INTRODUCTION:

Teneligliptin is an anti-diabetic drug categorised as a dipeptidyl peptidase-4 (DPP-4) inhibitor¹, also termed a "gliptin." Its chemical name is 3-(4-(4-(3-methyl-1-phenyl-1H-pyrazol-5-yl) piperazin-1-yl) pyrrolidin-2-ylcarbonyl) thiazolidine (figure 1). Blood sugar levels are lowered, which helps to treat type 2 diabetes mellitus. To do this, the enzyme DPP-4 is inhibited, which stops GLP-1 from breaking down and raises the blood level of active GLP-1.

Figure 1. Structure of Teneligliptin

Pioglitazone, chemically named (±)-5-((4-(2-(5-ethyl-2-pyridinyl) ethoxy) phenyl) methyl)-2,4-thiazolidinedione (figure 2), is a thiazolidinedione that functions by activating peroxisome proliferator-activated receptors (PPARs) in the nuclei of adipocyte cells. This activation influences gene transcription, enhancing tissue sensitivity to insulin and promoting glucose uptake in muscle and fat tissues. Additionally, pioglitazone reduces glucose production in the liver and increases hepatic glucose uptake¹.

Figure 2. Structure of Pioglitazone

Metformin HCl, chemically identified as 3-(diaminomethylidene)-1,1-dimethylguanidine hydrochloride (Figure 3), primarily targets mitochondria by inhibiting the respiratory chain, which raises AMP levels. In doing so, AMP-activated protein kinase (AMPK), an essential cellular energy sensor that controls food intake, is activated⁽¹⁾.

Figure 3. Structure of Metformin HCl

There are no documented methods for the simultaneous analysis of this particular medication combination, according to a review of the literature and a patent search. While spectrophotometric and chromatographic techniques are available for individual drugs or their combinations with other medications, none address this combination. As a result, an easy-to-use, accurate, and stable stability-indicating RP-HPLC method is required for the simultaneous measurement of these medications in their combination dose form. This led to the creation and verification of a novel technique that was thought to be highly intriguing.

MATERIALS AND METHODS:

Materials:

The combination of Teneligliptin, Pioglitazone, and Metformin HCl (Zita-PioMet), along with the pure active pharmaceutical ingredients (APIs) of Teneligliptin, Pioglitazone, and Metformin HCl. The following materials were used in the study: ortho-phosphoric acid, methanol, acetonitrile, phosphate buffer, and potassium dihydrogen phosphate buffer. Rankem provided all of the solvents and chemicals.

Instruments:

The equipment used in this study included an ultrasonicator, a Dionex HPLC system with a Force scientific C18 column, a Shimadzu SPD-20A VP UV-VIS detector, and an autosampler integrated with Chromeleon software. A Jasco spectrophotometer with specified bandwidths along with matched quartz cells equipped with a UV probe, were used to measure the absorbance of teneligliptin, pioglitazone, and metformin HCl.

Methods:

Preparation of primary stock solutions:

Metformin HCl:

· Primary stock-1 solution (1000ppm) was prepared by dissolving 100mg of Metformin HCl in methanol, sonicated for 5 minutes, and diluted to 100ml. Stock-2 solution (500ppm) was then diluted accordingly.

Teneligliptin:

· Primary stock-1 solution (1000ppm) was prepared by dissolving 100mg of Teneligliptin in methanol, sonicated for 5 minutes, and diluted to 100ml. Stock-2 solution (50ppm) was then diluted accordingly.

Pioglitazone:

· Primary stock-1 solution (1000ppm) was prepared by dissolving 100mg of Pioglitazone in methanol, sonicated for 5 minutes, and diluted to 100ml. Stock-2 solution (15ppm) was then prepared by further dilution.

Preparation of Final Solution:

A 10ml volumetric flask was used to combine 1ml each of Metformin HCl standard stock-2 solution, Teneligliptin stock-2 solution, and Pioglitazone stock-2 solution. The volume was adjusted with the mobile phase used for trials to prepare a solution containing 50ppm of Metformin HCl, 2ppm of Teneligliptin, and 1.5ppm of Pioglitazone. The mobile phase was then chosen by injecting this functional standard solution.

Method Development:

By modifying different mobile phase ratios, buffers, and other parameters, the method was developed^2,3. It was then validated in compliance with ICH guidelines⁴.

Stability Studies

The stability-indicating features of the medication in bulk and the marketed formulation were assessed by forced degradation tests. Degradation was tested under various stress conditions, including acid, alkaline, oxidative hydrolysis, thermal treatment, photolytic, and neutral degradation (Table 4), to evaluate the drug's capacity to be separated from the products of its breakdown by the procedure. The findings showed that Teneligliptin degraded under acidic, oxidative, and photolytic conditions, while Pioglitazone experienced mild degradation in alkaline and oxidative conditions⁵. Metformin showed degradation under acidic and photolytic conditions, but no degradation occurred under thermal or neutral conditions. Table 5 outlines the extent of degradation for both the drug substance and the marketed product under different stress conditions. Chromatograms of the degraded samples, shown in Figures 6 to 11, confirmed that the degradation products were effectively separated from the drug, validating the method’s stability-indicating properties⁶.

RESULT AND DISCUSSION:

Teneligliptin, Pioglitazone, and Metformin HCl were eluted at 6.692, 10.883, and 2.265 minutes, respectively, with excellent resolution⁷. The plate count and tailing factor met all necessary criteria⁸, leading to the optimization and validation of this method (Table 1) (Figure 4).

Table 1: Chromatographic conditions are optimized

Trial No.	Mobile Phase⁹	Ratio (%v/v)	Remark
1.	Methanol: Water	60:40	Obtaining peak splitting and peak tailing
2.	Phosphate buffer (pH 3): Methanol: Acetonitrile	50:35:15	Three peak obtain in which 1 peak splitting observed with less resolution.
3.	Phosphate buffer (pH 3): Methanol: Acetonitrile	55:35:10	Three peak obtain with sharp peak but less resolution is observed
4.	Phosphate buffer (pH 3): Methanol: Acetonitrile	60:35:05	Three peaks are obtained with greater resolution and a sharp peak than three trials.
5.	Phosphate buffer (pH 3): Methanol: Acetonitrile	70:25:05	Compared to four trials, three peaks produced a sharp peak, higher resolution, and a longer retention period.

Figure 4. Optimized chromatogram

System suitability:

Every system suitability parameter was within acceptable bounds and satisfied the ICH standards⁴ (Table 2).

Table 2. Parameters of system suitability

System Suitability Parameter	Metformin HCl	Teneligliptin	Pioglitazone
Retention time (min)	2.265	6.692	10.883
Resolution (R)	0.0	9.2	18.7
Tailing factor (T)	0.94	1.74	1.8
Theoretical plate number (N)	9835	4720	3547

LOD and LOQ:

The detection limit is defined as the smallest quantity of analyte that can be identified, though it may not always be quantifiable. To calculate the limit of detection (LOD), three calibration curves were utilized. The LOD can be expressed using the formula LOD = 3.3 × (SD/Slope). Similarly, the limit of quantification (LOQ) was calculated using the same three calibration curves, with the formula LOQ = 10 × (SD/Slope).

Linearity:

The linearity of response for Teneligliptin, Pioglitazone, and Metformin HCl was assessed by analyzing three distinct levels of calibration curve, specifically within ranges of 0.5-3 µg/ml, 0.375-2.25 µg/ml, and 12.5-75 µg/ml, respectively. The average areas obtained are detailed below, with the linearity equations for Teneligliptin, Pioglitazone, and Metformin HCl being y = 52885x + 361.69, y = 57333x - 641.08, and y = 19077x + 6656.9, respectively. The correlation coefficients for these medications were found to be 0.9998, 0.9998, and 0.9997, respectively (figure 5).

Figure 5: Overlain chromatogram of Teneligliptin, Pioglitazone & Metformin HCl

Precision¹⁰:

Repeatability:

Aliquots of standard stock-2 solutions were measured into three 10 ml volumetric flasks for Metformin, Teneligliptin, and Pioglitazone at 50%, 100%, and 150% concentrations. The final solutions were diluted to specified concentrations using the mobile phase, injected into the chromatographic system, and analysed for percent relative standard deviation (RSD).

Intra-Day Precision:

Aliquots of standard stock-2 solutions for Metformin, Teneligliptin, and Pioglitazone were added to three 10 ml flasks at 50%, 100%, and 150% concentrations. After diluting the final solutions with the mobile phase, they were injected into the chromatographic apparatus and their percent relative standard deviation (RSD) was determined.

Inter-Day Precision:

Three 10ml volumetric flasks were prepared by adding aliquots of Metformin, Teneligliptin, and Pioglitazone stock-2 solutions at 50%, 100%, and 150%. The final concentrations were adjusted with the mobile phase, injected into the chromatographic system, and analyzed on different days to calculate the percent relative standard deviation (RSD).

Accuracy:

Three different levels of accuracy samples were created using the conventional addition approach. For each level, three injections were performed, resulting in recoveries for Teneligliptin, Pioglitazone, and Metformin HCl within the ranges of 99.06% to 100.14%, 99.67% to 100.42%, and 98.72% to 99.06%, respectively.

Robustness:

By adjusting parameters including flow rate, mobile phase pH, and their ratios while injecting samples twice, the method's resilience was evaluated. There was no significant effect on the system suitability parameters, and all criteria were satisfied. The limit for percent relative standard deviation (%RSD) was achieved.

Assay:

The proposed method's applicability was tested on the tablet formulation Zita-PioMet. The powder content was determined by weighing twenty tablets, then Teneligliptin (5 mg), Pioglitazone (3 mg), and Metformin HCl (100 mg) were combined in a 100 ml volumetric flask. For fifteen minutes, the mixture was subjected to sonication using methanol to dissolve completely, then filtered and adjusted to volume. Sample solutions were diluted to concentrations of Teneligliptin (20 µg/ml), Pioglitazone (15 µg/ml), and Metformin HCl (500 µg/ml) for HPLC analysis¹¹.

Table 3. Summarise all validated data

Sr. No.	Parameters	Metformin HCl	Teneligliptin	Pioglitazone
1.	LOD (n=3) (μg/ml)	3.46	0.12	0.08
2.	LOQ (n=3) (μg/ml)	10.5	0.35	0.24
3.	Repeatability (n=3) %RSD	0.003 – 0.036	0.108 – 0.360	0.08 – 0.693
4.	Intraday Precision (n=3) %RSD	0.02 – 0.033	0.219 – 0.241	0.260 – 0.69
5.	Interday Precision (n=3) %RSD	0.02 – 0.05	0.116- 0.26	0.107 – 0.363
6.	Accuracy (n=3) % recovery	75%	98.72	99.06
		100%	99.41	99.19
		125%	99.06	100.14
7.	Robustness (n=3) (%RSD)	pH (+0.2 units)	0.03	0.18
		pH (‒0.2 units)	0.01	0.11
		F.R (+0.2 units)	0.01	0.21
		F.R (‒0.2 units)	0.03	0.33
		M.P (+2%)	0.04	0.15
		M.P (‒2%)	0.03	0.26
8.	% Assay ± SD (n=3)		99.96	99.73

Stability Studies:

Table 4. Stability Condition

Sr. No.	Stress Type	Stress Condition	Neutralization
1.	Acidic degradation	0.1 N HCl at 60℃ for 8 hr.	Neutralize with 0.1 N NaOH
2.	Alkaline degradation	0.1 N NaOH at 60℃ for 8 hr.	Neutralize with 0.1 N HCl
3.	Oxidative degradation	3% H₂O₂ at 60℃ for 4 hr.	Mobile Phase
4.	Thermal degradation	80℃ for 2 hr	Mobile Phase
5.	Photolytic degradation	Presence in Sunlight for 3 hr	Mobile Phase
6.	Neutral degradation	H2O Refluxed at 40℃ for 4 hr	Mobile Phase

Figure 6. Acidic degradation

Figure 7. Alkaline degradation

Figure 8. Oxidative degradation

Figure 9. Thermal degradation

Figure 10. Photolytic degradation

Figure 11. Neutral degradation

Table 5. Summarise data of stability Study¹²

Degradation condition	Drug	Conc. of drug (µg/ml)	RT of observed peak*	AUC*	% Of drug	% Of degradation
Acidic	MET	50	2.182	898468.68	92.65	7.35
	MET	50	1.023 (I)	45867.13	-	4.73
	TENE	2	6.684	87661.18	83.05	16.95
	TENE	2	4.983 (I)	13879.21	-	13.15
	PIO	1.5	10.875	83861.67	98.01	1.99
Basic	MET	50	2.264	965122.55	99.52	0.48
	TENE	2	6.663	95767.12	90.73	9.27
	PIO	1.5	10.867	78472.84	91.71	8.29
	PIO	1.5	10.022 (I)	6279.79	-	7.34
Oxidative	MET	50	2.254	845688.66	87.21	12.79
	TENE	2	6.681	101426.49	96.10	3.90
	TENE	2	5.813 (I)	3987.16	-	3.78
	PIO	1.5	10.883	81855.68	95.66	4.34
	PIO	1.5	10.405 (I)	4147.55	-	4.85
Photolytic	MET	50	2.265	845887.32	87.23	12.77
			1.102 (I)	35571.76	-	3.67
			0.681 (II)	2466.46	-	0.25
	TENE	2	6.671	92575.57	87.71	12.29
			5.813 (I)	3246.61	-	3.08
			5.024 (II)	1247.79	-	1.18
	PIO	1.5	10.862	82916.12	96.90	3.10
Thermal	MET	50	2.235	917588.38	94.62	5.38
	TENE	2	6.681	97877.61	92.73	7.27
	PIO	1.5	10.883	84467.72	98.72	1.28
Neutral	MET	50	2.265	968461.76	99.87	0.13
	TENE	2	6.692	105871.66	100.31	-0.31
	PIO	1.5	10.883	85723.22	100.18	-0.18

CONCLUSION:

For the tablet form, a simple, fast, and exact method was created for the simultaneous measurement of TENE, PIO, and MET HCl. It was discovered that every validation parameter complied with ICH regulations. The quality control department can evaluate pharmaceutical preparation assays and stability samples using this stability-indicating approach that efficiently separates degradants.

CONFLICT OF INTEREST:

No conflicts of interest exist for the authors in relation to this study.

ACKNOWLEDGEMENT:

The facilities provided by Smt. B.N.B. Swaminarayan Pharmacy College in Salvav-Vapi. They also value the support from teaching and non-teaching staff, as well as Dr. Ashvinkumar V. Dudhrejiya's assistance. We would especially want to thank my parents for their support.

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Received on 01.10.2024 Revised on 15.02.2025

Accepted on 18.04.2025 Published on 15.05.2025

Available online from May 17, 2025

Research J. Science and Tech. 2025; 17(2):101-108.

DOI: 10.52711/2349-2988.2025.00014

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