Polar metabolomics using trichloroacetic acid extraction and porous graphitic carbon stationary phase

Accurately identifying and quantifying polar metabolites using untargeted metabolomics has proven challenging in comparison to mid to non-polar metabolites. Hydrophilic interaction chromatography and gas chromatography–mass spectrometry are predominantly used to target polar metabolites.

This study aims to demonstrate a simple one-step extraction combined with liquid chromatography–mass spectrometry (LC–MS) that reliably retains polar metabolites.

The method involves a MilliQ + 10% trichloroacetic acid extraction from 6 healthy individuals serum, combined with porous graphitic carbon liquid chromatography–mass spectrometry (LC–MS). The coefficient of variation (CV) assessed retention reliability of polar metabolites with logP as low as − 9. QreSS (Quantification, Retention, and System Suitability) internal standards determined the method's consistency and recovery efficiency.

The method demonstrated reliable retention (CV < 0.30) of polar metabolites within a logP range of − 9.1 to 5.6. QreSS internal standards confirmed consistent performance (CV < 0.16) and effective recovery (70–130%) of polar to mid-polar metabolites. Quality control dilution series demonstrated that ~ 80% of annotated metabolites could be accurately quantified (Pearson’s correlation coefficient > 0.80) within their concentration range. Repeatability was demonstrated through clustering of repeated extractions from a single sample.

This LC–MS method is better suited to covering the polar segment of the metabolome than current methods, offering a reliable and efficient approach for accurate quantification of polar metabolites in untargeted metabolomics.