High-performance liquid chromatographic analysis of amphotericin B in rat plasma using α-naphthol as an internal standard Original Research Article

A simple, sensitive and accurate reverse phase high-performance liquid chromatographic (RP-HPLC) method with photo-diode array detector (PDA) was developed and validated for the determination of amphotericin B (AMB) in the rat plasma using a new internal standard (IS) α-naphthol. The plasma samples were subjected to protein precipitation with methanol prior to a HPLC analysis. Chromatographic separations were achieved on a Nucleosil® 100-5C18 (150 mm × 4.6 mm) column. The mobile phase consisted of acetonitrile and sodium acetate buffer (pH 4; 10 mM) in a gradient mode. Detection was carried out at a wavelength of 407 and 294 nm for AMB and IS, respectively. The retention times of AMB and IS were about 6.8 and 7.8 min, respectively. The calibration curve was linear in the range of 10–2000 ng mL−1 for AMB (r2 > 0.998). No significant matrix effect was observed on quantification of AMB or IS. At three quality control concentrations of 20, 500, and 2000 ng mL−1, the intra-day and inter-day relative standard deviation ranged from 1.13% to 4.91%. The limit of detection (LOD) was 5 ng mL−1 and the limit of quantification (LOQ) was 10 ng mL−1 for AMB in rat plasma. This method is simple, sensitive, rapid and does not require any extensive sample purification before injecting into HPLC.