Plasmodium falciparum is the causative agent of malaria, a deadly infectious 
disease for which treatments are scarce and drug-resistant parasites are now 
increasingly found. A comprehensive method of identifying and quantifying 
metabolites of this intracellular parasite could expand the arsenal of tools to 
understand its biology, and be used to develop new treatments against the 
disease. Here, we present two methods based on liquid chromatography tandem mass 
spectrometry for reliable measurement of water-soluble metabolites involved in 
phospholipid biosynthesis, as well as several other metabolites that reflect the 
metabolic status of the parasite including amino acids, carboxylic acids, 
energy-related carbohydrates, and nucleotides. A total of 35 compounds was 
quantified. In the first method, polar compounds were retained by hydrophilic 
interaction chromatography (amino column) and detected in negative mode using 
succinic acid-(13)C(4) and fluorovaline as internal standards. In the second 
method, separations were carried out using reverse phase (C18) ion-pair liquid 
chromatography, with heptafluorobutyric acid as a volatile ion pairing reagent 
in positive detection mode, using d(9)-choline and 4-aminobutanol as internal 
standards. Standard curves were performed in P. falciparum-infected and 
uninfected red blood cells using standard addition method (r(2)>0.99). The 
intra- and inter-day accuracy and precision as well as the extraction recovery 
of each compound were determined. The lower limit of quantitation varied from 
50pmol to 100fmol/3×10(7)cells. These methods were validated and successfully 
applied to determine intracellular concentrations of metabolites from uninfected 
host RBCs and isolated Plasmodium parasites.