Simple method to detect triclofos and its metabolites in plasma of children by combined use of liquid chromatography tandem-mass spectrometry and gas chromatography-mass spectrometry.

Triclofos sodium (TCS) and chloral hydrate (CH) are widely used as sedatives for children, but no analytical method to simultaneously monitor concentrations of blood TCS, CH and their metabolites, trichloroacetic acid (TCA) and trichloroethanol (TCEOH), has been reported. The present study aimed to develop a simple analytical method for TCS and its metabolites (TCA, TCEOH and CH) in small-volume plasma from children. After acidification of specimens, TCS formic acid adduct or the metabolites derivatized using water/sulfuric acid/methanol (6:5:1, v/v) were measured by combined use of liquid chromatography tandem-mass spectrometry and gas chromatography mass-spectrometry. The limits of detection and quantification levels (µg/ml) were 0.10 and 0.29 for TCS, 0.24 and 0.72 for TCA, 0.10 and 0.31 for TCEOH, and 0.25 and 0.76 for CH, respectively. The mean recoveries were 82.8-107% for TCS, 85.4-101% for TCA, 91.6-107% for TCEOH, and 88.9-109% for CH. Within-run and between-run precision (percent of relative standard deviation, %RSD) using this method ranged from 1.1 to 15.7% and 3.6 to 13.5%, respectively, for TCS and all of its metabolites. The calibration curves were obtained with standard spiked plasma, and all of the coefficients of determination were more than 0.975. Subsequently, we applied the present method to plasma taken from five children after sedation induced by CH and TCS. In addition to TCS and CH, elevated TCA and TCEOH concentrations were detected. This new method can be applied for the pharmacokinetic analysis of TCS and its metabolites and the determination of the optimal TCS dosage in children.

[Simultaneous determination of trichloroethylene and trichloroethanol in blood by liquid-liquid extraction-gas chromatography].

Objective: To establish a method for determing the trichloroethylene(TCE)and trichloroethanol(TCOH)in blood samples by liquid-liquid extraction-gas chromatography with electron capture detector. Methods: With this method,ether was used as extraction solvent and trichloromethane was used as an internal standard. The whole blood sample was extracted with ether, and dehydrated by anhydrous sodium sulfate. Then the analytes were separated on HP-5 capillary column(30m×0.32mm×0.15µm)and detected byECD.The retention time was for qualitative analysis and the internal standard was for quantitation. Results: The standard curves of TCE and TCOH showed significant linearity between 95.5µg/L-7640.0µg/L(r=0.9997)and 19.0µg/L-1520.0µg/L(r=0.9992). The average recovery was 95.5%-103.6%.The intra-day and inter-day precisions(RSD)were 2.5%-6.8%(n=6)and 1.6%-4.3%(n=6) respectively. The detect limit of TCE and TCOH were 2.10 µg/L and 0.56µg/L(S/N=3)respectively.The blood can be kept 7 days at-20℃ refrigerator without significantly loss. Conclusion: This method is proved to be simple,practical and highly sensitive. It can satisfy the request for the determination of blood samples of humans exposed to TCE.

In vivo effects of naproxen, salicylic acid, and valproic acid on the pharmacokinetics of trichloroethylene and metabolites in rats.

It was recently demonstrated that some drugs modulate in vitro metabolism of trichloroethylene (TCE) in humans and rats. The objective was to assess in vivo interactions between TCE and three drugs: naproxen (NA), valproic acid (VA), and salicylic acid (SA). Animals were exposed to TCE by inhalation (50 ppm for 6 h) and administered a bolus dose of drug by gavage, equivalent to 10-fold greater than the recommended daily dose. Samples of blood, urine, and collected tissues were analyzed by headspace gas chromatography coupled to an electron capture detector for TCE and metabolites (trichloroethanol [TCOH] and trichloroacetate [TCA]) levels. Coexposure to NA and TCE significantly increased (up to 50%) total and free TCOH (TCOHtotal and TCOHfree, respectively) in blood. This modulation may be explained by an inhibition of glucuronidation. VA significantly elevated TCE levels in blood (up to 50%) with a marked effect on TCOHtotal excretion in urine but not in blood. In contrast, SA produced an increase in TCOHtotal levels in blood at 30, 60, and 90 min and urine after coexposure. Data confirm in vitro observations that NA, VA, and SA affect in vivo TCE kinetics. Future efforts need to be directed to evaluate whether populations chronically medicated with the considered drugs display greater health risks related to TCE exposure.

[To the question of the optimization of methods for detection of vinyl chloride and 1,2-dichloroethane, and their metabolites in biological fluids in workers involved in production of polyvinyl chloride].

There is considered the improvement of methodological approaches to the gas chromatographic methods- of the detection of vinyl chloride and 1,2-dichloroethane and their metabolites--chloroethanol and monochloroacetic acid in biological fluids. There were evaluated such metrological characteristics of methods, as repeatability, interlaboratoty precision, relevance and accuracy. The value of relative expanded uncertainty does not exceed 30%. There are reported optimal regimes of gas chromatographic analysis, conditions for sample preparation. The results of the contents ofthese chemical compounds and their metabolites in biological fluids from persons working in contact with chlorinated hydrocarbons are presented These techniques can be used for the detection ofthe fact of exposure to toxic substances, assessment of the level of exposure and biomonitoring.

Chloral hydrate intoxication in a 3-month-old child: avoidance of hemodialysis by an immediate determination of trichloroethanol.

BACKGROUND: Chloral hydrate is used worldwide as a first-line agent for procedural sedation in paediatric patients undergoing painless diagnostic investigations. Chloral hydrate overdoses in children and adults have been reported to cause various toxicities, including central nervous system, respiratory and cardiac depression with sometimes fatal outcome. PATIENT AND METHODS: A 3-month-old girl was admitted after an unintentional administration of a 10-fold dose of chloral hydrate (667 mg/kg). She showed respiratory insufficiency in need of intubation and ventilation. Gastric endoscopy revealed esophagitis and gastric ulcerations. To assess the need for hemodialysis, serum trichloroethanol (TCE) was determined using a mass spectrometric quantification after a methyl tertiary butyl ether extraction using an external standard method. The serum TCE level 6 h after administration was 89 mg/L and declined to 20 mg/L within 24 h. The child could be extubated the next day; her further course was uneventful. CONCLUSION: The repeated determination of serum TCE levels prevented a technically difficult and risky hemodialysis in this very young patient.

Kinetics of chloral hydrate and its metabolites in male human volunteers.

Chloral hydrate (CH) is a short-lived intermediate in the metabolism of trichloroethylene (TRI). TRI, CH, and two common metabolites, trichloroacetic acid (TCA) and dichloroacetic acid (DCA) have been shown to be hepatocarcinogenic in mice. To better understand the pharmacokinetics of these metabolites of TRI in humans, eight male volunteers, aged 24-39, were administered single doses of 500 or 1,500 mg or a series of three doses of 500 mg given at 48 h intervals, in three separate experiments. Blood and urine were collected over a 7-day period and CH, DCA, TCA, free trichloroethanol (f-TCE), and total trichloroethanol (T-TCE=trichloroethanol and trichloroethanol-glucuronide [TCE-G]) were measured. DCA was detected in blood and urine only in trace quantities (<2 microM). TCA, on the other hand, had the highest plasma concentration and the largest AUC of any metabolite. The TCA elimination curve displayed an unusual concentration-time profile that contained three distinct compartments within the 7-day follow-up period. Previous work in rats has shown that the complex elimination curve for TCA results largely from the enterohepatic circulation of TCE-G and its subsequent conversion to TCA. As a result TCA had a very long residence time and this, in turn, led to a substantial enhancement of peak concentrations following the third dose in the multiple dose experiment. Approximately 59% of the AUC of plasma TCA following CH administration is produced via the enterohepatic circulation of TCE-G. The AUC for f-TCE was found to be positively correlated with serum bilirubin concentrations. This effect was greatest in one subject that was found to have serum bilirubin concentrations at the upper limit of the normal range in all three experiments. The AUC of f-TCE in the plasma of this individual was consistently about twice that of the other seven subjects. The kinetics of the other metabolites of CH was not significantly modified in this individual. These data indicate that individuals with a more impaired capacity for glucuronidation may be very sensitive to the central nervous system depressant effects of high doses of CH, which are commonly attributed to plasma levels of f-TCE.

Dose-dependent liver regeneration in chloroform, trichloroethylene and allyl alcohol ternary mixture hepatotoxicity in rats.

The present study was designed to examine the hypothesis that liver tissue repair induced after exposure to chloroform (CF) + trichloroethylene (TCE) + allyl alcohol (AA) ternary mixture (TM) is dose-dependent similar to that elicited by exposure to these compounds individually. Male Sprague Dawley (S-D) rats (250-300 g) were administered with fivefold dose range of CF (74-370 mg/kg, ip), and TCE (250-1250 mg/kg, ip) in corn oil and sevenfold dose range of AA (5-35 mg/kg, ip) in distilled water. Liver injury was assessed by plasma alanine amino transferase (ALT) activity and liver tissue repair was measured by (3) H-thymidine incorporation into hepatonuclear DNA. Blood and liver levels of parent compounds and two major metabolites of TCE [trichloroacetic acid (TCA) and trichloroethanol (TCOH)] were quantified by gas chromatography. Blood and liver CF and AA levels after TM were similar to CF alone or AA alone, respectively. However, the TCE levels in blood and liver were substantially decreased after TM in a dose-dependent fashion compared to TCE alone. Decreased plasma and liver TCE levels were consistent with decreased production of metabolites and elevated urinary excretion of TCE. The antagonistic interaction resulted in lower liver injury than the summation of injury caused by the individual components at all three-dose levels. On the other hand, tissue repair showed a dose-response leading to regression of injury. Although the liver injury was lower and progression was contained by timely tissue repair, 50% mortality occurred only with the high dose combination, which is several fold higher than environmental levels. The mortality could be due to the central nervous system toxicity. These findings suggest that exposure to TM results in lower initial liver injury owing to higher elimination of TCE, and the compensatory liver tissue repair stimulated in a dose-dependent manner mitigates progression of injury after exposure to TM.

Evaluation of dynamic headspace with gas chromatography/mass spectrometry for the determination of 1,1,1-trichloroethane, trichloroethanol, and trichloroacetic acid in biological samples.

A sensitive and reproducible method is described for the analysis of trichloroacetic acid in urine and 1,1,1-trichloroethane in blood using dynamic headspace GC/MS. Samples were analyzed using the soil module of a modified purge and trap autosampler to facilitate the use of disposable purging vessels. Coefficients of variation were below 3.5% for both analytes, and response was linear in the range of 0.01-7.0 microg/ml for trichloroacetic acid and 0.9 ng/ml-2.2 microg/ml for 1,1,1-trichloroethane. Attempts at using dynamic headspace for the analysis of trichloroethanol in urine were unsuccessful.

Renal toxicity after chronic inhalation exposure of rats to trichloroethylene.

Male Long-Evans rats were exposed to 0 (controls) or 500 ppm trichloroethylene (TRI) for 6 months, 6 h daily, and 5 days a week. The TRI metabolites trichloroethanol (TCE) in blood and trichloroacetic acid (TCA) in urine were measured. Specific parameters related to the renal damage were determined in urine [biomarker for glomerular damage: high molecular weight proteins (HMW), albumin (ALB); for proximal tubular damage: N-acetyl-beta-D-glucosaminidase (NAG), low-molecular-weight-proteins (LMW)]. Significantly increased concentrations of NAG and LMW in urine of exposed rats were detected. No DNA-strand breaks in kidney cells could be detected using the comet assay, and histological examinations were performed. Histological alterations were observed in glomeruli and tubuli of exposed rats. The release of biomarkers for nephrotoxicity suggested alterations preferably in the proximal tubules of the exposed rats.

Fatal intoxications with chloral hydrate.

An alcoholic man, treated with chloral hydrate (CH) syrup to which he was dependent, was discovered comatose and in respiratory arrest. Death occurred on the ninth day of hospitalization following cerebral oedema. A woman, alcohol addicted, depressed, and epileptic was admitted in the Intensive Care Unit with heart and respiratory failure following CH absorption. She died three days later after a deep coma. In these two cases, CH intoxication was confirmed by toxicological analysis: CH and its major metabolite, trichloroethanol (TCE), were identified and determined in serum and urine using headspace-capillary gas chromatography-mass spectrometry. The concentrations measured were compared with those found in previously published fatalities. The analytical method used can be proposed for both clinical and forensic cases.

Tetrachloroethylene and trichloroethylene fatality: case report and simple headspace SPME-capillary gas chromatographic determination in tissues.

We describe a simple, precise, and sensitive assay of tetrachloroethylene and trichloroethylene in tissues, suitable both for emergency cases and forensic medicine. The method employs headspace solid phase microextraction-capillary gas chromatography and electron capture detection. The case is relative to a 45-year-old woman discovered unconscious in a laundry area. The concentrations of the solvents in tissues were determined and compared to other previously published fatalities.

Determination of chloral hydrate metabolites in human plasma by gas chromatography-mass spectrometry.

Chloral hydrate (CH) is a widely used sedative. Its pharmacological and toxicological effects are directly related to its metabolism. Prior investigations of CH metabolism have been limited by the lack of analytical techniques sufficiently sensitive to identify and quantify metabolites of CH in biological fluids. In this study a gas chromatography mass spectrometry (GC/MS) method was developed and validated for determining CH and its metabolites, monochloroacetate (MCA), dichloroacetate (DCA), trichloroacetate (TCA) and total trichloroethanol (free and glucuronidated form, TCE and TCE-Glu) in human plasma. Of these, DCA and MCA are newly identified metabolites in humans. The drug, its plasma metabolites and an internal standard, 4-chlorobutyric acid (CBA), were derivatized to their methyl esters by reacting with 12% boron trifluoride-methanol complex (12% BF3-MeOH). The reaction mixture was extracted with methylene chloride and analyzed by GC/MS, using a selected ion monitoring (SIM) mode. The quantitation limits of MCA, DCA, TCA, and TCE were between 0.12 and 7.83 microM. The coefficients of variation were between 0.58 and 14.58% and the bias values ranged between -10.03 and 14.37%. The coefficients of linear regression were between 0.9970 and 0.9996.

Enterohepatic recirculation of trichloroethanol glucuronide as a significant source of trichloroacetic acid. Metabolites of trichloroethylene.

Trichloroacetic acid (TCA) is a metabolite of trichloroethylene (TRI) thought to contribute to its hepatocarcinogenic effects in mice. Recent studies have shown that peak blood concentrations of TCA do not occur until approximately 12 hr after an oral dose of TRI; however, blood concentrations of TRI reach a maximum within 1 hr and is nondetectable after 2 hr. The objective of this study was to examine quantitatively enterohepatic recirculation of trichloroethanol (TCEOH) and TCA as a possible mechanism responsible for the delayed production of TCA. Jugular vein, duodenum, and bile duct-cannulated Fischer 344 rats were used, with the collection of blood, bile, urine, and feces samples after intraduodenal and intravenous dosing of animals with TRI, TCEOH, and TCA. Samples were analyzed by GC for TCA, total TCEOH, and free TCEOH. The results show that, after an intravenous dose of TCEOH (100 mg/kg), 36% of the TCEOH in blood is attributable to enterohepatic recirculation. With the same treatment, 76% of the TCA in blood is attributable to enterohepatic recirculation of metabolites. Peak concentrations of total TCEOH in bile, after an intraduodenal dose of TRI, are over 5 times higher than peak concentrations of total TCEOH in systemic blood. Peak concentrations of TCEOH glucuronide in bile are approximately 200 times higher than peak concentrations of TCEOH glucuronide in systemic blood.

Combining physiologically based pharmacokinetic modeling with Monte Carlo simulation to derive an acute inhalation guidance value for trichloroethylene.

Using the Monte Carlo method and physiologically based pharmacokinetic modeling, an occupational inhalation exposure to trichloroethylene consisting of 7 h of exposure per day for 5 days was simulated in populations of men and women of 5000 individuals each. The endpoint of concern for occupational exposure was drowsiness. The toxicologic condition leading to drowsiness was assumed to be high levels of both trichloroethanol and trichloroethylene. Therefore, the output of the simulation or dose metric was the maximum value of the sum of the concentration of trichloroethylene in blood and the concentration of trichloroethanol within its volume of distribution occurring within 1 week of exposure. The distributions of the dose metric in the simulated populations were lognormal. To protect 99% of a worker population, a concentration of 30 ppm over a 7-h period of the work day should not be exceeded. Subjecting a susceptible individual (the 99th percentile of the dose metric) to 200 ppm (the ACGIH short-term exposure limit or STEL) for 15 min twice a day over a work week necessitates a 2.5-h rest in fresh air following the STEL exposure to allow the blood concentrations of trichloroethylene and trichloroethanol to drop to levels that would not cause drowsiness. Both the OSHA PEL and the ACGIH TLV are greater than the value of 30 ppm derived here. As well as suggesting a new occupational guidance value, this study provides an example of this method of guidance value derivation.

Concentrations of trichloroethylene and its metabolites in blood and urine after acute poisoning by ingestion.

A 58-year-old man fell into a trichloroethylene reservoir bath head first, during a maintenance degreasing bath and accidentally ingested the solvent. Although he showed deep coma, chemical burns and pneumonia on admission, these symptoms gradually subsided. The concentrations of trichloroethylene (TRI) and its metabolites, trichloroethanol (TCE) and trichloroacetic acid (TCA) in blood and urine were measured during hospitalization. Eight hours after the accident, the concentrations of TRI and its metabolites in serum were 31.4 micrograms/ml TRI, 16.5 micrograms/ml TCE and 79.5 micrograms/ml TCA. The serum TRI concentration decreased to 4.3 micrograms/ml on the following day. Elimination of TCE and TCA from serum occurred biphasically, the estimated half-lives of each metabolites being about 52.6 and 50.4 h in an initial fast phase and 268.3 and 277.2 h in a subsequent slow phase, respectively. Urinary TRI excretion persisted for the first 2 days. The urinary TCE and TCA excretions were longer than that of TRI with a biphasic decrease and the total amount of TCE excreted during the first 2 days was about two times that of TCA. The half-life of urinary TCE excretion (t1/2 25.7 h) was shorter than that of TCA (t1/2 52.1 h) in the fast phase but did no difference during the slow phase, with each half-time being about 166.3 h. The kinetics of TRI metabolites in blood and urine in this case were in slight agreement with the results following inhalation exposure previously reported in the literature.

Determination of chloral hydrate and its metabolites (trichloroethanol and trichloracetic acid) in human plasma and urine using electron capture gas chromatography.

Capillary gas chromatography with electron capture detection is described for the quantification of chloral hydrate (CH) and is metabolites trichloroethanol (TCE) and trichloroacetic acid (TCA) in 0.1-1 mL of plasma samples. The method, with 2,2'-dichloroethanol (DCE) as internal standard, involved extraction of chloral hydrate and trichloroethanol with diethyl ether and methylation of trichloroacetic acid with 3-methyl-1-tolyltriazene (MTT), followed by diethyl ether extraction. The method has a detection limit of 5 ng/mL for CH and TCE and 10 ng/mL for TCA and also allows the determination of TCE-glucuronide in 0.1-1 mL of plasma samples. It exhibits good linearity and precision. The method was applied to samples of plasma from a neonate after a single dose of 40 mg/kg of chloral hydrate and from an adult after a single dose of 6.25 mg/kg.

Determination of 2,2,2-trichloroethanol in plasma and urine by ion-exclusion chromatography.

A simple and rapid chromatographic method has been developed for the determination of 2,2,2-trichloroethanol (TCEOH) and its glucuronide in plasma and urine. A glass column (150 x 6.6 mm i.d.) packed with Aminex A-5 cation-exchange resin (potassium form) following the slurry method was used as the analytical column, and an admixture of 10 mmol l-1 potassium sulfate and 10 mmol l-1 potassium hydroxide solution as the eluent (pH 12.2). Diluted plasma samples and urine samples were directly injected into the chromatograph through a 0.45 micron membrane filter without deproteinization. The amount of TCEOH conjugated to glucuronide was determined following treatment with beta-glucuronidase (200 U) for 30 min at 37 degrees C. This allowed the concentration of free, total, and conjugated TCEOH to be determined. The calibration graph was rectilinear from 5 to 500 mg l-1 of TCEOH, with a detection limit of 3 mg l-1, 2 sigma, being the signal-to-noise ratio. The analytical recovery of TCEOH, obtained by analysing spiked plasma and urine samples, was in the range 98.4-102% and the relative standard deviation was less than 3.5%.

Electroretinal responses are modified by chronic exposure to trichloroethylene.

Using an inhalation chamber, New Zealand albino rabbits were exposed to 350 ppm (n = 6) and 700 ppm (n = 8) of trichloroethylene (TRI) 4 hrs/day, 4 days/week for 12 weeks. Electroretinograms (ERG) and oscillatory potentials (OPs) were recorded weekly under mesopic conditions. Blood samples were also collected weekly to determine the concentration of TRI and its main metabolites. Recordings from the 350 and 700 ppm exposed groups showed a significant (p < 0.01) increase in the amplitude of the a- and b-waves (ERG), while the amplitude of the OPs was significantly (p < 0.01) decreased at 350 ppm and increased at 700 ppm. These electroretinal changes were reversed to the baseline value within six weeks after the inhalation stopped. The observed variations in a-wave and OP amplitudes were related to plasmatic level of trichloroethanol, while the effects on the b-wave were related to the blood level of TRI. These results confirm the neuro-ophthalmotoxicity of TRI and support the hypothesis that trichloroethanol is the major neurotoxic metabolite of TRI.

Chloral hydrate toxicity in a term infant.

Chlorate hydrate is commonly used for neonatal sedation, but blood levels are infrequently monitored, reflecting an underemphasis of acute toxic effects. This report describes a case of chloral hydrate toxicity in a term infant with cardiac, renal, neurologic, bladder and gastrointestinal dysfunction. The effects of exchange transfusion are described as well as pharmacokinetics.

Circadian variations in trichloroethylene toxicity under a 12:12 hr light-dark cycle and their alterations under constant darkness in rats.

In order to investigate the circadian variations in acute toxicity of trichloroethylene (TRI), TRI (1.2 g/kg weight) or saline was injected intraperitoneally in a total of 88 male Wistar rats at four circadian stages (03.00, 09.00, 15.00, and 21.00:hr.min) under two different lighting regimens of a 12:12 hr light-dark cycle (LD; light from 06.00 to 18.00) and of constant darkness (DD). Circadian variations in TRI toxicity were confirmed in both LD and DD. The toxicity of TRI evaluated by the increase in glutamic-pyruvate transaminase activity (GPT) was greatest when injected at 09.00 in LD while at 21.00 in DD. The increases in blood urea nitrogen, serum total cholesterol and triglyceride concentrations reached peaks when injected at 09.00 in LD and 03.00 in DD. The circadian variations in serum trichloroethanol concentration were very similar to those in GPT in both LD and DD, showing a significant correlation (p less than 0.05). The present study revealed that circadian variations in TRI toxicity existed in LD and that these variations persisted in a free-running condition. The peak phase of TRI toxicity was located in a trough phase (09.00) in LD and in a peak phase (21.00 or 03.00) in DD of temperature rhythm. Thus, the phase relationship changed in DD, showing a desynchronization between TRI toxicity rhythm and temperature rhythm, which is an unusual phenomenon. This means that an unexpected potentiation of TRI toxicity during active phase which is not a critical phase in a well-synchronized state could occur in a free-running condition.