Plasma Free Amino Acid Analysis

Blood for amino acid analysis was drawn from a fresh catheter in the forearm in non-fasting adults immediately following study enrolment and usually before a first dose of antimalarials was provided. Blood samples was collected in lithium-heparin tubes that were immediately centrifuged and flash-frozen in liquid-nitrogen. Samples were shipped on dry ice and stored at −80 °C. In brief, amino acids were extracted by solid-phase extraction using sorbent tips (Phenomenex EZ:faast™), as described previously^{38 (link)}. Plasma free amino acids (PFAA) were separated on an AAA-MS™ column (250 × 3.0 mm, Phenomenex, Torrance, California) placed in a Thermo LC system. Mass spectrometry with multiple reaction monitoring was done in positive ion mode with electrospray ionization on an ABSciex API 5000 Triple Quadrupole LC/MS/MS Mass Spectrometer. Inter-assay variation scores were determined for three amino acid standards and the individual amino acids. Concentrations were based on interpolation on a linear standard curve. We excluded amino acids if they could be measured in less than 50 cases. The final amino acid data set is publicly available on GitHub, found at: https://github.com/Stije/PFAA.