Development of a UHPLC-MS/MS method for the quantification of DMPC and DMPG in rats and its pharmacokinetic and tissue distribution application

The aim of this study was to develop and fully validate a sensitive and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous quantification of Dimyristoyl phosphatidylcholine (DMPC) and Dimyristoyl phosphatidylglycerol (DMPG) in biological matrices of rats after intravenous administration of amphotericin B phospholipid complex suspension injection and excipients (DMPC and DMPG). DMPC, DMPG and Loratadine (internal standard, IS) were separated on an Agilent ZORBAX Extend-C18 column (2.1 mm × 50 mm, 3.5 μm particle size) by using gradient elution consisting of isopropanol-acetonitrile (90:10, v/v, 10 mM ammonium formate and 0.1 % formic acid) and water (10 mM ammonium formate and 0.1 % formic acid) at a flow rate of 0.3 mL/min in 4.0 min. Multiple reaction monitoring (MRM) mode was performed to quantify data under monitoring precursor-product ion transitions of m/z 678.9→184.0 (DMPC, ESI+), 665.5→227.2 (DMPG, ESI-) and 383.0→337.1 (loratadine, IS, ESI+), respectively. The method was developed at linearity ranging from 10.0 to 1000 ng/mL for all analytes. The inter-day accuracy of DMPC and DMPG was observed to range between -12.8-12.7 %, while the precision was found to be within 15.0 %. DMPC and DMPG demonstrated stability during sample storage, preparation and analytic procedures. Furthermore, this method was successfully applied in the investigation of the pharmacokinetic profile and tissue distribution of DMPC and DMPG in rats after intravenous administration. Dose-dependent increases were observed for DMPC and DMPG in both areas under the curve (AUC) and maximum plasma concentration (Cmax), demonstrating positive correlations across all tested dose levels. The biological half-lives (t₁/₂) of DMPC and DMPG following intravenous administration of an amphotericin B phospholipid complex suspension were 1.2-3.4 h and 4.6-8.1 h, respectively. However, after intravenous administration of the excipients, their half-lives were 3.5 h and 1.1-2.3 h. The observed prolongation of DMPC's half-life and concomitant shortening of DMPG's half-life are attributed to the distinct physicochemical properties of the nanoparticles.

DMPC; DMPG; Pharmacokinetics; Tissue distribution; UHPLC-MS/MS.