AUTHOR=Abu-Izneid Tareq , Abbas Muhammad , Watson David G. , Shah Yasar , Shah Sayyed I. , Khuda Fazli 

TITLE=Estimation of dihydroartemisinin in human plasma using a highly sensitive LTQ Orbitrap mass spectrometer with Xcalibur software

JOURNAL=Frontiers in Pharmacology

VOLUME=Volume 14 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2023.1157604

DOI=10.3389/fphar.2023.1157604

ISSN=1663-9812

ABSTRACT=Background
Artemether which is the O-methyl ether prodrug of dihydroartemisinin (DHA) is considered as a first-line antimalarial agent. Artemether metabolized extensively in vivo into its active metabolite DHA and therefore its determination offers considerable difficulties. In present study, DHA identification and estimation was performed by the accurate mass spectrometric analysis, using high-resolution liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) LTQ-Orbitrap hybrid mass spectrometer
Methods
The plasma samples taken from healthy volunteers and the spiked plasma was extracted by adding 1 ml of a mixture of dichloromethane and tert.-Methyl butyl ether (8:2 v/v) to 0.5 ml of plasma. Internal standard solution (artemesinin 500 ng/ml) was added to the plasma samples. After vertexing and centrifugation the organic layer was separated and transferred into another tube and dried under nitrogen. The residue was reconstituted in 100 μL of acetonitrile and was injected onto the LC–MS system for analysis. Measurement of standards and samples was carried out isocratically on a Surveyor HPLC system combined with an LTQ Orbitrap mass spectrometer using an ACE 5 C18-PFP column. Mobile phase A  consisted of 0.1%  v/v formic acid in water and Mobile phase B consisted of acetonitrile only  and isocratic elution was carried out with A:B 20:80, v/v. The flow rate was 500 µl/min.   The ESI interface was operated in a positive ion mode with a spray voltage of 4.5 kV.
Results 
Artemether is not a very biologically stable compound and is immediately metabolized to its active metabolite dihydroartemisinin, so no clear peak was observed for artemether. Both artemether and DHA after ionization undergo neutral losses of methanol and water respectively in the source of the mass spectrometer. The ions observed were (MHᶧ-H2O) m/z 267.15 for DHA and (MHᶧ) m/z 283.15 for internal standard artemisinin. The method was validated according to international guidelines.
Discussion
The validated method was applied successfully for the determination and quantification of dihydroartemisinin (DHA) in plasma samples. This method works well for the extraction of drugs and the Orbitrap system with the help of X calibur software accurately and precisely determine the concentration of DHA in spiked as well as in volunteer’s plasma