- Email: [email protected]
Formation of the diuretic chlorazanil from the antimalarial drug proguanil—Implications for sports drug testing
Accepted Manuscript Title: Formation of the diuretic chlorazanil from the antimalarial drug proguanil – implications for sports drug testing Author: Mario Thevis Hans Geyer Andreas Thomas Laura Tretzel Isabelle Bailloux Corinne Buisson Francoise Lasne Maximilian S. Schaefer Peter Kienbaum Irmela Mueller-Stoever Wilhelm Sch¨anzer PII: DOI: Reference:
S0731-7085(15)30071-6 http://dx.doi.org/doi:10.1016/j.jpba.2015.07.017 PBA 10173
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-6-2015 15-7-2015 18-7-2015
Please cite this article as: Mario Thevis, Hans Geyer, Andreas Thomas, Laura Tretzel, Isabelle Bailloux, Corinne Buisson, Francoise Lasne, Maximilian S.Schaefer, Peter Kienbaum, Irmela Mueller-Stoever, Wilhelm Sch¨anzer, Formation of the diuretic chlorazanil from the antimalarial drug proguanil ndash implications for sports drug testing, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.07.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Formation of the diuretic chlorazanil from the antimalarial drug proguanil – implications for sports drug testing Mario Thevis1,2*, Hans Geyer1, Andreas Thomas1, Laura Tretzel1, Isabelle Bailloux3, Corinne Buisson3, Francoise Lasne3, Maximilian S. Schaefer4, Peter Kienbaum4, Irmela Mueller-Stoever5, and Wilhelm Schänzer1
1Center
for Preventive Doping Research - Institute of Biochemistry, German Sport
University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany 2European 3Agence
Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany
Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro,
92290 Châtenay-Malabry, France 4Department
of Anaesthesiology, University Hospital Duesseldorf, Moorenstr. 5,
40225 Duesseldorf, Germany 5Tropical
Medicine Unit, University Hospital for Gastroenterology, Hepatology and
Infectious Diseases, Heinrich-Heine-University Duesseldorf, Germany
*corresponding author:
Mario Thevis, PhD Institute of Biochemistry - Center for Preventive Doping Research German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne, Germany Tel: +49 221 4982 7070 Fax: +49 221 4982 7071 Email: [email protected] Graphical abstract
1
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 AntiDoping 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. 2
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 4chlorophenyl-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. Keywords:
doping, sport, mass spectrometry, diuretics
Introduction Based on findings by Lipschitz and co-workers concerning the diuretic activity of triazine derivatives in the mid-1940s [1], chlorazanil (Ordipan, N-(4-chlorophenyl)-
3
1,3,5-triazine-2,4-diamine, Figure 1, 4) was introduced as one of several new nonmercurial diuretic agents [2, 3] approximately a decade thereafter. The drug was commonly administered at 2 mg/kg of bodyweight, resulting in a significant increase of the individuals’ diuresis [3]. Due to their potential to illicitly reduce an athlete’s bodyweight and to mask other doping agents’ presence in sports drug testing urine samples, diuretic agents have been prohibited in sports since 1988. Since then, diuretics have frequently been detected in doping controls worldwide, ranking third among the internationally reported adverse analytical findings in 2013 [4]; however, chlorazanil was never identified in any doping control sample until 2014. This, the fact that chlorazanil has meanwhile become an obsolete therapeutic, and the credible denial of both athletes who were tested positive for the diuretic have been the motives for follow-up studies that potentially allow identifying another source of chlorazanil in athletes urine samples. A medication declared by two individuals with adverse analytical findings for chlorazanil was the antimalarial prophylactic drug proguanil (Figure 1, 1). It has been in clinical use since 1946 [5] and, in combination with chloroquine, it has represented a mainstay in malarial chemoprophylaxis ever since [6]. Proguanil is metabolized in vivo into the active drug cycloguanil (Figure 1, 2) and other metabolites, most importantly 4-chlorophenyl biguanide (Figure 1, 3) [7]. The formation of chlorazanil as a proguanil metabolite or a drug impurity was however never confirmed. Notwithstanding, two adverse analytical findings presenting with similar general circumstances (vide infra) existed that necessitated further investigations to provide best possible scientific data for a well-grounded anti-doping result management.
4
Case vignette In January and February 2014, three urine samples (pH 5.5-6.0) analyzed in the World Anti-Doping Agency (WADA)-accredited doping control laboratory in ChâtenayMalabry (France) yielded initial testing results suspicious for the presence of chlorazanil. The samples were collected from 2 female and one male elite athletes, competing in team and endurance sport, respectively. Testing locations were Senegal and Cameroon, and all three individuals indicated, amongst other medications and dietary supplements, the use of Malarone that contains 100 mg of proguanil hydrochloride and 250 mg of atovaquone. Confirmatory analyses of the urine samples verified the presence of proguanil, its main metabolites cycloguanil and N-(4chlorophenyl)-biguanide in all three cases. Further, chlorazanil was confirmed at approximately 0.3 and 1.3 ng/mL of urine in two cases, which were consequently reported as adverse analytical findings (AAFs). Follow-up studies were initiated that aimed at probing for a potential correlation between the malarial chemoprophylaxis and the AAFs for chlorazanil, including investigations into metabolic and chemical reactions of proguanil, its urinary metabolites, and ‘reactants’ commonly found in human urine.
Experimental 3.1 Drugs, chemicals and reagents The antimalarial drug Malarone (PZN: 8602632) was obtained from GlaxoSmithKline (Munich, Germany), N-(4-chlorophenyl)-biguanide, formaldehyde, formic acid, and 5
formic acid methyl ester were bought from Sigma (Steinheim, Germany), chlorazanil was from Heumann Pharma (Nuremberg, Germany), and human liver microsomes were from BD Gentest (Woburn, MA, USA).
3.2 Doping control urine samples analysis More than 9,000 doping control urine samples are tested each year for diuretics including chlorazanil in the WADA-accredited doping control laboratory in ChâtenayMalabry (France). Analytical initial testing and confirmation procedures enabling detection limits of 0.1 ng/mL for chlorazanil are applied. The initial testing method utilizes a total of 2 mL of urine, which is extracted twice with ethyl acetate, first at pH 5.2 and then at pH 14. The two organic extracts are combined, concentrated, and reconstituted in liquid chromatography (LC) mobile phase solvents. Four mL of urine is extracted twice with ethyl acetate at pH 14 in the confirmation methodology used. The combined extracts are concentrated and the residue is re-dissolved in LC mobile phase solvents for subsequent LC-MS/MS analysis.
3.3 Liquid chromatography – low resolution tandem mass spectrometry (LC-MS/MS) Sports drug testing analyses for diuretics are conducted using liquid chromatographictandem mass spectrometric (LC-MS/MS) approaches. The instrument used consisted of an Acquity UPLC (Waters, Guyancourt, France) LC equipped with an Agilent (Waldbronn, Germany) Zorbax SB-C8 column (2.1 100 mm, 1.8 m particle size). The solvents used were: water containing 10 mM of ammonium formate and acetic acid (pH 4) (eluent A) and acetonitrile (eluent B). The gradient program started at 10 % B 6
and increased to 55 % in 8 min, then increased to 100 % in 0.1 min and decreased to starting conditions within 0.5 min. The column was re-equilibrated at 10 % for 2 min. The flow rate was set at 400 L/min and the column temperature was 20 °C. The mass spectrometric identification of chlorazanil was performed using a Xevo TQMS triple quadrupole system (Waters, Guyancourt, France) with positive electrospray ionization using a gas flow desolvation of 800 L/h and a gas flow cone of 50 L/h. Source and desolvation temperatures were set to 150°C and 450°C, respectively. The apparatus was operated in selected reaction monitoring (SRM) acquisition mode using diagnostic precursor/product ion pairs of m/z 222 – 178, m/z 222 – 153, m/z 222 – 99, and m/z 222 – 45, with m/z 222 representing the protonated molecule [M+H]+. Collisioninduced dissociation (CID) was conducted at optimized collision energies, and nitrogen was used as collision gas at 3.7x10-3 mBar.
3.4 Patients’ urine sample analysis Spot urine samples collected approximately 16 h post-administration of Malarone were obtained via the University Hospital Duesseldorf (Heinrich-Heine University Duesseldorf, Germany) from four different patients (three male and one female) undergoing malaria chemoprophylaxis. The volunteers provided written consent, and the collection and use of the urine samples for research purposes were approved by the local ethics committee of the medical faculty of the Heinrich-Heine University Duesseldorf (study number 4749). The urine samples’ pH ranged from 5.5-8.0 and all specimens were subjected to analyses concerning chlorazanil and the potential synthetic precursor of chlorazanil, N-(4-chlorophenyl)-biguanide (Figure 1, 3) using an alternative test method employing solid-phase extraction [8, 9] combined with LC7
HRMS (vide infra).
3.5 Liquid chromatography – high resolution (tandem) mass spectrometry (LCHRMS/MS) LC-HRMS analyses were conducted on a Thermo Dionex Ultimate 3000 liquid chromatograph interfaced to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. The Dionex system was equipped with a Hypersil Gold analytical column, 2.1 50 mm, 1.9 µm particle size (Thermo Scientific, Bremen, Germany). The eluents consisted of A: 5 mM ammonium acetate (containing 0.1% acetic acid, pH = 3.5), and B: acetonitrile. Following injection of 5 µL of the sample, a gradient elution program was used, starting with 100% A, decreasing to 0% A in 7 min, followed by an isocratic step for 3 min and re-equilibration at starting conditions for 5 min. The total run time was 15 min, applying a flow rate of 200 µL/min. Measurements were conducted in positive ion mode. The HESI source temperature was 300 °C, the temperature of the transfer capillary was set to 320 °C, and the applied spray voltage was 3.75 kV. Nitrogen generated by a CMC nitrogen generator (CMC instruments, Eschborn, Germany) was used for collision-induced dissociation experiments in the HCD cell and furthermore as damping gas in the curved linear ion trap. The resolution of the mass spectrometer was set to 30,000 full width at half maximum (FWHM) (at m/z 200), and the precursor isolation window was adjusted to 1.5 Da. In order to ascertain mass errors below 5 ppm, the instrument was externally calibrated in positive mode using the manufacturer’s calibration solution (containing caffeine, Ultramark and the tetrapeptide MRFA). The system was operated in targeted MS/MS mode with
8
optimized collision energies.
3.6 Analysis of antimalarial drug (Malarone) for chlorazanil residues In order to sensitively probe for the potential presence of chlorazanil in the antimalarial therapeutic Malarone, three tablets containing 250 mg of atovaquone and 100 mg of proguanil each were dissolved in methanol and diluted to final concentrations of 100 µg/mL of proguanil. The samples were subsequently analyzed using the aforementioned LC-HRMS/MS test method, focusing on the accurate mass (± 5 ppm) of the protonated species [M+H]+ of diuretic agent at m/z 222.0541. With standard solutions of chlorazanil yielding detectable signals at concentrations of 0.01 ng/mL, the obtained solution of Malarone was tested for trace amounts of chlorazanil, accounting for 0.0001% of the structurally related proguanil.
3.7 In vitro production of chlorazanil from N-(4-chlorophenyl)-biguanide The possibility of artificial chlorazanil production in human urine was assessed by incubating reference standard solutions of N-(4-chlorophenyl)-biguanide with different reagents such as methanol, formaldehyde, formic acid, and methyl formate [10]. One mL aliquots of a methanolic solution containing 10 µg of N-(4-chlorophenyl)biguanide each were fortified with a) formaldehyde (50 µL of a 3% methanolic solution), b) formaldehyde (same as for a)) plus 20 mg of potassium carbonate, c) 10 µL of formic acid, and d) 10 µL of methyl formate, respectively. Further, ten different blank urine samples (1 mL, pH 5.5-8.0) enriched with 10 µg of N-(4-chlorophenyl)biguanide each were prepared with and without the addition of methyl formate, formaldehyde, or formic acid as described above. All specimens were incubated at room temperature for 72 h. 9
Methanolic solutions were analyzed concerning the formation of chlorazanil using the aforementioned LC-HRMS/MS method without further sample preparation while urine samples were prepared as described above for the patient’s urine specimens.
3.8 In vitro metabolism of proguanil using human liver microsomes Metabolic products of proguanil were enzymatically generated with human liver microsomes according to established in vitro assay protocols [11]. In brief, a substrate stock solution of 100 µM proguanil in dimethyl sulfoxide (DMSO) and a 50 mM phosphate buffer (pH 7.4) containing 5 mM MgCl2 as incubation buffer (IB) were prepared and stored at 4 °C. Samples were prepared in 1.5 mL test tubes. The reaction mixture used for phase-I metabolism contained 10 µL of the proguanil stock solution (1 mM), 10 µL of NADPH (50 mM in IB), and 10 µL of microsomes (200 µg protein/incubation) dissolved in IB, which was topped up with 70 µL of IB. The metabolic reaction was accomplished within 4 h at 37 °C. To probe for non-enzymatic reactions, an enzyme blank consisting of all reaction components except for the microsomes was prepared also. The incubation was terminated by adding 100 µL of ice-cold acetonitrile (ACN). Precipitated proteins were removed from the reaction mixture by centrifugation at 17,000 × g for 5 min at 4 °C and the supernatant was transferred to vials for subsequent LC-HRMS analysis.
Results and discussion Initiated by the observation that doping control urine samples of three athletes undergoing antimalarial chemoprophylaxis contained trace amounts of the obsolete but
10
nevertheless prohibited diuretic agent chlorazanil, follow-up studies on the potential origin of the drug were conducted. First, although conducted earlier with different techniques [12], the drug Malarone containing 250 mg of atovaquone and 100 mg of proguanil was analyzed for potential contaminations, specifically focusing on chlorazanil using LC-HRMS. None of the analyzed tablets returned findings for chlorazanil, indicating that the antimalarial medication was not the source of diuretic. Second, the possibility of a metabolic conversion of the structurally related product proguanil to chlorazanil was considered. The metabolism of proguanil has been subject of substantial research in the past, specifically because proguanil is converted in the human organism to its bioactive derivative cycloguanil as mediated mainly by CYP2C19 [13, 14]. Besides cycloguanil, N-(4-chlorophenyl)-biguanide was identified as major metabolite with urinary concentrations reaching µg/mL levels [15, 16], but no reference to the formation of chlorazanil has ever been reported. However, the aforementioned metabolite N-(4-chlorophenyl)-biguanide was referred to as chemical intermediate and reactant in the production process of chlorazanil [10], and individuals referred to as poor metabolizers (due to a null allele genotype regarding CYP2C19) were shown to produce significantly elevated urinary concentrations of N-(4chlorophenyl)-biguanide as a result of the limited ability of converting proguanil to cycloguanil [17, 18]. Consequently, the possibility of a metabolic or artificial generation of the diuretic agent at low amounts warranted consideration. The formation of chlorazanil from N-(4-chlorophenyl)-biguanide would necessitate an adequate reaction partner such as formaldehyde, formamide, formic acid ester, etc. [10], some of which have been described as compounds regularly eliminated into human urine under specific dietary, environmental/occupational, and/or health 11
conditions [19-24]. In order to test the hypothesis, the reactivity of N-(4chlorophenyl)-biguanide with different chemicals was assessed in methanol in accordance to literature protocols [10] at ambient temperatures. While formamide, formaldehyde, and formic acid appeared to necessitate reflux conditions for the formation of chlorazanil, the incubation of N-(4-chlorophenyl)-biguanide with formic acid methyl ester yielded approximately 10% of the desired product (chlorazanil). Subsequently, ten different urine specimens were enriched each with 10 µg of N-(4chlorophenyl)-biguanide per mL and incubated for 72 h at room temperature before analysis by means of LC-HRMS. None of these samples showed the formation of chlorazanil. Only after repetition of the experiment with the additions of formaldehyde (with and without potassium carbonate), formic acid, or formic acid methyl ester also here the conversion of N-(4-chlorophenyl)-biguanide to chlorazanil was verified. Predominantly in the presence of formaldehyde under alkaline conditions (pH 9.5 as a result of the potassium carbonate) but also with formaldehyde or formic acid (pH 4.04.5) only, chlorazanil was observed. A reference standard containing all relevant analytes, a blank urine sample, and a blank urine specimen enriched with N-(4chlorophenyl)-biguanide, formaldehyde (plus potassium carbonate) incubated for 72 h is illustrated in Figure 2. This suggests that an artificial production of the diuretic from a major urinary metabolite of proguanil is principally possible, especially when circumstances
of increased urinary formaldehyde elimination prevail.
Such
circumstances include e.g. creatine supplementation [19, 20], which is a common scenario in the world of sport. Third, urine samples collected from patients undergoing malaria chemoprophylaxis with proguanil were analyzed for the presence of chlorazanil. In two out of four individuals, chlorazanil was detected (Figure 3, left and middle pane), which further 12
corroborated the possibility of an artificial formation of the diuretic under yet not fully clarified conditions. As none of the patients was receiving any other medication than proguanil hydrochloride and atovaquone, the presence of the diuretic agent chlorazanil was conclusively linked to the administration of the antimalarial drug combination. Fourth, to provide further information on a potential metabolic production of chlorazanil from proguanil, in vitro metabolism simulation experiments using human liver microsomal preparations were conducted. After 4.5 h of incubation, proguanil, the main metabolites cycloguanil and N-(4-chlorophenyl)-biguanide but no chlorazanil were observed (Figure 3, right pane), indicating that the diuretic is not an immediate metabolite of the antimalarial therapeutic.
Conclusion Circumstantial evidence and analytical data from authentic elimination studies with an antimalarial medication have contributed to a plausible explanation, other than an intentional anti-doping rule violation, why low amounts of the obsolete diuretic chlorazanil were detected in athletes’ doping control samples. In the given scenario, no one involved was acting negligent or engaged in any misconduct, i.e. the athletes’ medication was indicated, the drugs used were declared, and the analytical results were correct. Nevertheless, adverse analytical findings occurred, for which comprehensive follow-up studies were conducted, convincingly suggesting the artificial production of chlorazanil from a major metabolite of proguanil in urine. Consequently, none of the athletes was sanctioned, and for future chlorazanil findings in sports drug testing samples data interpretation in the light of the herein presented cases is recommended
13
to avoid unwarranted accusations of athletes.
Acknowledgments The project was conducted with support of the World Anti-Doping Agency (WADA, Montreal, Grant #T14M03WS) and the Federal Ministry of the Interior of the Federal Republic of Germany (Bonn, Germany). The authors thank Dr. George Ruijsch van Dugteren for fruitful discussions, and Oliver Krug and Josef Dib are thankfully acknowledged for technical support.
References [1]
W.L. Lipschitz, Z. Hadidian, Amides, amines and related compounds as diuretics, J. Pharmacol. Exp. Ther. 81 (1944) 84-94.
[2]
G. Kühn, Comparison of effects of various diuretics, with special consideration of N-p-chlorophenyl-2, 4-diamino-S-triazine (orpidan), Klin. Wochenschr. 35 (1957) 346-347.
[3]
I. Esch, H. Heeger, G. Saiko, Observations on the diuretic effect of orpidan, Klin. Wochenschr. 35 (1957) 855-857.
[4]
World Anti-Doping Agency. 2013 Anti-Doping Testing Figures 2015, https://www.wada-ama.org/en/resources/laboratories/2013-anti-doping-testingfigures-laboratory-report (27-05-2015)
[5]
J.F. Chaulet, G. Grelaud, P. Bellemin-Magninot, C. Mounier, J.L. Brazier, Simultaneous determination of chloroquine, proguanil and their metabolites in human biological fluids by high-performance liquid chromatography, J. Pharm. Biomed. Anal. 12 (1994) 111-117.
14
[6]
R.B. Taylor, R. Behrens, R.R. Moody, J. Wangboonskul, Assay method for the simultaneous determination of proguanil, chloroquine and their major metabolites in biological fluids, J. Chromatogr. 527 (1990) 490-497.
[7]
Y. Bergqvist, L. Funding, B. Krysen, T. Leek, K. Yvell, Improved validated assay for the determination of proguanil and its metabolites in plasma, whole blood, and urine using solid-phase extraction and high-performance liquid chromatography, Ther. Drug Monit. 20 (1998) 325-330.
[8]
W. Schänzer (1987). The problem of diuretics in doping control, in International Athletic Foundation World Symposium on Doping in Sport (Bellotti, P., Benzi, G., and Ljungqvist, A., Eds.), 89-106, FIDAL Centro Studi&Richerche, Florence.
[9]
M. Thevis, M. Kamber, W. Schänzer, Screening for metabolically stable arylpropionamide-derived selective androgen receptor modulators for doping control purposes, Rapid Commun. Mass Spectrom. 20 (2006) 870-876.
[10]
C.G. Overberger, S.L. Shapiro, Monoer Synthesis. Triazines. The Reaction of Phenylbiguanide with Ethyl Oxalate and Ethyl Formate, J. Am. Chem. Soc. 76 (1954) 93-96.
[11]
A. Knoop, O. Krug, M. Vincenti, W. Schänzer, M. Thevis, In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatographymass spectrometry, Eur. J. Mass. Spectrom. 21 (2015) 27-36.
[12]
P. Hommerson, A.M. Khan, T. Bristow, M.W. Harrison, G.J. de Jong, G.W. Somsen,
Drug
impurity
profiling
by
capillary
electrophoresis/mass
spectrometry using various ionization techniques, Rapid Commun. Mass Spectrom. 23 (2009) 2878-2884. 15
[13]
J.D. Wright, N.A. Helsby, S.A. Ward, The role of S-mephenytoin hydroxylase (CYP2C19) in the metabolism of the antimalarial biguanides, Br. J. Clin. Pharmacol. 39 (1995) 441-444.
[14]
J.K. Coller, A.A. Somogyi, F. Bochner, Association between CYP2C19 genotype and proguanil oxidative polymorphism, Br. J. Clin. Pharmacol. 43 (1997) 659-660.
[15]
R. Setiabudy, M. Kusaka, K. Chiba, I. Darmansjah, T. Ishizaki, Metabolic disposition of proguanil in extensive and poor metabolisers of S-mephenytoin 4'-hydroxylation recruited from an Indonesian population, Br. J. Clin. Pharmacol. 39 (1995) 297-303.
[16]
E. Petersen, H. Flachs, B. Hogh, A.P. Hanson, A. Bjorkman, E.F. Hvidberg, Plasma, erythrocyte and urine concentrations of chlorproguanil and two metabolites in man after different doses, J. Trop. Med. Hyg. 94 (1991) 199205.
[17]
N.A. Helsby, G. Edwards, A.M. Breckenridge, S.A. Ward, The multiple dose pharmacokinetics of proguanil, Br. J. Clin. Pharmacol. 35 (1993) 653-656.
[18]
N.A. Helsby, S.A. Ward, G. Edwards, R.E. Howells, A.M. Breckenridge, The pharmacokinetics and activation of proguanil in man: consequences of variability in drug metabolism, Br. J. Clin. Pharmacol. 30 (1990) 593-598.
[19]
J.R. Poortmans, A. Kumps, P. Duez, A. Fofonka, A. Carpentier, M. Francaux, Effect of oral creatine supplementation on urinary methylamine, formaldehyde, and formate, Med. Sci. Sports Exerc. 37 (2005) 1717-1720.
[20]
A. Nasseri, A. Jafari, Effects of creatine supplementation along with resistance training on urinary formaldehyde and serum enzymes in wrestlers, J. Sports Med. Phys. Fitness (2014) in press. 16
[21]
M. Berode, T. Sethre, T. Laubli, H. Savolainen, Urinary methanol and formic acid as indicators of occupational exposure to methyl formate, Int. Arch. Occup. Environ. Health 73 (2000) 410-414.
[22]
A. Takeuchi, T. Takigawa, M. Abe, T. Kawai, Y. Endo, T. Yasugi, G. Endo, K. Ogino, Determination of formaldehyde in urine by headspace gas chromatography, Bull. Environ. Contam. Toxicol. 79 (2007) 1-4.
[23]
L.C. Short, T. Benter, Selective measurement of HCHO in urine using direct liquid-phase fluorimetric analysis, Clin. Chem. Lab. Med. 43 (2005) 178-182.
[24]
P. Spanel, D. Smith, T.A. Holland, W. Al Singary, J.B. Elder, Analysis of formaldehyde in the headspace of urine from bladder and prostate cancer patients using selected ion flow tube mass spectrometry, Rapid Commun. Mass Spectrom. 13 (1999) 1354-1359.
Figures
Figure 1:
Chemical structures of proguanil (1, mol wtmonoisotopic = 253 Da), cycloguanil (2, mol wtmonoisotopic = 251 Da), N-(4-chlorophenyl)-biguanide (3, mol wtmonoisotopic = 211 Da), and chlorazanil (4, mol wtmonoisotopic = 221 Da) with suggested as well as proven metabolic and chemical interrelations.
17
Figure 2:
Extracted ion chromatograms of a mixture of reference substances (left), a blank urine sample containing the internal standard mefruside only (middle), and a blank urine specimen enriched with N-(4-chlorophenyl)biguanide and formaldehyde (plus potassium carbonate) after 72 h of incubation (right). Chlorazanil is obtained by incubation of the proguanil metabolite with formaldehyde as reactant as evidenced by the corresponding product ion mass spectra of chlorazanil.
Figure 3:
Extracted ion chromatograms of urine samples collected from individuals undergoing anti-malarial chemoprophylaxis with proguanil resulting in negative (left) and positive (middle) findings for chlorazanil (middle). In vitro metabolism experiments using human liver microsomal preparations yielded the metabolic products cycloguanil and 4-chlorophenyl-biguanide but no chlorazanil (right), supporting the conclusion that chlorazanil is not a direct metabolite of proguanil. *HLM = human liver microsomes
1
18
2
19
3
20
21
22
S0731-7085(15)30071-6 http://dx.doi.org/doi:10.1016/j.jpba.2015.07.017 PBA 10173
To appear in:
Journal of Pharmaceutical and Biomedical Analysis
Received date: Revised date: Accepted date:
15-6-2015 15-7-2015 18-7-2015
Please cite this article as: Mario Thevis, Hans Geyer, Andreas Thomas, Laura Tretzel, Isabelle Bailloux, Corinne Buisson, Francoise Lasne, Maximilian S.Schaefer, Peter Kienbaum, Irmela Mueller-Stoever, Wilhelm Sch¨anzer, Formation of the diuretic chlorazanil from the antimalarial drug proguanil ndash implications for sports drug testing, Journal of Pharmaceutical and Biomedical Analysis http://dx.doi.org/10.1016/j.jpba.2015.07.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Formation of the diuretic chlorazanil from the antimalarial drug proguanil – implications for sports drug testing Mario Thevis1,2*, Hans Geyer1, Andreas Thomas1, Laura Tretzel1, Isabelle Bailloux3, Corinne Buisson3, Francoise Lasne3, Maximilian S. Schaefer4, Peter Kienbaum4, Irmela Mueller-Stoever5, and Wilhelm Schänzer1
1Center
for Preventive Doping Research - Institute of Biochemistry, German Sport
University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany 2European 3Agence
Monitoring Center for Emerging Doping Agents, Cologne/Bonn, Germany
Française de Lutte contre le Dopage (AFLD), 143 avenue Roger Salengro,
92290 Châtenay-Malabry, France 4Department
of Anaesthesiology, University Hospital Duesseldorf, Moorenstr. 5,
40225 Duesseldorf, Germany 5Tropical
Medicine Unit, University Hospital for Gastroenterology, Hepatology and
Infectious Diseases, Heinrich-Heine-University Duesseldorf, Germany
*corresponding author:
Mario Thevis, PhD Institute of Biochemistry - Center for Preventive Doping Research German Sport University Cologne Am Sportpark Müngersdorf 6 50933 Cologne, Germany Tel: +49 221 4982 7070 Fax: +49 221 4982 7071 Email: [email protected] Graphical abstract
1
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 AntiDoping 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. 2
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 4chlorophenyl-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. Keywords:
doping, sport, mass spectrometry, diuretics
Introduction Based on findings by Lipschitz and co-workers concerning the diuretic activity of triazine derivatives in the mid-1940s [1], chlorazanil (Ordipan, N-(4-chlorophenyl)-
3
1,3,5-triazine-2,4-diamine, Figure 1, 4) was introduced as one of several new nonmercurial diuretic agents [2, 3] approximately a decade thereafter. The drug was commonly administered at 2 mg/kg of bodyweight, resulting in a significant increase of the individuals’ diuresis [3]. Due to their potential to illicitly reduce an athlete’s bodyweight and to mask other doping agents’ presence in sports drug testing urine samples, diuretic agents have been prohibited in sports since 1988. Since then, diuretics have frequently been detected in doping controls worldwide, ranking third among the internationally reported adverse analytical findings in 2013 [4]; however, chlorazanil was never identified in any doping control sample until 2014. This, the fact that chlorazanil has meanwhile become an obsolete therapeutic, and the credible denial of both athletes who were tested positive for the diuretic have been the motives for follow-up studies that potentially allow identifying another source of chlorazanil in athletes urine samples. A medication declared by two individuals with adverse analytical findings for chlorazanil was the antimalarial prophylactic drug proguanil (Figure 1, 1). It has been in clinical use since 1946 [5] and, in combination with chloroquine, it has represented a mainstay in malarial chemoprophylaxis ever since [6]. Proguanil is metabolized in vivo into the active drug cycloguanil (Figure 1, 2) and other metabolites, most importantly 4-chlorophenyl biguanide (Figure 1, 3) [7]. The formation of chlorazanil as a proguanil metabolite or a drug impurity was however never confirmed. Notwithstanding, two adverse analytical findings presenting with similar general circumstances (vide infra) existed that necessitated further investigations to provide best possible scientific data for a well-grounded anti-doping result management.
4
Case vignette In January and February 2014, three urine samples (pH 5.5-6.0) analyzed in the World Anti-Doping Agency (WADA)-accredited doping control laboratory in ChâtenayMalabry (France) yielded initial testing results suspicious for the presence of chlorazanil. The samples were collected from 2 female and one male elite athletes, competing in team and endurance sport, respectively. Testing locations were Senegal and Cameroon, and all three individuals indicated, amongst other medications and dietary supplements, the use of Malarone that contains 100 mg of proguanil hydrochloride and 250 mg of atovaquone. Confirmatory analyses of the urine samples verified the presence of proguanil, its main metabolites cycloguanil and N-(4chlorophenyl)-biguanide in all three cases. Further, chlorazanil was confirmed at approximately 0.3 and 1.3 ng/mL of urine in two cases, which were consequently reported as adverse analytical findings (AAFs). Follow-up studies were initiated that aimed at probing for a potential correlation between the malarial chemoprophylaxis and the AAFs for chlorazanil, including investigations into metabolic and chemical reactions of proguanil, its urinary metabolites, and ‘reactants’ commonly found in human urine.
Experimental 3.1 Drugs, chemicals and reagents The antimalarial drug Malarone (PZN: 8602632) was obtained from GlaxoSmithKline (Munich, Germany), N-(4-chlorophenyl)-biguanide, formaldehyde, formic acid, and 5
formic acid methyl ester were bought from Sigma (Steinheim, Germany), chlorazanil was from Heumann Pharma (Nuremberg, Germany), and human liver microsomes were from BD Gentest (Woburn, MA, USA).
3.2 Doping control urine samples analysis More than 9,000 doping control urine samples are tested each year for diuretics including chlorazanil in the WADA-accredited doping control laboratory in ChâtenayMalabry (France). Analytical initial testing and confirmation procedures enabling detection limits of 0.1 ng/mL for chlorazanil are applied. The initial testing method utilizes a total of 2 mL of urine, which is extracted twice with ethyl acetate, first at pH 5.2 and then at pH 14. The two organic extracts are combined, concentrated, and reconstituted in liquid chromatography (LC) mobile phase solvents. Four mL of urine is extracted twice with ethyl acetate at pH 14 in the confirmation methodology used. The combined extracts are concentrated and the residue is re-dissolved in LC mobile phase solvents for subsequent LC-MS/MS analysis.
3.3 Liquid chromatography – low resolution tandem mass spectrometry (LC-MS/MS) Sports drug testing analyses for diuretics are conducted using liquid chromatographictandem mass spectrometric (LC-MS/MS) approaches. The instrument used consisted of an Acquity UPLC (Waters, Guyancourt, France) LC equipped with an Agilent (Waldbronn, Germany) Zorbax SB-C8 column (2.1 100 mm, 1.8 m particle size). The solvents used were: water containing 10 mM of ammonium formate and acetic acid (pH 4) (eluent A) and acetonitrile (eluent B). The gradient program started at 10 % B 6
and increased to 55 % in 8 min, then increased to 100 % in 0.1 min and decreased to starting conditions within 0.5 min. The column was re-equilibrated at 10 % for 2 min. The flow rate was set at 400 L/min and the column temperature was 20 °C. The mass spectrometric identification of chlorazanil was performed using a Xevo TQMS triple quadrupole system (Waters, Guyancourt, France) with positive electrospray ionization using a gas flow desolvation of 800 L/h and a gas flow cone of 50 L/h. Source and desolvation temperatures were set to 150°C and 450°C, respectively. The apparatus was operated in selected reaction monitoring (SRM) acquisition mode using diagnostic precursor/product ion pairs of m/z 222 – 178, m/z 222 – 153, m/z 222 – 99, and m/z 222 – 45, with m/z 222 representing the protonated molecule [M+H]+. Collisioninduced dissociation (CID) was conducted at optimized collision energies, and nitrogen was used as collision gas at 3.7x10-3 mBar.
3.4 Patients’ urine sample analysis Spot urine samples collected approximately 16 h post-administration of Malarone were obtained via the University Hospital Duesseldorf (Heinrich-Heine University Duesseldorf, Germany) from four different patients (three male and one female) undergoing malaria chemoprophylaxis. The volunteers provided written consent, and the collection and use of the urine samples for research purposes were approved by the local ethics committee of the medical faculty of the Heinrich-Heine University Duesseldorf (study number 4749). The urine samples’ pH ranged from 5.5-8.0 and all specimens were subjected to analyses concerning chlorazanil and the potential synthetic precursor of chlorazanil, N-(4-chlorophenyl)-biguanide (Figure 1, 3) using an alternative test method employing solid-phase extraction [8, 9] combined with LC7
HRMS (vide infra).
3.5 Liquid chromatography – high resolution (tandem) mass spectrometry (LCHRMS/MS) LC-HRMS analyses were conducted on a Thermo Dionex Ultimate 3000 liquid chromatograph interfaced to a Q Exactive Plus mass spectrometer (Thermo Scientific, Bremen, Germany), using a heated electrospray ionization (HESI-II) source. The Dionex system was equipped with a Hypersil Gold analytical column, 2.1 50 mm, 1.9 µm particle size (Thermo Scientific, Bremen, Germany). The eluents consisted of A: 5 mM ammonium acetate (containing 0.1% acetic acid, pH = 3.5), and B: acetonitrile. Following injection of 5 µL of the sample, a gradient elution program was used, starting with 100% A, decreasing to 0% A in 7 min, followed by an isocratic step for 3 min and re-equilibration at starting conditions for 5 min. The total run time was 15 min, applying a flow rate of 200 µL/min. Measurements were conducted in positive ion mode. The HESI source temperature was 300 °C, the temperature of the transfer capillary was set to 320 °C, and the applied spray voltage was 3.75 kV. Nitrogen generated by a CMC nitrogen generator (CMC instruments, Eschborn, Germany) was used for collision-induced dissociation experiments in the HCD cell and furthermore as damping gas in the curved linear ion trap. The resolution of the mass spectrometer was set to 30,000 full width at half maximum (FWHM) (at m/z 200), and the precursor isolation window was adjusted to 1.5 Da. In order to ascertain mass errors below 5 ppm, the instrument was externally calibrated in positive mode using the manufacturer’s calibration solution (containing caffeine, Ultramark and the tetrapeptide MRFA). The system was operated in targeted MS/MS mode with
8
optimized collision energies.
3.6 Analysis of antimalarial drug (Malarone) for chlorazanil residues In order to sensitively probe for the potential presence of chlorazanil in the antimalarial therapeutic Malarone, three tablets containing 250 mg of atovaquone and 100 mg of proguanil each were dissolved in methanol and diluted to final concentrations of 100 µg/mL of proguanil. The samples were subsequently analyzed using the aforementioned LC-HRMS/MS test method, focusing on the accurate mass (± 5 ppm) of the protonated species [M+H]+ of diuretic agent at m/z 222.0541. With standard solutions of chlorazanil yielding detectable signals at concentrations of 0.01 ng/mL, the obtained solution of Malarone was tested for trace amounts of chlorazanil, accounting for 0.0001% of the structurally related proguanil.
3.7 In vitro production of chlorazanil from N-(4-chlorophenyl)-biguanide The possibility of artificial chlorazanil production in human urine was assessed by incubating reference standard solutions of N-(4-chlorophenyl)-biguanide with different reagents such as methanol, formaldehyde, formic acid, and methyl formate [10]. One mL aliquots of a methanolic solution containing 10 µg of N-(4-chlorophenyl)biguanide each were fortified with a) formaldehyde (50 µL of a 3% methanolic solution), b) formaldehyde (same as for a)) plus 20 mg of potassium carbonate, c) 10 µL of formic acid, and d) 10 µL of methyl formate, respectively. Further, ten different blank urine samples (1 mL, pH 5.5-8.0) enriched with 10 µg of N-(4-chlorophenyl)biguanide each were prepared with and without the addition of methyl formate, formaldehyde, or formic acid as described above. All specimens were incubated at room temperature for 72 h. 9
Methanolic solutions were analyzed concerning the formation of chlorazanil using the aforementioned LC-HRMS/MS method without further sample preparation while urine samples were prepared as described above for the patient’s urine specimens.
3.8 In vitro metabolism of proguanil using human liver microsomes Metabolic products of proguanil were enzymatically generated with human liver microsomes according to established in vitro assay protocols [11]. In brief, a substrate stock solution of 100 µM proguanil in dimethyl sulfoxide (DMSO) and a 50 mM phosphate buffer (pH 7.4) containing 5 mM MgCl2 as incubation buffer (IB) were prepared and stored at 4 °C. Samples were prepared in 1.5 mL test tubes. The reaction mixture used for phase-I metabolism contained 10 µL of the proguanil stock solution (1 mM), 10 µL of NADPH (50 mM in IB), and 10 µL of microsomes (200 µg protein/incubation) dissolved in IB, which was topped up with 70 µL of IB. The metabolic reaction was accomplished within 4 h at 37 °C. To probe for non-enzymatic reactions, an enzyme blank consisting of all reaction components except for the microsomes was prepared also. The incubation was terminated by adding 100 µL of ice-cold acetonitrile (ACN). Precipitated proteins were removed from the reaction mixture by centrifugation at 17,000 × g for 5 min at 4 °C and the supernatant was transferred to vials for subsequent LC-HRMS analysis.
Results and discussion Initiated by the observation that doping control urine samples of three athletes undergoing antimalarial chemoprophylaxis contained trace amounts of the obsolete but
10
nevertheless prohibited diuretic agent chlorazanil, follow-up studies on the potential origin of the drug were conducted. First, although conducted earlier with different techniques [12], the drug Malarone containing 250 mg of atovaquone and 100 mg of proguanil was analyzed for potential contaminations, specifically focusing on chlorazanil using LC-HRMS. None of the analyzed tablets returned findings for chlorazanil, indicating that the antimalarial medication was not the source of diuretic. Second, the possibility of a metabolic conversion of the structurally related product proguanil to chlorazanil was considered. The metabolism of proguanil has been subject of substantial research in the past, specifically because proguanil is converted in the human organism to its bioactive derivative cycloguanil as mediated mainly by CYP2C19 [13, 14]. Besides cycloguanil, N-(4-chlorophenyl)-biguanide was identified as major metabolite with urinary concentrations reaching µg/mL levels [15, 16], but no reference to the formation of chlorazanil has ever been reported. However, the aforementioned metabolite N-(4-chlorophenyl)-biguanide was referred to as chemical intermediate and reactant in the production process of chlorazanil [10], and individuals referred to as poor metabolizers (due to a null allele genotype regarding CYP2C19) were shown to produce significantly elevated urinary concentrations of N-(4chlorophenyl)-biguanide as a result of the limited ability of converting proguanil to cycloguanil [17, 18]. Consequently, the possibility of a metabolic or artificial generation of the diuretic agent at low amounts warranted consideration. The formation of chlorazanil from N-(4-chlorophenyl)-biguanide would necessitate an adequate reaction partner such as formaldehyde, formamide, formic acid ester, etc. [10], some of which have been described as compounds regularly eliminated into human urine under specific dietary, environmental/occupational, and/or health 11
conditions [19-24]. In order to test the hypothesis, the reactivity of N-(4chlorophenyl)-biguanide with different chemicals was assessed in methanol in accordance to literature protocols [10] at ambient temperatures. While formamide, formaldehyde, and formic acid appeared to necessitate reflux conditions for the formation of chlorazanil, the incubation of N-(4-chlorophenyl)-biguanide with formic acid methyl ester yielded approximately 10% of the desired product (chlorazanil). Subsequently, ten different urine specimens were enriched each with 10 µg of N-(4chlorophenyl)-biguanide per mL and incubated for 72 h at room temperature before analysis by means of LC-HRMS. None of these samples showed the formation of chlorazanil. Only after repetition of the experiment with the additions of formaldehyde (with and without potassium carbonate), formic acid, or formic acid methyl ester also here the conversion of N-(4-chlorophenyl)-biguanide to chlorazanil was verified. Predominantly in the presence of formaldehyde under alkaline conditions (pH 9.5 as a result of the potassium carbonate) but also with formaldehyde or formic acid (pH 4.04.5) only, chlorazanil was observed. A reference standard containing all relevant analytes, a blank urine sample, and a blank urine specimen enriched with N-(4chlorophenyl)-biguanide, formaldehyde (plus potassium carbonate) incubated for 72 h is illustrated in Figure 2. This suggests that an artificial production of the diuretic from a major urinary metabolite of proguanil is principally possible, especially when circumstances
of increased urinary formaldehyde elimination prevail.
Such
circumstances include e.g. creatine supplementation [19, 20], which is a common scenario in the world of sport. Third, urine samples collected from patients undergoing malaria chemoprophylaxis with proguanil were analyzed for the presence of chlorazanil. In two out of four individuals, chlorazanil was detected (Figure 3, left and middle pane), which further 12
corroborated the possibility of an artificial formation of the diuretic under yet not fully clarified conditions. As none of the patients was receiving any other medication than proguanil hydrochloride and atovaquone, the presence of the diuretic agent chlorazanil was conclusively linked to the administration of the antimalarial drug combination. Fourth, to provide further information on a potential metabolic production of chlorazanil from proguanil, in vitro metabolism simulation experiments using human liver microsomal preparations were conducted. After 4.5 h of incubation, proguanil, the main metabolites cycloguanil and N-(4-chlorophenyl)-biguanide but no chlorazanil were observed (Figure 3, right pane), indicating that the diuretic is not an immediate metabolite of the antimalarial therapeutic.
Conclusion Circumstantial evidence and analytical data from authentic elimination studies with an antimalarial medication have contributed to a plausible explanation, other than an intentional anti-doping rule violation, why low amounts of the obsolete diuretic chlorazanil were detected in athletes’ doping control samples. In the given scenario, no one involved was acting negligent or engaged in any misconduct, i.e. the athletes’ medication was indicated, the drugs used were declared, and the analytical results were correct. Nevertheless, adverse analytical findings occurred, for which comprehensive follow-up studies were conducted, convincingly suggesting the artificial production of chlorazanil from a major metabolite of proguanil in urine. Consequently, none of the athletes was sanctioned, and for future chlorazanil findings in sports drug testing samples data interpretation in the light of the herein presented cases is recommended
13
to avoid unwarranted accusations of athletes.
Acknowledgments The project was conducted with support of the World Anti-Doping Agency (WADA, Montreal, Grant #T14M03WS) and the Federal Ministry of the Interior of the Federal Republic of Germany (Bonn, Germany). The authors thank Dr. George Ruijsch van Dugteren for fruitful discussions, and Oliver Krug and Josef Dib are thankfully acknowledged for technical support.
References [1]
W.L. Lipschitz, Z. Hadidian, Amides, amines and related compounds as diuretics, J. Pharmacol. Exp. Ther. 81 (1944) 84-94.
[2]
G. Kühn, Comparison of effects of various diuretics, with special consideration of N-p-chlorophenyl-2, 4-diamino-S-triazine (orpidan), Klin. Wochenschr. 35 (1957) 346-347.
[3]
I. Esch, H. Heeger, G. Saiko, Observations on the diuretic effect of orpidan, Klin. Wochenschr. 35 (1957) 855-857.
[4]
World Anti-Doping Agency. 2013 Anti-Doping Testing Figures 2015, https://www.wada-ama.org/en/resources/laboratories/2013-anti-doping-testingfigures-laboratory-report (27-05-2015)
[5]
J.F. Chaulet, G. Grelaud, P. Bellemin-Magninot, C. Mounier, J.L. Brazier, Simultaneous determination of chloroquine, proguanil and their metabolites in human biological fluids by high-performance liquid chromatography, J. Pharm. Biomed. Anal. 12 (1994) 111-117.
14
[6]
R.B. Taylor, R. Behrens, R.R. Moody, J. Wangboonskul, Assay method for the simultaneous determination of proguanil, chloroquine and their major metabolites in biological fluids, J. Chromatogr. 527 (1990) 490-497.
[7]
Y. Bergqvist, L. Funding, B. Krysen, T. Leek, K. Yvell, Improved validated assay for the determination of proguanil and its metabolites in plasma, whole blood, and urine using solid-phase extraction and high-performance liquid chromatography, Ther. Drug Monit. 20 (1998) 325-330.
[8]
W. Schänzer (1987). The problem of diuretics in doping control, in International Athletic Foundation World Symposium on Doping in Sport (Bellotti, P., Benzi, G., and Ljungqvist, A., Eds.), 89-106, FIDAL Centro Studi&Richerche, Florence.
[9]
M. Thevis, M. Kamber, W. Schänzer, Screening for metabolically stable arylpropionamide-derived selective androgen receptor modulators for doping control purposes, Rapid Commun. Mass Spectrom. 20 (2006) 870-876.
[10]
C.G. Overberger, S.L. Shapiro, Monoer Synthesis. Triazines. The Reaction of Phenylbiguanide with Ethyl Oxalate and Ethyl Formate, J. Am. Chem. Soc. 76 (1954) 93-96.
[11]
A. Knoop, O. Krug, M. Vincenti, W. Schänzer, M. Thevis, In vitro metabolism studies on the selective androgen receptor modulator (SARM) LG121071 and its implementation into human doping controls using liquid chromatographymass spectrometry, Eur. J. Mass. Spectrom. 21 (2015) 27-36.
[12]
P. Hommerson, A.M. Khan, T. Bristow, M.W. Harrison, G.J. de Jong, G.W. Somsen,
Drug
impurity
profiling
by
capillary
electrophoresis/mass
spectrometry using various ionization techniques, Rapid Commun. Mass Spectrom. 23 (2009) 2878-2884. 15
[13]
J.D. Wright, N.A. Helsby, S.A. Ward, The role of S-mephenytoin hydroxylase (CYP2C19) in the metabolism of the antimalarial biguanides, Br. J. Clin. Pharmacol. 39 (1995) 441-444.
[14]
J.K. Coller, A.A. Somogyi, F. Bochner, Association between CYP2C19 genotype and proguanil oxidative polymorphism, Br. J. Clin. Pharmacol. 43 (1997) 659-660.
[15]
R. Setiabudy, M. Kusaka, K. Chiba, I. Darmansjah, T. Ishizaki, Metabolic disposition of proguanil in extensive and poor metabolisers of S-mephenytoin 4'-hydroxylation recruited from an Indonesian population, Br. J. Clin. Pharmacol. 39 (1995) 297-303.
[16]
E. Petersen, H. Flachs, B. Hogh, A.P. Hanson, A. Bjorkman, E.F. Hvidberg, Plasma, erythrocyte and urine concentrations of chlorproguanil and two metabolites in man after different doses, J. Trop. Med. Hyg. 94 (1991) 199205.
[17]
N.A. Helsby, G. Edwards, A.M. Breckenridge, S.A. Ward, The multiple dose pharmacokinetics of proguanil, Br. J. Clin. Pharmacol. 35 (1993) 653-656.
[18]
N.A. Helsby, S.A. Ward, G. Edwards, R.E. Howells, A.M. Breckenridge, The pharmacokinetics and activation of proguanil in man: consequences of variability in drug metabolism, Br. J. Clin. Pharmacol. 30 (1990) 593-598.
[19]
J.R. Poortmans, A. Kumps, P. Duez, A. Fofonka, A. Carpentier, M. Francaux, Effect of oral creatine supplementation on urinary methylamine, formaldehyde, and formate, Med. Sci. Sports Exerc. 37 (2005) 1717-1720.
[20]
A. Nasseri, A. Jafari, Effects of creatine supplementation along with resistance training on urinary formaldehyde and serum enzymes in wrestlers, J. Sports Med. Phys. Fitness (2014) in press. 16
[21]
M. Berode, T. Sethre, T. Laubli, H. Savolainen, Urinary methanol and formic acid as indicators of occupational exposure to methyl formate, Int. Arch. Occup. Environ. Health 73 (2000) 410-414.
[22]
A. Takeuchi, T. Takigawa, M. Abe, T. Kawai, Y. Endo, T. Yasugi, G. Endo, K. Ogino, Determination of formaldehyde in urine by headspace gas chromatography, Bull. Environ. Contam. Toxicol. 79 (2007) 1-4.
[23]
L.C. Short, T. Benter, Selective measurement of HCHO in urine using direct liquid-phase fluorimetric analysis, Clin. Chem. Lab. Med. 43 (2005) 178-182.
[24]
P. Spanel, D. Smith, T.A. Holland, W. Al Singary, J.B. Elder, Analysis of formaldehyde in the headspace of urine from bladder and prostate cancer patients using selected ion flow tube mass spectrometry, Rapid Commun. Mass Spectrom. 13 (1999) 1354-1359.
Figures
Figure 1:
Chemical structures of proguanil (1, mol wtmonoisotopic = 253 Da), cycloguanil (2, mol wtmonoisotopic = 251 Da), N-(4-chlorophenyl)-biguanide (3, mol wtmonoisotopic = 211 Da), and chlorazanil (4, mol wtmonoisotopic = 221 Da) with suggested as well as proven metabolic and chemical interrelations.
17
Figure 2:
Extracted ion chromatograms of a mixture of reference substances (left), a blank urine sample containing the internal standard mefruside only (middle), and a blank urine specimen enriched with N-(4-chlorophenyl)biguanide and formaldehyde (plus potassium carbonate) after 72 h of incubation (right). Chlorazanil is obtained by incubation of the proguanil metabolite with formaldehyde as reactant as evidenced by the corresponding product ion mass spectra of chlorazanil.
Figure 3:
Extracted ion chromatograms of urine samples collected from individuals undergoing anti-malarial chemoprophylaxis with proguanil resulting in negative (left) and positive (middle) findings for chlorazanil (middle). In vitro metabolism experiments using human liver microsomal preparations yielded the metabolic products cycloguanil and 4-chlorophenyl-biguanide but no chlorazanil (right), supporting the conclusion that chlorazanil is not a direct metabolite of proguanil. *HLM = human liver microsomes
1
18
2
19
3
20
21
22