Stability indicating for Quality Control Assessment of three antidiabetic molecules using HPLC technique: Stability Assessment of three antidiabetic molecules

: Diabetes mellitus responds better to co-formulated tablet dosages of dapagliflozin (DFZN), metformin (MFMN), and vildagliptin (VDGN). For the purpose of studying stability and quantifying DFZN, VDGN, and MFMN in bulk forms and in dosage forms, an efficient and fast HPLC method of analysis is currently developed. The mobile phase featured 80:20 ( v/v ) 0.2 M, pH 3.0, ammonium acetate buffer and acetonitrile mixed together and Luna's HPLC C-18 column, named Phenyl hexyl, was utilised for DFZN, VDGN, and MFMN separation and its quantification. The PDA type detector operating at 235 nm wavelength was deployed. The “International Conference on Harmonisation” recommendations were strictly adhered throughout the validation process. A strong linear association between response and quantity in the range of 2.5–15 µg/ml (DFZN), 25–150 µg/ml (VDGN), and 125–750 µg/ml (MFMN) is supported by the regression information for the DFZN, VDGN, and MFMN calibration plots. The precision, selectivity, accuracy, sensitivity, ruggedness and robustness were satisfactory for the method. The tablet sample of DFZN, VDGN, and MFMN was subjected to acid, water, base, sodium bisulfite, light, dry heat and peroxide degradations. Significant differences in retention times were observed between the well-resolved peaks of the degradants and the primary peaks (DFZN, VDGN, and MFMN). Thus, the assay might be characterised as stability indicating. The contents of DFZN, VDGN, and MFMN in dosage forms were assessed precisely and accurately by currently developed HPLC technique. This technique may be applied to ensure the quality of formulation doses for DFZN, VDGN, and MFMN contents.


Introduction
A chronic metabolic disease titled diabetes mellitus is marked by continuing hyperglycemia [1].It has been reported that 415 million persons aged 20 to 79 had diabetes mellitus during 2015.This information comes from the "International Diabetes Federation" [2,3].Since this number is predicted to increase to an additional 200 million by 2040, diabetes is proving to pose a burden on worldwide public healthcare [2,3].In those suffering from diabetes mellitus, chronic hyperglycaemia can exacerbate other metabolic abnormalities and harm multiple organ systems, including retinopathy, nephropathy, and neuropathy.It may additionally result in macrovascular complications, which may elevate the chances of cardiovascular disorders by two to four times [4,5].
In diabetic individuals, the increases in blood glucose quantities can usually be multifactorial [6,7].As such, attempting to restore normal blood glucose quantities by influencing the activity of a single hypoglycemic mechanism is challenging.As a result, various single medication combinations are now utilized as an appealing strategy for the medical management of diabetes [8].Getting blood glucose quantities into the acceptable range without experiencing uncomfortable side effects is the main focus of every antidiabetic therapy, and this could be achieved by combining medications with multiple strategies of action.The need to develop appropriate and effective analytical techniques for the simultaneous assessment of the coadministered pharmaceutical molecules increases due to this treatment trend.
Int. Res.J. Multidiscip.Technovation, 6(4) (2024) 308-324 | 309 The hypoglycemic effects of these three medications (DFZN, VDGN, and MFMN) are produced by distinct mechanisms.With the DFZN, blood glucose quantities are lowered through increasing urine glucose excretion [13].By raising the body's incretin levels, which induce the pancreas to build up more insulin, the VDGN aids with blood glucose regulation [14].In cases of hyperglycemia, the VDGN further instructs the liver to cease synthesising glucose.Food-derived glucose absorption and hepatic glucose synthesis are both reduced by the MFMN [15,16].Additionally, the MFMN strengthens the body's responsiveness to insulin, a naturally occurring chemical that manages blood glucose quantities.
As a result, it's critical to possess a reliable analytical methodology that could be implemented to analyze various medications together.Also, the analysis need to hold applicable to pharmaceutical dose formulations.Additionally, the analysis must be reliable when respective degradation products are present [17][18][19][20].Separations using HPLC offer a number of benefits, including quick analytical times.Furthermore, it only needs less injection quantities (in microliters quantity).Additionally, minimal solvent is used, and no previous extraction processes need to be done [21][22][23].To far, no study has yet been published on the combined analysis of DFZN, VDGN, and MFMN using any kind of strategy.Thus, the key objective of this research is to establish an efficient RP-HPLC technique that uses simple mobile phase components to determine DFZN, VDGN, and MFMN simultaneously despite the presence of respective degradation products and in their tablet forms, resulting in a safe analytical approach.It should therefore be implemented for regular quality control examination of DFZN, VDGN, and MFMN.It can additionally be exploited to indicate stability in DFZN, VDGN, and MFMN analyses.

Instruments
The "Waters" HPLC system was the device put to use for DFZN, VDGN, and MFMN analysis.The "Waters" PDA system was the device that was executed to detect DFZN, VDGN, and MFMN.Selected reactions were conducted in solution while the stress degradation analysis by using an accurate water bath fitted with an internal temperature controller.An investigation of thermal stability on DFZN, VDGN, and MFMN was done in a thermostatic enclosed hot air oven.Analytical balances and sonicators were among the other instruments employed.For data processing in the current study project, "Waters" Empower software 2® was utilized.

Chromatographic conditions
The mobile phase featured 80:20 (v/v) 0.2 M, pH 3.0, ammonium acetate buffer and acetonitrile mixed together and filtered using an 0.45 µ membrane sieve.Luna's HPLC C-18 column, named Phenyl hexyl, was utilised; its physical dimensions were 4.6 mm × 250 mm and 5 µ.With a 10 µl injection volume, the mobile phase transfer rate was preset at 1.0 ml/min and the column's temperature stayed constant with ambient.The PDA type detector operating at 235 nm wavelength was deployed to measure the analyte (DFZN, VDGN, and MFMN) concentrations.

Forced Degradation Studies
Stress experiments were conducted pursuant to ICH-recommended circumstances to assess the stability indicating feature of the newly proposed HPLC technique [24].The tablet Vylda DM® sample (DFZN -100 µg/ml, VDGN -1000 µg/ml and MFMN -5000 µg/ml) was tested against the following kinds of stress conditions in an attempt to cause intentional degradation: acid (1.0 N HCl at 60 °C for reaction time of 15 min; 30 min; 45 min; 60 min), base (1.0 N NaOH at 60 °C for reaction time of 15 min; 30 min; 45 min; 60 min), oxidation (10% H2O2 at 60 °C for reaction time of 15 min; 30 min; 45 min; 60 min), water (distilled water at 60 °C for reaction time of 15 min; 30 min; 45 min; 60 min) dry heat (105 °C, for exposure period of 2 hr; 4 hr; 6 hr; 8 hr), reduction (sodium busulfite at 60 °C for reaction time of 15 min; 30 min; 45 min; 60 min) and UV light (320 nm to 400 nm range, for exposure period of 1 hr; 2 hr; 3 hr; 4 hr).It was investigated if the suggested strategy could evaluate the analytes (DFZN, VDGN, and MFMN) responses while its degradation products were present.Additionally, this will be applied for testing the consistency of DFZN, VDGN, and MFMN when administered with deliberate degradations.

Assay of DFZN, VDGN, and MFMN marketed formulation
After precisely weighing twenty Vylda DM® pills to determine their mean weight, they were crushed in a pestle and mortar around ten minutes to turn them into a fine powder.A precisely measured amount of tablet powder, about equal to 10 mg of DFZN, 100 mg of VDGN, and 500 mg of MFMN, was put into a 100 ml volumetric flask, into which 30 ml of mobile phase was included.The flask's contents were thereafter sonicated for approximately 15 minutes, and mobile phase was then included to bring the volume equal to the desired level.This is stock Vylda DM® solution (DFZN -100 µg/ml, VDGN -1000 µg/ml and MFMN -5000 µg/ml).A membrane sieve with a thickness of 0.45 μ was used to filter the stock Vylda DM® solution.Using mobile phase again, an aliquot holding 10 ml of stock Vylda DM® solution was diluted to 100 ml.To acquire the results, the resultant solution (10 μl) was put on to HPLC column, chromatograms were established and an assessment of DFZN, VDGN, and MFMN in Vylda DM® tablets using PDA was carried out.

Placebo solution
A placebo constituted of a homogenous powder comprising 40 mg each of stearate (a lubricant), lactose (a binder), hydroxyl cellulose (a binder), magnesium sodium alginate (a lubricant), starch (a binder), ferric oxide yellow (a colorant), and talc (a lubricant).A precise 20 mg placebo homogenous powder was utilised, and the extract was made according to the tablet extract preparation.

Optimization Outcomes
Before the methodology validation, HPLC assay parameters for DFZN, VDGN, and MFMN were optimised ensuring a smoother elution procedure with improved separation performance.Two separate columns were put into use to investigate DFZN, VDGN, and MFMN peaks separation.One is Luna's HPLC C-18 column, named Phenyl hexyl; its physical dimensions were 4.6 mm × 250 mm and 5 µ.Another one is Scimadzu's HPLC C-18 column, named Symmetry; its physical dimensions were 4.6 mm × 250 mm and 5 µ.Luna's HPLC C-18 column adequately separated all peaks (Figure .2), consequently it was picked for investigation.
The mobile phase combination was assessed by varying the solvents ratio.

Int. Res. J. Multidiscip. Technovation, 6(4) (2024) 308-324 | 311
The solvent mixtures investigated were: acetonitrile with 0.1% (pH 3.0) trifluoroacetic acid and acetonitrile with 0.2 M (pH 3.0) ammonium acetate buffer.The mobile phase featured 80:20 (v/v) 0.2 M, pH 3.0, ammonium acetate buffer and acetonitrile mixed together resulted in good sensitivity and resolution (Figure .2).It became apparent that using ambient temperature and flow rate of 1.0 ml a min produced finest peak separations (Figure .2) as well as good reproducibility.A scan of DFZN, VDGN, and MFMN solutions was performed over a wavelength spanning of 200 to 400 nm.The isobestic point of the DFZN, VDGN, and MFMN was spotted at 235 nm (Figure .3), consequently it was picked for DFZN, VDGN, and MFMN investigation.

Method validation
The validation elements were tested in tandem with the ICH's suggested directives for completing the method validation procedures [25].

Robustness
Essential investigative parameters, such as the column chromatographic temperature and the proportion of organic modifier solvent, were changed so as to show robustness.This resulted in a change in the flow rate to 1.1 ml/min and 0.9 ml/min from 1.0 ml/min and a change in the volume percent ratio of acetonitrile to 15% vol.and 25% vol.from 20% vol.As a means to evaluate robustness, amounts of DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) in diluent solution were used.The effects were expressed in assay percentile of DFZN, VDGN, and MFMN in modified procedures.By modifying the volume percent ratio of acetonitrile, the percent assays were 100.434 and 100.329% for DFZN, 100.381 and 99.837% for VDGN, and 100.252 and 100.933% for MFMN.During change in flow rate, the percent assays were 100.704 and 100.534% for DFZN, 100.198 and 100.469% for VDGN, and 100.970 and 100.030% for MFMN (Table 1).By demonstrating that observed values remain consistent even as chromatographic parameters like column chromatographic temperature and the proportion of organic modifier solvent are changed, robustness can be verified.2), 99.733% for VDGN (Table 3), and 100.367% for MFMN (Table 4).Good recoveries in a sample of spiked tablets demonstrated unequivocally that other excipient components of tablets have no influence on the extraction as well as evaluation of DFZN, VDGN, and MFMN.

Precision
The precision of the chromatographic evaluation of DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) in diluent solution (n = 6) and in tablet Vylda DM® sample (n = 6) was assessed.The RSD% of measurements (n = 6) made in a single day is used to express precision.The novel method's precision for DFZN, VDGN, and MFMN was found to be inside of the acceptable range of less than 2.0%, with an RSD percentage ranging from 0.4400% to 0.8234 % (Table 5) for system precision and 0.3295% to 0.7342% (Table 5) for method precision.

Ruggedness
The ruggedness of the chromatographic evaluation of DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) in diluent solution (n = 6) was assessed.By analysing the DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) in diluent solution six times over the course of two days in two separate labs (n = 6 per day, two days), ruggedness was evaluated.The RSD% of all measures (n = 12) serves as an indication of ruggedness.The novel method's ruggedness for DFZN, VDGN, and MFMN analysis was found to be inside of the acceptable range of less than 2.0%, with an RSD percentage ranging from 0.3833% to 0.7387% (Table 6).

Selectivity
We assessed the selectivity for chromatographic evaluation of DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) using the interference experiment parameter.It became apparent that the mobile phase nor excipients (placebo) were not the cause of any noteworthy or credible disturbances (as depicted in Figure . 5).The method's selectivity for the assessment of DFZN, VDGN, and MFMN is evident.

System suitability
The plate count, resolution, and tailing factor were evaluated using the same injection concentrations of DFZN (10 µg/ml), VDGN (100 µg/ml), and MFMN (500 µg/ml) in six repetitions, and the system suitableness was assessed.Following an analysis of six analytical replicates, it appears that there have been no statistically noteworthy variations in the responses of MFMN (Table 7), DFZN (Table 8), and VDGN (Table 9).The RSD (%) for system suitableness was assessed to be less than 2 (0.0798% to 1.9578% for DFZN; 0.0798% to 1.9578% for VDGN; 0.0443% to 1.9698% for MFMN), confirming that the device has a high level of precision.10-12), DGFN and VDGN appear to be far more susceptible to oxidation (10% H2O2 at 60 °C), whereas MFMN proved to be more sensitive towards dry heat (105 °C) conditions.DGFN and MFMN appear to be far more stable to reduction (sodium bisulfite at 60 °C) condition, whereas VDGN proved to be more stable towards dry heat (105 °C) conditions.

Forced degradation study
Forced degradation was also used to measure method's specificity for the assessment of DFZN, VDGN, and MFMN.There was no of any additional peaks corresponding to the degradants generated across any of the stressed sample chromatograms (Figure .6, Chromatograms A-G).Empower software was used to conduct a peak purity study in the 200-400 nm region.The spectral purity of the MFMN, VDGN, and DFZN peaks in all stress Vylda DM® tablet samples were verified by analysis.In every tablet samples, the peak purity values (purity threshold > purity angle, Table 13) of DFZN, VDGN, and MFMN showed that no other peaks were co-eluting with those three peaks.The new method's capacity to indicate stability was confirmed as there was no observable co-elution of substances from degradation with DFZN, VDGN, and MFMN within any of the stressed Vylda DM® tablet samples.

Conclusion
The HPLC approach for DGFN, VDGN and MFMN assay seemed advantageous for both qualitative and quantitative examination.The HPLC approach seemed like a reliable, ideal technique for determining DGFN, VDGN, and MFMN simultaneously.The current strategy is also quite accurate, simple, and precise enough.All of these factors therefore point to the possibility of using the proposed and validated approach as a basis for quality control of DGFN, VDGN, and MFMN in the dosage forms as well as bulk forms.Degradation products that were obtained under the influence various stress circumstances illustrated good separation from DGFN, VDGN, and MFMN, showing that the approach represents stability-indicating.

Figure 1 .
Figure 1.Structures and chemical names of drugs of investigation

Figure 2 .Figure 3 .
Figure 2. DFZN, VDGN, and MFMN chromatogram with optimized HPLC: DFZN, VDGN, and MFMN -assay parameters By repeatedly (n = 3 per level) evaluating the tablet Vylda DM® sample spiked with different three measures of DFZN, VDGN, and MFMN, the recovery of DFZN, VDGN, and MFMN in a simulated tablet Vylda DM® sample shows the accuracy of DFZN, VDGN, and MFMN extraction from tablet as well as sample processing.Percentage recovery is a means for reporting accuracy characteristics.The percent mean (n = 3 per level) recovery was 100.633% for DFZN (Table

Table 1 .
DFZN, VDGN, and MFMN robustness reports a -Average of three values; b -standard deviation of three values; c -relative standard deviation

Table 9 .
VDGN system suitability reportsIt provides details on how stable the pharmaceuticals (DFZN, VDGN, and MFMN) are in situations of acidity, basicity, light, thermal, hydrolysis, reduction, and oxidation.It has a direct impact on the choice of formulation development, packaging, preservation, shelf life, drug stability, and drug product stability.

Table 10 .
Stabilities of DFZN in forced degradation analysis

Table 12 .
Stabilities of MFMN in forced degradation analysis