Characterization of rapid extraction protocols for high-throughput metabolomics.

RATIONALE: In the last five years, high-throughput metabolomics has significantly advanced scientific research and holds the potential to promote strides in the fields of clinical metabolomics and personalized medicine. While innovations in the field of flow-injection mass spectrometry and three-minute metabolomics methods now allow investigators to process hundreds to thousands of samples per day, time-sensitive clinical applications, particularly in the emergency department, are limited by a lack of rapid extraction methods.

METHODS: Here we characterized the efficacy of fast liquid-liquid extractions for characterization of hydrophilic compounds through ultra-high-pressure liquid chromatography/mass spectrometry. Internal stable-isotope-labeled standards were used to quantitatively characterize markers of energy and oxidative metabolism in human whole blood, plasma and red blood cells - three common matrices of clinical relevance.

RESULTS: For all the tested matrices, vortexing time (4-60 min) did not significantly affect extraction yields for the tested hydrophilic metabolites. Coefficients of variations <20% for all tested compounds, except for the redox-sensitive metabolite cystine (accumulating over time). Internal standards and second extractions confirmed recoveries >80% for all tested metabolites, except for basic amino acids and polyamines, which showed reproducible yields ranging from 50 to 75%. Global profiling and absolute quantitation of 24 metabolites revealed similarities between the plasma and red blood cell metabolomes.

CONCLUSIONS: Rapid extraction (~4 min) of hydrophilic compounds is a viable and potentially automatable strategy to perform quantitative analysis of whole blood, plasma and red blood cells for research or clinical applications.