A fast and sensitive method for quantifying lumefantrine and desbutyl-lumefantrine using LC–MS/MS

• A bioanalytical method for lumefantrine and its main metabolite desbutyl-lumefantrine was developed.
• Two isotopically labeled internal standards give robust quantification.
• Sensitivite and short analysis times is applicable to large volume studies.
• The method was successfully applied for clinical samples.

A sensitive liquid chromatography tandem mass spectrometry (LC–MS/MS) method was developed for quantification of lumefantrine (LUM) and its metabolite desbutyl-lumefantrine (DBL) in human plasma. Sample preparation was done by protein precipitation using acetonitrile containing deuterated lumefantrine (LUM-d18) and desbutyl-lumefantrine (DBL-d9) as internal standards. Total chromatography time was 2.2 min using an Hypersil Gold C18 column (20 × 2.1 mm, 1.9 μm), with a gradient using 0.5% formic acid in water (mobile phase A) and 0.5% formic acid in methanol (mobile phase B) at a flow rate of 0.5 mL/min. The mass spectrometric quantification was performed in positive electro spray ionization (ESI+) mode using selected reaction monitoring (SRM). Measuring range was 21–529 ng/mL for LUM and 1.9–47 ng/mL for DBL in plasma. Inter- and intra-assay precision was within 10% coefficient of variation (CV) for all levels of both LUM and DBL. Accuracy was within ±10% for all levels of both LUM and DBL. This method requires 100 μL plasma volume and its short retention times allow a high throughput. Samples were stable for a week at +5 °C, and up to six months −20 °C. The method was successfully applied for plasma LUM and DBL determination in children under 5 years of age with uncomplicated malaria, up to 28 days after a standard 3-day treatment with artemether-lumefantrine.