Susanne Römsing

Assay for screening for six antimalarial drugs and one metabolite using dried blood spot sampling, sequential extraction and ion-trap detection.

Background: More parasites are becoming resistant to antimalarial drugs, and in many areas a change in first-line drug treatment is necessary. The aim of the developed assay is to help determine drug use in these areas and also to be a complement to interviewing patients, which will increase reliability of surveys. Results: This assay detects quinine, mefloquine, sulfadoxine, pyrimethamine, lumefantrine, chloroquine and its metabolite desethylchloroquine in a 100-µl dried blood spot. Most of the drugs also have long half-lives that make them detectable at least 7 days after administration. The drugs are extracted from the dried blood spot with sequential extraction (due to the big differences in physicochemical properties), solid-phase extraction is used as sample clean-up and separation is performed with gradient-LC with MS ion-trap detection. Conclusion: Detection limits (S/N > 5:1) at 50 ng/ml or better were achieved for all drugs except lumefantrine (200 ng/ml), and thus can be used to determine patient compliance. A major advantage of using the ion-trap MS it that it will be possible to go back into the data and look for other drugs as needed.

Determination of tafenoquine in dried blood spots and plasma using LC and fluorescence detection

BACKGROUND The growing problem of parasites developing resistance to the traditional antimalarial drugs makes the development of new effective and safe drugs crucial. Tafenoquine is a new promising antimalarial drug for prophylaxis and treatment. RESULTS A bioanalytical method for the determination of tafenoquine in 100 µl of capillary blood applied onto sampling paper and in 100 µl of plasma has been developed and validated. The Whatman 31 ET Chr paper was treated with 0.6 mol/l tartaric acid to improve the extraction recovery and solid-phase extraction was used for cleanup procedure of the blood samples. Plasma samples were precipitated with methanol. Tafenoquine and internal standard were separated on a Zorbax SB-CN column by reversed-phase LC and detected with fluorescence detection at 262 and 470 nm. The within- and between-day variations were below 10 and 14%, respectively, over the range 50-200 nmol/l for capillary blood on sampling paper and below 6 and 10% for plasma samples. The LLOQ of the method was 50 nmol/l. CONCLUSION The developed method has adequate sensitivity and is highly suitable for clinical studies in dried blood spots and plasma.

Determination of melatonin in human plasma with solid-phase extraction, high-performance liquid chromatography and fluorescence detection

A new bioanalytical method for the determination of melatonin in plasma with high‐performance liquid chromatography (HPLC) and fluorescence detection preceded by solid‐phase extraction has been developed and validated. Melatonin was extracted from 3 mL plasma using a Waters Oasis HLB solid‐phase extraction cartridge and the elute was evaporated to dryness and dissolved in 200 μl mobile phase; acetonitrile‐phosphate buffer, 0.01 M pH 7.2 (25:75, v/v). 125 μL was injected into the HPLC system and separation was carried out on a Waters SymmetryShield™ RP18 column 5 μm (250×4.6 mm). Excitation and emission wavelengths were set to 285 nm and 345 nm, respectively. The HPLC system was able to separate melatonin and internal standard (5‐fluorotryptamine) from other endogenous indole compounds such as serotonin and tryptophan. Determination down to 0.10 nmol/L was possible, with an intra‐assay precision of about 13%. Melatonin was stable in plasma for at least 30 days at about 23°C.