Development and validation of a liquid chromatography-mass spectrometry assay for the determination of pyronaridine in human urine.

A reliable method has been developed for the determination of pyronaridine in human urine using amodiaquine as an internal standard. Liquid-liquid extraction was used for sample preparation. Analysis was performed on a Shimadzu LCMS-2010 in single ion monitoring positive mode using atmospheric pressure chemical ionization (APCI) as an interface. The extracted ion for pyronaridine was m/z 518.20 and for amodiaquine was m/z 356.10. Chromatography was carried out using a Gemini 5 microm C18 3.0 mmx150 mm column using 2 mM perflurooctanoic acid and acetonitrile mixture as a mobile phase delivered at a flow rate of 0.5 mL/min. The mobile phase was delivered in gradient mode. The retention times of pyronaridine and amodiaquine were 9.1 and 8.1 min respectively, with a total run time of 14 min. The assay was linear over a range of 14.3-1425 ng/mL for pyronaridine (R2>or=0.992, weighted 1/Concentration). The analysis of quality control samples for pyronaridine at 28.5, 285, 684 and 1140 ng/mL demonstrated excellent precision with relative standard deviation of 5.1, 2.3, 3.9 and 9.2%, respectively (n=5). Recoveries at concentrations of 28.5, 285, 684 and 1140 ng/mL were all greater than 85%. This LC-MS method for the determination of pyronaridine in human urine has excellent specifications for sensitivity, reproducibility and accuracy and can reliably quantitate concentrations of pyronaridine in urine as low as 14.3 ng/mL. The method will be used to quantify pyronaridine in human urine for pharmacokinetic and drug safety studies.

Liquid chromatographic assay of ivermectin in human plasma for application to clinical pharmacokinetic studies.

There is a need for an accurate, sensitive and selective high-performance liquid chromatography (HPLC) method for the quantitation of ivermectin in human plasma that separates the parent drug from metabolites. Ivermectin and the internal standard, moxidectin, were extracted from 0.2 ml of human plasma using Oasis HLB solid phase extraction cartridges. After extraction, fluorescent derivatives of ivermectin and moxidectin were made by reaction with trifluoroacetic anhydride and N-methylimidazole. Separation was achieved on a Alltech Ultrasphere C18 5mu column with a mobile phase composed of tetrahydrofuran-acetonitrile-water (40:38:22 v/v/v). Detection is by fluorescence, with an excitation of 365 nm and emission of 475 nm. The retention times of ivermectin and internal standard, moxidectin are approximately 24.5 and 12.5 min, respectively. The assay is linear over the concentration range of 0.2-200 ng/ml of ivermectin in human plasma (r = 0.9992, weighted by 1/concentration). Recoveries of ivermectin are greater than 80% at all concentrations. The analysis of quality control samples for ivermectin 0.2, 25, and 200 ng/ml demonstrated excellent precision with coefficient of variation of 6.1, 3.6 and 2.3%, respectively (n = 6). The method is accurate with all intra-day (n = 6) and interday (n = 12) mean concentration within 10% of nominal values at all quality control sample concentrations. Storage stability for 30 days at -80 degrees C and after three freeze-thaw cycles are within acceptable limits. The method separates ivermectin from multiple less and more polar unidentified metabolites. This method is robust and suitable for clinical pharmacokinetic studies. The analytical procedure has been applied to a pharmacokinetic study of ivermectin in healthy volunteers and to the analysis of plasma specimens from patients with disseminated strongyloidiasis.