Olodaterol and vilanterol detection in sport drug testing.

The possibility of the detection of olodaterol and vilanterol, two novel ß2 -agonists, in human urine for the purpose of sport drug testing was investigated. Compounds of interest were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) employing methods commonly used in the World Anti-Doping Agency (WADA) accredited laboratories. For both substances, the respective parent compound was found to be a suitable target analyte for monitoring therapeutic dose administration.

Tissue distribution, excretion, and the metabolic pathway of 2,2',4,4',5-penta-chlorinated diphenylsulfide (CDPS-99) in ICR mice.

The tissue distribution, excretion, and metabolic pathway of 2,2',4,4',5-penta-chlorinated diphenylsulfide (CDPS-99) in ICR mice were investigated after oral perfusion at 10mg/kg body weight (b.w.). Biological samples were extracted and separated and, for the first time, were determined by a novel, sensitive, and specific GC-MS method under the full scan and selected ion monitoring (SIM) modes. The results showed that the concentrations of CDPS-99 in the liver, kidneys, and serum reached a maximum after a one-day exposure and that the CDPS-99 concentration in the liver was the highest (3.43µg/g). The increase in the concentration of CDPS-99 in muscle, skin, and adipose tissue was slower, and the concentrations of CDPS-99 achieved their highest levels after 3 days of exposure. It was observed that the CDPS-99 concentration in adipose tissue was still very high (0.71µg/g) after 21 days of exposure, which suggested that CDPS-99 was able to accumulate in adipose tissue. In addition, mouse feces accounted for approximately 75% of the total gavage dose, indicating that CDPS-99 was mainly excreted via mouse feces. Metabolism analysis demonstrated that there were three possible metabolic pathways of CDPS-99 in mice: dechlorination reactions with the formation of tetra-CDPS and hydroxylation and oxidation reactions with the formation of OH-CDPS-99 and chlorinated diphenylsulfone. The present study will help to develop a better understanding of mammalian metabolism of CDPS-99.

Formation of the diuretic chlorazanil from the antimalarial drug proguanil--implications for sports drug testing.

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.

An intake of C14-labelled dichlorobenzene.

An intake of C14 in the form of dichlorobenzene was followed up with 90 spot urine samples over a period of almost 2 weeks. This dataset has been fitted by a model consisting of three exponential terms. The intake and effective dose have been calculated. This case has been used to examine the effects of recent proposals by ICRP concerning the calculation of effective dose and the use of non-standard biokinetic models.

Simple isotope dilution headspace-GC-MS analysis of naphthalene and p-dichlorobenzene in whole blood and urine.

A very simple and sensitive method for the simultaneous analysis of naphthalene and p-dichlorobenzene in human whole blood and urine by headspace capillary gas chromatography-mass spectrometry (GC-MS) is presented. The advantages of the method were that as much as 1 mL of headspace vapor could be injected into a GC port in the splitless mode, and that the addition of deuterated naphthalene and p-dichlorobenzene as internal standards resulted in much better headspace extraction efficiencies, which resulted in high sensitivity. The detection limits for both naphthalene and p-dichlorobenzene were 1 ng mL(-1) for whole blood and 0.5 ng mL(-1) for urine. Validation data, such as the linearity of calibration curves, reproducibility and recovery rates, were all satisfactory. Using this method, both compounds could actually be detected from whole blood samples of a male volunteer after the inhalation of each gas of the compounds.

Characterization of cytochrome P450-mediated bioactivation of a compound containing the chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-(4-chlorophenyl amide), to a chemically reactive p-chlorophenyl isocyanate intermediate in human liver microsomes.

Compound A (Cmpd A) was previously reported to form p-chlorophenyl isocyanate (CPIC), which was trapped by GSH to yield S- (N- [p-chlorophenyl] carbamoyl) glutathione adduct (SCPG) in the presence of human liver microsomes. In this study, P450 3A4 and 2C9 were demonstrated to be the enzymes mediating the activation of Cmpd A to CPIC in human liver microsomes based on inhibitory and correlation studies. Enzyme kinetics studies indicated that P450 3A4 was the primary enzyme involved in the activation of Cmpd A. In silico P450 3A4 active site docking of Cmpd A exhibited a low energy pose that orientated the pyrazole ring proximate to the heme iron atom, in which the distance between the C-3 and potential activated oxygen species was shown to be 3.4 A. Quantum molecular calculations showed that the electron density on C-3 was relatively higher than those on C-4 and C-5. These measurements suggested that the C-3 of Cmpd A was the preferred site of oxidation and hence predisposed Cmpd A in forming CPIC as previously proposed. The in silico prediction was corroborated by studies with the C-3 substituted analogue (methyl at C-3), which showed minimal conversion to CPIC in human liver microsomes. These results demonstrated a pivotal role for P450 3A4 in bioactivating Cmpd A by oxidizing at C-3 of the pyrazoline, hence facilitating the CPIC formation. Evidence of the bioactivation to CPIC in vivo was obtained by liquid chromatography-mass spectrometry (LC/MS) analysis of urine samples from human subjects administered a structural analogue of Cmpd A. The presence of S-(N-[p-chlorophenyl] carbamoyl) N-acetyl l-cysteine (SCPAC) as well as p-chlorophenyl aniline (CPA) was unequivocally demonstrated in the urine samples. The chemical scaffold, 4,5-dihydropyrazole-1-carboxylic acid-[(4-chlorophenyl)-amide], was demonstrated to possess potential metabolic liability in forming a reactive intermediate, CPIC, in humans. Bioactivation to CPIC may cause undesirable side effects through its reactivity and subsequent conversion to CPA, an established rodent carcinogen.

Inhalation toxicokinetics of p-dichlorobenzene and daily absorption and internal accumulation in chronic low-level exposure to humans.

Inhalation toxicokinetics of p-dichlorobenzene ( p-DCB) in humans was evaluated, and the amounts of daily absorption and internal accumulation were estimated in order to obtain fundamental data for the risk assessment of chronic low-level exposure in the general population. Seven male subjects continuously inhaled about 2.5 ppm of p-DCB vapor for 1 h, and the concentration-time courses of p-DCB in their exhaled air and serum and of urinary 2,5-dichlorophenol (2,5-DCP), a major metabolite of p-DCB, were examined. The toxicokinetics of p-DCB was evaluated on the basis of the time courses using a linear two-compartment model. The amounts of p-DCB absorbed daily and the internal accumulation in chronic low-level exposure were extrapolated using the estimated toxicokinetic parameters. p-DCB was transferred from inhaled air to the body with a constant high absorption rate during exposure. The major route for elimination from the body was urinary excretion followed by metabolism, not exhalation. However, during 9-11 h after the start of exposure, the fraction of p-DCB excreted in urine was only 5-16% of the amount absorbed. Furthermore, most of the absorbed p-DCB seemed to be distributed rapidly to the tissues, such as fat, according to toxicokinetic analysis. Consequently, p-DCB seems to require a long time to be completely eliminated from the body. The amounts of daily absorption and internal accumulation were extrapolated to average 0.27 mg/day and 2.9 mg, respectively, in the subjects exposed chronically to 1 ppb of p-DCB. The amount absorbed daily agreed approximately with that extrapolated from rats which inhaled p-DCB in our previous study.

Human toxicokinetics of inhaled monochlorobenzene: latest experimental findings regarding re-evaluation of the biological tolerance value.

OBJECTIVE: The aim of this study was to obtain toxicokinetic data on the absorption and elimination of monochlorobenzene (MCB) in blood and its main metabolite 4-chlorocatechol (4-ClCat) as well as on the isomeric chlorophenols (o-ClPh, m-ClPh, and especially p-CIPh as the main ClPh metabolite) in urine for reevaluation of the biological tolerance (BAT) value of MCB. METHODS: Eight subjects performed 8-h inhalation tests daily over five successive days in an exposure chamber, at a maximum allowable concentration at the workplace (MAK) value of 10 ppm MCB. Five and two probands carried out the test series during physical activity levels of 75 and 50 W, respectively, for 10 min/h on a bicycle ergometer, and one subject was exposed continuously while at rest. MCB and its metabolites were analyzed by gas chromatography in combination with mass spectrometry. RESULTS: The mean MCB blood concentration of the five subjects exposed during physical activity of 75 W was 217 +/- 42 microg/l. The relationship of the mean blood concentration measured under the conditions of rest or 50 and 75 W activity levels was in a ratio of about 1:1.7:2.8. The half-life values in the first hour after ending the exposures were 53 min and 150 min for the ensuing period, with steady-state being reached after 45 min. The mean 4-ClCat concentration in urine at the end of the five days was 150 +/- 13 mg/g creatinine in the case of the subjects exposed at 75 W, which decreased to 25 mg/g creatinine at the beginning of the next exposure. The analogous p-ClPh concentrations were 25 +/- 2 and 9 +/- 2 mg/g creatinine. The elimination half-life values of the ClPh isomers ranged from 12.4 to 16.5 h, and the half-life of 4-ClCat was 6.4 h. There was no apparent tendency for MCB and its metabolites to accumulate in blood or urine. CONCLUSIONS: The results are in accordance with relevant field and laboratory studies. Taken into consideration with the 95th percentile, the evaluated BAT values should be set at levels of 300 microg MCB/l blood, 175 mg 4-ClCat/g creatinine or alternatively at 30 mg p-ClPh/g creatinine in urine after the end of a shift. At the beginning of the next shift, the BAT values of the metabolites should be 35 and 15 mg/g creatinine, respectively.

Comparative metabolism of the renal carcinogen 1,4-dichlorobenzene in rat: identification and quantitation of novel metabolites.

1. The metabolism of 1,4-dichlorobenzene has been studied in the male and female Fisher 344 rat over 72 h after oral administration of 14C-1,4-dichlorobenzene (900 mg = 96.8 microCi/kg). No covalent binding of radioactivity could be detected in samples of liver, kidney, lung and spleen. The major route of excretion was with urine accounting for 41.3% of the dose for male and 37.8% of the dose for female rat within 72 h after dosing. 2. Urinary metabolites of 1,4-dichlorobenzene were identified and quantified. The major metabolites identified in the urine of both the male and female rat, were the sulphate and glucuronide of 2,5-dichlorophenol. Minor amounts of 2,5-dichlorohydroquinone were excreted as an unidentified conjugate. 3. 2-(N-acetyl-cysteine-S-yl)-1,4-dichlorobenzene and 2-(N-acetyl-cysteine-S-yl)-2,3-dihydro-3-hydroxy-1,3-hydroxy-1,4-dich lorobenzen e were minor metabolites excreted in the urine of both sexes. 4. A novel biotransformation pathway for 1,4-dichlorobenzene may be postulated, leading to the urinary excretion of a mercapturic acid of chlorophenol. 5. No marked differences in the distribution and excretion of metabolites of 1,4-dichlorobenzene were observed between the male and female Fisher 344 rat.

Comparison of the urinary metabolite profiles of hexachlorobenzene and pentachlorobenzene in the rat.

The urinary metabolite profile of hexachlorobenzene (HCB) and pentachlorobenzene (PCBz) in the rat is compared after dietary exposure for 13 weeks. Both HCB and PCBz are oxidized to pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ), which were the only two mutual metabolites formed. Additional urinary metabolites of HCB are N-acetyl-S(pentachlorophenyl)cysteine (PCTP-NAC), which appeared to be quantitatively the most important product, and mercaptotetrachlorothioanisole (MTCTA), which was excreted as a glucuronide. PCBz is more extensively metabolized to the major metabolites 2,3,4,5-tetrachlorophenol (TCP), mercaptotetrachlorophenol (MTCP) and the glucuronide of pentachlorothiophenol (PCTP), and the minor metabolites methylthiotetrachlorophenol (MeTTCP), hydroxytetrachlorophenyl sulphoxide (HTCPS), and bis(methylthio)-trichlorophenol (bis-MeTTriCP). The biotransformation of HCB and PCBz was modulated by selective inhibition of cytochrome P450IIIA in rats which received combined treatment of HCB or PCBz with triacetyloleandomycin (TAO). Rats receiving this diet had a strongly diminished excretion of both PCP and TCHQ, as compared to rats fed HCB or PCBz alone, indicating the involvement of P450IIIA in the oxidation of both compounds. However, the excretion of 2,3,4,5-TCP was not diminished by co-treatment of rats with PCBz and TAO, indicating that: (i) the oxidation of PCBz to PCP and 2,3,4,5-TCP does not proceed via a common intermediate; and (ii) oxidation of PCBz to 2,3,4,5-TCP is not mediated by P450IIIA. Co-treatment of rats with PCBz and TAO had a differential effect on the excretion of sulphur-containing metabolites, resulting in a decrease in the excretion of PCTP glucuronide, whereas no change was observed in the excretion of MTCP, as compared to rats receiving PCBz alone. The observed differences in HCB and PCBz metabolites clearly deserve further in vitro studies to elucidate their origin.

Glutathione-mediated methylthio-turnover and sex differences in the metabolism of pentachlorothioanisole by rat.

1. Sex differences observed in the metabolism of pentachlorothioanisole in rat were due to: (1) greater excretion in urine by females, and greater biliary excretion by males; (2) formation of pentachlorophenyl mercapturic acid pathway metabolites by females; and (3) redox-cycling between methylthio and methylsulphoxyl oxidation congeners in intermediary metabolites by females. 2. Three methylthio-turnover processes are proposed in the intermediary metabolism of pentachlorothioanisole.

Effect of dietary protein and sulfur-containing amino acids on urinary metabolites in rats injected with chlorobenzene.

Rats were fed for 10 days a 10 or 20% casein diet, or the diets with 0.3% DL-methionine or L-cystine. Rats fed the 20% casein exceeded those fed the 10% casein in growth, hepatic glutathione (GSH) levels, and microsomal drug-metabolizing activities including cytochrome P-450, aminopyrine N-demethylase, and aniline hydroxylase. Supplement with methionine or cystine had significantly elevated hepatic GSH, regardless of the casein content. After the feeding, rats were intraperitoneally injected with chlorobenzene (0.5 mmol/kg body weight), and the urinary metabolites (4-chlorophenylmercapturic acid (4-CPMA), 2-, 3- and 4-chlorophenol (CPs), 4-chlorocatechol (4-CC), and 2-, 3- and 4-chlorophenylmethylsulfide (CPMSs) ) were measured for 24 hours post-injection. Rats fed the 20% casein exceeded those fed the 10% casein in 4-CPMA, CPs, and in total urinary metabolites. Supplement with methionine or cystine to the 10% casein significantly increased 4-CPMA and decreased 4-CC. Supplement with methionine or cystine to the 20% casein also significantly increased 4-CPMA excretion, but had no effect on urinary 4-CC. The highest urinary excretion of CPMSs was observed in rats fed the 10% casein. Both increase of dietary protein and addition of the sulfur-containing amino acids decreased urinary CPMSs. These results indicate that total urinary metabolites are strongly associated with the microsomal drug-metabolizing activity, formation of the mercapturic acid is dependent on the hepatic GSH level, and the urinary CPMS level is independent on the mercapturic acid formation.

Pentachlorophenol and hexachlorobenzene in serum and urine of the population of Barcelona.

1 Urinary chlorophenols of the general population of Barcelona, Spain were determined. Pentachlorophenol (PCP: 25.0 +/- 3.9 ng/ml; mean +/- s.e.m., n = 50) and tetrachlorophenol (TCP: 6.2 +/- 1.6 ng/ml; mean +/- s.e.m., n = 25) were found in all samples. 2 Pentachlorophenol and hexachlorobenzene were also determined in serum. Both were present in all samples (PCP: 21.9 +/- 1.9 ng/ml; HCB: 11.1 +/- 1.1 ng/ml; mean +/- s.e.m., n = 100). Their concentrations do not show any correlation, suggesting no metabolic relation between them.

Tissue distribution and elimination of trichlorobenzenes in the rat.

The tissue distribution and excretion of three trichlorobenzene isomers (TCB) were investigated in the rat. Single doses of TCBs were administered orally to groups of 5 fasted rats at 10 mg/kg body weight. Serial sacrifices were carried out and the radioactivity contents were determined in tissues and blood. For all three TCB isomers, radioactivity appeared in the blood and tissues at 0.5 h, and peaked around 2-4 h after dosing. Fat, skin, and liver had high concentrations of the parent compound while kidney and muscle had high levels of metabolites. Elimination of TCB from tissues and blood can best be described by a two-compartmental open pharmacokinetic model. The terminal half-lives were 145, 93 and 68 h for 1,2,3-, 1,2,4 and 1,3,5-TCB isomer respectively. Ninety-five percent of the administered 1,2,3- and 89% of the 1,3,5-isomers were eliminated within 48 h in the urine and feces with the former being the major route.

Metabolism of 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-tetrachlorobenzene in the squirrel monkey.

The metabolism of three tetrachlorobenzene isomers (TeCB) was investigated in the squirrel monkey. The animals were administered orally 6 single doses of 14C-labeled 1,2,3,4-, 1,2,4,5-, or 1,2,3,5-tetrachlorobenzene over a 3-wk period at levels ranging from 50 to 100 mg/kg body weight (b.w.) and kept in individual metabolism cages to collect urine and feces for radioassay. Approximately 38% (1,2,3,4-TeCB), 36% (1,2,3,5-TeCB), and 18% (1,2,4,5-TeCB) of the doses were excreted respectively in the feces 48 h postadministration. In monkeys dosed with 1,2,3,4-TeCB, unchanged compound accounted for 50% of the fecal radioactivity; its fecal metabolites were identified as 1,2,4,5-tetrachlorophenol (TeCP, 22%), N-acetyl-S-(2,3,4,5-tetrachlorophenyl) cysteine (18%), 2,3,4,5-tetrachlorophenyl sulfinic acid (3%), 2,3,4-trichlorophenyl methyl sulfide (0.6%), and 2,3,4,5-tetrachlorophenyl methyl sulfide (0.2%). As was the case with 1,2,3,4-TeCB, unchanged compound accounted for more than 50% of the fecal radioactivity found in the monkeys dosed with 1,2,3,5-TeCB. The fecal metabolites of 1,2,3,5-TeCB consisted of 2,3,4,5-TeCP (2%), 2,3,4,6-TeCP (14%), 2,3,5,6-TeCP (9%), and 2,3,5,6-tetrachlorophenyl sulfinic acid (15%). No metabolites were detected in the feces of monkeys dosed with 1,2,4,5-TeCB. While the fecal route represented the major route of excretion for 1,2,3,4-TeCB, the other two isomers were eliminated exclusively in the feces. The above data in the squirrel monkey are different from those obtained with the rat and the rabbit, and demonstrate the different metabolic pathways for the isomers.

Excretion, distribution and metabolism of 1,2,4-trichlorobenzene in rats.

1,2,4-Trichlorobenzene (TCB) labeled with C-14 was given perorally to rats at a dosage of 50 mg/kg for excretion and distribution studies. About 66% and 17% of the oral dose was excreted in the urine and feces, respectively, within 7 days. Trapped radioactivity in the expired air amounted to 2.1% of the dose, but production of labeled carbon dioxide was negligible. Tissue residues were evenly distributed throughout the organs and tissues examined, except for the adipose tissue which consistently had a little higher concentration. The urinary, fecal and expiratory metabolites were identified. Free 2,4,5- and 2,3,5-trichlorophenol (TCP) and their conjugates were mainly detected in the urine. 5- or 6-Sulfhydryl, methylthio, methylsulfoxide and methylsulfone derivatives of TCB were also detected as minor metabolites. Dichlorobenzenes and unchanged TCB were confirmed in the expired air. Reductive dechlroination seems to be catalysed by intestinal microflora enzymes.

Urinary elimination of p-dichlorobenzene (p-DCB) and weighted exposure concentration.

The aim of the present work is to determine p-DCB concentration in the urine of exposed workers and to verify a possible correlation with the environmental exposure. The authors studied four subjects exposed to different p-DCB environmental concentrations during a working week. The measurement of the substances was performed by means of a Hewlett-Packard 5880 A gas-chromatograph supplied with a Hewlett-Packard 5970 A Mass Selective Detector. The analysis was performed by a head space method (after determining the urine/air partition coefficient (lambda) by the multiple phase equilibration method). The lambda value (urine/air) of p-DCB is 10.8. The Authors found a significant relationship between the difference of p-DCB urinary concentration at the beginning and end of a daily work delta Cu (microgram/l) and the p-DCB environmental concentration C-I (mg/m3) (r = 0.64) (P less than 0.01); delta Cu = 0,48C-I + 15.61. We found that with an environmental exposure of 44.7 mg/m3 median value (geometrical deviation 1.15) there is an increase of p-DCB concentration in the urine of workers during the working week. For a daily environmental exposure of 450 mg/m3 (ACGIH TLV, 1984), we think that it is possible to propose a Biological Exposure Index (B.E.I.) of 250 micrograms/l as difference between beginning and end of a daily work.