ABSTRACT
A sensitive and selective liquid chromatography-tandem mass spectrometry method was developed and validated for the determination of ofloxacin in 20 µl human plasma over the concentration range of 0.078-20 µg/ml. Sample preparation was achieved by protein precipitation with acetonitrile and methanol containing the internal standard (Gatifloxacin). Chromatographic separation was achieved on a Luna 5 µm PFP (110 A, 50 × 2 mm) column with acetonitrile and water containing 0.1% formic acid (50:50, v/v) as the mobile phase, at a flow rate of 400 µl/ml. The within-day and between-day precision determinations for ofloxacin, expressed as the percentage coefficient of variation, were lower than 7% at all test concentrations. Recovery of ofloxacin was greater than 70% and reproducible at the low, medium and high end of the dynamic range. No significant matrix effects were observed for the analyte or internal standard. The assay was successfully used to examine the pharmacokinetics of ofloxacin as part of a study to characterize the pharmacokinetics of a number of anti-tuberculosis drugs utilized in the treatment of multi-drug resistant tuberculosis (MDR-TB).
Subject(s)
Anti-Bacterial Agents/blood , Chromatography, Liquid/methods , Ofloxacin/blood , Tandem Mass Spectrometry/methods , Acetonitriles/chemistry , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacokinetics , Calibration , Drug Stability , Humans , Methanol/chemistry , Ofloxacin/chemistry , Ofloxacin/pharmacokinetics , Reference Standards , Reproducibility of Results , Sensitivity and Specificity , Spectrometry, Mass, Electrospray Ionization/methods , Water/chemistryABSTRACT
A solvent extraction method was developed and validated for the determination of the antimalarial drug, artemether and its active metabolite dihydroartemisinin (DHA) in malaria patient plasma samples. An AB Sciex 4000 triple quadrupole mass spectrometer in the multiple reaction monitoring (MRM) mode was used for detection in the positive ionisation mode. Liquid-liquid extraction was followed by PFP liquid chromatography and tandem mass spectrometry. Stable isotope labelled artemether and DHA was used as internal standards. The calibration range was between 2.00 and 500 ng/ml for both artemether and DHA during the original validation and the upper limit was lowered to 200 ng/ml during a re-instatement validation, prior to sample analysis. The assay was used to measure artemether and DHA in human plasma samples, which were generated from a safety and efficacy clinical trial in Mbarara, Uganda; as well as for a pharmacokinetic interaction study between the antimalarial combination artemether/lumefantrine and combination antiretroviral therapy including nevirapine in HIV-infected adults.
Subject(s)
Antimalarials/therapeutic use , Artemisinins/therapeutic use , Malaria/drug therapy , Tandem Mass Spectrometry/methods , Antimalarials/blood , Antimalarials/pharmacokinetics , Artemisinins/blood , Artemisinins/pharmacokinetics , Calibration , Humans , Malaria/blood , Reference StandardsABSTRACT
Endocytosis is a fundamental process of eukaryotic cells and fulfills numerous functions, most notably, that of macromolecular nutrient uptake. Malaria parasites invade red blood cells and during their intracellular development endocytose large amounts of host cytoplasm for digestion in a specialized lysosomal compartment, the food vacuole. In the present study we have examined the effects of artemisinin and the quinoline drugs chloroquine and mefloquine on endocytosis in Plasmodium falciparum. By using novel assays we found that mefloquine and artemisinin inhibit endocytosis of macromolecular tracers by up to 85%, while the latter drug also leads to an accumulation of undigested hemoglobin in the parasite. During 5-h incubations, chloroquine inhibited hemoglobin digestion but had no other significant effect on the endocytic pathway of the parasite, as assessed by electron microscopy, the immunofluorescence localization of hemoglobin, and the distribution of fluorescent and biotinylated dextran tracers. By contrast, when chloroquine was added to late ring stage parasites, followed by a 12-h incubation, macromolecule endocytosis was inhibited by more than 40%. Moreover, there is an accumulation of transport vesicles in the parasite cytosol, possibly due to a disruption in vacuole-vesicle fusion. This fusion block is not observed with mefloquine, artemisinin, quinine, or primaquine but is mimicked by the vacuole alkalinizing agents ammonium chloride and monensin. These results are discussed in the light of present theories regarding the mechanisms of action of the antimalarials and highlight the potential use of drugs in manipulating and studying the endocytic pathway of malaria parasites.
