A quantitative liquid chromatography tandem mass spectrometry method for metabolomic analysis of Plasmodium falciparum lipid related metabolites

• Published in: Analytica chimica acta Vol. 739; pp. 47 - 55
• Main Authors: Vo Duy, S., Besteiro, S., Berry, L., Perigaud, C., Bressolle, F., Vial, H.J., Lefebvre-Tournier, I.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20-08-2012; Elsevier; Elsevier Masson
• Subjects: Amino acids; Amino Acids - analysis; Amino Alcohols - analysis; Analytical chemistry; Carboxylic Acids - analysis; Chemical Sciences; Chemistry; Choline - analysis; Chromatographic methods and physical methods associated with chromatography; Chromatography, High Pressure Liquid - methods; Erythrocytes; Exact sciences and technology; Extraction; Humans; Hydrophobic and Hydrophilic Interactions; Limit of Detection; Linear Models; Lipids - analysis; Liquid chromatography; Liquid chromatography tandem mass-spectrometry; Malaria, Falciparum - parasitology; Mass spectrometry; Medical services; Metabolites; Metabolome; Metabolomics; Metabolomics - instrumentation; Metabolomics - methods; Nucleotides - analysis; Organic chemistry; Other chromatographic methods; Parasites; Phospholipid; Plasmodium falciparum; Plasmodium falciparum - chemistry; Reference Standards; Reproducibility of Results; Spectrometric and optical methods; Succinic Acid - analysis; Tandem Mass Spectrometry - methods; Valine - analysis; Phospholipid; Metabolomics; Plasmodium falciparum; Liquid chromatography tandem mass-spectrometry; Water; Protozoal disease; Standard addition method; Metabolite; Internal standard; Lipids; Biology; Biosynthesis; Separation method; Accuracy; Carboxylic acid; Standard curve; Carbohydrate; Tool; Drug; Protozoa; Apicomplexa; Malaria; Use; Ion pair; Red blood cell; Liquid chromatography; Parasitosis; Polar compound; Infection; Ion chromatography; Analysis method; Treatment; Aminoacid; Mass spectrometry MS/MS; Reagents; Succinic acid; Nucleotide; Intracellular; Detection; Column chromatography; Liquid chromatography tandem; mass-spectrometry
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2012.06.016

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► Plasmodium falciparum is the causative agent of malaria. ► Two analytical techniques using LC/MS/MS developed. ► 35 compounds were quantified. ► The methods were validated and applied to determine intracellular concentrations of metabolites.
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-13C4 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 d9-choline and 4-aminobutanol as internal standards. Standard curves were performed in P. falciparum-infected and uninfected red blood cells using standard addition method (r2>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/3×107cells. These methods were validated and successfully applied to determine intracellular concentrations of metabolites from uninfected host RBCs and isolated Plasmodium parasites.