An eco-friendly bioanalytical RP-HPLC method coupled with fluorescence detection for simultaneous estimation of felodipine and metoprolol

An ecofriendly, sensitive, selective, precise, and accurate HPLC method with fluorescence detection (FD) has been developed and validated for simultaneous determination of two hypertensive drugs, felodipine and metoprolol in their pure samples, combined pharmaceutical dosage form and in spiked human plasma. Separation was carried out on Inertsil C₁₈ column (150 mm × 4.6 ID; Particle size 5 µm) and a mobile phase composed of ethanol and 30mM potassium dihydrogen phosphate buffer, adjusted to pH 2.5 using ortho-phosphoric acid (40:60, v/v) was used. A constant flow rate of 1.0 mL/min at an ambient temperature was adopted. Method validation was performed according to the ICH Q2 R2 and bioanalytical method validation was done according to FDA guidelines for determination of both drugs simultaneously in biological matrices. The method demonstrated excellent linearity over the concentration range of 0.01-1.00 µg/mL and 0.003-1.00 µg/mL that were selected with accordance to the C_max of both drugs with a correlation coefficient (r²) of 0.9998 and 0.9999 for felodipine and metoprolol, respectively in human plasma. The intra-day and inter-day precision were ≤ 2% in their pure forms and in spiked human plasma, and the accuracy was within ± 2% of the nominal concentration for both drugs in their pure forms and within ± 10% of the nominal concentration in human plasma. A statistical comparison was conducted between the proposed method and the reported method; it showed no significant difference between both methods regarding both accuracy and precision. The proposed method proved to be eco-friendly with three green assessment tools (AGREE calculator, MoGAPI, RGBfast study).