| 1. |
- Kip, A E, et al.
(author)
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Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry.
- 2015
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In: Journal of chromatography. B. - : Elsevier BV. - 1570-0232 .- 1873-376X. ; 998-999, s. 57-62
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Journal article (peer-reviewed)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.
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| 2. |
- Kip, A E, et al.
(author)
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Validation and clinical evaluation of a novel method to measure miltefosine in leishmaniasis patients using dried blood spot sample collection
- 2016
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In: Antimicrobial Agents and Chemotherapy. - 0066-4804 .- 1098-6596. ; 60:4, s. 2081-2089
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Journal article (peer-reviewed)abstract
- To facilitate future pharmacokinetic studies of combination treatments against leishmaniasis in remote endemic regions, a simple and cheap sampling methodology was required for miltefosine quantification. The aim of this study was to validate a liquid chromatography-tandem mass spectrometry method to quantify miltefosine in dried blood spots (DBS) and to validate its use in Ethiopian visceral leishmaniasis (VL) patients. Since hematocrit (Ht) values are typically severely decreased in VL patients, regressing to normal during treatment, the method was evaluated over a range of clinically relevant Ht values.Miltefosine was extracted from DBS using a simple pre-treatment method with methanol, resulting in >97% recovery. The method was validated over a calibration range of 10-2,000 ng/mL and accuracy and precision were within ±11.2% and ≤7.0% (≤19.1% at LLOQ), respectively. The method was accurate and precise for blood spot volumes between 10-30 μL and for an Ht of 20-35%, though a linear effect of Ht 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's r=0.946). Correcting for patient-specific Ht did not further improve the concordance between the sampling methods.This successfully validated method to quantify miltefosine in DBS was demonstrated to be a valid and practical alternative to venous blood sampling which can be applied in future miltefosine pharmacokinetic studies in leishmaniasis patients, without Ht-correction.
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