Simultaneous Determination of Amlodipine and Naringin in Rat Plasma by LC-MS: Method Development and Validation

Naringin, a citrus flavanone glycoside, inhibits CYP3A4 and P-glycoprotein, making it a clinically relevant modulator of drug bioavailability. Amlodipine, a widely prescribed calcium channel blocker, metabolised by CYP3A4, is susceptible to pharmacokinetic interactions with citrus-derived flavonoids. A LC-MS bioanalytical method was developed and fully validated for simultaneous quantification of amlodipine and naringin in rat plasma per ICH M10(2022) guidelines. Separation was achieved on a Waters C18 column (250 * 4.6 mm, 3 μm) with gradient elution using 10 mM ammonium acetate (pH 3.0) and acetonitrile. Detection employed positive electrospray ionisation at m/z 409.02,579.2 and 271.1 for amlodipine, naringin and naringenin (internal standard), respectively. Protein precipitation by addition of acetonitrile was employed for sample preparation. Calibration curves were linear over 24.01-2020.6 ng/mL (amlodipine) and 94.01-2012 ng/mL (naringin). Precision (%CV ≤ 15%), accuracy (85%-115%), and stability under all tested conditions met acceptance criteria. The validated method was utilized to analyse the non-compartmental pharmacokinetics study in Wistar albino rats after oral amlodipine (1 mg/kg), naringin (15 mg/kg), and their combination. Co-administration led to increased amlodipine C_max (~2.2-fold; p < 0.01), AUC 0-∞ (~2.0-fold; p < 0.01), and decreased oral clearance. This is the first simultaneous LC-MS approach in conformity with ICH M10 (202) that provides a reliable preclinical citrus flavonoid-drug interaction tool.