Extraction strategies for tackling complete hair metabolome using LC-HRMS-based analysis

Abstract

Over recent years, metabolomics has been featured as the state-of-the-art technology that successfully opens the paths to understanding biological mechanisms and facilitating biomarker discovery. However, the inherent dynamic and sensitive nature of the metabolome have been challenging the accuracy of capturing the timepoints of interest while using biofluids such as urine and blood. Hair has thus emerged as a valuable analytical specimen for the long-term and retrospective determinations. Unfortunately, notwithstanding the apparent interest on global hair metabolomics, very few studies have engaged in the optimisation of the extraction strategy. In this study, we systemically investigated the extraction procedures for hair metabolome using a single factor experimental design. Three pH values (acidic, neutral, and basic) in aqueous solution, six extraction solvents (methanol, acetonitrile, acetone, phosphate-buffered saline, deionised water, and dichloromethane), different compositions of selected solvent mixtures and their sequential extraction, and a series of extraction times (15, 45, 60, 120, 240, and 480 min) were evaluated. The ideal condition for hair extraction is ultrasonic-assisted extraction with methanol:phosphate-buffered saline 50:50 (v/v) under +55 °C for 240 min. This strategy may secure the true composition of the metabolome, maximise the signal abundance, and guarantee a high coverage of wide-range metabolites in a straightforward approach. The optimised extraction strategy was then coupled with structure annotation tools for hair metabolome profiling. After a single RPLC-HRMS run, hair metabolite identification was achieved as the annotations of 171 probable structures and 853 tentative structures as well as the assignments of 414 unequivocal molecular formulae. In conclusion, we established an efficient extraction strategy for untargeted hair metabolomics, which the method is deliverable to any analytical laboratories and the sample can be directly profiled by means of a conventional RPLC-HRMS gradient.

Keywords: Complementary matrices; High-resolution mass spectrometry; Metabolome; Non-targeted; Optimisation; Untargeted.