Development and application of UPLC-Q-Orbitrap HRMS methods for the determination of eight metabolites of p-chloronitrobenzene in human urine.

Accurate, reliable, and sensitive methods for the determination of eight metabolites of p-chloronitrobenzene (p-CNB) were developed based on ultra-performance liquid chromatography - quadrupole - orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS). The free and conjugated forms of metabolites were determined before and after urine samples were hydrolyzed with acid. Subsequently, three solid phase extraction steps were used for concentration and purification. The calibration curves of the eight metabolites exhibited good linearity with an R2 of >0.999, and the precision was good as well, with the coefficient of variations of intra-day and inter-day being lower than 7.0 % and 8.5 %, respectively. Analytical accuracy for all metabolites varied within ranges of 76.0-102.9 %, and the limit of detection and limit of quantification of all the metabolites varied within ranges of 0.2-7.7 µg/L and 0.6-25.6 µg/L in human urine, respectively. In addition, the application potential of the proposed methods were evaluated by applying them to the determination of metabolites in the urine of workers exposed to p-CNB, and these results showed that these methods were accurate, reliable, and sensitive, which makes them an excellent choice for detecting the metabolites of p-CNB in the urine of exposed workers.

Alterations in the activities of ornithine and histidine decarboxylases provoked by testosterone in mice.

1. The urinary excretion of putrescine has been determined in female mice before and during repeated injections of testosterone.2. Testosterone administration effected a striking increase in the excretion of free putrescine.3. Ornithine decarboxylase (L-ornithine carboxy-lyase; E.C. 4.1.1.17) and histidine decarboxylase (L-histidine carboxy-lyase; E.C. 4.1.1.22) activities of mouse kidney and liver were examined. In the kidney, following testosterone administration, ornithine decarboxylase activity was found to be substantially elevated, whereas that of histidine decarboxylase was depressed. In the liver, by contrast, the activity levels of these enzymes were not significantly altered by testosterone treatment.4. The possibility of a functional interrelation between putrescine and histamine, via the two enzyme activities investigated, is discussed.

Different metabolic pathways of 2,5-difluoronitrobenzene and 2,5-difluoroaminobenzene compared to molecular orbital substrate characteristics.

The in vivo metabolite patterns of 2,5-difluoroaminobenzene and of its nitrobenzene analogue, 2,5-difluoronitrobenzene, were determined using 19F NMR analysis of urine samples. Results obtained demonstrate significant differences between the biotransformation patterns of these two analogues. For the aminobenzene, cytochrome P450 catalysed aromatic hydroxylation presents the main metabolic pathway. 2,5-Difluoronitrobenzene was predominantly metabolised through glutathione conjugation leading to excretion of 5-fluoro-2-(N-acetylcysteinyl)-nitrobenzene and fluoride anions, and, to a minor extent, through cytochrome P450 catalysed hydroxylation and nitroreduction. Pretreatment of the rats with various inducers of cytochrome P450 enzymes, known also to influence glutathione S-transferase enzyme patterns, followed by exposure to the 2,5-difluoroamino- or 2,5-difluoronitrobenzene, generally resulted in metabolite patterns that varied only to a small (< or = 12%) extent. Based on these results it was concluded that the biotransformation enzyme pattern is not the predominant factor in determining the metabolic route of these two model compounds. Additional in vitro microsomal and cytosolic incubations with 2,5-difluoroaminobenzene and 2,5-difluoronitrobenzene qualitatively confirmed the in vivo results. NADPH/oxygen supported microsomal cytochrome P450 catalysed hydroxylation was observed only for 2,5-difluoroaminobenzene whereas cytosolic GSH conjugation occurred only in incubations with 2,5-difluoronitrobenzene as the substrate. Outcomes from molecular orbital calculations provided a working hypothesis that can explain the difference in metabolic pathways of the nitro- and aminobenzene derivative on the basis of their chemical characteristics. This hypothesis states that the chances for a nitro- or aminobenzene derivative to enter either a cytochrome P450 or a glutathione conjugation pathway are determined by the relative energy levels of the frontier orbitals of the compounds. The aminobenzene derivative has relatively high energy molecular orbitals leading to an efficient reaction of its highest occupied molecular orbital (HOMO) with the singly occupied molecular orbital of the cytochrome P450 (FeO)3+ intermediate, but a low reactivity of its lowest unoccupied molecular orbital (LUMO) with the HOMO of glutathione. The nitrobenzene, on the other hand, has molecular orbitals of relatively low energy, explaining the efficient interaction, and, thus, reaction between its LUMO and the HOMO electrons of glutathione, but resulting in low reactivity with the SOMO electron of the cytochrome P450 (FeO)3+ reaction intermediate.

Urinary metabolites and health effects in workers exposed chronically to chloronitrobenzene.

For workers exposed to 4-chloronitrobenzene (4CNB), the major metabolites were determined. Urine were analysed before and after acid hydrolysis to qualify the free and conjugated metabolites of 4CNB. Three conjugated metabolites were identified in exposed workers: the mercapturic acid N-acetyl-S-(4-nitrophenyl)-L-cysteine (NANPC) was the only metabolite detected in non-hydrolysed urine, and accounted for approximately 51% of the total metabolites detected. The two remaining metabolites 4-chloroaniline (4CA) and 2-chloro-5-nitrophenol (CNP) were identified as cleavage products in hydrolysed urine, and accounted for approximately 18 and 30% of the total metabolites detected, respectively. No metabolites were found in factory controls within the limits of quantitation (LOQ) of the assay. There is a moderate correlation between NANPC and both 4CA and CNP. The correlation between 4CA and CNP is minor. The correlation between the total metabolites and both 4CA and CNP are good. The best correlation was found between the total metabolites and NANPC. There is a moderate inverse correlation between age and the creatinine levels. The raw metabolite levels CNP and NANPC decrease with age. The urine metabolites increase and correlate significantly with the creatinine levels. 4CA, NANPC and the total metabolite levels correlate with the haemoglobin adduct levels. NANPC is the most appropriate biomarker in the urine for a recent absorbed dose of 4CNB, since NANPC reflects the levels of 4CA and CNP and is the most prevalent metabolite detected in all the exposed workers.

Tissue distribution and elimination of N-methyl-N-2,4,6-tetranitroaniline (tetryl) in rats.

The elimination of tetryl was studied using ring-labeled 14C-tetryl. Tetryl was given subcutaneously to male Sprague-Dawley rats at doses of 25, 100, and 300 mg kg(-1), and urine and feces were collected 24 h post-injection. Percent urinary elimination was observed to be 10.02 +/- 2.48, 11.2 +/- 1.66, and 13.24 +/- 5.79 (mean +/- SEM) respectively. Percent fecal elimination was 15.68 +/- 6.13, 9.41 +/- 1.52, and 8.45 +/- 1.81 respectively. At 24 h post-injection, tissues from male Sprague-Dawley rats were collected from animals that received 100 mg kg(-1) 14C-tetryl. Tetryl was found to be poorly absorbed with approximately 65% of the administered dose remaining at the site of subcutaneous injection. Blood was found to be the principal depot of radioactivity, followed by muscle, liver, and kidney. Analysis of the tissue to blood radioactivity ratio revealed that the liver had the highest ratio (1.2), followed by brain (0.45), kidney (0.38), and testes (0.35). All other tissues analyzed had ratios less than 0.30. Urine of animals receiving 14C-tetryl (100 mg kg(-1)) was analyzed using HPLC coupled with UV detection (200-600 nm; 1.2 nm resolution). During HPLC analysis, 1 min fractions were collected and radioactivity measured. Two major peaks of radioactivity were identified at approximately 5 and 14 min retention times, respectively. The 14 min peak had the same retention time and UV spectrum as picric acid and 5 min peak had the same retention time and UV profile as picramic acid. The data presented demonstrates that that there is little retention of tetryl in specific tissue depots and that tetryl is eliminated in roughly equal amounts in both urine and feces. The major urinary metabolites identified picric acid and picramic acid (a known urinary metabolite observed in rabbits). From microsomal fraction studies, a major metabolite, NMPA, was identified. The formation of this metabolite was found to be dependent on at least two enzymes. One enzyme is dependent on NAD+ for NMPA formation and is likely to be NADP(H):quinone oxidoreductase. The second metabolite is NADP+ dependent and is probably related to NADPH:cytochrome-P450 reductase.

Determination of urinary nitrobenzene by the microdiffusion method.

A simple, sensitive and specific analytical method based on microdiffusion of urinary nitrobenzene into a nitrating mixture and its subsequent determination by the butanone method is described in this paper. The method is sensitive to detect 0.2 mg/L of urinary nitrobenzene.

Identification of urinary metabolites in rats treated with p-chloronitrobenzene.

Urinary metabolites in rats treated with p-chloronitrobenzene were identified by gas chromatography-mass spectrometry. A single dose of 100 mg/kg body wt p-chloronitrobenzene was administered intraperitoneally to male Sprague-Dawley rats and urine samples were collected from the 8th to 24th hour after the administration. Urinary metabolites were extracted with diethylether at pH 1.0 and pH 10.0 from urine samples hydrolyzed with acid and base and from intact urine samples. Aliquots of the ethereal extracts were injected into a gas chromatograph-mass spectrometer. Nine substances were identified: p-chloroaniline, 2,4-dichloroaniline, p-nitrothiophenol, 2-chloro-5-nitrophenol, 2-amino-5-chlorophenol, p-chloroformanilide, 4-chloro-2-hydroxyacetanilide, a small amount of p-chloroacetanilide and traces of unchanged p-chloronitrobenzene.

A simple method of determination of nitrobenzene and nitrochlorobenzene in air and urine.

An analytical method based on the direct coupling of the dye sodium salt of 1,2-naphthoquinone-4-sulphonic acid with the chemically converted nitrobenzene (NB) and p-nitrochlorobenzene (NCB) to aniline and p-chloroaniline in aqueous medium (pH 7.0-9.0) is presented. The coupled dye is extracted in carbon tetrachloride and spectrophotometrically determined at 450 nm wavelength. Carbon tetrachloride is found to be a selective solvent for extraction of aniline and its analogous coupled derivative of the dye, thus the interference of the other urinary nitro and amine compounds normally present is removed. Ethanol and isopropyl alcohol are efficient sampling media for trapping airborne NB and NCB vapors. The sensitivity and precision of the method are 10 micrograms and 3.5%, respectively, for NB and NCB in air samples. The sensitivity and precision of this method for urinary NB and NCB estimation is 0.8 mg/L and 0.6 mg/L; and 6.1% and 4.0%, respectively.

Identification of urinary metabolites of human subjects acutely poisoned by p-chloronitrobenzene.

1. Urinary metabolites from human subjects acutely poisoned with p-chloro-nitrobenzene (p-CNB) were identified by g.l.c.-mass spectrometry. 2. Eight substances, namely, a very large amount of N-acetyl-S-(4-nitrophenyl)-L-cysteine, relatively large quantities of p-chloroaniline, 2-chloro-5-nitrophenol and p-chloroformanilide produced by pyrolysis of a substance originating from p-CNB, small amounts of 2-amino-5-chlorophenol and 2,4-dichloroaniline, and traces of p-chloroacetanilide and 4-chloro-2-hydroxyacetanilide, were detected in urine samples. 3. All of the absorbed p-CNB was metabolized prior to excretion, as the parent compound was not found in urine. 4. N-Acetylated metabolites of p-chloroaniline and 2-amino-5-chlorophenol, resulting from p-CNB by metabolism, were found in only one of eight individuals indicating that this pathway is weak or may be absent in some humans. 5. A scheme for the pattern of metabolic pathways of p-CNB is proposed, and chlorination was considered to be a possible novel metabolic pathway.

Reversed-phase ion-pair chromatography of straight- and branched-chain dicarboxylic acids in urine as their 2-nitrophenylhydrazides.

The derivatization of urinary dicarboxylic acids with 2-nitrophenylhydrazine hydrochloride produced corresponding monohydrazides, which were separated from monocarboxylic acid hydrazides by two step extraction with ethyl acetate at different pH values. Monohydrazides of 11 straight- and branched-chain dicarboxylic acids were eluted isocratically on reversed-phase ion-pair chromatography within 24 min by the combination of pH, the polarity of mobile phase, and the size of counter ion. The analytical results showed good recovery and reproducibility using 3,3-dimethyglutaric acid as an internal standard. The present method provides a notable HPLC method with precolumn derivatization for the analysis of urinary dicarboxylic acids.

Pharmacokinetic study of p-chloronitrobenzene in rat.

The pharmacokinetics of p-chloronitrobenzene (p-CNB) was evaluated in rats to propose an index for monitoring p-CNB exposure of humans exposed to it. After a single dose of 30, 100, or 333 mg/kg body weight, p-CNB was administered intraperitoneally to male Sprague-Dawley rats; blood and urine were collected periodically. p-CNB in plasma and its five major metabolites--2-chloro-5-nitrophenol, N-acetyl-S-(4-nitrophenyl)-L-cysteine,2,4-dichloroaniline,p-chloroanilin e, and 2-amino-5-chlorophenol--in urine were measured by reversed-phase HPLC methods. Pharmacokinetics was evaluated by moment analysis and compartment model analysis of the p-CNB concentration in plasma vs. time curves and of the urinary excretion rate of its metabolites vs. time curves. Urinary excretion was considered to be the most important pathway for disappearance of p-CNB, because the fraction of p-CNB metabolites excreted in urine was ca.2/3 of the dose level. N-Acetyl-S-(4-nitrophenyl)-L-cysteine was the most abundant urinary metabolite of p-CNB and comprised ca.1/2 of the total amount of the five metabolites excreted into the urine. The urinary excretion of N-acetyl-S-(4-nitrophenyl)-L-cysteine was considered to be proportional to the dose of p-CNB over a wide range of doses, because the process of metabolism of p-CNB to N-acetyl-S-(4-nitrophenyl)-L-cysteine was linear in the dose range studied. Consequently, urinary N-acetyl-S-(4-nitrophenyl)-L-cysteine was considered to be suitable as an index for monitoring p-CNB exposure.

Determination of p-chloronitrobenzene and its metabolites in urine by reversed-phase high-performance liquid chromatography.

A simple, accurate and precise isocratic reversed-phase high-performance liquid chromatographic method (HPLC) using ultraviolet detection was developed for the determination of p-chloronitrobenzene (p-CNB) and seven of its metabolites in rat urine. Analysis was performed before and after hydrolysis of the urine samples with acid to determine both free and conjugate forms of the metabolites. An equal volume of methanol was added to the urine sample and after centrifugation the mixed solution was injected into a high-performance liquid chromatograph. A column packed with 5-microns octadecylsilane (ODS) spherical particles was used at 30 degrees C. The metabolites were divided into three groups, and each group was subjected to three different mobile phase and detection wavelength conditions as follows: water-methanol (60:40, v/v) and 250 nm for p-CNB and 2,4-dichloroaniline; 0.005 M phosphate buffer (pH 3.6)-methanol (76:24, v/v) containing 1.2 mM sodium 1-octanesulphonate and 240 nm for p-chloroaniline, 2-chloro-5-nitrophenol, 2-amino-5-chlorophenol, p-chloroacetanilide and 4-chloro-2-hydroxyacetanilide; and 0.005 M phosphate buffer (pH 6.0)-methanol (80:20, v/v) and 340 nm for N-acetyl-S-(4-nitrophenyl)-L-cysteine. The response was linear at concentrations less than 200.0 micrograms/ml (r = 0.9998) for all metabolites, and the detection limits of each metabolite were between 0.05 and 0.2 micrograms/ml in non-hydrolysed urine. Analysis of the spiked samples demonstrated good accuracy and precision of the method in both intra- and inter-day assays. Storage stabilities of p-CNB and its metabolites at -20 degrees C, 4 degrees C and room temperature were examined for both neutral and acidic urine samples. This method was also shown to be applicable to toxicokinetic study of p-CNB following administration to rats.

Pharmacokinetic study of p-chloronitrobenzene in humans suffering from acute poisoning.

The pharmacokinetics of p-chloronitrobenzene (p-CNB) in human subjects suffering from acute poisoning was studied from the urinary excretion of p-CNB metabolites. The time course for excretion of five metabolites--2-chloro-5-nitrophenol,N-acetyl-S-(4-nitrophenyl)- L-cysteine, 2,4-dichloroaniline, p-chloroaniline, and 2-amino-5-chlorophenol-were determined by reversed-phase HPLC analysis of urine collected from six subjects during their hospitalization after accidental exposure to p-CNB. The cumulative excretion amount at infinite time (X infinity) and mean residence time for each metabolite were estimated by moment analysis of excretion rate vs. the time curve. The rate constants for p-CNB metabolism were calculated by nonlinear least squares fitting of the time course data using a one-compartment model. The results indicated that the average values of the ratio of excreted amount of each metabolite to the total amount of the five metabolites (X infinity metabolite/X infinity Total) and mean residence time in the six subjects were, respectively: 12.2% and 6.7 days for 2-chloro-5-nitrophenol, 48.0% and 7.0 days for N-acetyl-S-(4-nitrophenyl)-L-cysteine, 1.2% and 3.7 days for 2,4-dichloroaniline, 29.9% and 10.0 days for p-chloroaniline, and 8.7% and 6.0 days for 2-amino-5-chlorophenol. The average values of rate constants in the metabolism of p-CNB to 2-chloro-5-nitrophenol, N-acetyl-S-(4-nitrophenyl)-L-cysteine, and p-chloroaniline were 0.021, 0.082, and 0.067 day-1, respectively. Similarly those in further metabolism of p-chloroaniline for C-chlorination, C-hydroxylation, and N-conjugation were 0.205, 1.206, and 3.526 day-1, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of molecular-secondary-ion mass spectrometry for drug metabolism studies. I. Direct analysis of conjugates by thin-layer chromatography-secondary-ion mass spectrometry.

Molecular-secondary-ion mass spectrometry (SIMS) is a suitable method for the analysis of nonvolatile substances such as conjugated metabolites of drugs. We have developed a simple method for the direct SIMS measurement of conjugates following thin-layer chromatography without any extraction procedure. After separation with a butanol-acetic acid-ethanol-water (3:1:1:1, v/v) system, the spot was cut out and attached to a SIMS probe. The conjugates of p-nitrophenol and 4-hydroxyantipyrine were measured. The quantitative application of the method is also discussed, using deuterium-labelled internal standards for p-nitrophenol conjugates.

Simultaneous high-performance liquid chromatographic determination of urinary metabolites of benzene, nitrobenzene, toluene, xylene and styrene.

A high-performance liquid chromatographic method is described for the simultaneous determination of six urinary metabolites of several aromatic chemicals: phenol (from benzene), hippuric acid (from toluene), 3-methylhippuric acid (from xylene), mandelic and phenylglyoxylic acid (from styrene) and 4-nitrophenol (from nitrobenzene). Reversed-phase liquid chromatography was performed in an isocratic mode at 1 ml/min on a 5-microns C18 column using two mobile phases: (A) acetonitrile-1% phosphoric acid (10:90); (B) acetonitrile-1% phosphoric acid (30:70). Phase A separates the six metabolites well, but phase B allows to a more rapid and reproducible simultaneous determination of phenolic compounds than phase A. For these compounds a prior enzymic hydrolysis step using Helix pomatia juice is performed to hydrolyse their sulphate and glucuronate conjugates. The reproducibility and the specificity are both excellent. Furthermore, the method is rapid, economical and easily automated. The proposed method appears very suitable for the routine monitoring of workers exposed to these chemicals on the basis of the biological threshold limit values.

Metabolic studies on pentachloronitrobenzene (PCNB) in rats.

The metabolism of PCNB in rats was studied. Metabolites isolated from rat excreta and identified were: N-acetyl-S-(pentachlorophenyl)cysteine, pentachlorothiophenol, pentachlorothioanisole, 2,3,4,5-tetrachlorothiophenol, 2,3,4,5-tetrachlorothioanisole, 2,3,4,6- and/or 2,3,5,6-tetrachloro-thiophenol and -thioanisole, 1,4-bis(methylthio)tetrachlorobenzene, 1,4-dimercapto-tetrachlorobenzene and/or 4-methylthio-tetrachlorothiophenol, pentachlorophenol, pentachloroanisole, 2,3,4,5-tetrachlorophenol, 2,3,4,5-tetrachloroanisole, 2,3,4,6- and/or 2,3,5,6-tetrachloro-phenol and -anisole, pentachlorobenzene, 2,3,4,5-tetrachloronitrobenzene, pentachloroaniline and 2,3,4,5-tetrachloroaniline.

Efficient cleavage of conjugates of drugs or poisons by immobilized beta-glucuronidase and arylsulfatase in columns.

BACKGROUND: Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures. METHODS: beta-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns. RESULTS: In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples. CONCLUSIONS: Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.