estimation of Lobeglitazone sulfate and Glimeperide. The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) emphasizes the importance of the QbD approach in pharmaceutical development, including HPLC method development. QbD principles provide a systematic framework for designing, developing, and validating analytical methods to ensure their robustness, accuracy, and reliability. QbD approach was utilized to build an efficient and robust RP-HPLC technique for simultaneous determination of Lobeglitazone sulfate and Glimeperide. An Ishikawa diagram, also known as a Fishbone or Cause-and-Effect diagram, is a visual tool used to identify and analyse potential causes of a problem or to understand the factors influencing a particular outcome. Box-Behnken Design (BBD) is a statistical experimental design technique used to systematically explore the effects of multiple factors on a response variable. BBD is a powerful approach to optimize method parameters and achieve robust and efficient separation. Using BBD, the chromatographic conditions were optimized by examining the interactions and quadratic effects of important factors on the chosen responses. The optimized conditions are Acetonitrile as to Water (% OPA) in ratio of 80:20, flow rate 1 ml/min and pH 2.5 shows the suitability for estimation of Lobeglitazone sulfate and Glimeperide. The method chosen was validated using the (ICH) International Conference on Harmonization Q2R (1). Linearity for Lobeglitazone sulfate and Glimeperide was found within range of 5 μg/ml-25 μg/ml and 10 μg/ml-30 μg/ml, respectively.  Percentage % recovery was found within range of 98.5-101.3% and 99.82-100.37% for Lobeglitazone sulfate and Glimeperide. RSD was found not more than 2 that indicate satisfactory precision of method. This newly developed HPLC method represents a significant advancement in analytical sciences.

5. ACKNOWLEDGEMENT:

The authors are grateful to MVP’S College of Pharmacy, Nashik for motivating us to carry out this research.

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GRAPHICAL ABSTRACT:

Received on 16.07.2025      Revised on 10.10.2025

Accepted on 08.12.2025 Published on 27.01.2026

Available online from February 02, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(1):1-8.

DOI: 10.52711/2231-5675.2026.00001

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