The triple combination is becoming increasingly common in the management of type 2 diabetes, and for good reason. Each drug attacks high blood sugar through a different biological route: metformin suppresses glucose production in the liver and improves insulin sensitivity, gliclazide prods the pancreas to release more insulin, and dapagliflozin forces the kidneys to flush excess glucose out through the urine. Combining them in a single pill improves patient compliance and delivers better glycaemic control than any one agent alone. But that therapeutic synergy creates an analytical headache, because a quality-control laboratory must be able to verify the exact amount of each ingredient in every batch before the tablets reach patients.

The difficulty lies in chemistry. Metformin is a small, strongly polar, highly hydrophilic molecule with a log P value of roughly minus 2.64, which means it barely interacts with the oily, hydrophobic surface of a reverse-phase column and tends to zip through unretained. Dapagliflozin and gliclazide, by contrast, are far more lipophilic, with log P values around 2 or higher, so they cling to the stationary phase much longer. On top of that, the dose disparity is enormous: commercial tablets contain 500 to 1000 milligrams of metformin, 40 to 80 milligrams of gliclazide, and a mere 5 to 10 milligrams of dapagliflozin, so any single method must accurately quantify compounds whose concentrations in solution differ by orders of magnitude.

Led by Rajendra Patel, with Nishith Teraiya and Anjali Prajapati, all of the K.B. Institute of Pharmaceutical Education and Research in Gandhinagar, the team systematically tuned every variable that governs chromatographic behavior. They tested water-methanol and water-acetonitrile mixtures in various ratios, but unbuffered systems produced broad, asymmetrical peaks with poor resolution. The breakthrough came from adding a volatile 20 millimolar ammonium acetate buffer adjusted to pH 3.0 with ortho-phosphoric acid. At that acidity, metformin remains fully protonated and water-loving, so it elutes first, while the more lipophilic dapagliflozin and gliclazide linger progressively longer on the hydrophobic C18 stationary phase, producing clean, well-resolved peaks.

The final recipe is elegantly simple. Separation occurs on a Kromasil C18 column, 250 millimeters long with a 4.6 millimeter internal diameter and 5 micrometer particles, using a mobile phase of the acetate buffer and acetonitrile mixed 35:65 by volume, pumped at a constant 0.8 milliliters per minute in isocratic mode. A UV detector set at 224 nanometers, a wavelength where the absorption spectra of all three drugs overlap acceptably, monitors the eluate. Under these conditions metformin appears at 2.47 minutes, dapagliflozin at 3.90 minutes, and gliclazide at 6.46 minutes. Because the solvent composition never changes during the run, the method avoids the complexity, variability, and extra solvent consumption of gradient elution, keeping both analysis time and operating costs low.

Validation followed the International Council for Harmonisation’s Q2 (R2) guidelines, the global benchmark for analytical procedures. Calibration curves were linear across 125 to 375 micrograms per milliliter for metformin, 2.5 to 7.5 for dapagliflozin, and 15 to 45 for gliclazide, with correlation coefficients all above 0.997. Recovery experiments, in which known amounts of standard drug were spiked into tablet samples at 80, 100, and 120 percent of target, yielded recoveries between 98 and 102 percent for every analyte, demonstrating negligible interference from tablet excipients. Precision testing, repeated within a single day and across three separate days, produced relative standard deviations consistently below 2 percent, the accepted threshold for repeatability in pharmaceutical analysis.

The robustness trials are particularly reassuring for routine laboratories. The researchers deliberately nudged the method’s settings, slightly altering mobile phase composition, flow rate, detection wavelength, buffer pH, and column temperature, and found that the separation held up every time, with peaks remaining symmetrical, well resolved, and statistically unchanged. Sample solutions stored for 48 hours at room temperature and at refrigerator temperature showed no degradation and no loss of assay value, meaning laboratories can prepare samples in advance without compromising results. Limits of detection and quantification, calculated from the regression statistics of the calibration lines, confirmed the method is sensitive enough for the low-dose dapagliflozin component.

The most dramatic evidence for the method’s stability-indicating power came from forced degradation studies, which regulators require to prove that an analytical method can spot decomposition before it spots nothing at all. The team exposed the drugs to 0.1 normal hydrochloric acid for one hour, 0.1 normal sodium hydroxide for two hours, 3 percent hydrogen peroxide for one hour, 40 degrees Celsius heat for one hour, and direct sunlight for two hours. Each stressor told a chemical story: gliclazide degraded most under acid, consistent with hydrolysis of its sulfonylurea linkage; dapagliflozin proved most vulnerable to light, its aromatic glucoside structure susceptible to photo-oxidation; and metformin, shielded by its highly polar biguanide scaffold, stayed comparatively stable even under heat. Crucially, in every case the degradation products eluted as separate peaks that never overlapped the three drug peaks.

Applied to a real commercial tablet containing 10 milligrams of dapagliflozin, 60 milligrams of gliclazide, and 500 milligrams of metformin, the method returned assay values of 101.52, 98.19, and 99.64 percent respectively, comfortably within pharmacopoeial expectations and confirming that the technique works outside the pristine world of pure standards. That real-world performance matters because regulators and manufacturers increasingly rely on such methods for batch release, shelf-life determination, and post-market surveillance. A method that is simultaneously accurate, robust, and stability-indicating gives quality-control chemists a single, streamlined workflow where they previously might have needed multiple runs or separate methods for each drug pair.

The broader significance extends past one formulation. As fixed-dose combinations multiply across modern pharmacotherapy, the analytical community faces growing pressure to verify ever more complex mixtures with fewer resources. This study demonstrates that careful exploitation of fundamental properties, lipophilicity, ionization state, and spectral overlap, can tame even a three-drug combination spanning a hundredfold dose range in a simple isocratic run. For the millions of patients taking triple antidiabetic therapy, the work represents an invisible but vital layer of assurance: every tablet’s contents can now be confirmed, and its chemical deterioration detected, in under seven minutes of chromatography.

Subject of Research: Development and validation of a stability-indicating RP-HPLC method for simultaneous quantification of dapagliflozin, gliclazide, and metformin in antidiabetic combination tablets

Article Title: Development of a robust stability-indicating RP-HPLC method for simultaneous estimation of triple antidiabetic combination containing dapagliflozin, gliclazide and metformin

Article References: Patel, R., Teraiya, N., & Prajapati, A. (2026). Development of a robust stability-indicating RP-HPLC method for simultaneous estimation of triple antidiabetic combination containing dapagliflozin, gliclazide and metformin. Discover Chemistry, 3(1), Article 518. https://doi.org/10.1007/s44371-026-00978-x

Image Credits: AI Generated

DOI: 10.1007/s44371-026-00978-x

Keywords: RP-HPLC, dapagliflozin, gliclazide, metformin, type 2 diabetes, stability-indicating method, forced degradation, pharmaceutical analysis, ICH Q2 (R2), chromatography, quality control, fixed-dose combination