Development and Validation of a Stability Indicating HPLC Method for the Simultaneous Determination of Metformin, Hydrochlorothiazide and Acetylsalicylic Acid in an On-Demand Fixed-Dose Combination Dosage Form

The ever-growing novel on-demand dose combinations require innovative and resourceful analytical methods for the simultaneous assay of combined doses. A novel and efficient stability-indicating reversed-phase high performance liquid chromatographic method was developed and validated for the determination of metformin, hydrochlorothiazide and acetylsalicylic acid in a combination oral dosage form. The method was developed by assessing the impact of ion-pairing reagent, chromatographic column and gradient elution and validated according to ICH (R1) Q2 guideline and following the acceptance criteria suggested from the FDA laboratory manual for method validation and verification. Forced degradation studies were performed by subjecting the standards and samples to hydrolysis (acid and base), oxidative and thermal stress conditions. The determination was achieved with a Waters Sunfire C18 (250 × 4.6 mm i.d., 5 µm) column at ambient temperature using 5 mM sodium dihydrogen phosphate and 10 mM pentane-1-sulphonic acid sodium salt buffer pH 3.0 (solution A) and acetonitrile (solution B) as mobile phases in gradient elution at a flow rate of 0.7 mL/min and UV detection at 260 nm. The linearity was proven for the three active pharmaceutical ingredients (APIs) over a concentration range of 995–3240 µg/mL ( r 2 = 0.9985), 50–160 µg/mL ( r 2 = 0.9993) and 120–380 µg/mL ( r 2 = 0.9997), respectively. The method was accurate within 5% of recovery, precise and reproducible with RSD below 2.0%. The robustness was achieved at recovery rate of 98.0 to 102.0%. The validated method is suitable for the quantitative analysis of bulk, stability, and oral dosage form. Furthermore, this work paves the way for providing a platform of robust analytical methods for the simultaneous assay of innovative on-demand new dose combinations.