19 F-NMR and directly coupled 19 F/ 1 H-HPLC-NMR spectroscopic investigations of the metabolism of the model ecotoxin 3-trifluoromethylaniline in the earthworm species Eisenia veneta

1. The metabolic fate of the model ecotoxin 3-trifluoromethylaniline (3-TFMA) in earthworm was studied by 19 F- and directly coupled 19 F/ 1 H-HPLC-NMR spectroscopy. Earthworms of Eisenia veneta spp. were subjected to the ecotoxin during a filter papercontact toxicity test at exposure levels of 1000, 100, 10, 1 and 0.1 µg cm -2. A metabolic profile was obtained previously by 19 F-NMR spectroscopy and metabolites were observed at all the exposure levels. 2. Identification of metabolites in individual worm extracts at the (lethal) exposure levels of 1000 and 100 µg cm -2 could be achieved on-line without sample preparation by 19 F/ 1 H-HPLC-NMR spectroscopy. 19 F-HPLC-NMR spectroscopy was used in the continuous-flow mode, which enabled the HPLC chromatographic retention times (t R) of the metabolites to be established in a single analytical step. 3. In total, three 19 F-NMR signals could be detected, of which one was identified as the parent compound. Two earlier eluting metabolites were identified to be α - and β -glucoside conjugates of 3-TFMA. 4. Metabolites at the lower (sublethal) exposure levels of 10, 1 and 0.1 µg cm -2 escaped identification by 19 F/ 1 H-HPLC-NMR spectroscopy as outlined here and will require concentration prior to analysis.     

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy, HPLC-MS/MS, HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50 mg kg(-1) using a combination of HPLC-MS, HPLC-MS/MS, (19)F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with (35)Cl and (34)S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5 mg kg(-1) with [(14)C]-labelled compound enabled the comparison of standard radioassay analysis methods with (19)F-NMR spectroscopy. (19)F-NMR resonances were only readily detectable in the 0-12 h post-dose samples. Dosing at 50 mg kg(-1) allowed the facile and specific detection and quantification of metabolites by (19)F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with (35)Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.     

The metabolism of 2-trifluormethylaniline and its acetanilide in the rat by 19F NMR monitored enzyme hydrolysis and 1H/19F HPLC-NMR spectroscopy

The urinary excretion profile and identity of the metabolites of 2-trifluoromethyl aniline (2-TFMA) and 2-trifluoromethyl acetanilide (2-TFMAc), following i.p. administration to the rat at 50 mg kg(-1), were determined using a combination of 19F NMR monitored enzyme hydrolysis, SPEC-MS and 19F/1H HPLC-NMR. A total recovery of approximately 96.4% of the dose was excreted into the urine as seven metabolites. The major routes of metabolism were N-conjugation (glucuronidation), and ring-hydroxylation followed by sulphation (and to a lesser extent glucuronidation). The major metabolites excreted into the urine for both compounds were a labile N-conjugated metabolite (a postulated N-glucuronide) and a sulphated ring-hydroxylated metabolite (a postulated 4-amino-5-trifluoromethylphenyl sulphate) following dosing of 2-TFMA. These accounted for approximately 53.0 and 31.5% of the dose, respectively. This study identifies problems on sample component instability in the preparation and analysis procedures.     

Metabolism of 3-chloro-4-fluoroaniline in rat using [14C]-radiolabelling, 19F-NMR spectroscopy,HPLC-MS/MS,HPLC-ICPMS and HPLC-NMR

The metabolic fate of 3-chloro-4-fluoroaniline was investigated in rat following intraperitoneal (i.p.) administration at 5 and 50?mg?kg^?1 using a combination of HPLC-MS, HPLC-MS/MS, ¹⁹F-NMR spectroscopy, HPLC-NMR spectroscopy and high-pressure liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS) with ³⁵Cl and ³⁴S detection. The metabolism of 3-chloro-4-fluoroaniline at both doses was rapid and extensive, to a large number of metabolites, with little unchanged compound excreted via the urine. Dosing at 5?mg?kg^?1 with [¹⁴C]-labelled compound enabled the comparison of standard radioassay analysis methods with ¹⁹F-NMR spectroscopy. ¹⁹F-NMR resonances were only readily detectable in the 0–12?h post-dose samples. Dosing at 50?mg?kg^?1 allowed the facile and specific detection and quantification of metabolites by ¹⁹F-NMR spectroscopy. Metabolite profiling was also possible at this dose level using HPLC-ICPMS with ³⁵Cl-specific detection. The principal metabolites of 3-chloro-4-fluoroaniline were identified as 2-amino-4-chloro-5-fluorophenyl sulfate and 2-acetamido-4-chloro-5-fluorophenyl glucuronide. N-acetylation and hydroxylation followed by O-sulfation were the major metabolic transformations observed.     

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using (19)F-NMR spectroscopy, HPLC-MS, and HPLC-ICPMS ((127)I)

A combination of (19)F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by (19)F-NMR spectroscopy and HPLC-(127)I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.     

Quantitative studies on the urinary metabolic fate of 2-chloro-4-trifluoromethylaniline in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS

1. The metabolism and urinary excretion of 2-chloro-4-trifluoromethylaniline has been studied in the rat using 19F-NMR spectroscopy and directly coupled HPLC-NMR-MS methods. The compound was dosed to three male Sprague-Dawley rats (50 mg kg(-1) i.p.) and urine collected over 0-8, 8-24 and 24-48 h post-dosing. 2. A total urinary recovery of 56.3+/-2.2% of the dose was achieved up to 48 h after dosing. The major metabolite in the urine was identified as 2-amino-3-chloro-5-trifluoromethylphenylsulphate accounting for a total of 33.5+/-2.2% of the dose. 3. Further metabolites detected and characterized included 2-chloro-4-trifluoromethylphenylhydroxylamine glucuronide (13.2+/-0.5% of the dose), 2-amino-3-chloro-5-trifluoromethylphenylglucuronide (3.8+/-0.4% of the dose) and 2-chloro-4-trifluoromethylaniline-N-glucuronide (3.6+/-0.1% of the dose). Several minor metabolites were also found and identified, including 2-chloro-4-trifluoromethylphenylsulphamate, which together accounted for 2.1+/-0.4% of the dose. 4. Directly coupled HPLC-NMR-MS and 19F-NMR spectroscopy is shown to provide an efficient approach for the unequivocal and rapid determination of the quantitative urinary metabolic fate and excretion balance of a fluorinated xenobiotic without the necessity for specific radiolabelling.     

Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients

1: The use of fluorine-19 nuclear magnetic resonance (19F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored. 2: Ten patients were anaesthetized by sevoflurane for 135-660 min at a flow rate of 6 l min(-1). Urine samples were collected before, directly after and 24 h after discontinuation of anaesthesia. 3: 19F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the 19F-NMR spectrum. HFIP concentrations after beta-glucuronidase treatment were quantified by (19)F-nuclear magnetic resonance. Concentrations directly after and 24 h after discontinuation of anaesthesia were 131 +/- 41 (mean +/- SEM) and 61 +/- 19 mol mg(-1) creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure. 4: Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from beta-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24 h. 5: Since 19F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 +/- 0.7 and 1.4 +/- 0.4 mol mg(-1) creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not. 6. The results show that 19F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. 19F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.     

A comparison of quantitative NMR and radiolabelling studies of the metabolism and excretion of Statil (3-(4-bromo-2-fluorobenzyl)-4-oxo-3H-phthalazin-1-ylacetic acid) in the rat

The identification and quantitation of the metabolites of Statil in rat bile and urine were investigated by 1H- and 19F-NMR spectroscopy and liquid scintillation counting. Male Wistar rats received a single oral dose of 100 mg/kg of radiolabelled Statil. Statil is known to produce glucuronide conjugates which are predominantly excreted into the bile in male rats. The complex multiphasic matrix of bile has been shown to make identification of the resonances by 1H-NMR spectroscopy very difficult as Statil appeared to be micellar bound giving rise to very broad signals. This not only impaired unambiguous signal characterisation but also quantification. The partial separation by SPEC-(1)H-NMR spectroscopy enabled the disruption of the micellar matrices and hence enabled the identification of Statil predominantly as aglycone, and to a lesser extent as glucuronide conjugate. In addition, minor acyl migration products of Statil glucuronide could also be detected as they were separated during the SPEC-process. 19F-NMR spectroscopic measurements on whole bile confirmed their presence as a number of overlapped signals could be observed. The selectivity, simplicity and signal dispersion characteristic of 19F-NMR spectroscopy also enabled the calculation of dose related recoveries of Statil related material in the bile and urine samples without the need for a radiolabel. The aim of this work was to investigate the usefulness and limitations of NMR spectroscopy of intact bile and urine as a means of quantifying levels of drug metabolites. The results obtained from NMR spectroscopy are compared with those obtained using scintillation techniques. Scintillation counting yields unequivocal quantification results, provided the label is preserved in metabolites as has been the case here. In general, quantification by 19F-NMR results similar to those obtained by scintillation counting (in agreement within about 20%). However, discrepancies have been observed with very small and broad 19F-NMR signals in bile. Nevertheless, 19F-NMR spectroscopy of bile is a rapid and facile method for assessing metabolite levels of fluorinated drugs.     

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf) in rabbits

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as refrigerant. Due to lethality observed after high concentration inhalation exposures of HFO-1234yf in a developmental toxicity study with rabbits, the biotransformation of HFO-1234yf was investigated in this species. Female New Zealand White rabbits were exposed to air containing 2000; 10,000; or 50,000 ppm (n = 3/concentration) HFO234yf. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. Animals were individually housed in metabolic cages after the end of the exposures and urines were collected at 12 h intervals for 60 h. For metabolite identification, urine samples were analyzed by ¹H-coupled and ¹H-decoupled ¹⁹F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by ¹⁹F-NMR chemical shifts, signal multiplicity, ¹H-¹⁹F coupling constants and by comparison with synthetic reference compounds. In urine samples of rabbits exposed to 2000; 10,000; or 50,000 ppm HFO-1234yf, the predominant metabolite was N-acetyl-S-(3,3,3-trifluoro-2-hydroxypropanyl)-l-cysteine and accounted for app. 48% of total ¹⁹F-NMR signal intensities. S-(3,3,3-Trifluoro-2-hydroxypropanyl)mercaptolactic acid, 3,3,3-trifluoro-1,2-dihydroxypropane, 3,3,3-trifluoro-2-propanol and inorganic fluoride were also present as urinary metabolites. In incubations of rabbit liver S9 fractions containing glutathione, NADPH and HFO-1234yf, 3,3,3-trifluoro-1,2-dihydroxypropane, S-(3,3,3-trifluoro-2-hydroxypropanyl)glutathione, 3,3,3-trifluoro-2-propanol and inorganic fluoride were identified as metabolites of HFO-1234yf by ¹⁹F-NMR. The quantity of recovered metabolites in urine suggest a low extent (< 0.1% of dose received) of biotransformation of HFO-1234yf in rabbits, and 95% of all metabolites were excreted within 12 h after the end of the exposures (t_1/2 app. 9.5 h). The obtained results indicate that HFO-1234yf is metabolized in rabbits by a CYP450-mediated epoxidation at low rates and glutathione conjugation of the epoxide. The differences in urinary metabolite patterns between rats and rabbits seen with HFO-1234yf are likely due to species-specific processing of glutathione S-conjugates. Rabbits also show a larger extent of biotransformation of HFO-1234yf.     

Biotransformation of trans-1,1,1,3-tetrafluoropropene (HFO-1234ze)

trans-1,1,1,3-Tetrafluoropropene (HFO-1234ze) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential and is developed as foam blowing agent. The biotransformation of HFO-1234ze was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000; 10,000; or 50,000 ppm (n = 5/concentration) HFO-1234ze. Male B6C3F1 mice were only exposed to 50,000 ppm HFO-1234ze. All inhalation exposures were conducted for 6 h in a dynamic exposure chamber. After the end of the exposures, animals were individually housed in metabolic cages and urines were collected at 6 or 12 h intervals for 48 h. For metabolite identification, urine samples were analyzed by ¹H-coupled and ¹H-decoupled ¹⁹F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by ¹⁹F-NMR chemical shifts, signal multiplicity, ¹H-¹⁹F coupling constants and by comparison with synthetic reference compounds. In urine samples of rats exposed to 50,000 ppm HFO-1234ze, the predominant metabolite was S-(3,3,3-trifluoro-trans-propenyl)-mercaptolactic acid and accounted for 66% of all integrated ¹⁹F-NMR signals in urines. No ¹⁹F-NMR signals were found in spectra of rat urine samples collected after inhalation exposure to 2000 or 10,000 ppm HFO-1234ze likely due to insufficient sensitivity. S-(3,3,3-Trifluoro-trans-propenyl)-l-cysteine, N-acetyl-S-(3,3,3-trifluoro-trans-propenyl)-l-cysteine and 3,3,3-trifluoropropionic acid were also present as metabolites in urine samples of rats and mice. A presumed amino acid conjugate of 3,3,3-trifluoropropionic acid was the major metabolite of HFO-1234ze in urine samples of mice exposed to 50,000 ppm and related to 18% of total integrated ¹⁹F-NMR signals. Quantification of three metabolites in urines of rats and mice was performed, using LC/MS-MS and GC/MS. The quantified amounts of the metabolites excreted with urine in both mice and rats, suggest only a low extent (< 1% of dose received) of biotransformation of HFO-1234ze and 95% of all metabolites were excreted within 18 h after the end of the exposures (t_1/2 app. 6 h). The obtained results suggest that HFO-1234ze is likely subjected to an addition-elimination reaction with glutathione and to a CYP 450 mediated epoxidation at low rates.     

Biotransformation of 2,3,3,3-tetrafluoropropene (HFO-1234yf)

2,3,3,3-Tetrafluoropropene (HFO-1234yf) is a non-ozone-depleting fluorocarbon replacement with a low global warming potential which has been developed as refrigerant. The biotransformation of HFO-1234yf was investigated after inhalation exposure. Male Sprague-Dawley rats were exposed to air containing 2000, 10,000, or 50,000 ppm HFO-1234yf for 6 h and male B6C3F1 mice were exposed to 50,000 ppm HFO-1234yf for 3.5 h in a dynamic exposure chamber (n=5/concentration). After the end of the exposure, animals were individually housed in metabolic cages and urines were collected at 6 or 12-hour intervals for 48 h. For metabolite identification, urine samples were analyzed by (1)H-coupled and decoupled (19)F-NMR and by LC/MS-MS or GC/MS. Metabolites were identified by (19)F-NMR chemical shifts, signal multiplicity, (1)H-(19)F coupling constants and by comparison with synthetic reference compounds. In all urine samples, the predominant metabolites were two diastereomers of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine. In (19)F-NMR, the signal intensity of these metabolites represented more than 85% (50,000 ppm) of total (19)F related signals in the urine samples. Trifluoroacetic acid, 3,3,3-trifluorolactic acid, 3,3,3-trifluoro-1-hydroxyacetone, 3,3,3-trifluoroacetone and 3,3,3-trifluoro-1,2-dihydroxypropane were present as minor metabolites. Quantification of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine by LC/MS-MS showed that most of this metabolite (90%) was excreted within 18 h after the end of exposure (t(1/2) app. 6 h). In rats, the recovery of N-acetyl-S-(3,3,3-trifluoro-2-hydroxy-propyl)-l-cysteine excreted within 48 h in urine was determined as 0.30+/-0.03, 0.63+/-0.16, and 2.43+/-0.86 micromol at 2000, 10,000 and 50,000 ppm, respectively suggesting only a low extent (<1% of dose received) of biotransformation of HFO-1234yf. In mice, the recovery of this metabolite was 1.774+/-0.4 mumol. Metabolites identified after in vitro incubations of HFO-1234yf in liver microsomes from rat, rabbit, and human support the metabolic pathways of HFO-1234yf revealed in vivo. The obtained results suggest that HFO-1234yf is subjected to a typical biotransformation reaction for haloolefins, likely by a cytochrome P450 2E1-catalyzed formation of 2,3,3,3-tetrafluoroepoxypropane at low rates, followed by glutathione conjugation or hydrolytic ring opening.     

19F and 1H magnetic resonance strategies for metabolic studies on fluorinated xenobiotics: application to flurbiprofen [2-(2-fluoro-4-biphenylyl)propionic acid

Strategies for the use of 1H and 19F nuclear magnetic resonance (NMR) spectroscopy as an aid to the study of the metabolic fate of fluorinated drugs are discussed with reference to the application of these methods to flurbiprofen metabolism in man. 1H and 19F NMR analysis of untreated urine enabled the detection of two major and eight minor metabolites of the drug. The two major metabolites were identified using a combination of NMR spectroscopy, solid-phase extraction chromatography with 19F and 1H NMR detection and chemical hydrolysis to a flurbiprofen glucuronide and the glucuronide of the 4-hydroxy metabolite. 1H-19F 2D shift correlated spectroscopy and spin-echo difference experiments are discussed in relation to their use in the structural identification of drug metabolites.     

Metabolism of 2-fluoro-4-iodoaniline in earthworm Eisenia veneta using 19F-NMR spectroscopy,HPLC-MS,and HPLC-ICPMS (127I)

A combination of ¹⁹F-NMR spectroscopy, HPLC-MS/MS, HPLC-MS with constant neutral loss scanning of 127, and HPLC-ICPMS with iodine detection has enabled the profiling, quantification, and limited characterization of the metabolites produced in the earthworm Eisenia veneta, following exposure to 2-fluoro-4-iodoaniline. Mass spectrometric analysis of the worm tissue and coelomic fluid afforded the identification of two Phase II metabolites, N-glutamyl and N-glucoside conjugates, indicating the importance of these pathways in the detoxification of xenobiotics for earthworms. Several further metabolites were observed and quantified by ¹⁹F-NMR spectroscopy and HPLC-¹²⁷I-ICPMS, although these were of low abundance and their structures were not unequivocally identified. The parent compound and the glutamyl conjugate were found to be the major xenobiotic components of both the coelomic fluid and the worm tissue, representing approximately 23 and approximately 35%, respectively, of the dose that was recovered from the earthworm tissue extract.     

Alternate strategies to obtain mass balance without the use of radiolabeled compounds: application of quantitative fluorine (19F) nuclear magnetic resonance (NMR) spectroscopy in metabolism studies

Nuclear magnetic resonance (NMR) spectroscopy is playing an increasingly important role in the quantitation of small and large molecules. Recently, we demonstrated that (1)H NMR could be used to quantitate drug metabolites isolated in submilligram quantities from biological sources. It was shown that these metabolites, once quantitated by NMR, were suitable to be used as reference standards in quantitative LC/MS-based assays, hence circumventing the need for radiolabeled material or synthetic standards to obtain plasma exposure estimates in humans and preclinical species. The quantitative capabilities of high-field NMR is further demonstrated in the current study by obtaining the mass balance of fluorinated compounds using (19)F-NMR. Two fluorinated compounds which were radio-labeled with carbon-14 on metabolically stable positions were dosed in rats and urine and feces collected. The mass balance of the compounds was obtained initially by counting the radioactivity present in each sample. Subsequently, the same sets of samples were analyzed by (19)F-NMR, and the concentrations determined by this method were compared with data obtained using radioactivity counting. It was shown that the two methods produced comparable values. To demonstrate the value of this analytical technique in drug discovery, a fluorinated compound was dosed intravenously in dogs and feces and urine collected. Initial profiling of samples showed that this compound was excreted mainly unchanged in feces, and hence, an estimate of mass balance was obtained using (19)F-NMR. The data obtained by this method was confirmed by additional quantitative studies using mass spectrometry. Hence cross-validations of the quantitative (19)F-NMR method by radioactivity counting and mass spectrometric analysis were demonstrated in this study. A strategy outlining the use of fluorinated compounds in conjunction with (19)F-NMR to understand their routes of excretion or mass balance in animals is proposed. These studies demonstrate that quantitative (19)F-NMR could be used as an alternate technique to obtain an estimate of the mass balance of fluorinated compounds, especially in early drug development where attrition of the compounds is high, and cost savings could be realized through the use of such a technique rather than employing radioactive compounds. The potential application of qNMR in conducting early human ADME studies with fluorinated compounds is also discussed.     

Use of 19F-nuclear magnetic resonance and gas chromatography-electron capture detection in the quantitative analysis of fluorine-containing metabolites in urine of sevoflurane-anaesthetized patients

1.?The use of fluorine-19 nuclear magnetic resonance (¹⁹F-NMR) and gas chromatography-electron capture detection (GC-ECD) in the analysis of fluorine-containing products in the urine of sevoflurane-exposed patients was explored.

2.?Ten patients were anaesthetized by sevoflurane for 135–660?min at a flow rate of 6 l?min^?1. Urine samples were collected before, directly after and 24?h after discontinuation of anaesthesia.

3.?¹⁹F-NMR analysis of the urines showed the presence of several fluorine-containing metabolites. The main oxidative metabolite, hexafluoroisopropanol (HFIP)-glucuronide, showed two strong quartet signals in the ¹⁹F-NMR spectrum. HFIP concentrations after β-glucuronidase treatment were quantified by ¹⁹F-nuclear magnetic resonance. Concentrations directly after and 24?h after discontinuation of anaesthesia were 131 ± 41 (mean ± SEM) and 61 ± 19?mol?mg^?1 creatinine, respectively. Urinary HFIP excretions correlated with sevoflurane exposure.

4.?Longer scanning times enabled the measurement of signals from two compound A-derived metabolites, i.e. compound A mercapturic acid I (CAMA-I) and compound A mercapturic acid II (CAMA-II), as well as products from β-lyase activation of the respective cysteine conjugates of compound A. The signals of the mercapturic acids, 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid were visible after combining and concentrating the patient urines. CAMA-I and -II excretions in patients were completed after 24?h.

5.?Since ¹⁹F-nuclear magnetic resonance is not sensitive enough, urinary mercapturic acids concentrations were quantified by gas chromatography-electron capture detection. CAMA-I and -II urinary concentrations were 2.3 ± 0.7 and 1.4 ± 0.4?mol?mg^?1 creatinine, respectively. Urinary excretion of CAMA-I showed a correlation with sevoflurane exposure, whereas CAMA-II did not.

6.?The results show that ¹⁹F-nuclear magnetic resonance is a very selective and convenient technique to detect and quantify HFIP in non-concentrated human urine. ¹⁹F-nuclear magnetic resonance can also be used to monitor the oxidative biotransformation of sevoflurane in anaesthetized patients. Compound A-derived mercapturic acids and 3,3,3-trifluoro-2-(fluoromethoxy)-propanoic acid and 3,3,3-trifluorolactic acid, however, require more sensitive techniques such as gas chromatography-electron capture detection and/or gas chromatography-mass spectrometry for quantification.     

Studies of metabolism and disposition of potent human immunodeficiency virus (HIV) integrase inhibitors using 19F-NMR spectroscopy

(19)F-nuclear magnetic resonance (NMR) has been extensively used in a drug-discovery programme to support the selection of candidates for further development. Data on an early lead compound, N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(4-methylmorpholin-3-yl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide (compound A (+)), and MK-0518 (N-(4-fluorobenzyl)-5-hydroxy-1-methyl-2-(1-methyl-1-{[(5-methyl-1,3,4-oxadiazol-2-yl)carbonyl]amino}ethyl)-6-oxo-1,6-dihydropyrimidine-4-carboxamide), a potent inhibitor of this series currently in phase III clinical trials, are described. The metabolic fate and excretion balance of compound A (+) and MK-0518 were investigated in rats and dogs following intravenous and oral dosing using a combination of (19)F-NMR-monitored enzyme hydrolysis and solid-phase extraction chromatography and NMR spectroscopy (SPEC-NMR). Dosing with the (3)H-labelled compound A (+) enabled the comparison of standard radiochemical analysis with (19)F-NMR spectroscopy to obtain quantitative metabolism and excretion data. Both compounds were eliminated mainly by metabolism. The major metabolite identified in rat urine and bile and in dog urine was the 5-O-glucuronide.     

Two-generation reproduction and cross-foster studies of perfluorooctanesulfonate (PFOS) in rats

Perfluorooctanesulfonate (PFOS) is a persistent acid found widely distributed in wildlife and humans. To understand the potential reproductive and developmental effects of PFOS, a two-generation reproduction study was conducted in rats. Male and female rats were dosed via oral gavage at dose levels of 0, 0.1, 0.4, 1.6, and 3.2 mg/(kg day) for 6 weeks prior to mating, during mating, and, for females, through gestation and lactation, across two generations. Due to substantial F1 neonatal toxicity observed in the 1.6 and 3.2 mg/(kg day) groups, continuation into the second generation was limited to F1 pups from the 0, 0.1, and 0.4 mg/(kg day) groups. No adverse effects were observed in F0 females or their fetuses upon caesarean sectioning at gestation day 10. Statistically significant reductions in body-weight gain and feed consumption were observed in F0 generation males and females at dose levels of 0.4 mg/(kg day) and higher, but not in F1 adults. PFOS did not affect reproductive performance (mating, estrous cycling, and fertility); however, reproductive outcome, as demonstrated by decreased length of gestation, number of implantation sites, and increased numbers of dams with stillborn pups or with all pups dying on lactation days 1-4, was affected at 3.2 mg/(kg day) in F0 dams. These effects were not observed in F1 dams at the highest dose tested, 0.4 mg/(kg day). Neonatal toxicity in F1 pups, as demonstrated by reduced survival and body-weight gain through the end of lactation, occurred at a maternal dose of 1.6 mg/(kg day) and higher while not at dose levels of 0.1 or 0.4 mg/(kg day) or in F2 pups at the 0.1 or 0.4 mg/(kg day) dose levels tested. In addition to these adverse effects, slight yet statistically significant developmental delays occurred at 0.4 (eye opening) and 1.6 mg/(kg day) (eye opening, air righting, surface righting, and pinna unfolding) in F1 pups. Based on these data, the NOAELs were as follows: reproductive function: F0> or =3.2 and F1> or =0.4 mg/(kg day); reproductive outcome: F0=1.6 and F1> or =0.4 mg/(kg day); overall parental effects: F0=0.1 and F1> or =0.4 mg/(kg day); offspring effects: F0=0.4 and F1> or =0.4 mg/(kg day). To distinguish between maternal and pup influences contributing to the perinatal mortality observed in the two-generation study, a follow-up cross-foster study was performed. Results of this study indicated that in utero exposure to PFOS causally contributed to post-natal pup mortality, and that pre-natal and post-natal exposure to PFOS was additive with respect to the toxic effects observed in pups.     

Detection of new metabolites of trifluridine (F3TdR) using 19F NMR spectroscopy.

The metabolism of 5-trifluoromethyl-2'-deoxyuridine (trifluridine, F3TdR) in male BALB/C mice has been studied by 19F NMR spectroscopy. Contrary to previous reports, a number of fluorinated metabolites were observed in urine, whole livers and blood samples taken from mice after i.p. injection of F3TdR. The present study describes the identification of two new metabolites in mouse urine using the 19F NMR technique. The NMR of crude urine showed the presence of F3TdR 5-trifluorothymine (F3T), the newly-identified metabolites, 5-trifluoromethyl-5,6-dihydrouracil (DHF3T) and 5-trifluoromethyl-5,6-dihydroxyracil (DOHF3T), and several new, as yet unidentified fluorinate metabolites. These two new metabolites were characterized by comparison to authentic compounds prepared synthetically from F3T.     

19F NMR spectroscopy study of the metabolites of flucloxacillin in rat urine

19F NMR spectroscopy was used to monitor the metabolites of flucloxacillin in the urine of a rat dosed with its sodium salt. 19F NMR signals were detected and quantified from flucloxacillin and three metabolites. The 19F NMR method involves minimal sample preparation and is free from interference by endogenous urine components. High-field spin-echo 1H NMR spectroscopy and HPLC were used to confirm the results.     

UPLC-MS,HPLC-radiometric,and NMR-spectroscopic studies on the metabolic fate of 3-fluoro-[U-14C]-aniline in the bile-cannulated rat

1. A study of the rates and routes of excretion of 3-fluoro-[U-¹⁴C]aniline following intraperitoneal administration to male bile-cannulated rats by liquid scintillation counting (LSC) gave a total recovery of ～ 90% in the 48?h following dosing, with the majority of the dose being excreted in the urine during the first 24?h (～ 49%).

2. The total recovery as determined by ¹⁹F-nuclear magnetic resonance (¹⁹F-NMR) was ～ 49%, with the majority of the dose excreted in the first 24?h (～ 41%). The comparatively low recovery in comparison to that obtained from LSC was due to matrix effects in bile and a contribution from metabolic defluorination.

3. High-performance liquid chromatography with radiometric profiling of urine and bile revealed a complex pattern of metabolites with the bulk of the dose excreted as a single peak.

4. Ultra-performance liquid chromatography-orthogonal acceleration time of flight mass spectrometry profiling also showed a complex pattern of metabolites, detecting ～ 21 metabolites of 3-fluoroaniline (3-FA) with six of these detected only in urine and four solely in bile.

5. ¹⁹F-NMR revealed the presence of the parent compound and 15 metabolites in urine collected during the first 24?h after -dosing. The matrix effects of bile on ¹⁹F-NMR spectroscopy made metabolite profiling impractical for this biofluid.

6. The major metabolite of 3-FA was identified as 2-fluoro-4-acetamidophenol-sulfate.