title: Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry.
abstract: Phagocytes, the physiological compartment in which Leishmania parasites reside, are the main site of action of the drug miltefosine, but the intracellular pharmacokinetics of miltefosine remain unexplored. We developed a bioanalytical method to quantify miltefosine in human peripheral blood mononuclear cells (PBMCs), expanding from an existing high performance liquid chromatography-tandem mass spectrometry method for the quantification of miltefosine in plasma. The method introduced deuterated miltefosine as an internal standard. Miltefosine was extracted from PBMC pellets by addition of 62.5% methanol. Supernatant was collected, evaporated and reconstituted in plasma. Chromatographic separation was performed on a reversed phase C18 column and detection with a triple-quadrupole mass spectrometer. Miltefosine was quantified using plasma calibration standards ranging from 4 to 1000ng/mL. This method was validated with respect to its PBMC matrix effect, selectivity, recovery and stability. No matrix effect could be observed from the PBMC content (ranging from 0.17 to 26.3×10(6)PBMCs) reconstituted in plasma, as quality control samples were within 3.0% of the nominal concentration (precision less than 7.7%). At the lower limit of quantitation of 4 ng/mL plasma, corresponding to 0.12ng/10(6) PBMCs in a typical clinical sample, measured concentrations were within 8.6% of the nominal value. Recovery showed to be reproducible as adding additional pre-treatment steps did not increase the recovery with more than 9%. This method was successfully applied to measure intracellular miltefosine concentrations in PBMC samples from six cutaneous leishmaniasis patients up to one month post-treatment.
authors.affiliation: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital/Slotervaart Hospital, Amsterdam, The Netherlands; Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Utrecht, The Netherlands. Electronic address: anke.kip@slz.nl.
authors.forename: A E
authors.initials: AE
authors.lastname: Kip
authors.affiliation: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital/Slotervaart Hospital, Amsterdam, The Netherlands.
authors.forename: H
authors.initials: H
authors.lastname: Rosing
authors.affiliation: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital/Slotervaart Hospital, Amsterdam, The Netherlands.
authors.forename: M J X
authors.initials: MJ
authors.lastname: Hillebrand
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM), Cali, Colombia.
authors.forename: M M
authors.initials: MM
authors.lastname: Castro
authors.affiliation: Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM), Cali, Colombia.
authors.forename: M A
authors.initials: MA
authors.lastname: Gomez
authors.affiliation: Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Utrecht, The Netherlands; Department of Clinical Pharmacology, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
authors.forename: J H M
authors.initials: JH
authors.lastname: Schellens
authors.affiliation: Department of Pharmacy & Pharmacology, Antoni van Leeuwenhoek Hospital/Slotervaart Hospital, Amsterdam, The Netherlands; Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Utrecht, The Netherlands; Department of Clinical Pharmacology, Antoni van Leeuwenhoek Hospital, Amsterdam, The Netherlands.
authors.forename: J H
authors.initials: JH
authors.lastname: Beijnen
authors.affiliation: Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences (UIPS), Faculty of Science, Utrecht University, Utrecht, The Netherlands.
authors.forename: T P C
authors.initials: TP
authors.lastname: Dorlo
mesh_terms: D000981:Antiprotozoal Agents / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000032:analysis; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000981:Antiprotozoal Agents; D010767:Phosphorylcholine; C039128:miltefosine