Detection of sildenafil and its 9 metabolites in a post-race horse urine sample: A case report.

The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control Institute Doping Control Laboratory through a screening analysis performed with Liquid Chromatography Triple Quadrupole Mass Spectrometry. These results were confirmed by Q Exactive Orbitrap Mass Spectrometry and follow-up analyses were performed. As a result of these analyses; simultaneous detection of 9 metabolites in horse urine was reported, two of them for the first time. In addition, the pioneer and comprehensive data resulting from this study provide preliminary data for future studies and anti-doping analyses.

Broad-spectrum and sensitive screening of more than 1000 compounds in equine urine using liquid chromatography/high-resolution mass spectrometry.

RATIONALE: To uphold the integrity of horseracing and equestrian sports, it is critical for an equine doping control laboratory to develop a comprehensive screening method to cover a wide range of target substances at the required detection levels in equine urine. METHODS: The procedure involved the enzymatic hydrolysis of 3 mL urine samples followed by solid-phase extraction using HF Bond Elut C18 cartridge. The resulting extracts were then separated on a C18 reversed-phase column and analyzed using liquid chromatography/high-resolution mass spectrometry (LC/HRMS) in both electrospray ionization positive and negative modes in two separate injections. The analytical data were obtained in full scan and product ion scan (PIS) modes in an 11 min LC run. RESULTS: The method can detect 1011 compounds (in both positive and negative ion modes). Over 95% of the target compounds have limits of detections (LODs) ≤10 ng/mL, and more than 50% of the LODs are ≤0.5 ng/mL. The lowest LOD can reach down to 0.01 ng/mL. The applicability of the method was demonstrated by the successful detection of prohibited substances in overseas and domestic equine urine samples. CONCLUSIONS: We have successfully developed a regular screening method for equine urine samples that can detect more than 1000 compounds at sub-ppb levels in both positive and negative ion modes with full scan and PIS using LC/HRMS. Furthermore, this method can theoretically be expanded to accommodate an unlimited number of prohibited substances in full-scan mode.

Sondagem da pelve renal guiada por cistoscopia em éguas / Catheterization of the renal pelvis guided by cistoscopy in mare

O objetivo deste trabalho foi padronizar a técnica de acesso à pelve renal por meio de sondagem ureteral guiada por cistoscopia em éguas. Foram utilizados oito animais, de raças variadas, com peso médio de 439kg. As éguas foram sedadas e mantidas em tronco de contenção para a realização da cistoscopia com endoscópio flexível. Após identificação do óstio ureteral esquerdo, uma sonda de polietileno foi introduzida em seu lume, até a pelve renal. A localização da sonda no rim foi confirmada por meio de ultrassonografia transcutânea. Foram coletados 3mL de urina, de forma asséptica, para citologia e cultura bacteriana. Todas as amostras obtiveram resultados negativos na cultura e análise do sedimento urinário. Nenhum dos animais apresentou quaisquer complicações após a sondagem. Este estudo demonstrou que a coleta de urina diretamente da pelve renal em éguas, com auxílio da cistoscopia na realização da sondagem ureteral, consiste em um procedimento viável e seguro.(AU)

The objective of this study was to standardize the technique of access to the renal pelvis by means of ureteral catheterization guided by cystoscopy in mares. Eight animals of different races were used, with an average weight of 439kg. The mares were sedated and contained in containment trunk for the accomplishment of cystoscopy with flexible endoscope. After the identification of the left ureteral ostium, from where inside a catheter was introduced, which went through the entire extension of the ureter up to the renal pelvis. After identification of the left ureteral ostium, from where inside a catheter was introduced, into its lumen until reaching the renal pelvis. The location of the probe in the kidney was confirmed by transcutaneous ultrasonography. Three ml of urine was aspirated aseptically for cytology and microbiological culture. All the samples obtained negative results in the culture and sedimentation. None of the animals had any complications after catheterization. This study demonstrated that the collection of urine directly from the renal pelvis in mares, with the assistance of cystoscopy in the realization of the ureteral catheter, consists of a viable and safe procedure.(AU)

Quantification of caffeine in horse urine and saliva by gas chromatography and ELISA

Foram pesquisados resíduos organoclorados em sangue humano, colhido de 122 indivíduos. Foram compostos dois grupos: um urbano e outro residente em áreas de intensa atividade agrícola. Os resíduos organoclorados pesquisados foram: ∞ - HCH; γ - HCH; Aldrin; Diedrin; Edrin; Hepatocloro; Hepatocloro epóxido; HCB; MIrex: o,p DDD; Oxiclordane; p.p Metoxicloro; p,p DDE; p,p DDD e Tranonacloro (limite de detecção = 0,010μg/L). Todos os indivíduos responderam a questionário com quesitos sobre idade, sexo, peso, atividade, local de residência, contato ou não praguidas. Todas as amostras urbanas foram negativas para organoclorados analisados. 10,5 por cento das amostras obtidas nas áreas de atividade agrícola foram positivas. Os resultados das análises do presente trabalho foram comparados com resultados pre-existentes no Rio grande do Sul. Entre a pesquisa atual e a pesquisas anteriores usada para comparação, verificou-se diminuição da incidência de organoclorados...

Combined chromatographic methodology for antidoping control in horse urine

A combination of chromatographic techniques is described for the screening of drugs in horse urine for antidoping control purposes. The procedure consists of liquid-liquid base extraction with analysis by HPTLC and GC-NPD and liquid-liquid acidic extraction followed by HPTLC and HPLC. The results of 86 drugs analyzed by these procedures, as well as limits of detection of the drugs in each technique are presented, The use of ELISA tests are recommended for the screening of drugs which would not be detected by the chromatographic proposed procedure. The results show that the proposed combination of techniques enables an efficient antidoping control of racing horses.

Detección de metabolitos de cocaina en orina humana con kits inmunologicos y confirmacion por GC/MSD / Detection of cocaine metabolites in human urine with immunological kits and confirmation by GC/MSD

Los kits ELISA de NEOGEN para el screening de metabolitos de cocaína en orina de caballo, de una de una sensibilidad de 0.3 ng/mL para la benzoilecgonina, son utilizados en el Laboratorio de control Antidoping con un límite de corte de 3 ng/mL, en orinas humanas diluídas al décimo. Este límite tan bajo nos ha dado un alto número de positivos, difíceles de confirmar po GC-MS. Por esta razón, hemos probado diferentes técnicas para alcanzar en la confirmación un límite de detección acorde con el screening inmunológico. Para ello se realizaron ensayos con diferentes métodos de extracción, solventes de extracción y agentes de derivatización. La técnica seleccionada consiste en una extracción por columnas Bond Elut Certify y derivatización con PFPA/PFPOH, con um límite de deteccíon de 5 ng/mL. El uso de estos kits en estas condiciones produjo algunos resultados falso-positivos, la gran mayoria debidos a orinas conteniendo butilescopolamina y sus metabolitos.