Clenbuterol in the horse: confirmation and quantitation of serum clenbuterol by LC-MS-MS after oral and intratracheal administration.

This research presents a newly developed method used to accurately and specifically identify and measure the presence of clenbuterol, a popular bronchodilator, in horse serum. This method will help enforce regulations surrounding the use of this substance in horses.

Objective and Methodology

• The aim of this study was to devise a precise and highly sensitive method for quantifying clenbuterol in horse serum. Accurate detection of clenbuterol is crucial as its presence in post-race samples can lead to sanctions.
• As part of this method, clenbuterol-d9 was synthesized to serve as an internal standard.
• An automated solid-phase extraction process was developed and used alongside a multiple reaction monitoring liquid chromatography-tandem mass spectrometry (LC-MS-MS) method. This combo was employed to affirmatively identify and quantify clenbuterol concentration in 2 mL of serum at levels as low as 10 pg/mL.

Implementation and Results

• The researchers administered clenbuterol orally to five horses over a 10-day period. The serum was then collected for the following 14 days.
• The average serum clenbuterol concentrations were akin to trough levels of approximately 150 pg/mL.
• Following the last dose on the tenth day, the concentration of serum clenbuterol peaked at roughly 500 pg/mL and started to decrease with a half-life of about 7 hours.
• The clenbuterol level was measured at 30 pg/mL and 10 pg/mL 48 and 72 hours after dosing, respectively. After 96 hours, the concentration was below 4 pg/mL, which is the limit of detection for this method. This comparison revealed that using serum for clenbuterol detection is more reliable than traditional urine tests.

Intratracheal Administration Results

• In a separate test, clenbuterol was administered intratracheally to five horses. The peak serum levels were about 230 pg/mL, which were detected 10 minutes after administration.
• The serum concentration declined to approximately 50 pg/mL within 30 minutes and reduced slowly thereafter. These findings indicated that the new method could detect clenbuterol in horses that received an intratracheal dose before racing.

Conclusions

• Equine drug testing has traditionally depended on urine samples due to the small serum volume and low drug concentrations.
• However, the LC-MS-MS method allows for unequivocal identification and quantitation of drugs in serum, even at low concentrations of 10 pg/mL.
• The potential of this method, the speed of new test development it allows, and the confidence with which the results can be applied forensically offer significant scientific and regulatory benefits over conventional urine testing methods.