Development and Validation of RP HPLC Method for The Simultaneous Estimation of Dapagliflozin and Metformin Tablet Formulation

Diabetes mellitus belongs to a category of metabolic disorder, characterized by chronic hyperglycemia due to deficiency in insulin secretion or action or both people with type 2 diabetes mellitus are susceptible to various short-term complications including premature death and coma.1Dapagliflozin is a highly selective, orally active and reversible inhibitor of the human SodiumGlucose Co-Transporter 2 (SGLT2), the major transporter responsible for the renal glucose reabsorption. It improves glyceamic control in patients with Type 2 Diabetes Mellitus by inhibiting the Sodium-Glucose Co-Transporter 2, intern by reducing glucose reabsorption. Dapagliflozin is a first generation selective SGLT inhibitor that blocks for SGLT 2 over SGLT1.2 Chemically Dapagliflozin is called as (2S,3R,4R,5S,6R)-2-{4-chloro-3-[(4-ethoxyphenyl) methyl] phenyl}-6 hydroxymethyl) oxane-3,4,5-triol. Dapagliflozin retard the sodium glucose cotransporter 2 (SGLT2) which is essentially present in the primary part of the nephron. Since SGLT2 aides in 92% of the kidneys' ability to uptake glucose, it's blockage enables glucose to be released in urine. Patient with type 2 diabetes may experience better glucose control and possible weight loss due to this excretion. Analytical chemistry helps for the determination of quality, purity, safety of the chemicals and drugs by separating, quantifying and identifying the sample using certain methods and instruments. It helps in both quantitative as well as qualitative analysis of the sample, in qualitative analysis, it determines the purity and quality of the sample whereas, in quantitative analysis the concentration of the sample i.e., the amount of the expected content present within the sample can be found HPLC is High-Performance Liquid Chromatography is mainly used for the separation, identification and quantification of the components present in a mixture. Till 1960 liquid chromatography in which only glass columns were used and it was used to work in low pressure was developed to HPLC later with metal columns and high pressure. The basic working principle of HPLC is that it separates the constituent of the mixture based on relative affinities of the constituents for the stationary phase and mobile phase, which are used for the separation. Mainly there are two types of HPLC they are; reverse phase uses polar mobile phase and the non-polar stationary phase and the normal phase uses the non-polar mobile phase and polar stationary phase. Major applications of HPLC areanalysing drugs, pollutants and synthetic polymers, isolation of components and used in industries in quality control departments to check the purity of the drug samples and chemicals.

3. Analytical method validation:

all the requirements. Method validation provides evidence in documented form and gives high degree of assurance whether the developed method is suitable to produce a consistent result or not. ICH (International Conference on Harmonization) guidelines are issued by regulatory authorities to validate the newly developed method by using parameters such as accuracy, precision, intermediate precision(ruggedness), specificity, linearity and range, system suitability, robustness to test whether the developed method posess

4. Metformin HCL

Metformin HCl is an antidiabetic drug with a chemical name, 1-carbamimidamido-N, N-dimethylmethanimidamide hydrochloride falls under biguanide class of antihyperglycemic agent. Works by reducing production of hepatic glucose and also increases glucose uptake, it also helps to prevent complications related to cardiovascular system. Its also used for PCOS problems and also decreases levels of triglycerides and low-density lipoprotein cholesterol. As per pharmacopeia only potentiometry and UV methods are mentioned for Metformin, as per European pharmacopeia and Indian pharmacopeia and as per literature HPTLC [5], HPLC [6, 8], UV [6, 9, 10], LC-MS/MS [11] were reported. Even in the mentioned HPLC method, there was a need for an improved and stable method.

5. Drug Profile

IUPAC NAME:[2S,3R,4R,5S,6R]-2{4-C holoro-3-{(4ethoxyphenyl)methyl]phenyl}-6-(hydroxymethyl)oxane-3,4,5-triol Common Synonym:(2S,3R,4R,5S,6R)-2-(4-Chloro-3-(4ethoxybenzyl)phenyl)-6-(hydroxymethyl)tetrahydro-2H-Pyran-3,4,5-triol

Chemical name:

1-carbamimidamido-N,

N-dimethylmethanimidamide hydrochloride

Molecular Formula: C4H12ClN5

Molecular Weight: 165.62

Appearance: White or almost white crystalline powder

State: solid

Melting point: 223-226 °C

pKa: 12.4

Category: Biguanide class of anti-hyperglycemic agent

RESULT AND DISCUSSION

The absorption spectra of DAPA greatly overlap; so conventional determination of these compounds in mixture is not possible. To optimize the LC parameters, several mobile phase compositions were tried. A satisfactory separation and good peak symmetry for DAPA were obtained with a mobile phase consisting of Acetonitrile: Water: Methanol (10:20:70 v/v), pH 4.8 adjusted using o-phosphoric acid Buffer. Quantification of the drugs was performed at 234nm. Resolution of the components with clear baseline separation was obtained New method was developed for the assay studies of Metformin HCl tablet (500 mg); the following are conditions stabilized.

8. Accuracy

Recovery studies were carried out by adding a known amount of pure drugs DAPA to pre analyzed sample solution. These studies were carried out by spiking 80%, 100% and 120% respective drug. The recovery studies showed that the results were within acceptable limits, above 99% and below 101%. The results are given in (Table 2). The accuracy was carried at 3 levels, at 50%, 100% and 150%. The % recovery at 50%, 100% and 150% levels is within the acceptance criteria 98.0% to 102% Based on the below-obtained recovery results as in table 8, it is concluded that the method for assay by HPLC is accurate.

9. Robustness

Robustness of the method was determined by making slight changes in chromatographic conditions. Effect of % of methanol (59, 60 and 61%) in mobile phase on the retention time and slight changes in flow rate were applied as variable parameters. Flow rate varied at three levels (-1, 0, 1). One factor at the time was changed to estimate the effect. Thus standard solution at varied pH ( pH 4.6, 4.7 and 4.8) three pH levels was perf. Robustness was carried out on standard solution by making the following alterations:

* By changing wavelength

* By changing the oven temperature

* By changing the flow rate

10. Preparation of mobile phase

mixing an organic solvent (acetonitrile or methanol) with water or an aqueous buffer (such as 0.1% orthophosphoric acid or triethylamine) in specific ratios, such as 60:40 or 50:50, followed by filtration through a 0.45μm membrane filter and sonicating to degas. Mobile phase was prepared by mixing the buffer and aceto trile in the ratio of 70:30.

11. System suitability test

The tertiary solution of 10 µg/ml of DAPA was prepared and same was injected, then the system suitability parameters were calculated from the chromatogram. The parameters, retention times, resolution factor, tailing factor and theoretical plates were evaluated. Standard solution was prepared as per the methodology and injected into the HPLC system before starting every validation parameter. The percentage RSD for 5 replicate injections, tailing factor and theoretical plates of standard solution are considered.The tailing factor of Metformin HCl fulfills the acceptance criteria of not more than 2.0. The theoretical plates of Metformin HCl fulfills the acceptance criteria of more than 2000.

12. Linearity

To evaluate the linearity of the method, 5 levels of concentrations 50%, 80%, 100%, 120% and 150% of Metformin HCl were taken. The correlation coefficient for Metformin HCl was found to be within the acceptable limit of not less than 0.999, which passes the linearity parameter. Dapagliflozin demonstrates linear analytical behavior (concentration vs. detector response) in pharmaceutical analysis over various ranges, most commonly reported between 1–25 µg/mL in RP-HPLC methods, with correlation coefficients (r²) often. It exhibits dose-proportional pharmacokinetics, meaning its plasma concentration increases proportionally with dose.

13. Range

Dapagliflozin is typically administered at a dosage of 10 mg orally once daily, which is the standard dose for type 2 diabetes, chronic kidney disease (CKD), and heart failure (HFrEF). For some patients with type 2 diabetes, a lower starting dose of 5 mg per day may be used. For range, injected six replicates of lower and higher concentration levels of the linearity level and calculated the Mean and relative standard deviation. The relative standard deviation for Metformin HCl range levels was found to be within acceptable limits of not more 2.0%.

Table no. 1 Common Dapagliflozin+ Metformin Tablet Strengths

14. Repeatability

Dapagliflozin demonstrates high analytical repeatability, validated through RP-HPLC methods with %RSD values typically less than 1–2%, indicating precise, consistent results in quality control studies. It shows consistent performance for both API and combination tablet formulations (e.g., with Metformin). Six sample preparations were prepared and repeatability was checked. The relative standard deviation for Metformin HCl was found to be within acceptable limits of not more2.0%. The % Assay of Metformin HCl was within the acceptable limit. i.e.98%-102% of label claim.

Fig No.1First derivative UV Spectrum of powder mixture of Dapagliflozin and metformin

Table No.3:  Linearity Result For Dapagliflozin

Correlation Coefficient 0.999

Table No .4: Linearity Result for Metformin