Simple Cost-Effective Stability Indicating HPLC Method For Simultaneous Estimation of Linagliptin and Dapagliflozin in Marketed Formulation

Abstract

The development of a robust and reliable RP-HPLC method for the simultaneous quantification of Linagliptin (LGT) and Dapagliflozin (DGF) required systematic optimization of mobile phase composition, chromatographic conditions, and instrumental parameters to achieve ideal resolution, peak symmetry, reproducibility, and sensitivity. Initially, several combinations of aqueous buffer systems and organic solvents were screened in varying proportions to determine a suitable mobile phase that would ensure selective separation of both analytes. Different ratios of methanol, acetonitrile, phosphate buffers, and acidic modifiers such as orthophosphoric acid (OPA) were evaluated. However, many of these initial trials resulted in overlapping peaks, broad peak shapes, excessive tailing, or unsatisfactory retention characteristics. The difficulty in optimizing the method stemmed from the structural and physicochemical differences between LGT and DGF; Linagliptin is more polar and elutes earlier under reversed-phase conditions, whereas Dapagliflozin is comparatively less polar and requires stronger elution strength. After extensive method development trials, the mobile phase comprising 25 mM potassium dihydrogen phosphate (KH?PO?) buffer and acetonitrile in the ratio of 22:78 v/v, with the buffer pH adjusted to 3.5 using orthophosphoric acid (OPA), was found to be the most effective in providing well-resolved, sharp, and symmetrical peaks for both drugs. A variation of ±10% in the ratio was also tested to evaluate robustness, and results consistently demonstrated that the 22:78 v/v composition offered optimal resolution and peak characteristics without significant deviation in retention time or peak area. This mobile phase resulted in shorter run times, improved peak shapes, and enhanced reproducibility, making it ideal for routine analysis. Before use, the mobile phase was filtered through a 0.45 ?m membrane filter to eliminate particulate matter and degassed using sonication to prevent bubble formation and baseline noise during chromatographic runs.

Keywords

Introduction

Table 1: Selection of mobile phase

Linearity and Calibration Graph

Table 2: Linearity of LGT

Standard Concentration (µg/ml)	Area Under Curve (AUC)						Mean
	Rep-1	Rep-2	Rep-3	Rep-4	Rep-5	Rep-6
1	224.845	226.784	235.156	227.452	231.684	235.879	230.300
2	442.685	449.315	454.798	437.542	450.122	447.968	447.072
3	659.874	667.218	650.684	658.312	646.587	672.843	659.253
4	884.963	885.742	890.568	894.684	877.945	879.384	885.548
5	1093.452	1089.874	1084.657	1095.786	1079.865	1091.652	1089.881

Figure 1: Calibration Curve of LGT

Figure 2: Chromatogram of LGT

Table 3: Linearity of DGF

Standard Conc. (µg/ml)	Area Under Curve (AUC)						Mean
	Rep-1	Rep-2	Rep-3	Rep-4	Rep-5	Rep-6
5	790.265	785.432	781.564	776.845	779.624	767.458	780.865
10	1556.874	1528.436	1522.985	1532.478	1547.215	1578.968	1544.826
15	2376.542	2352.864	2346.587	2341.478	2352.698	2342.985	2352.526
20	3161.745	3156.894	3146.325	3141.856	3140.985	3142.658	3148.744
25	3952.475	3946.352	3942.685	3953.864	3948.256	3941.795	3947.905

Figure 3: Calibration Curve of DGF

Figure 4: Chromatogram of DGF

CONCLUSION: - The development of a robust and reliable RP-HPLC method for the simultaneous quantification of Linagliptin (LGT) and Dapagliflozin (DGF) required systematic optimization of mobile phase composition, chromatographic conditions, and instrumental parameters to achieve ideal resolution, peak symmetry, reproducibility, and sensitivity. Initially, several combinations of aqueous buffer systems and organic solvents were screened in varying proportions to determine a suitable mobile phase that would ensure selective separation of both analytes. Different ratios of methanol, acetonitrile, phosphate buffers, and acidic modifiers such as orthophosphoric acid (OPA) were evaluated. However, many of these initial trials resulted in overlapping peaks, broad peak shapes, excessive tailing, or unsatisfactory retention characteristics. The difficulty in optimizing the method stemmed from the structural and physicochemical differences between LGT and DGF; Linagliptin is more polar and elutes earlier under reversed-phase conditions, whereas Dapagliflozin is comparatively less polar and requires stronger elution strength.

Overall, the optimized RP-HPLC method successfully fulfilled all criteria for a validated analytical method, demonstrating excellent peak resolution, retention reproducibility, system suitability, and analytical sensitivity for simultaneous estimation of Linagliptin and Dapagliflozin. The use of a C18 stationary phase, buffer–organic mobile phase, and UV detection at 254 nm resulted in an efficient, reliable, and fast method suitable for analytical quality control, routine pharmaceutical analysis, and stability studies. The carefully optimized chromatographic conditions ensure that the method can be confidently employed in research, pharmaceutical industries, and regulatory environments for accurate estimation of these antidiabetic agents.

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