Liquid chromatography/electrospray ionization tandem mass spectrometric screening and confirmation methods for beta2-agonists in human or equine urine.

The researchers conducted an investigation on the electrospray ionization (ESI) mass spectra of 19 common beta2-agonists, focusing on their fragmentation and behavioral dissociation. The data obtained was used to formulate liquid chromatographic/ESI tandem mass spectrometric (LC/ESI-MS/MS) methodologies that could test for the presence of these beta2-agonists in human and equine urine for doping control purposes.

Fragmentation and Dissociation Behavior

• The experiment was undertaken to understand in detail how beta2-agonists break down and separate under the influence of ESI.
• Particular emphasis was laid on understanding the charge-driven and charge-remote fragmentation mechanisms of the analytes.
• This deep understanding of fragmentation and dissociation of beta2-agonists under ESI was used in developing a reliable detection method based on liquid chromatography and ESI tandem mass spectrometry (LC/ESI-MS/MS).

Development of LC/ESI-MS/MS Screening Method

• Using information obtained from the fragmentation and dissociation behavior of beta2-agonists, the LC/ESI-MS/MS screening and confirmation methods were developed.
• This screening method uses acidic or enzymatic hydrolysis, alkaline extraction and, in the case of equine urine samples, an acidic re-extraction of the analytes, as part of its sample preparation steps.
• An interesting finding was the identification of a degradation product of formoterol that occurs due to acidic hydrolysis during sample preparation. This compound could be utilized as a target in the screening and confirmation methodology.

Performance of the Developed Screening Method

• The method was effective in detecting between 18 or 19 beta2-agonists.
• The estimated limits of detection for equine and human urine samples were found to vary between 2 and 100 ng ml(-1) and between 2 and 50 ng ml(-1), respectively.
• In terms of duration, a single LC/ESI-MS/MS analysis using this methodology can be completed within 9 minutes, indicating the potential for efficient, high-throughput screening.