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The study of the metabolome within tissues, organisms, cells or biofluids can be carried out by several bioanalytical techniques. Among them, nuclear magnetic resonance (NMR) is one of the principal spectroscopic methods. This is due to a sample rotation technique, high-resolution magic angle spinning (HR-MAS), which targets the analysis of heterogeneous specimens with a bulk sample mass from 5 to 10 mg. Recently, a new approach, high-resolution micro-magic angle spinning (HR-μMAS), has been introduced. It opens, for the first time, the possibility of investigating microscopic specimens (&lt;500 μg) with NMR spectroscopy, strengthening the concept of homogeneous sampling in a heterogeneous specimen. As in all bioanalytical approaches, a clean and reliable sample preparation strategy is a significant component in designing metabolomics (or -omics, in general) studies. The sample preparation for HR-μMAS is consequentially complicated by the μg-scale specimen and has yet to be addressed. This report details the strategies for three specimen types: biofluids, fluid matrices and tissues. It also provides the basis for designing future μMAS NMR studies of microscopic specimens.

General Guidelines for Sample Preparation Strategies in HR-µMAS NMR-based Metabolomics of Microscopic Specimens