We compared three solvent addition strategies: stepwise, two-step, and all solvents simultaneously. Previously, we concluded that methanol/chloroform/water extraction is preferable for metabolomics, and we further optimized this here using fish liver and an automated Precellys 24 bead-based homogenizer, allowing rapid extraction of multiple samples without carryover. Here we optimize tissue extraction methods compatible with high-throughput, reproducible nuclear magnetic resonance (NMR) spectroscopy- and mass spectrometry (MS)-based metabolomics. This is time-consuming and difficult to automate, and the multiple steps can introduce variability. In metabolomics, tissues typically are extracted by grinding in liquid nitrogen followed by the stepwise addition of solvents.
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