Abstract

Furosemide was a loop diuretic drug with extensive use for the treatment of various indications so many copy products had been manufactured. A copy product of furosemide should pass bioequivalence requirements which need a validated analytical method, but the previous methods still too complicated. For this reason, a simple and rapid RP-HPLC method was developed and validated for quantifying furosemide in human plasma. Chromatographic separation was performed under isocratic elution on Luna Phenomenex® C18 (250 x 4.6 mm, 5 μm) column. The mobile phase was comprised of methanol, acetonitrile, and phosphate buffer 20 mM pH 3.0 (10:30:60 v/v/v) and pumped at flow rate of 1.6 mL/min. The UV detector was operated at 233 nm. Protein precipitation was conducted using methanol and propranolol was used as an internal standard. This method was validated according to the FDA guidelines. Calibration curve were linear (r = 0.9997) in the ranges of 400-1400 ng/mL. The lower limit of quantification (LLOQ) was established at 400 ng/mL with errors and RSD at this concentration did not exceed 20% and 8% respectively. The limit of detection (LOD) was found at concentration of 100 ng/mL. The within-run and between-run precision did not exceed 10%, whereas the errors did not exceed 6% at concentration above LLOQ. So the method was proved reliable for quantifying furosemide in human plasma.