Detection and confirmation of 60 anabolic and androgenic steroids in equine plasma by liquid chromatography-tandem mass spectrometry with instant library searching.

The research article provides an account of how a quick and efficient method for testing sixty types of anabolic and androgenic steroids (AAS) in horse plasma was developed using liquid chromatography and tandem mass spectrometry. The method was developed to help enforce a ban on the use of these substances in equine sports.

Methodology

• The researchers used liquid chromatography-tandem mass spectrometry (LC-MS/MS), a popular technique used in drug detection. This analysis method helps scientists separate components of a mixture and accurately measure each one.
• The researchers recovered AAS from the horse’s blood (plasma) through a process known as liquid-liquid extraction (LLE) where they employed methyl tert-butyl ether. The AAS were then separated on a C₁₈ column, commonly used for reversed-phase high performance liquid chromatography.
• The separated substances were then analysed by electrospray ionization mass spectrometry, which is a technique used to produce ions out of solution which can be detected and measured.

Use of Multiple Reaction Monitoring

• A scan method, multiple-reaction monitoring (MRM), was employed for initial screening of the substances. If the MRM signal went over 1000 counts per second, an enhanced product ion scan of the AAS was generated using a system called information-dependent acquisition (IDA).
• Simultaneously, a library of the AAS was created using the enhanced product ion spectra, facilitating future comparisons.
• An AAS was unmistakably identified by matching the MRM response within the correct time window and comparison between the test substance’s spectrum with the reference spectrum in the library.

Results and Conclusions

• The method proved to be efficient as it only took a total analysis time of 7 minutes.
• The limit of detection for the substances ranged between 0.01-2 ng/mL and the limit of confirmation ranged between 0.1-10 ng/mL, making the method highly sensitive and reliable.
• The discovery rate was also satisfactory with ranges from 74% to 138% when the substances were recovered from the plasma through LLE.
• The method was successfully tested and is now routinely used in post-race screenings for the presence of the 60 AAS substances in equine plasma samples.