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

• Published in: Analytica chimica acta Vol. 634; no. 1; pp. 110 - 114
• Main Authors: Italia, J.L., Singh, D., Ravi Kumar, M.N.V.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 16-02-2009; Elsevier
• Subjects: Amphotericin B; Amphotericin B - blood; Analytical chemistry; Animals; Calibration; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid - methods; Exact sciences and technology; Naphthols - chemistry; Other chromatographic methods; Plasma; Rats; Reference Standards; Reverse phase; Sensitivity and Specificity; α-Naphthol; α-Naphthol; Plasma; Reverse phase; Amphotericin B; Rat; Internal standard; HPLC chromatography; Retention time; Precipitation; Detection limit; Standard curve; Diode array; Standard deviation; Quantitative analysis; Methanol; Purification; Rodentia; Matrix effect; Protein; Vertebrata; Reversed phase chromatography; Mammalia; Quality control; Naphthol; Sensor array
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2008.12.006

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 (
r
2
>
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.