Simultaneous detection of recombinant growth hormones in equine plasma by liquid chromatography/high-resolution tandem mass spectrometry for doping control.

The research article discusses a new method for detecting the use of prohibited growth hormones in horse racing with increased efficiency and precision. The new method makes use of liquid chromatography and high-resolution tandem mass spectrometry to simultaneously detect different types of recombinant growth hormones in equine plasma.

Overview of the Research

• This research study focuses on prohibited use of growth hormones, especially recombinant growth hormones, in horse racing. These hormones, capable of enhancing physical performance and facilitating growth, are banned from use in equine sports due to potential advantages they provide.
• The researchers have developed an effective method for detection of three different types of recombinant growth hormones – recombinant equine GH (reGH), recombinant human GH (rhGH), and recombinant porcine GH (rpGH) – simultaneously, in equine plasma.
• Previous detection methods often require more time and resources, and struggle to distinguish between different types of GHs, making this new method a significant advancement.

Detection Method

• The researchers make use of liquid chromatography and high-resolution tandem mass spectrometry (LC-MS/HRMS) for their detection process. The methodology involves extraction of these hormones from equine plasma, cleanup, and analysis of the proteotypic peptides through LC-MS/HRMS.
• Recombinant chicken GH (rcGH) is used as an internal standard for the procedure. The hormones are extracted following ammonium sulfate precipitation and then cleaned up by chloroform/methanol precipitation before trypsination.
• The detection limits are estimated to be approximately 0.5ng/mL for reGH, 2.5ng/mL for rhGH, and 1.25ng/mL for rpGH. Comparing the retention times and relative abundances of three major product-ions in accordance with industry criteria allows for confirmation at different concentration levels for each hormone.

Significance and Advantages

• The new detection method requires less plasma for analysis (around 2mL) and has a shorter turnaround time compared to other methods. This increases its efficiency, making it a potential preferred choice for doping control in horse racing.
• Also, the method exhibits reliable precision and reproducibility, further strengthening its potential application in doping control.
• According to the research team, this method represents the first known instance of a detection method capable of simultaneously detecting reGH, rhGH and rpGH at such low concentration levels in horse plasma samples.