# Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography–High-Resolution Mass Spectrometry

## TL;DR

This study presents a new method to detect synthetic oligonucleotides in horse serum, which are hard to spot with current anti-doping tests.

## Contribution

A non-targeted LC-HRMS approach for detecting synthetic oligonucleotides in equine serum is developed and validated.

## Key findings

- The method successfully detected specific product ions from three unique oligonucleotide sequences.
- Detection limits, accuracy, and precision were validated using a 13mer oligonucleotide.
- The method is effective for identifying non-naturally occurring ions in RNA.

## Abstract

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.