Determination of Encapsulated and Unencapsulated Amphotericin B in Dog Plasma by LC–MS/MS Coupled With a Simple and Efficient Solid‐Phase Extraction: Application to a Pharmacokinetic Study of Liposomal Amphotericin B

A convenient and reliable SPE method coupled with a specific and sensitive LC-MS/MS technique was developed and validated for the separation and determination of unencapsulated amphotericin B (F-AMB) and encapsulated amphotericin B (L-AMB) in dog plasma. L-AMB and F-AMB in the biomatrix were simultaneously separated by Oasis HLB SPE column using natamycin as the internal standard (IS). Chromatographic separation was achieved on a ZORBAX Eclipse XDB C18 column with gradient elution at a flow rate of 0.5 mL/min. The mobile phase consisted of methanol (0.1% formic acid) and a 5-mM ammonium acetate solution (0.1% formic acid). Mass spectrometry detection was performed in positive ion mode with an electrospray ionization source. Apart from regular quality control (QC) samples, a series of cross-QCs was adopted to verify the specificity and reproducibility of the quantification. We showed that F-AMB and L-AMB were completely separated without mutual interference in the quantitative linearity ranges of F-AMB and L-AMB, which were 10.0-800 and 100-25,000 ng/mL, respectively. The method was then applied to a pharmacokinetic study of liposomal amphotericin B in beagle dogs, and excellent ISR results were obtained for both L-AMB and F-AMB assays.