Molecular characterization of pathogens such as the malaria parasite can lead to improved biological understanding and novel treatment strategies. However, the distinctive biology of the Plasmodium parasite, including its repetitive genome and the requirement for growth within a host cell, hinders progress toward these goals. Untargeted metabolomics is a promising approach to learn about pathogen biology. By measuring many small molecules in the parasite at once, we gain a better understanding of important pathways that contribute to the parasite’s response to perturbations such as drug treatment. Although increasingly popular, approaches for intracellular parasite metabolomics and subsequent analysis are not well explored. The findings presented in this report emphasize the critical need for improvements in these areas to limit misinterpretation due to host metabolites and to standardize biological interpretation. Such improvements will aid both basic biological investigations and clinical efforts to understand important pathogens.

20 Due to improved instrument sensitivity and access, the use of metabolomics is gaining 21 traction for the study of many organisms and pathogens. were most appropriate for normalization as they separate sample groups and reduce 32 noise within the data set. However, these post-analysis steps did not remove the 33 contribution from the host erythrocyte, in the form of membrane rich 'ghosts', and levels 34 of technical sample variation persisted. In fact, we found that host contamination is as 35 influential on the metabolome as sample treatment. This analysis also identified 36 metabolites with potential to be used as markers to quantify host contamination levels.

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