The SIMPLIFY Protocol: A Monophasic Extraction System Suitable for Exposomics, Metabolomics, Lipidomics, and Proteomics Research

Advancing our understanding of human health and disease
requires
comprehensive analytical approaches capable of capturing the complex
interplay between endogenous metabolism and environmental exposures.
A major challenge in clinical research is the ability to capture multidimensional
data, particularly a broad range of biochemical profiles, due to limitations
of biological resources, time, and budget. In this study, we introduce
the SIMPLIFY Protocol, a unified monophasic extraction method that
enables the simultaneous extraction of chemical exogenous products
and endogenous molecules. The method was evaluated against in-house
extraction techniques, including protein precipitation with methanol
(MeOH) and acetonitrile (ACN), and the Folch method using various
sample types, particularly certified reference materials. We demonstrate
that the SIMPLIFY Protocol not only performs comparably to our in-house
methods but also offers enhanced versatility for additional applications
such as derivatization and proteomics. The analyte abundances and
reproducibility with this method strongly correlate with those from
in-house-established techniques across diverse sample types. The method
encompasses a broad spectrum of compounds, effectively profiling approximately
800 identified compounds, including polar compounds (e.g., amino acids),
semipolar compounds (e.g., polyfluorinated compounds, bile acids,
and lysophophatidylcholine), and nonpolar compounds (e.g., cholesteryl
ester), with some limitations in extracting triacylglycerols. By maintaining
simplified workflow and minimizing biological and resource consumption
of multiple extractions, this method supports high-throughput exposomics/metabolomics
and lipidomics studies. Furthermore, its streamlined design facilitates
(semi)automation, making it highly suitable for large-scale clinical
studies, where efficiency, cost-effectiveness, and sample availability
are critical factors.