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

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.