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A convenient and rapid LC-MS/MS method for determination of free and liposomal amphotericin B in human plasma by simultaneous separation using SPE.

Yi Jin,Bowen Wu,Yuting Gong,Huanyu Wei,R. Ma,Yue Wang,Min Yuan,Haiyan Xu

Published 2025 in Journal of Pharmaceutical and Biomedical Analysis

ABSTRACT

Effective separation and specific detection of free and encapsulated drugs in bio-samples are critical to the pharmacokinetic investigation of liposomal medicine. In this study, a simple, convenient, reliable, and selective SPE separation method coupled with sensitive LC-MS/MS technique was developed and fully validated for the detection of liposomal amphotericin B (L-AMB) and non-liposomal amphotericin B (F-AMB) in human plasma. The simultaneous separation between L-AMB and F-AMB in plasma were realized using Oasis HLB SPE cartridge. L-AMB was collected in the aqueous eluate and F-AMB was eluted from the cartridge by methanol containing 1 % formic acid. The analyte and internal standard (natamycin) were separated on a ZORBAX Eclise XDB C18 column (2.1 × 50 mm, 3.5 μm) with gradient elution at a flow rate of 0.5 mL/min, employing a mobile phase that consisted of methanol (0.1 % formic acid) and 5 mM ammonium acetate solution (0.1 % formic acid). Mass spectrometry detection was performed in positive ion mode with electrospray ionization (ESI) interface by multiple reaction monitoring (MRM) method. The linearity range was 200-50000 ng/mL for L-AMB and 10.0-1600 ng/mL for F-AMB. A series of cross quality control samples was adopted to verify the selectivity, stability, and reproducibly of the quantification. Using our methodology, we quantified F-AMB and L-AMB without mutual interferences and obtained excellent incurred sample reanalysis (ISR) results for both analytes. The method was also successfully applied to a clinical study in healthy volunteers.

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