Detection of singly- and doubly-charged quaternary ammonium drugs in equine urine by liquid chromatography/tandem mass spectrometry.

The research paper presents an efficient method for detecting both singly- and doubly-charged quaternary ammonium drugs (QADs) in horse urine. This method, which uses liquid chromatography/tandem mass spectrometry (LC/MS/MS), could detect these drugs at extremely low levels after solid-phase extraction.

Background of the Research

• The researchers first discuss quaternary ammonium drugs (QADs), substances that act as anticholinergic agents and have been documented to be abused or misused in equine sports.
• Reviewing existing research, it is discovered that traditional screening methods primarily focus on detecting singly-charged QADs. Doubly-charged QADs, however, are highly polar substances that are difficult to extract and poorly retained on reversed-phase columns.
• The researchers aim to develop a comprehensive method that can accurately detect both singly- and doubly-charged QADs simultaneously.

The New LC/MS/MS Method

• The report then details the development and implementation of an effective LC/MS/MS method designed to detect and confirm the presence of 38 different singly- and doubly-charged QADs at sub-parts-per-billion (ppb) to low-ppb levels in equine urine following a process called solid-phase extraction.
• The QADs were extracted from horse urine using solid-phase extraction (SPE) with a specific type of SPE column. They were then analyzed using LC/MS/MS in positive electrospray ionisation mode.
• The separation of the 38 QADs was then achieved using a polar group embedded C18 LC column, with a specific mixture of aqueous ammonium formate and acetonitrile serving as the mobile phase.

Results and Implementation

• Utilizing selected reaction monitoring (SRM), the scientists were able to detect and confirm the presence of the 38 QADs at sub-ppb to low-ppb levels in horse urine within 16 minutes.
• There were no observed matrix interferences of the target transitions at expected retention times. Other method validation data, such as precision and recovery, was validated and found to be acceptable.
• The researchers have successfully applied this method to the analysis of actual drug-administration samples, confirming its effectiveness and potential for practical use.