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. 2018 Oct 29;11(1):770.
doi: 10.1186/s13104-018-3885-5.

Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control

Teruaki Tozaki  1 , Kaoru Karasawa  2 , Yohei Minamijima  3 , Hideaki Ishii  3 , Mio Kikuchi  4 , Hironaga Kakoi  4 , Kei-Ichi Hirota  4 , Kanichi Kusano  5 , Shun-Ichi Nagata  4
Affiliations

Detection of phosphorothioated (PS) oligonucleotides in horse plasma using a product ion (m/z 94.9362) derived from the PS moiety for doping control

Teruaki Tozaki et al. BMC Res Notes. .

Abstract

Objective: Clinical research on gene therapy has advanced the field of veterinary medicine, and gene doping, which is the illegal use of gene therapy, has become a major concern in horseracing. Since the International Federation of Horseracing Authorities defined the administration of oligonucleotides and its analogues as a genetic therapy in 2017, the development of therapeutic nucleotide-detection techniques has become an urgent need. Most currently marketed and developed oligonucleotide therapeutics for humans consist of modified nucleotides to increase stability, and phosphorothioate (PS) modification is common.

Results: We demonstrated the specific detection of phosphorothioated oligonucleotides (PSOs) using LC/MS/MS. PSOs produce the specific product ion (m/z 94.9362) derived from PS moiety. PS is not derived from endogenous substances in animal body, and the product ion is a suitable marker for the detection of PSOs. With our strategy, reproducible target analyses were achieved for identifying the specific substances, with a LOD of 0.1 ng/mL and a quantification rage of 0.1-200 ng/mL in deproteinated plasma. Non-target analyses could also detect the presence of PSOs selectively with 100 ng/mL in the same matrix. These results suggested that the detection of PSOs in horse blood is possible by targeting the product ion using LC/MS/MS.

Keywords: Gene doping; LC/MS/MS; Mass spectrometry; Phosphorothioated oligonucleotides (PSOs); Therapeutic nucleotides; Thoroughbred.

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Figures

Fig. 1
Fig. 1
Structure of the product ion (m/z 94.9362) derived from phosphorothioated oligonucleotides (PSOs)
Fig. 2
Fig. 2
Non-target analyses of phosphorothioated oligonucleotides (PSOs) in deproteined plasma. a Total ion chromatogram (TIC), data dependent acquisition (DDA) Explorer (Time versus Precursor MS/Charge for DDA dependents), DDA Explorer filtered by m/z 94.936 with 10 ppm mass tolerance of deproteinated plasma with 4.5 µg/mL oligo dT. b TIC, DDA Explorer, and DDA Explorer filtered by m/z 94.936 with 10 ppm mass tolerance of 100 ng/mL oligo 1 in deproteinated plasma with 4.5 µg/mL oligo dT. Phosphorothioated (PS) oligomers were selectively detected in a heavy biological matrix containing a 45 times higher concentration of a non-PS oligomer without any sequence information
See this image and copyright information in PMC

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