Development of high throughput 96-blade solid phase microextraction-liquid chromatrography-mass spectrometry protocol for metabolomics

• In vivo 96 blade system was optimized for bacteria metabolomics.
• Escherichia coli was used as model bacteria.
• Different coating chemistries were evaluated as the stationary phase for 96-blade system.
• Over 1000 cellular metabolites were extracted ranging within −7 < log P < 22.6.
• Applying 96-blade autosampler provides high-throughput sample preparation.

In metabolomics, the workflow for quantitative and comprehensive metabolic mapping of cellular metabolites can be a very challenging undertaking. Sampling and sample preparation play a significant role in untargeted analysis, as they may affect the composition of the analyzed metabolome. In the current work, different solid phase microextraction (SPME) coating chemistries were developed and applied to provide simultaneous extraction of a wide range of both hydrophobic and hydrophilic cellular metabolites produced by a model organism, Escherichia coli. Three different LC-MS methods were also evaluated for analysis of extracted metabolites. Finally, over 200 cellular metabolites were separated and detected with widely varying hydrophobicities ranging within −7 < log P < 15, including amino acids, peptides, nucleotides, carbohydrates, polycarboxylic acids, vitamins, phosphorylated compounds, and lipids such as hydrophobic phospholipids, prenol lipids, and fatty acids at the stationary phase of the E. coli life cycle using the developed 96-blade SPME-LC-MS method.

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