# Development of an Optimized Two-Step Solid-Phase Extraction Method for Urinary Nucleic Acid Adductomics

## TL;DR

Researchers developed a new method to extract DNA and RNA damage markers from urine, which could help study how environmental exposures contribute to diseases like cancer.

## Contribution

A novel two-step solid-phase extraction method was developed for improved recovery of nucleic acid adducts in urine.

## Key findings

- The combination of ENV+ and PHE SPE columns provided optimal recovery of nucleic acid adduct standards.
- Approximately 500 adducts were detected in mouse and human urine using high-resolution mass spectrometry.
- Isotopically labeled standards helped identify and characterize endogenous urinary nucleic acid adducts.

## Abstract

The exposome represents the totality of endogenous and exogenous exposures across the lifespan. These exposures may result in DNA and RNA damage, in the form of adducts, which is a key factor in the etiology of a variety of human diseases, including cancer. It is understood that, following their repair, nucleic acid adducts are excreted into the urine, making urine an ideal, non-invasive matrix in which to study the whole-body nucleic acid adductome (the totality of nucleic acid adducts). However, the measurement of these adducts in urine presents challenges due to matrix interference and the variety of the chemical nature across the spectrum of nucleic adducts making their “one-size-fits-all” extraction by solid-phase extraction (SPE) challenging. Here, different types of SPE sorbents, and their combination, were evaluated for maximal recovery of nucleic acid adducts from urine. The SPE column combination of ENV+ coupled with PHE provided the best retention of a cocktail of 20 nucleic acid adduct standards. An untargeted high resolution mass spectrometry approach incorporating FeatureHunter 1.3 software was used to demonstrate the ability of this SPE method to successfully recover endogenous urinary nucleic acid adducts in addition to those represented by the cocktail of isotopically labeled standards. Using our approach, FeatureHunter 1.3 recognized approximately 500 adducts in both mouse and human urine samples. Isotopically labeled standards were used to identify a selection of the endogenous adducts and begin the characterization of the urinary nucleic acid adductome of mice and humans.