When developing a sample preparation protocol for LC–MS untargeted metabolomics of a new sample matrix unfamiliar to the laboratory, selection of a suitable injection concentration is rarely described. Here we developed a simple workflow to address this issue prior to untargeted LC–MS metabolomics using pig adipose tissue and liver tissue. Bi-phasic extraction was performed to enable simultaneous optimisation of parameters for analysis of both lipids and polar extracts. A series of diluted pooled samples were analysed by LC–MS and used to evaluate signal linearity. Suitable injected concentrations were determined based on both the number of reproducible features and linear features. With our laboratory settings, the optimum concentrations of tissue mass to reconstitution solvent of liver and adipose tissue lipid fractions were found to be 125 mg/mL and 7.81 mg/mL respectively, producing 2811 (ESI+) and 4326 (ESI−) linear features from liver, 698 (ESI+) and 498 (ESI−) linear features from adipose tissue. For analysis of the polar fraction of both tissues, 250 mg/mL was suitable, producing 403 (ESI+) and 235 (ESI−) linear features from liver, 114 (ESI+) and 108 (ESI−) linear features from adipose tissue. Incorrect reconstitution volumes resulted in either severe overloading or poor linearity in our lipid data, while too dilute polar fractions resulted in a low number of reproducible features (<50) compared to hundreds of reproducible features from the optimum concentration used. Our study highlights on multiple matrices and multiple extract and chromatography types, the critical importance of determining a suitable injected concentration prior to untargeted LC–MS metabolomics, with the described workflow applicable to any matrix and LC–MS system.
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