In vivo comparative study of hemp straw exposure and cannabidiol oil administration in horse urine.

The non-psychoactive cannabinoids cannabidiol (CBD) and cannabidiolic acid (CBDA) are available on the market in different forms, mostly for their anti-inflammatory and potential analgesic properties. These substances are prohibited during equine competitions. CBD and CBDA are naturally present in hemp straw, commonly used as a bedding substitute for wheat straw. Unfortunately, horses can eat it, which therefore could lead to a possible risk of positive findings for CBD/CBDA in biological samples after doping control tests. The goals of this study were, first, to provide recommendations on the use of hemp straw before competition and, second, to assess if discrimination between hemp bedding exposure and CBD oil administration is possible. Several CBD equine in vivo studies have been conducted, including one on hemp straw used as bedding and one after administration of CBD oil by topical and sublingual routes. In hemp straw, CBDA was detected in higher quantities than CBD, and other cannabinoids have been observed. After hemp straw exposure, CBDA was also detected in higher quantities than CBD in all urine samples. It appeared that hemp straw should not be used as bedding for equine competition except if a delay of at least 48 h is respected. Regarding the CBD oil product analysis, CBD was the main compound detected. After administration, 7-hydroxy CBD was identified in the urine. In conclusion, based on these data, we highlighted that it could be possible to discriminate the exposure of a horse to hemp straw from an administration of a CBD oil product thanks to the main presence of CBDA.

LC-HRMS/MS study of the prodrug ciclesonide and its active metabolite desisobutyryl-ciclesonide in plasma after an inhalative administration to horses for doping control purposes.

Ciclesonide (CIC) is the first inhaled highly potent corticosteroid that does not cause any cortisol suppression. It has been developed for the treatment of asthma in human and more recently in equine. CIC is the active compound of Aservo® EquiHaler® (Boehringer Ingelheim Vetmedica GmbH), the pre-filled inhaler generating a medicated mist based on Soft Mist™ technology. This prodrug is rapidly converted to desisobutyryl-ciclesonide (des-CIC), the main pharmacologically active compound. Due to its anti-inflammatory properties, CIC is prohibited for use in horse competitions. To set up an appropriate control, the determination of detection times and screening limits are required. Therefore, a highly sensitive analytical method based on supported liquid extraction (SLE) combined with liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) was developed to detect CIC and its active metabolite des-CIC in plasma. The lower limit of detection of CIC and des-CIC was approximately 1 pg/ml in plasma. After a pilot study conducted on a single horse at the recommended dose (eight actuations twice daily corresponding to 5.5 mg/day for the first 5 days, followed by 12 actuations once daily corresponding to 4.1 mg/day in the last 5 days), the same protocol was applied in the main study using six horses. In all horses, CIC and des-CIC levels were less than 5 and 10 pg/ml, respectively, at 36 h after the end of the administration. The outcome of this risk assessment study should be useful to draw any recommendations for horse competitions.

Comprehensive characterization of the peroxisome proliferator activated receptor-δ agonist GW501516 for horse doping control analysis.

According to international sport institutions, the use of peroxisome proliferator activated receptor (PPAR)-δ agonists is forbidden at any time in athlete career due to their capabilities to increase physical and endurance performances. The (PPAR)-δ agonist GW501516 is prohibited for sale but is easily available on internet and can be used by cheaters. In the context of doping control, urine is the preferred matrix because of the non-invasive nature of sampling and providing broader exposure detection times to forbidden molecules but often not detected under its native form due to the organism's metabolism. Even if urinary metabolism of G501516 has been extensively studied in human subjects, knowledge on GW501516 metabolism in horses remains limited. To fight against doping practices in horses' races, GW501516 metabolism has to be studied in horse urine to identify and characterize the most relevant target metabolites to ensure an efficient doping control. In this article, in vitro and in vivo experiments have been conducted using horse S9 liver microsome fractions and horse oral administration route, respectively. These investigations determined that the detection of GW501516 must be performed in urine on its metabolites because the parent molecule was extremely metabolized. To maximize analytical method sensitivity, the extraction conditions have been optimized. In accordance with these results, a qualitative analytical method was validated to detect the abuse of GW501516 based on its most relevant metabolites in urine. This work enabled the Laboratoire des Courses Hippiques (LCH) to highlight two cases of illicit administration of this forbidden molecule in post-race samples.

MetIDfyR: An Open-Source R Package to Decipher Small-Molecule Drug Metabolism through High-Resolution Mass Spectrometry.

With recent advances in analytical chemistry, liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) has become an essential tool for metabolite discovery and detection. Even if most of the common drug transformations have already been extensively described, manual search of drug metabolites in LC-HRMS/MS datasets is still a common practice in toxicology laboratories, complicating metabolite discovery. Furthermore, the availability of free open-source software for metabolite discovery is still limited. In this article, we present MetIDfyR, an open-source and cross-platform R package for in silico drug phase I/II biotransformation prediction and mass-spectrometric data mining. MetIDfyR has proven its efficacy for advanced metabolite identification in semi-complex and complex mixtures in in vitro or in vivo drug studies and is freely available at github.com/agnesblch/MetIDfyR.

Use of benchtop exactive high resolution and high mass accuracy orbitrap mass spectrometer for screening in horse doping control.

Liquid chromatography-mass spectrometry (LC-MS) has been widely used in doping control laboratories over the last two decades. Currently, simple quadrupole, triple quadrupole and ion trap are the most commonly employed analyzers in toxicological analysis. Nevertheless, the main lack of these technologies is the restricted number of target compounds simultaneously screened without loss of sensitivity. In this article we present an innovative screening approach routinely applied in the French horse doping control laboratory based on high resolution (50000) and high mass accuracy (<5 ppm) in full scan MS mode for more than 235 target analytes screened from an initial volume of 5 mL of urine. The sample preparation was classically founded on solid phase extraction by means of reverse phase C18 cartridges. LC-MS analyses were carried out on a Shimadzu binary HPLC pumps linked to a C18 Sunfire column associated with the high resolution exactive benchtop orbitrap mass spectrometer. This screening was performed alternatively in positive-negative ionization mode during the same run. Thus, the identification of compounds of interest was made using their exact mass in positive-negative ionization mode at their expected retention time. All data obtained were processed by ToxID software (ThermoFisherScientific) which is able to identify a molecule by theoretical mass and retention time. In order to illustrate this innovative technology applied in our laboratory, sample preparation, validation data performed on 20 target compounds from 16 different horse urine samples, chromatograms and spectra will be discussed in this paper.

Countercurrent chromatographic isolation of lolitrem B from endophyte-infected ryegrass (Lolium perenne L.) seed.

This paper describes a new method of purification of the Lolitrem B, a tremorgenic mycotoxin produced in planta by the endophytic fungus Neotyphodium lolii. The method is based on the large-scale isolation of the toxin by countercurrent chromatography (CCC). The lolitrem B content in endophyted ryegrass seed, 11 microg/g or 11 ppm, is extracted by stirring finely ground seeds with ethanol for 3 h at room temperature. The concentrated crude extract contains about 0.6 mg/g or 600 ppm of lolitrem B. It is then submitted to CCC purification with a biphasic four-solvent liquid system. A 160-fold enrichment was obtained in one step producing a raffinate containing 10% or 100 mg/g of the toxin. Further purifications were then performed by thin layer and low pressure liquid chromatography. Twenty-eight micrograms of lolitrem B with a 96% purity grade were obtained from 8 kg of seeds (yield 32%).