Bisphosphonates are prohibited drugs according to Article 6 of the International Agreement on Breeding, Racing and Wagering of the International Federation of Horseracing Authorities (IFHA) and the International Equestrian Federation (FEI). These compounds are used for the treatment of lameness, navicular and bone diseases in horses and are divided into two groups: non-nitrogen-containing bisphosphonate drugs (e.g. clodronic acid) and nitrogen-containing bisphosphonate drugs (e.g. zoledronic acid). Their hydrophilic properties and the high affinity for the bone matrix make the control of their use quite difficult. Current analytical strategies to detect such compounds often rely on a solid phase extraction (SPE) followed by detection by means of UHPLC-MS/MS after methylation with chemical reagents. To improve the analysis throughput and to eliminate the need for chemical derivatization, an innovative 96-well SPE followed by ion chromatography-mass spectrometry was developed. Analyses are conducted on an ICS-6000 HPIC system coupled to a TSQ Altis™ (Thermo Scientific™). The use of a 96-well SPE allowed 5-fold sample increase and a 6-fold throughput improvement. While preliminary results conducted on horse plasma exhibited similar performances to the method for the detection of non-nitrogen-containing bisphosphonates, the analytical performances of nitrogencontaining bisphosphonates were greatly improved.

Many innovative biotherapeutics have been marketed in the last decade. Monoclonal antibodies (mAbs) and Fc‐fusion proteins (Fc‐proteins) have been developed for the treatment of diverse diseases (cancer, autoimmune diseases, and inflammatory disorders) and now represent an important part of targeted therapies. However, the ready availability of such biomolecules, sometimes characterized by their anabolic, anti‐inflammatory, or erythropoiesis‐stimulating properties, raises concerns about their potential misuse as performance enhancers for human and animal athletes. In equine doping control laboratories, a method has been reported to detect the administration of a specific human biotherapeutic in equine plasma; but no high‐throughput method has been described for the screening without any a priori knowledge of human or murine biotherapeutic. In this context, a new broad‐spectrum screening method involving UHPLC‐HRMS/MS has been developed for the untargeted analysis of murine or human mAbs and related macromolecules in equine plasma. This approach, consisting of a “pellet digestion” strategy performed in a 96‐well plate, demonstrates reliable performances at low concentrations (pmol/mL range) with high‐throughput capability (≈100 samples/day). Targeting species‐specific proteotypic peptides located within the constant parts of mAbs enables the “universal” detection of human biotherapeutics only by monitoring 10 peptides. As proof of principle, this strategy successfully detected different biotherapeutics in spiked plasma samples, and allowed, for the first time, the detection of a human mAb up to 10 days after a 0.12 mg/kg administration to a horse. This development will expand the analytical capabilities of horse doping control laboratories towards protein‐based biotherapeutics with adequate sensitivity, throughput, and cost‐effectiveness.