Development and Validation of Stability Indicating HPTLC Methods for Estimation of Vildaglitpin and Pioglitazone with Greenness Assessment using AGREE And AES

One of the most prevalent chronic metabolic disorders, diabetes mellitus continues to have long-term negative effects on the vascular, renal, retinal, and neurological systems. Combination therapy is increasingly seen as a strategy to improve results through complementary techniques and lessen the negative effects of high-dose monotherapy because many patients find it difficult to achieve stable glycemic control with one therapeutic agent alone [1,2]. Vildagliptin hydrochloride (VIH) is chemically represented as (2S)-1-{2-[(3-hydroxyadamantan-1yl)amino]acetyl}pyrrolidine-2 carbonitrile. This new oral anti-hyperglycemic medication is a member of the class of medications known as dipeptidyl peptidase-4 inhibitors. It functions to prevent glucagon-like peptide-1 (GLP-1) and gastric inhibitory polypeptide (GIP) from being destroyed by the dipeptidyl peptidase 4 (DPP-4) inhibitor, which in turn increases insulin secretion by beta cells and prevents alpha cells in the islets of the pancreas of Langerhans from releasing blood sugar [3]. Pioglitazone hydrochloride (PIO) has the chemical name 5-[[4-[2-(5-ethylpyridin-2yl)ethoxy]phenyl]methyl]. A potent and highly selective agonist of the nuclear peroxisome proliferator-activated receptor (PPAR) is thiazolidinedione (1,3-thiazolidine-2,4-dione). PPARs are found in tissues like skeletal muscle, the liver, and adipose tissue that are vital to the action of insulin. When PPAR-nuclear receptors are activated, the transcription of several insulin-responsive genes involved in the control of glucose and lipid metabolism is altered. Alpha-glucosidase inhibitors, sulfonylureas, and biguanides have no chemical or functional affinity for it. It is utilized because it addresses insulin resistance, the main pathophysiological issue [4].  Chemical structure of both drugs shown in figure 1.

Figure 1: Chemical structure of VIL and PIH

Pharmaceutical analytical chemistry is beginning to apply green chemistry concepts to reduce the hazards that lab labor poses to the environment and human health. Greenness tools quantify the environmental friendliness of analytical techniques. The Analytical Eco-Scale, for instance, assigns penalty points for consuming more energy, waste, or reagents than is optimal. Penalty points are subtracted from scores, which range from poor to good green. The twelve green chemistry principles are converted into a single score (0–1) using the AGREE metric; a higher score indicates greater greenness [5–9].

A comprehensive review of the literature revealed a limited number of analytical methods reported for PIO and VIH, both as individual agents and in combination with each other or alongside other drugs. PIO and VIH have been measured by HPLC [10–14], UV spectrophotometry [15], and HPTLC [16,17]. Simple HPLC techniques have also been reported in combination [18,19] as well as stability indicating HPLC and HPTLC methods [20,21]. HPTLC has significant advantages over HPLC because of its simple instrumentation, low maintenance requirements, and simultaneous analysis of different samples. Its only small amounts of mobile-phase solvents are employed, which reduces cost and chemical waste. It also reduces its energy usage since it does not depend on high-pressure systems or continuous column heating. These operational advantages support HPTLC for daily lab work. From a greenness perspective, its reduced solvent usage, minimal waste generation, and lower power requirement allow it to align well with modern green analytical chemistry principles, giving it favorable scores in established greenness assessment tools [7,22].

Extensive literature review reveals that there is no HPTLC method available for the simultaneous estimation of PIO and VIH with a comprehensive greenness assessment using AGREE, analytical Eco-Scale assessment. Therefore, a thought of interest to develop and validate stability indication HPTLC method for the fixed dose combination of PIO and VIH in bulk and laboratory prepared mixture. Quantitative greenness assessment confirmed the method’s reduced environmental burden while retaining the robustness, accuracy, and precision required for routine quality-control analysis of combined dosage forms.

EXPERIMENTAL

Standard API, chemicals and materials

VIH and PIO active pharmaceutical ingredients were supplied by a reputable pharmaceutical company. Methanol, glacial acetic acid, ethyl acetate, toluene is analytical grade (Rankem) was used.

Wavelength selection

VIH and PIO were freely soluble in Methanol and individual solutions at a concentration of 10 µg/ ml are prepared. Using a UV–Visible double beam spectrometer, both solutions were scanned from 400 to 200 nm in wavelength. Both the drugs appreciable absorption was found to be at common wavelength 215 nm after scanning.

HPTLC system

The analysis was carried out using a twin-trough glass development chamber (CAMAG, Muttenz, Switzerland), a Linomat 5 sample applicator (CAMAG), a UV Cabinet 4 (CAMAG), and a TLC Scanner 3 (CAMAG) operated with winCATS software (version 1.4.3.6336, CAMAG). Chromatographic separation was performed on precoated silica gel 60 F??? aluminum plates (10 × 10 cm, layer thickness 200 µm) supplied by E. Merck (Darmstadt, Germany). Sample application was achieved using a 100 µL microsyringe (Hamilton, Bonaduz, Switzerland). 

Linear ascending chromatographic development was performed using a mobile phase consisting of methanol, toluene, ethyl acetate, and ammonia in the proportion of 3.0:4.5:2.5:0.5 (v/v/v/v). The development was carried out in a twin-trough chamber previously saturated for 30 min under controlled laboratory conditions (25 ± 2 °C and 60 ± 5% relative humidity), and the solvent front was allowed to migrate up to a distance of 8 cm. Densitometric evaluation was conducted at 270 nm using a CAMAG TLC Scanner 3 operated with winCATS software, with a deuterium lamp serving as the radiation source. Quantitative analysis was based on peak area responses obtained by linear regression, employing slit dimensions of 5 × 0.45 mm and a scanning rate of 20 mm s?¹.

Standard solution preparation

Weigh accurately 50 mg of VIH and 15 mg of PIO was transferred separately into 100 ml volumetric flask, and dissolved in small volume of methanol. Then the volume was diluted up to the mark with methanol to get the concentration of VIH (500μg/ml) and PIO concentrations (150μg/ml).

Calibration curve determination

A 5 μL aliquot of each solution was applied onto TLC plates using a CAMAG Linomat 5 auto-sampler with a CAMAG microliter syringe (100.0 μL), resulting in final spot concentrations of 250, 500, 750, 1000, 1250, 1500 ng per band for VIH and 75, 150, 225, 300, 375, 450 ng per band for PIO.

Validation

The HPTLC method was validated in accordance with the Q2 (R2) guideline of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH).

Linearity

Six calibrations were conducted in the concentration range of 250 ng/band to 1500 ng/band and 75 ng/band to 450 ng/band to determine the linearity of Vildagliptin and pioglitazone. A straight-line equation was used to plot Peak Area v/s Concentration on the graph to determine the calibration cure.

Precision

The repeatability of peak area measurements was evaluated by analyzing VIH (750ng/band) and PIO (225ng/band) six times without altering the plate's position. To assess injection repeatability, seven tracks of VIH and PIO were applied on the same plate. The peak area for a fixed concentration was measured six times, and the percentage relative standard deviation (% RSD) was calculated.

Precision was examined in terms of intra-day and inter-day variations using three different concentrations from the calibration curve of VIH and PIO, with % RSD serving as the evaluation parameter. Intra-day precision was determined by analyzing sample solutions of VIH (250, 750, and 1500 ng/band) and PIO (75, 225, and 450 ng/band) at different intervals on the same day. Inter-day precision was assessed by analyzing the same concentrations over three consecutive days, following ICH guidelines. The obtained peak area values were used to calculate the mean, standard deviation (SD), and % RSD.

Accuracy

The accuracy studies were conducted using the usual spiking approach. Using this technique, the standard solution was spiked at 50%, 100%, and 150% concentrations in the VIH and PIO sample solution. There were three injections of each spiked sample. Calculations were made for mean area and recovery percentage.

Limit of quantification and detection

In accordance with the International Council for Harmonisation (ICH) criteria, the limit of detection (LOD) and limit of quantification (LOQ) of the developed method were determined using calibration curve, slope, and response of standard deviation: LOD = 3.3 σ/S; LOQ = 10 σ/S, where σ is the standard deviation and S is the slope of the calibration curve.

Robustness

The ability of an analytical technique to achieve the expected performance standards under typical use is known as its robustness. By purposefully changing the parameters of the analytical process, robustness is evaluated. To investigate the method's resilience, small adjustments to the saturation time and mobile phase composition were looked at.

Forced degradation study

The purpose of the forced deterioration study was to determine the intrinsic stability of both medications [9].

Preparation of stock solution

The stock solution was prepared by dissolving 250 mg of VIH and 75 mg of PIO methanol as diluents in 50 ml volumetric flask and then sonicated for 10 min and finally made up to the volume (Stock solution 5000μg/ml of VIH and 1500μg/ml of PIO).

Acid Hydrolysis

For acid-induced degradation from above stock solution 2 mL of the solution was mixed with 2 mL of 1 N HCl in a 10 mL volumetric flask and maintained at 70°C for 2 hours. The reaction mixture was then neutralized with 1N NaOH and filtered using a 0.45μm syringe filter to obtain a concentration of 1000 μg/mL VIH and 300μg/mL PIO. A 1 mL aliquot of this solution was diluted to 10 mL with the mobile phase, yielding final concentrations of 100 μg/mL VIH and 30μg/mL PIO. The same procedure was followed for preparing individual drug solutions. From the treated samples, 10μL was applied on to a TLC plate. The study was also conducted for the mixture, where the final concentrations on the TLC plate were 1000 ng/band for VIH and 300ng/band for PIO.

Base Hydrolysis

For alkaline degradation from above stock solution 2 mL of the solution was combined with 2 mL of 1 N NaOH same procedure followed.

Oxidative Degradation

Oxidative degradation was performed by from above stock solution adding 2 mL of the solution to 2 mL of 3% w/v hydrogen peroxide in a 10 mL volumetric flask. The mixture was kept at room temperature for 30 minutes before being filtered using a 0.45μm syringe filter, yielding a concentration of 1000 μg/mL VIH and 300 μg/mL PIO. A 1 mL aliquot was diluted to 10 mL with the mobile phase, achieving final concentrations of 100 μg/mL VIH and 30μg/mL PIO. A 10μL sample of the treated solution was spotted onto a TLC plate. The study was also conducted for the mixture, with final TLC plate concentrations of 1000 ng/band for VIH and 300 ng/band for PIO.

Thermal Degradation

For thermal degradation, solid drug samples (25 mg VIH and 7.5 mg PIO) were placed in a Petri dish and subjected to 110°C in a hot air oven for 3hours. The treated powders were then dissolved in a 10 mL volumetric flask with methanol to achieve concentrations of 2500 μg/mL VIH and 750 μg/mL PIO. A 0.4 mL aliquot was further diluted to 10 mL with the mobile phase, obtaining final concentrations of 100 μg/mL VIH and 30μg/mL PIO. The same method was followed for individual drug solutions. A 10μL sample was applied to a TLC plate, with final mixture concentrations on the TLC plate being 1000 ng/band for VIH and 300 ng/band for PIO.

Photo degradation

Photolytic degradation was conducted by spreading solid drug samples (25 mg VIH and 7.5 mg PIO) as a thin layer on a Petri plate and exposing them to direct UV light for 48 hours. The exposed powders were dissolved in a 10 mL volumetric flask with methanol to achieve concentrations of 2500 μg/mL VIH and 750μg/mL PIO. A 0.4 mL aliquot was diluted to 10 mL with the mobile phase to obtain final concentrations of 100 μg/mL VIH and 30μg/mL PIO. The same method was applied for individual drug solutions. A 10μL sample was spotted onto a TLC plate, with final mixture concentrations on the TLC plate being 1000 ng/band for VIH and 300 ng/band for PIO.

Assay of synthetic mixture

A synthetic mixture of VIH and PIO was prepared in a 50:15 mg ratio, along with common excipients such as croscarmellose sodium, lactose, hydroxypropyl methylcellulose, and magnesium stearate. The active ingredients and excipients were thoroughly mixed using a mortar and pestle, simulating the composition of 20 tablets. An accurately weighed 0.140 g portion of this synthetic mixture was transferred to a 10 mL volumetric flask containing a small amount of methanol and sonicated for 15 minutes to ensure complete dissolution. The solution was then filtered through Whatman filter paper No. 42, and the filtrate was collected in a 10 mL volumetric flask, with the volume adjusted to the mark using methanol, yielding a final concentration of 500 μg/mL for VIH and 150 μg/mL for PIO. A 1 mL aliquot of this stock solution was transferred to a new 10 mL volumetric flask and diluted to the mark with methanol to obtain a working concentration of 50 μg/mL for VIH and 15 μg/mL for PIO. This process was repeated three times for accuracy. A 10 µL volume of these solutions was applied onto HPTLC plates and analyzed using the proposed chromatographic method. The concentrations of VIH and PIO were determined using the regression equation derived from the calibration curve.

RESULTS AND DISCUSSION

Wavelength selection

Methanol was used as a solvent to create separate solutions of VIH and PIO at a concentration of 10 µg/ ml. using a UV–Visible double beam spectrometer, both solutions were scanned from 400 to 200 nm in wavelength. The iso-absorptive point was discovered to be 215 nm.

Mobile phase optimization

The mobile phase composition was systematically optimized by considering the polarity, solubility characteristics, and functional groups of the selected drugs. A total of six experimental trials were conducted to achieve adequate resolution, acceptable Rf values, and symmetrical peak shapes for both analytes. In the first trial, a mobile phase comprising ethyl acetate–toluene–methanol (1:7:2, v/v) was evaluated; however, poorly resolved and broadened peaks were obtained. In the second trial, ethyl acetate–methanol (5:5, v/v) was employed. The exclusion of toluene resulted in an increased peak height for PIO, while a reduction in peak height was observed for VIH and the Rf value of PIO fell outside the acceptable range. In the third trial, methanol–toluene–ethyl acetate (0.5:9:0.5, v/v) was tested. Although an increase in the peak height of VIH was noted due to the higher proportion of toluene, the corresponding Rf value was unsatisfactory. Next trial, methanol–toluene–ethyl acetate– ammonia (2:5:3:0.2, v/v) was investigated. Although an increase in the peak height of PIO was observed, the peak height and Rf value of VIH decreased, indicating suboptimal separation. Based on these observations, the final optimized mobile phase consisting of methanol, toluene, ethyl acetate, and ammonia in the ratio of 3.0:4.5:2.5:0.5 (v/v/v/v) was selected. This composition provided well-resolved, symmetrical, and selective peaks for vildagliptin (Rf=0.47) and pioglitazone (Rf=0.21), as illustrated in Fig. 2. The chamber saturation time was maintained at 25 min, and densitometric detection was carried out at 215 nm.

Figure 2: Chromatogram of VIH and PIO on optimized chromatographic condition

Validation

The proposed developed HPTLC method was validated in accordance with the Q2 (R1) guideline of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) and result and discussion were representing as follows.

Specificity:

The method's specificity was determined by evaluating reference pharmaceuticals as well as VIH and PIO samples. The presence of an excipient in the synthetic combination has no effect on the outcome. As a result of the findings, it appears that the proposed method is unique. Peak purity spectra of VIH and PIO in calibration curve shown in Figure 3.

Figure 3: Peak purity spectra of VIH and PIO in calibration curve

Linearity

The developed HPTLC method revealed that the correlation coefficients of 0.9991 for VIH and 0.9993 for PIO. VIH and PIO were linear within the specified concentration range of 250–1500 ng/band and 75-450 ng/band for VIH and PIO. Figure 4 shows the Overlay of the chromatogram of calibration range. The value of regression analysis is shown in Table 1.

Figure 4: Overlay Chromatogram of 250–1500 ng/band for VIH and 75–450 ng/band for PIO, respectively

Table 1: Regression analysis of VIH and PIO

Precision

VIH and PIO have percentage RSD value ranges for Inter-day precision of 1.15-1.77% and 1.27-1.62%, respectively, and intra-day precision of 1.31-1.51%, and 0.55-1.29%for VIH and PIO, respectively. When intraday and inter-day measurements are made, the precision result demonstrates that the values are near to one another. Table 3 shows the summary of validation parameters of proposed HPTLC method for VIH and PIO.

Accuracy

The accuracy studies were conducted using the usual spiking approach. The regression equation was used to compute the percentage recovery of VIH and PIO. The percentage recovery of VIH and PIO was found to be 99.03 – 100.46% and 99.58 – 100.32%, respectively (Table 3).

Robustness

Some deliberate modifications were made to the test parameters to carry out the robustness investigation. The percentage RSD was found to be less than 2% which demonstrates that VIH and PIO may be assessed using this method with minor adjustments to the optimized chromatographic conditions are shown in Tables 2.

Table 2: Robustness study for VIH and PIO by proposed HPTLC method

Table 3: Summary of validation parameters of for VIH and PIO

FORCED DEGRADATION

VIH and PIO were subjected to forced degradation studies, evaluating the stability of these compounds under different stress conditions. Forced degradation study results are summarized in Table 4.

Acidic hydrolysis

Exposure of the active pharmaceutical ingredients to acidic stress conditions resulted in noticeable degradation of both analytes. VIH exhibited an assay reduction of approximately 14.66%, with additional degradation bands detected at Rf? values of 0.72 and 0.84. Under the same conditions, PIO showed an assay loss of about 9.22%, accompanied by degradant peaks appearing at Rf? values of 0.32 and 0.55, as evident from the chromatogram (Figure 4(a)).

Alkaline hydrolysis

When subjected to alkaline stress, VIH demonstrated comparatively higher susceptibility, with an assay decrease of nearly 16.31%. Degradation products corresponding to Rf? values of 0.72 and 0.88 were observed. In contrast, PIO underwent a moderate extent of degradation, reflected by an assay loss of approximately 6.94%, with degradative peaks recorded at Rf? values of 0.57 and 0.62 (Figure 4(b)).

Oxidative degradation

Oxidative stress conditions led to significant degradation of both drugs. VIH showed an assay loss of around 13.76%, with a prominent degradant band observed at a Rf? value of 0.75. Similarly, PIO exhibited an assay reduction of approximately 12.74%, along with multiple degradation peaks appearing at Rf? values of 0.32, 0.58, and 0.61 (Figure 4(c)).

Photolytic degradation

Upon exposure to light, VIH underwent limited degradation, as indicated by an assay loss of about 8.27% and the presence of a degradant peak at a Rf? value of 0.72. PIO also displayed relatively low photodegradation, with an assay decrease of approximately 6.11% and a single degradation peak observed at a Rf? value of 0.37 (Figure 5(a)).

Thermal degradation

Thermal stress studies revealed moderate degradation of both analytes. VIH showed an assay loss of nearly 8.02%, with an additional peak corresponding to a Rf? value of 0.61. Under the same conditions, PIO exhibited an assay reduction of about 6.09%, accompanied by a degradation band detected at a Rf? value of 0.28 (Figure 5(b)).

Figure 4: Chromatogram of VIH and PIO (a) Acid stress (b) Alkaline stress (c) Oxidative degradation

Figure 5: Chromatogram of VIH and PIO (a) Light stree (b) Thermal stress

Table 4: Result of force degradation studies by proposed HPTLC method

Assay of Synthetic Mixture of VIH and PIO

The assay results demonstrated good agreement between the applied and measured concentrations for both analytes. VIH applied at 500 ng per band, was quantified as 498.99 ± 7.80 ng per band, corresponding to an assay value of 99.79 ± 1.55%. Similarly, PIO applied at 150 ng per band, showed a measured concentration of 151.17 ± 2.55 ng per band, with a percent assay of 100.78 ± 1.70%, indicating satisfactory accuracy and precision of the method (Table 3).

Greenness Evaluation

Greenness analysis showed that the proposed HPTLC method exhibited the highest cooperation between sustainability of the environment and analytical performance. Additionally, the simplified instrumentation, lower solvent use, and lower energy consumption of HPTLC than LC–MS and HPLC provide more justification for its reliability and compliance with green analytical chemistry principles.

The proposed method scored 85 (“excellent green”) on the Eco-Scale assessment; penalty points are decreased by decreasing the amount of moderately hazardous solvents used and by avoiding high-toxicity reagents totally shown in Table 5.

According to AGREE evaluation, the proposed method received a score of 0.64 — one of the highest values of any chromatographic technique, due to simple sample preparation, moderate solvent hazards and low waste shown in Table 5.

Table 5. Comparison of greenness evaluation scores (Analytical Eco-Scale and AGREE for the proposed HPTLC method.

Tools Name	Name of Reagents	Proposed HPTLC	AGREE Score
Analytical eco-scale		PP
	Methanol-m	3
	Methanol-s	3
	Toluene	3
	Ethyl Acetate	2
	Energy used for HPTLC	1
	Waste	3
	Occupational hazard	0
	Total penalty points	15
	ESA	85	0.64