Detection and elimination profile of cathinone in equine after norephedrine (Propalin®) administration using a validated liquid chromatography–tandem mass spectrometry method

Abstract

Cathinone is the principal psychostimulant present in the leaves of khat shrub, which are widely used in East Africa and the Arab peninsula as an amphetamine-like stimulant. Cathinone readily undergoes metabolism in vivo to form less potent cathine and norephedrine as the metabolites. However, the presence of cathine and norephedrine in biological fluids cannot be used as an indicator of cathinone administration. The metabolism of pseudoephedrine and ephedrine, commonly used in cold and allergy medications, also produces cathine and norephedrine, respectively, as the metabolites. Besides, cathine and norephedrine may also originate from the ingestion of nutritional supplemental products containing extracts of Ephedra species. In Canada, ephedrine and norephedrine are available for veterinary use, whereas cathinone is not approved for human or veterinary use. In this article, the detection of cathinone in equine after administration of norephedrine is reported. To the best of our knowledge, this is the first such report in any species where administration of norephedrine or ephedrine generates cathinone as the metabolite. This observation is quite significant, because in equine detection of cathinone in biological fluids could be due to administration of the potent stimulant cathinone or the nonpotent stimulant norephedrine. A single oral dose of 450 mg norephedrine was administered to four Standardbred mares. Plasma and urine samples were collected up to 120 h after administration. The amount of cathinone and norephedrine detected in post administration samples was quantified using a highly sensitive, specific, and validated liquid chromatography-tandem mass spectrometry method. Using these results, we constructed elimination profiles for cathinone and norephedrine in equine plasma and urine. A mechanism that generates a geminal diol as an intermediate is postulated for this in vivo conversion of norephedrine to cathinone. Cathinone was also detected in samples collected after a single intramuscular administration of 200 mg ephedrine and oral administration of 300 mg ephedrine in equine.

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

“…To achieve baseline chromatographic separation of pseudoephedrine and its metabolite, norpseudoephedrine, which are ephedrine and norephedrine diastereomers, respectively, only one dilution step is needed for LC-MS or LC-MS/MS [22,42,43]. For plasma and serum samples, protein precipitation requires dilution with methanol [44] or acetonitrile [45,46,49], or LLE [47,48].…”

“…For plasma and serum samples, protein precipitation requires dilution with methanol [44] or acetonitrile [45,46,49], or LLE [47,48]. RPLC is a method of choice for urine [22,43,50], plasma and serum ephedrine separations [43][44][45]49]. An insufficient diastereomeric separation may decrease quantification accuracy so for complete separation, mobile phases contain small amounts of formate to improve retain polar bases or organic additives for attaining acceptable peaks.…”

“…THCA and its glucuronide conjugate are major urinary cannabinoid metabolites and the WADA threshold for THCA is 150 ng/ml. Dilute-and-shoot method development and validation for LC-MS/MS to measure urinary THCA has been described in the literature [10][11][12] and this approach permitted more rapid sample preparation with fewer steps prior to LC-MS/ THCA 150 ng/ml 15 ng/ml 10 180 ng/ml [10][11][12][13][14][15][16][17][18][19][20][21] Salbutamol 1.0 μg/ml 0.1 μg/ml 10 1.2 μg/ml [22][23][24] Formoterol 40 ng/ml 6.0 ng/ml 15 50 ng/ml [10,[25][26][27] Glycerol 4.3 mg/ml 0.65 mg/ml 15 5.3 mg/ml [28][29][30] Morphine 1.0 μg/ml 0.15 μg/ml 15 1.3 μg/ml [31][32][33][34][35][36][37][38][39][40][41] Cathine 5.0 μg/ml 0.5 μg/ml 10 6.0 μg/ml [22] Ephedrine 10 μg/ml 0.5 μg/ml 5 11 μg/ml [42][43][44][45][46][47][48][49]…”

Challenges in detecting substances for equine anti‐doping

“…A significant increase in the abuse of synthetic cathinones (the β-keto analogues of amphetamine) has been reported. [77][78][79][80] Cathinone is the main psychoactive element of Khat (Cathis edulis), chewing the leaves of which is used for treatment of respiratory conditions due to relaxation of airway smooth muscle and, thus, it may be considered as an instant substitute for clenbuterol in racehorses. Psychostimulating and hallucinogenic properties are also attributed to cathinones.…”

“…[78] Except from cathinone derivatives, other new psychoactive substances (NPS) also continuously emerge on drug market. [77] The updated information on the second generation of NPS has recently been reported. [84] AH-7921 (referred to as doxylam) and MT-45 are new synthetic opioid analgesics, having recently appeared on the Internet market.…”