A simplified liquid chromatography-mass spectrometry assay for artesunate and dihydroartemisinin, its metabolite, in human plasma.
Artesunate (AS) is a potent antimalarial that is used worldwide for the treatment of malaria. A simple method with a total run time of 12 min was developed and validated for the quantification of AS and dihydroartemisinin (DHA), its active metabolite, in human (heparinized) plasma based on one-step protein precipitation in acetonitrile using artemisinin (ARN) as an internal standard, followed by liquid chromatography with a single quadrupole mass spectrometry system connected to a C18 column. Peak area ratio responses were fitted to the 2nd-order curve type, polynomial equation with weighting (1/concentration) over a quantification range between 3.20/5.33-3,000/5,000 nM (1.23/1.52-1153/1422 ng/mL) of AS/DHA showing linearity with very good correlation (r2>0.999). Single ion recordings of 5 µL injections of plasma extracts allowed for limits of detection of 1.02 nM (0.39 ng/mL) for AS and 0.44 nM (0.13 ng/mL) for DHA. The inter-assay and intra-assay accuracy and precision of the method was very good with an inaccuracy of ±12.4% and coefficients of variation of ≤10.7% at all tested concentrations. The recovery of the analytes from plasma was ≥95%. Other commonly used antimalarials including mefloquine, quinine, and chloroquine, did not interfere with the analysis. Post-preparative tests over 24 h in an autosampler (10 °C) showed that the DHA response was only 2.1% of AS from auto-hydrolysis, and β-DHA was the major, stable epimer that was used for quantification of DHA. In contrast, α-DHA increased steadily up to 600%. Artesunate and DHA in plasma were stable through three freeze/thaw cycles for up to 6 h at room temperature and up to one year at -80 °C.
Department of Immunology and Medicine, United States Army Medical Component, Armed Forces Research Institute of Medical Sciences, 315/6 Rajvithi Road, Bangkok 10400, Thailand. paktiyat@afrims.org
Paktiya
P
Teja-Isavadharm
Duangsuda
D
Siriyanonda
Raveewan
R
Siripokasupkul
Roongnapa
R
Apinan
Nitima
N
Chanarat
Apassorn
A
Lim
Srisombat
S
Wannaying
David
D
Saunders
Mark M
MM
Fukuda
Robert S
RS
Miller
Peter J
PJ
Weina
Victor
V
Meléndez
D000962:Antimalarials / Q000032:analysis* / Q000493:pharmacokinetics; D037621:Artemisinins / Q000032:analysis* / Q000493:pharmacokinetics; D000077332:Artesunate; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D010949:Plasma / Q000737:chemistry*; D012680:Sensitivity and Specificity; D013997:Time Factors
D000962:Antimalarials; D037621:Artemisinins; D000077332:Artesunate; C039060:artenimol