A Review on Development and Validation of Analytical Methods for Simultaneous Estimation of Dapagliflozin, Linagliptin and Metformin in Pharmaceutical Dosage Form

Diabetes mellitus is a chronic metabolic disorder characterized by persistent hyperglycaemia due to impaired insulin secretion, insulin action, or both. It affects carbohydrate, fat, and protein metabolism, leading to long-term complications such as cardiovascular disease, neuropathy, nephropathy, and retinopathy.

Diabetes Is Classified into Four Main Types:

1. Type 1 diabetes (autoimmune destruction of pancreatic beta cells):

Type 1 diabetes mellitus (T1DM) is a chronic autoimmune disease characterized by the destruction of insulin-producing beta cells in the pancreas. This leads to absolute insulin deficiency, requiring lifelong insulin therapy for survival. T1DM is commonly diagnosed in children and young adults but can occur at any age. It is associated with genetic susceptibility, environmental factors, and, potentially, viral triggers. Symptoms include polyuria, polydipsia, weight loss, and fatigue, with a risk of diabetic ketoacidosis if untreated. Management focuses on insulin administration, blood glucose monitoring, and education for maintaining glycemic control.

2. Type 2 diabetes (insulin resistance and relative insulin deficiency):

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disorder characterized by insulin resistance and a relative deficiency in insulin secretion. It accounts for 90-95% of all diabetes cases globally and is often associated with obesity, physical inactivity, and genetic predisposition. The condition results in hyperglycaemia, leading to complications such as cardiovascular disease, nephropathy, neuropathy, and retinopathy if left untreated. Management typically involves lifestyle modifications, oral hypoglycemic agents (e.g., metformin, sulfonylureas, DPP-4 inhibitors), and sometimes insulin therapy.

3. Gestational diabetes (occurring during pregnancy):

A form of glucose intolerance first recognized during pregnancy. It increases the risk of complications for both the mother and fetus and predisposes the mother to T2DM later in life.

4. Other specific types caused by genetic defects or secondary factors:

Includes diabetes due to genetic defects (e.g., MODY), diseases of the exocrine pancreas, drug- or chemical-induced diabetes, and endocrinopathies. Proper diagnosis and classification are critical for effective management and prevention of complications. For most patients, metformin is the first-line treatment, often combined with lifestyle modifications. If glycemic targets are not met, a second agent (e.g., GLP-1 agonist, SGLT-2 inhibitor, DPP-4 inhibitor) is typically added based on patient-specific factors.

Combined Dosgae Form (

Combined dosage form of Dapagliflozin propanediol monohydrate eq. to Dapagliflozin + Linagliptin + Metformin hydrochloride IP was approved by CDSCO on 8^th March, 2024. Drug combination Dapagliflozin propanediol monohydrate eq. to Dapagliflozin + Linagliptin + Metformin hydrochloride IP used for treatment of Type 2 Diabetes mellitus.  Dapagliflozin is chemically known as (2S, 3R, 4R, 5S, 6R)-2-[4-chloro-3-[(4-ethoxyphenyl) methyl] phenyls]-6-(hydroxymethyl) oxane-3,4,5-triol; (2S)-propane-1,2-diol; hydrate. Dapagliflozin is a C-glycosyl comprising beta-D-glucose in which the anomeric hydroxy group is replaced by a 4-chloro-3-(4-ethoxybenzyl)phenyl group. Used (in the form of its propanediol monohydrate) to improve glycemic control, along with diet and exercise, in adults with type 2 diabetes. It has a role as a hypoglycemic agent and a sodium-glucose transport protein subtype 2 inhibitor. It is a C-glycosyl compound, an aromatic ether and a member of monochlorobenzenes. Linagliptin is chemically known as 8-[(3R)-3-Aminopiperidin-1yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl) methyl]-3,7-dihydro-1H-purine-2,6-dione. It is a dipeptidyl peptidase-4 (DPP-4) inhibitor which is used in combination with diet and exercise in the therapy of type 2 diabetes, either alone or combination with oral hypoglycemic agents. Linagliptin has been link to rare instances of clinically apparent liver injury. Metformin is chemically known as 3-(diaminothylidene)-1,1-dimethylguanidine. It is a biguanide antihyperglycemic agent and first-line pharmacotherapy used in the management of type 2 diabetes. It is considered an antihyperglycemic drug because it lower blood glucose concentrations in type 2 diabetes without causing hypoglycaemia. It is commonly described as an “insulin sensitizer”, leading to a decrease in insulin resistance and a clinically significant reduction of plasma fasting insulin levels. Another well-known benefit of this drug is modest weight loss, making it an effective choice for obese patients type 2 diabetes.

Physical And Chemical Properties ^(2-4)

Dapagliflozin is white to off-white solid powder. IUPAC name of Dapagliflozin is (2S, 3R, 4R, 5S, 6R)-2-[4-chloro-3-[(4-ethoxyphenyl) methyl] phenyls]-6-(hydroxymethyl) oxane-3,4,5-triol; (2S)-propane-1,2-diol; hydrate. Chemical formula of Dapagliflozin is C₁₂H₂₅ClO₆. Molecular weight is 408.87 gm/mol. It is soluble in ethanol (95 %), slightly soluble in water and practically insoluble in hexane. The chemical structure of Dapagliflozin is shown in Figure 1. Linagliptin is white to yellowish, crystalline powder. IUPAC name of Linagliptin is 8-[(3R)-3-Aminopiperidin-1yl]-7-(but-2-yn-1-yl)-3-methyl-1-[(4-methylquinazolin-2-yl) methyl]-3,7-dihydro-1H-purine-2,6-dione. Chemical formula of Linagliptin is C₂₁H₂₅ClO₆. Molecular weight is 472.54 gm/mol. It is very slightly soluble in water, soluble in methanol and sparingly soluble in ethanol. The chemical structure of Linagliptin is shown in Figure 2. Metformin is white to yellowish, crystalline powder. IUPAC name of Metformin is 3-(diaminothylidene)-1,1-dimethylguanidine. Chemical formula of Metformin is C₄H₁₁N₅. Molecular weight is 165.62 gm/mol. It is freely soluble in water, slightly soluble in alcohol and practically insoluble in ether, chloroform, acetone, methylene chloride. The chemical structure of Metformin is shown in figure 3. 

Figure 1: Structure of Dapagliflozin

Figure 2: Structure of Linagliptin

Figure 3: Structure of Metformin

Mechanism Of Action ^(5-7)

Dapagliflozin: The drug dapagliflozin is a member of the sodium-glucose co-transporter 2 (SGLT2) inhibitor class and is mostly used to treat chronic kidney disease, heart failure, and type 2 diabetes. Its primary mode of action is inhibiting the SGLT2 protein, which is found in the kidneys' proximal convoluted tubule of the nephron. About 90% of the glucose that the glomeruli filter into the bloodstream is reabsorbed by SGLT2 under normal physiological conditions. Dapagliflozin lowers glucose reabsorption by specifically blocking this transporter, which increases glucose excretion in the urine (glycosuria) and lowers blood glucose levels as a result. The medication works even in insulin-resistant conditions because of its action, which is not dependent on insulin. Some people may lose weight as a result of the calories lost from the excretion of glucose in the urine. Moreover, osmotic diuresis brought on by glucose in the urine increases the excretion of salt and water. This diuretic impact helps lower blood pressure and plasma volume, which is especially helpful for heart failure patients. Dapagliflozin also enhances tubuloglomerular feedback and decreases glomerular hyperfiltration in the kidneys, two processes that are thought to contribute to its nephroprotective benefits. Dapagliflozin is a useful treatment choice for cardiovascular and renal protection because of these qualities, which also make it an excellent antihyperglycemic medication.

Linagliptin: An oral antidiabetic medication called linagliptin is used to treat type 2 diabetes. It is a member of the class of drugs known as Dipeptidyl Peptidase-4 (DPP-4) inhibitors, which function by improving the body's innate capacity to control blood sugar. Hormones known as incretins, primarily glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), are released by the gut after a meal and are essential for preserving glucose homeostasis. In a glucose-dependent way, these hormones cause pancreatic β-cells to secrete more insulin and α-cells to secrete less glucagon. However, the DPP-4 enzyme quickly breaks down incretins, reducing their effectiveness. Linagliptin increases and prolongs the levels of active GLP-1 and GIP by inhibiting the DPP-4 enzyme. When blood glucose levels are raised, this leads to increased insulin production and decreased glucagon release, which eventually lowers fasting and postprandial glucose levels. One of linagliptin's noteworthy characteristics is its glucose-dependent mode of action, which considerably lowers the risk of hypoglycaemia, particularly when taken alone without the use of insulin or sulfonylureas. Because linagliptin is mainly eliminated through the biliary and intestinal pathways rather than the kidneys, it differs from other DPP-4 inhibitors in terms of pharmacokinetics and does not necessitate dose modification in individuals with renal impairment. Because of its lengthy terminal half-life, once-daily dosage is convenient. Overall, linagliptin has a good safety and pharmacokinetic profile and enhances the body's endogenous incretin system to improve glucose control.

Metformin: Because of its efficacy, safety record, and minimal risk of hypoglycemia, metformin is a commonly used oral antidiabetic medication that is used as the first line of treatment for type 2 diabetes mellitus. Its main mode of action is lowering the amount of glucose produced by the liver, namely by preventing gluconeogenesis. The energy-sensing enzyme AMP-activated protein kinase (AMPK) is primarily responsible for this impact. Metformin mildly inhibits mitochondrial respiratory-chain complex I, which raises the intracellular AMP/ATP ratio and indirectly activates AMPK. The expression of important gluconeogenic enzymes like glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) is subsequently downregulated by activated AMPK, which significantly reduces the liver's production of glucose. Furthermore, through AMPK-independent mechanisms such inhibition of mitochondrial glycerophosphate dehydrogenase, which modifies the redox state inside hepatocytes and restricts the availability of substrates required for gluconeogenesis, metformin influences the synthesis of glucose in the liver. In addition to its effects on the liver, metformin increases peripheral insulin sensitivity, particularly in skeletal muscle and adipose tissue, which enhances the absorption and use of glucose. By enhancing insulin receptor signalling and facilitating the translocation of GLUT-4 glucose transporters to the cell membrane, it lowers insulin resistance. Metformin has significant impacts on the gastrointestinal system as well. It changes the gut microbiota's composition, delays intestinal glucose absorption, and increases the production of incretin hormones like GLP-1, which can help regulate blood sugar levels by encouraging the secretion of insulin and suppressing the release of glucagon in a glucose-dependent way. Interestingly, metformin has a low risk of producing hypoglycaemia when taken alone because it does not directly stimulate insulin secretion.

Table 1: Summary of Mechanism of Action

Figure 4: Mechanism of action of Dapagliflozin

Figure 5: Mechanism of action of Linagliptin

Figure 6: Mechanism of action of Metformin

Analytical Method Development ^(8-9)

A crucial step in chemical and pharmaceutical research is the development of analytical methods, which entails creating, refining, and confirming trustworthy techniques for identifying, detecting, and quantifying chemicals in a sample. This procedure guarantees the method's robustness, linearity, specificity, accuracy, and precision for the intended usage. Here, some key points for analytical method development:

Analytical technique selection is based on the analyte's physical and chemical characteristics, such as its solubility, volatility, and polarity.
Parameter optimization includes things like temperature, pH, flow rate, detection wavelength, and composition of the mobile phase.

Validation: Complies with ICH Q2(R1) standards, evaluating metrics like linearity, accuracy, precision, robustness, specificity, LOD, and LOQ.

Types of Methods Developed: These could include titration, gas chromatography, UV-Vis spectrophotometry, HPLC, and others.

Use: For impurity profiling, stability research, bioequivalence, dissolution testing, and quality control.

Regulatory Importance: Safe product development and regulatory compliance are guaranteed by appropriate technique development.

Literature Review:

Dapagliflozin and Linagliptin are currently not official in any major pharmacopoeia, although Metformin is an official medicine listed in various pharmacopoeias, including the Indian Pharmacopoeia (IP), United States Pharmacopeia (USP), and British Pharmacopoeia (BP). Numerous analytical techniques, including HPLC, UV-VIS spectrophotometry, and LC-MS, have been reported in the literature for the estimation of each drug separately as well as for their binary combinations, such as Dapagliflozin + Linagliptin, Linagliptin + Metformin, and Dapagliflozin + Metformin. However, there is currently no published or approved analytical technique for the simultaneous estimation of metformin, dapagliflozin, and linagliptin in a single formulation.

Table 2: Reported method for Dapagliflozin

Table 3: Reported Method For Linagliptin

Table 4: Reported method for Metformin

Table 5: Reported method for Dapagliflozin and Metformin

Table 6: Reported method for Dapagliflozin and Linagliptin

Table 7: Reported method for Metformin and Linagliptin