Multiplex PCR-Liquid Chromatography High-Resolution Tandem Mass Spectrometry for Simultaneous Detection of Multiple...

The development of gene therapy techniques introduces a potential risk of gene doping, which threatens the integrity of sport. In response to this challenge, we have developed a novel analytical method that employs a multiplex polymerase chain reaction (PCR) in conjunction with liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) for the simultaneous identification of multiple transgenes in equine plasma within a single reaction. The method targets three potential doping transgenes: equine growth hormone 1 (eGH1), equine growth hormone-releasing hormone (eGHRH), and equine interleukin 10 (eIL10), along with an internal control. The artificial internal control (AIC) has been developed to be coextracted, coamplified, and codetected with the transgenes, thereby reducing the risk of false negatives. We proposed design and optimization strategies for the multiplex PCR-LC-MS method to attain high sensitivity, with estimated limits of detection at 25 copies/mL and estimated limits of confirmation at 50 copies/mL for all targets. The method has been validated and successfully applied for the confirmation of the eIL10 transgene in plasma samples from horses administered with the transgene. The utilization of LC-HRMS/MS enabled unequivocal identification of transgenes and provided the flexibility to include multiple doping transgenes. This simple setup offers an alternative approach that enables sensitive and reliable multiplex detection of transgenes in the majority of doping control laboratories.