title: Validation and Clinical Evaluation of a Novel Method To Measure Miltefosine in Leishmaniasis Patients Using Dried Blood Spot Sample Collection.
abstract: To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote regions in which the disease is endemic, a simple cheap sampling method is required for miltefosine quantification. The aims of this study were to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spot (DBS) samples and to validate its use with Ethiopian patients with visceral leishmaniasis (VL). Since hematocrit (Ht) levels are typically severely decreased in VL patients, returning to normal during treatment, the method was evaluated over a range of clinically relevant Ht values. Miltefosine was extracted from DBS samples using a simple method of pretreatment with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10 to 2,000 ng/ml, and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at the lower limit of quantification), respectively. The method was accurate and precise for blood spot volumes between 10 and 30 μl and for Ht levels of 20 to 35%, although a linear effect of Ht levels on miltefosine quantification was observed in the bioanalytical validation. DBS samples were stable for at least 162 days at 37°C. Clinical validation of the method using paired DBS and plasma samples from 16 VL patients showed a median observed DBS/plasma miltefosine concentration ratio of 0.99, with good correlation (Pearson'sr= 0.946). Correcting for patient-specific Ht levels did not further improve the concordance between the sampling methods. This successfully validated method to quantify miltefosine in DBS samples was demonstrated to be a valid and practical alternative to venous blood sampling that can be applied in future miltefosine pharmacokinetic studies with leishmaniasis patients, without Ht correction.
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, Faculty of Science, Utrecht University, Utrecht, the Netherlands anke.kip@slz.nl t.p.c.dorlo@uu.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: Drugs for Neglected Diseases Initiative, Geneva, Switzerland.
authors.forename: S
authors.initials: S
authors.lastname: Blesson
authors.affiliation: Leishmaniasis Research and Treatment Center, University of Gondar, Gondar, Ethiopia.
authors.forename: B
authors.initials: B
authors.lastname: Mengesha
authors.affiliation: Department of Internal Medicine, University of Gondar, Gondar, Ethiopia.
authors.forename: E
authors.initials: E
authors.lastname: Diro
authors.affiliation: Department of Microbiology, Immunology, and Parasitology, School of Medicine, Addis Ababa University, Addis Ababa, Ethiopia.
authors.forename: A
authors.initials: A
authors.lastname: Hailu
authors.affiliation: Division of Pharmacoepidemiology & Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, Utrecht, the Netherlands Department of Clinical Pharmacology, Antoni van Leeuwenhoek Hospital/Netherlands Cancer Institute, 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, Faculty of Science, Utrecht University, Utrecht, the Netherlands Department of Clinical Pharmacology, Antoni van Leeuwenhoek Hospital/Netherlands Cancer Institute, 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, Faculty of Science, Utrecht University, Utrecht, the Netherlands Pharmacometrics Research Group, Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden anke.kip@slz.nl t.p.c.dorlo@uu.nl.
authors.forename: T P C
authors.initials: TP
authors.lastname: Dorlo
mesh_terms: D000981:Antiprotozoal Agents / Q000097:blood* / Q000627:therapeutic use; D002138:Calibration; D002853:Chromatography, Liquid; D060085:Coinfection; D059788:Dried Blood Spot Testing / Q000592:standards*; D004355:Drug Stability; D005002:Ethiopia; D006678:HIV / Q000502:physiology; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology; D006400:Hematocrit; D006801:Humans; D007893:Leishmania donovani / Q000187:drug effects; D007898:Leishmaniasis, Visceral / Q000097:blood / Q000188:drug therapy* / Q000469:parasitology; D057230:Limit of Detection; D059627:Liquid Phase Microextraction / Q000379:methods; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000097:blood / Q000627:therapeutic use; D053719:Tandem Mass Spectrometry
chemical_list: D000981:Antiprotozoal Agents; D010767:Phosphorylcholine; C039128:miltefosine