title: Formation of the diuretic chlorazanil from the antimalarial drug proguanil--implications for sports drug testing.
abstract: Chlorazanil (Ordipan, N-(4-chlorophenyl)-1,3,5-triazine-2,4-diamine) is a diuretic agent and as such prohibited in sport according to the regulations of the World Anti-Doping Agency (WADA). Despite its introduction into clinical practice in the late 1950s, the worldwide very first two adverse analytical findings were registered only in 2014, being motive for an in-depth investigation of these cases. Both individuals denied the intake of the drug; however, the athletes did declare the use of the antimalarial prophylactic agent proguanil due to temporary residences in African countries. A structural similarity between chlorazanil and proguanil is given but no direct metabolic relation has been reported in the scientific literature. Moreover, chlorazanil has not been confirmed as a drug impurity of proguanil. Proguanil however is metabolized in humans to N-(4-chlorophenyl)-biguanide, which represents a chemical precursor in the synthesis of chlorazanil. In the presence of formic acid, formaldehyde, or formic acid esters, N-(4-chlorophenyl)-biguanide converts to chlorazanil. In order to probe for potential sources of the chlorazanil detected in the doping control samples, drug formulations containing proguanil and urine samples of individuals using proguanil as antimalarial drug were subjected to liquid chromatography-high resolution/high accuracy mass spectrometry. In addition, in vitro simulations with 4-chlorophenyl-biguanide and respective reactants were conducted in urine and resulting specimens analyzed for the presence of chlorazanil. While no chlorazanil was found in drug formulations, the urine samples of 2 out of 4 proguanil users returned findings for chlorazanil at low ng/mL levels, similar to the adverse analytical findings in the doping control samples. Further, in the presence of formaldehyde, formic acid and related esters, 4-chlorophenyl-biguanide was found to produce chlorazanil in human urine, suggesting that the detection of the obsolete diuretic agent was indeed the result of artefact formation and not of the illicit use of a prohibited substance.
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany; European Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany. Electronic address: thevis@dshs-koeln.de.
authors.forename: Mario
authors.initials: M
authors.lastname: Thevis
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Geyer
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Laura
authors.initials: L
authors.lastname: Tretzel
authors.affiliation: Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro, 92290 Châtenay-Malabry, France.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bailloux
authors.affiliation: Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro, 92290 Châtenay-Malabry, France.
authors.forename: Corinne
authors.initials: C
authors.lastname: Buisson
authors.affiliation: Agence Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro, 92290 Châtenay-Malabry, France.
authors.forename: Francoise
authors.initials: F
authors.lastname: Lasne
authors.affiliation: Department of Anaesthesiology, University Hospital Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany.
authors.forename: Maximilian S
authors.initials: MS
authors.lastname: Schaefer
authors.affiliation: Department of Anaesthesiology, University Hospital Duesseldorf, Moorenstr. 5, 40225 Duesseldorf, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Kienbaum
authors.affiliation: Tropical Medicine Unit, University Hospital for Gastroenterology, Hepatology and Infectious Diseases, Heinrich-Heine-University Duesseldorf, Germany.
authors.forename: Irmela
authors.initials: I
authors.lastname: Mueller-Stoever
authors.affiliation: Center for Preventive Doping Research - Institute of Biochemistry, German Sport University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schänzer
mesh_terms: D000962:Antimalarials / Q000378:metabolism*; D002722:Chlorobenzenes / Q000652:urine*; D002853:Chromatography, Liquid; D004232:Diuretics / Q000652:urine*; D004300:Doping in Sports; D006801:Humans; D008862:Microsomes, Liver / Q000378:metabolism; D002727:Proguanil / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014227:Triazines / Q000652:urine*
chemical_list: D000962:Antimalarials; D002722:Chlorobenzenes; D004232:Diuretics; D014227:Triazines; C017837:chlorazanil; D002727:Proguanil
keywords: Diuretics; Doping; Mass spectrometry; Sport