53
Figure 4: DDA Explorer (time versus precursor MS/charge for DDA dependents) and DDA Explorer filtered by m/z 94.936 with 10 ppm mass tolerance of deproteinated plasma with 4.5 µg/mL oligo dT (blank sample), and with 10 ppm mass tolerance of 100 ng/mL PSOs in deproteinated plasma with 4.5 µg/mL oligo dT (spiked sample).
of m/z 94.9362 would be a good marker for detecting and monitoring PS compounds, such as those that may potentially be used in the gene doping of racehorses. The analytical method described may be of use in the testing of racehorses for gene doping, particularly as it can be applied using either a targeted or non-targeted analysis. This is especially pertinent as the oligonucleotides being used for gene doping may not be certified, and comprise unknown genetic sequences depending on the gene being manipulated, or any number of nucleic acids or nucleic acid analogues, making it difficult to identify the analyte for a targeted analysis. Another key factor for this methodology is the need for adequately rapid spectroscopic scan speeds, as well as the capability to capture comprehensive data with an information-dependent acquisition (IDA) and dynamic background subtraction (DBS). Therefore, the scan was able to identify all 95 phosphate groups without missing any detectable analytes, thus forming a complete study. The use of the SCIEX TripleTOF® system was therefore critical to the study design and the feasibility and overall success of this study.
Although it has yet to be investigated, this method may also perhaps be adapted for use in the detection and monitoring of gene doping in other sports, such as athletics, since PS compounds also do not naturally occur in humans. The direct detection of PSOs would also be a useful method to develop but until then, the use
of this method to detect the PS moiety as a surrogate marker for actual PSOs should be of some value. Further validation studies will need to be performed on samples from horses that have been doped in vivo but it is hoped that this would not be necessary because gene doping will not be realised in the sport of horseracing.
In conclusion, LC-MS/MS is a useful method for the detection of known and unknown compounds such as PSOs that may be used for doping, and the product ion of m/z 94.9362 is an effective marker for the detection of PSOs in equine plasma.
For those looking for more information, September’s Gene Doping Workshop in Japan (
https://biz.knt.co.jp/tour/2019/09/ aorc/
about.html) will look further into genetic laboratories used for gene doping detection, along with analyst and instrument requirements, and additional testing methods. The workshop will be organised and chaired by Dr Tozaki.
References
1. International Federation of Horseracing Authorities (IFHA). About IFHA. Committees – Gene Doping Control Subcommittee.
https://www.ifhaonline.org/default. asp?section=About%20IFHA&area=101.
2. G. Wood. Talking Horses: racing attempts to get out in front of gene doping. The Guardian. 24 April 2019. https://www.
theguardian.com/sport/blog/2019/apr/24/
talking-horses-bha-commits-1m-gene- doping.
3. Tozaki T, Gamo S, Takasu M, Kikuchi M, Kakoi H, Hirota KI, Kusano K, Nagata SI. Digital PCR detection of plasmid DNA administered to the skeletal muscle of a microminipig: a model case study for gene doping detection. BMC Res Notes. 2018 Oct 10;11(1):708
4. T. Wilkin, A. Baoutina, N. Hamilton. Equine performance genes and the future of doping in horseracing. Drug Test Anal. 9 (2017) 1456–71.
5. E.W.I. Neuberger, P. Simon. Gene and Cell Doping: The New Frontier – Beyond Myth or Reality. Med Sport Sci 62 (2017) 91–106.
6. A. Macedo, M. Moriggi, M. Vasso, S. De Palma, M. Sturnega, G. Friso, C. Gelfi, M. Giacca, S. Zacchigna. Enhanced Athletic Performance on Multisite AAV-IGF1 Gene Transfer Coincides with Massive Modification of the Muscle Proteome. Human Gene Therapy 23 (2012) 146–57.
7. M. Le Page. Gene doping in sport could make the Olympics fairer and safer. New Scientist. 5 August 2016. https://www.
newscientist.com/article/2100181-gene- doping-in-sport-could-make-the-olympics- fairer-and-safer/.
8. C.A. Stein, D. Castanotto. FDA-Approved Oligonucleotide Therapies in 2017. Molecular Therapy 25 (2017) 1069–75.
9. M. Krishnan. Drugging the
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