Analytical Quality by Design Approach To RP-HPLC Method Development and Validation of Dapagliflozin and Vildagliptin

Type 2 diabetes is a medical disorder resulting from impaired regulation and utilisation of sugar as an energy source by the body. This sugar is also referred to as glucose. This chronic process leads to excessive blood sugar levels. Prolonged elevation of blood glucose levels can ultimately result in dysfunctions of the circulatory, neurological, and immunological systems.

Primarily, type 2 diabetes is characterised by two challenges. The pancreas lacks sufficient production of insulin, a hormone responsible for controlling the transportation of sugar into cells. And cells exhibit decreased responsiveness to insulin and uptake of glucose.

While formerly referred to as adult-onset diabetes, both type 1 and type 2 diabetes can manifest in both childhood and maturity. Prevalence of type 2 is higher in elderly individuals. However, the rise in the prevalence of childhood obesity has resulted in an escalation of type 2 diabetes incidence among adults of younger age.¹

Type 2 Diabetes is a persistent endocrine disorder marked by increased levels of glucose in the bloodstream, accompanied with micro and macrovascular deficits ^2,3,4. Therefore, treatment with a combination of oral hypoglycemic drugs with distinct modes of action is often preferred for enhancing glycaemic control, as opposed to monotherapy. ^5,6

Metformin is almost often prescribed by endocrinologists as a therapeutic intervention for type II diabetes mellitus, except there's particular justifications for its avoidance ⁷ via inhibiting the mitochondrial respiratory chain of the liver, it stimulates the activation of AMP-activated protein kinase (AMPK), enhances insulin sensitivity (via influencing fat metabolism), and reduces cyclic adenosine monophosphate (cAMP) levels. Consequently, it decreases the activation of enzymes responsible for glucose synthesis and aids in the regulation of blood glucose levels. The compound dapagliflozin inhibits the function of sodium-glucose co-transporter 2 (SGLT2), therefore impeding the reabsorption of glucose that has undergone renal filtration. Consequently, enhanced excretion of glucose in the urine results in a reduction of blood glucose levels. The impact of pancreatic β cell activity or insulin sensitivity modification on the mechanism of action is negligible.⁸ By forming a robust chemical connection with the enzyme's active site of dipeptidyl peptidase-4 (DPP-4), vildagliptin induces permanent inhibition of the enzyme. ⁹ Increased amounts of intact glucagon-like peptide-1 (GLP-1) are observed both postprandial and during fasting periods. Experimental studies have shown that it efficiently stimulates the secretion of insulin while inhibiting the secretion of glucagon in reaction to glucose levels.¹⁰

Background:

Dapagliflozin and Vildagliptin demonstrate promise as a drug combination of sodium-glucose cotransporter type 2 inhibitors (SGT2i) and dipeptidyl peptidase-4 inhibitors (DPP4i)¹¹. SGLT2 inhibitors lower elevated blood glucose levels by enhancing the elimination of glucose in urine, while independent of insulin secretion or activity. DPP4 inhibitors, by impeding the breakdown of active incretin hormones, enhance glucose control and promote insulin secretion while decreasing glucagon liberation.

Dapagliflozin: Dapagliflozin is a sodium-glucose cotransporter 2 inhibitor used to treat type 2 diabetes mellitus. When used in conjunction with diet and exercise in adults, dapagliflozin enhances glycaemic management by blocking the absorption of glucose in the proximal tubule of the nephron and inducing the excretion of glucose with urine. It is chemically known as (2S,3R,4R,5S,6R)-2-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6- (hydroxymethyl) oxane -3,4,5-triol.¹² Administered in conjunction with diet and exercise in adults, dapagliflozin improves glycaemic control by inhibiting the reabsorption of glucose in the proximal tubule of the nephron, leading to the elimination of glucose in the urine. It has been investigated as a monotherapy and as an adjunctive therapy with insulin or other oral hypoglycemic drugs.^13,14

Vildagliptin: Vildagliptin (LAF237) is an orally administered antihyperglycemic medication that functions by specifically blocking the dipeptidyl peptidase-4 (DPP-4) enzyme. Its purpose is to control type II diabetes mellitus, a condition characterised by reduced GLP-1 production and insulinotropic effects. It is chemically known as (2S) -1-{2- [(3-hydroxyadamantan-1-yl)amino]acetyl} pyrrolidine-2-carbonitrile. ¹⁵

Figure No.1 structure of Dapagliflozin

Figure No.2 structure of Vildagliptin

Extensive literature research has unearthed a multitude of recorded analytical procedures, including the discovery of more economically efficient ways. Nevertheless, there is currently no documented approach for calculating stability studies. Hence, a reliable and cost-effective approach is suggested for assessing the stability of Dapagliflozin, Vildagliptin, and their medicinal dose form using RP-HPLC.16-19 must be validated and developed as per ICH guidelines

MATERIALS AND METHODS

Spectrum pharma Research Solution provide with Dapagliflozin and Vildagliptin pure drugs (API) gift samples and Combination Dapagliflozin and Vildagliptin tablets (Vildaily-DZ) received from local market. The chemicals and buffers utilized in this estimation were obtained from Rankem, an Indian supplier.

Instrumentation

The development and method validation were conducted using a WATERS HPLC, specifically the model 2695 SYSTEM, equipped with a Photo diode array detector. The system also included an automated sample injector and the Empower 2 software.

Objective:

The major aim of this work is to develop a precise, accurate, sensitive, specific, consistent, and efficient analytical technique for simultaneously measuring the amounts of Dapagliflozin and Vildagliptin in their pure state and tablet formulation.

Chromatographic Conditions:

Table No.1 chromatographic conditions

Preparation of Standard stock solutions: Accurately weighed 5mg of Dapagliflozin, 50mg of Vildagliptin and transferred to 100ml volumetric flask. 3/4th of diluents was added to the flasks and sonicated for 10 minutes. Flask were made up with diluents and labeled as Standard stock solution 1. (50µg/ml of Dapagliflozin and 500µg/ml of Vildagliptin)

Preparation of Standard working solutions (100% solution): 1ml from stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (5µg/ml of Dapagliflozin and 50µg/ml of Vildagliptin)

Preparation of Sample stock solutions: 10 tablets were weighed and the average weight of each tablet was calculated, then the weight equivalent to 1 tablet was transferred into a 100 ml volumetric flask, 50ml of diluents was added and sonicated for 25 min, further the volume was made up with diluent and filtered by HPLC filters.(100µg/ml of Dapagliflozin and 1000µg/ml of Vildagliptin)

Preparation of Sample working solutions (100% solution): 0.5ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (5µg/ml of Dapagliflozin and 50µg/ml of Vildagliptin)

System suitability parameters: The system suitability parameters were determined by preparing standard solutions of Dapagliflozin(5ppm) and Vildagliptin(50ppm) and the solutions were injected six times and the parameters like peak tailing, resolution and USP plate count were determined. The relative standard deviation (RSD) for the area of six standard injections should not exceed 2%. System suitability chromatogram was shown in figure 3 and values in table 1.

Specificity: Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific. The chromatogram were represented are shown in figure 4 and table 3.

Table No2: System suitability results

Figure 3: system suitability Chromatogram

Table No.3: Specificity data

    Figure 4: Specificity of Dapagliflozin and Vildagliptin

Linearity:

Calibration data is given in table 4 and regression data in table 5 and calibration curve in figure 5,6

Table No.4: Calibration data of Dapagliflozin and Vildagliptin

Figure No.5 Calibration curve of Dapagliflozin

Figure No.6 Calibration curve of Vildagliptin

Table No.5: regression data

Accuracy:

Recovery data shown in table 6

Table No.6: recovery data of Dapagliflozin and Vildagliptin

System precision was performed and the data was shown in table 7

Table No.7: System precision of Dapagliflozin and Vildagliptin

The % RSD for the peak areas of Dapagliflozin and Vildagliptin obtained from six replicate injections of standard solution was within the limit.

Method Precision: The precision of the method was determined by analyzing a sample of Dapagliflozin and Vildagliptin and shown in table 8.

Table No.8: Method Precision

From the above results, the % RSD of method precision study was within the limit for Dapagliflozin and Vildagliptin.

Robustness: Robustness conditions like Flow minus (0.7ml/min), Flow plus (0.9ml/min), mobile phase minus (65B:35A), mobile phase plus (75B:25A), temperature minus (27°C) and temperature plus(33°C) was maintained and samples were injected in duplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.

Table No.9: Robustness data for Dapagliflozin and Vildagliptin.

Force Degradation Studies: table 10 shows degradation conditions and table 10 shows the obtained degraded data and purity plot chromatogram in figure 7, 8

Table No.10: degradation conditions

Figure No.7 Purity plots for Acid Condition for Vildagliptin.

Figure No.8: Purity plots for Acid Condition for Dapagliflozin.

Assay: Vildaily, bearing the label claim Dapagliflozin 100mg, Vildagliptin 10mg. Assay was performed with the above formulation. Average % Assay for Dapagliflozin and Vildagliptin obtained was 100.00% and 100.02% respectively.

Table No.12: assay data

Assay was calculated by the formula:

Figure No.9 formula