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Rapid and Sensitive High Performance Liquid Chromatography Method for The Determination of Amphotericin B in Rat Plasma

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Author(s): Uday S Chakrabarty1, and Tapan K Pal*

Journal: Journal of Pharmacy Research
ISSN 0974-6943

Volume: 4;
Issue: 9;
Start page: 3194;
Date: 2011;
Original page

Keywords: Amphotericin B | Internal standard | Rat plasma | HPLC

ABSTRACT
A simple and sensitive reverse phase high-performance liquid chromatographic (RP-HPLC) method for the determination of amphotericin B (AMB) in ratplasma has been developed. An aliquot quantity of 225 μL plasma sample was taken and 25 μL internal standard (IS) was added to it and mixed well. 250 μLmethanol was added to it and sonicated for 10 minutes followed by 20 minutes centrifugation at 5000 rpm. The supernatant layer was separated and filteredthrough sample filtration unit (membrane filter, 0. 45 μm) and injected into the HPLC system consisting of Hypersil ODS, C18 (250×4.6 mm, 5 μm particlesize) column, using 10 mmol phosphate buffer : acetonitrile = 65:35 (v/v) as mobile phase and detected at 407 nm. Lower limit of detection (LOD) was 3 ng/mL and lower limit of quantitation (LOQ) was 10 ng/mL. Maximum between-run precision was 1.973%. Mean extraction recovery was found to be 91.764%to 96.457%. Stability study showed that after three freeze-thaw cycles the loss of three quality control samples were less than 15%. The processed samples werestable for at least 8 h. The method is simple, sensitive and rapid and applicable for pharmacokinetic study of amphotericin B formulation in rat model.
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