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.

A fatality due to an accidental methadone substitution in a dental cocktail.

A 6-year-old male child was scheduled for a dental procedure requiring conscious sedation. Prior to the procedure, the child was administered a dental cocktail containing chloral hydrate, hydroxyzine, and methadone. After returning from the dentist, the child appeared groggy and was allowed to sleep. A few hours later, he was found unresponsive, and following resuscitation attempts at a local medical center, he was pronounced dead. Toxicological analyses of femoral blood indicated the presence of hydroxyzine at less than 0.54 µg/mL, trichloroethanol (TCE) at 8.3 µg/mL, and methadone at 0.51 µg/mL. No meperidine was detected. The cause of death was reported to be due to the toxic effects of methadone. The toxicological analysis was corroborated by the analysis of the contents of the dental cocktail, which revealed the presence of hydroxyzine, chloral hydrate, and methadone. Residue from a control sample obtained from the same pharmacy, but administered to a different subject, was found to contain hydroxyzine, chloral hydrate, and meperidine. This report represents the first known fatality due to accidental substitution of methadone in a dental cocktail.

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.

Determination of chloral hydrate and its metabolites in blood plasma by capillary gas chromatography with electron capture detection.

A sensitive, accurate, and reliable method is described for the quantitative determination of chloral hydrate (CH) and its metabolites in blood plasma of mice and rats. Metabolites of CH include trichloroacetic acid (TCA), trichloroethanol (TCE), and trichloroethanol glucuronide (TCE-Glu). This new method uses capillary gas chromatography with electron-capture detection (GC/ECD). Procedures for improving sample stability and quality assurance are also described that were not mentioned in previous literature. Rat or mouse plasma (50 microl) is acidified (or treated enzymatically for TCE-Glu determination) and extracted with peroxide free methyl t-butyl ether. Distilled diazomethane (CH(2)N(2)) is added to derivatize TCA to its methyl ester. Detection limits were estimated at 0.2 microg/ml for CH and TCE, and 0.1 microg/ml for TCA. Detector response to TCA and TCE were shown to be linear in the range of 3.125-200 microg/ml (r> or =0.9996). For CH, the response fits a second-order equation in this same range (r=0.99994)

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.

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.

Kinetics and metabolism of chloral hydrate in children: identification of dichloroacetate as a metabolite.

Chloral hydrate was introduced into therapeutics more than 100 years ago, and since then a number of kinetic and metabolic studies have been conducted on this drug. Trichloroethanol, its glucuronide and trichloroacetic acid have been identified as the metabolites of chloral hydrate. We now report the identification of dichloroacetate as a major product of chloral hydrate metabolism in children, in addition to trichloroethanol and trichloroacetic acid. Furthermore, pretreatment of children with chloral hydrate appears to retard the plasma clearance of dichloroacetate.

A species comparison of chloral hydrate metabolism in blood and liver.

Chloral hydrate (CH), [302-17-0], is a human sedative useful in premature infants. No current epidemiological study supports increased cancer risk. CH is also a rodent toxicant and a P450-derived metabolite of trichloroethylene (TRI). P450 induction increases TRI toxicity in rodents. CH is very rapidly metabolized to trichloroacetic acid (TCA) and trichloroethanol (TCOH). Because TCA mediates some responses following TRI exposure, we assessed the metabolism of CH to TCA and TCOH by liver and blood of the rat, mouse, and human. Both TCA and TCOH are formed in blood and liver. The constants for hepatic TCA and TCOH formation are presented. The K(m) for hepatic TCOH formation is at least ten-fold lower than for TCA formation in these species. Clearance values for TCOH are higher than for TCA. These data support TCOH as the first major metabolite of TRI and CH in vivo.

Determination of chloral hydrate and metabolites in a fatal intoxication.

A young woman (32 years old) was found dead in her house. Screening of postmortem blood with enzyme multiplied immunoassay (EMIT) detected benzodiazepines, salicylic acid derivatives, and caffeine. These compounds were present in nontoxic concentrations as confirmed by thin-layer chromatography and high-performance liquid chromatography. The Fujiwara-Ross reaction on blood revealed the presence of chlorinated hydrocarbons in high concentrations. An optimized gas chromatographic method with electron capture detection allowed the identification and quantitation of chloral hydrate and both its metabolites, 2,2,2-trichloroethanol and trichloroacetic acid, in the available postmortem samples. The tissue concentrations indicated that chloral hydrate ingestion could be identified as the cause of this fatality.

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.

Chloral hydrate disposition following single-dose administration to critically ill neonates and children.

Although the metabolism and pharmacokinetics of chloral hydrate (CH) have been studied in healthy adults, no comprehensive studies have been done in neonates and young infants. Major physiological differences between these groups could greatly affect drug disposition. In this study the patient population (22 patients) was divided into three groups according to postconceptual age: group 1 = preterm infants (31-37 weeks), group 2 = fullterm infants (38-42 weeks) and group 3 = toddler-child patients (57-708 weeks). After receiving one 50 mg/kg oral dose of CH, the parent drug and its metabolites were determined by gas chromatography utilizing an electron capture detector. CH, contrary to what has been reported in the adult, was detectable for several hours after oral administration to patients in all three groups. A highly significant negative correlation was observed amongst the three groups for the half-life (t1/2) and area-under-the-curve for 0 to infinity values for trichloroethanol (TCE), the active metabolite responsible for the sedation effect. The t1/2 value for TCE in group 3 (9.67 h) was similar to that reported for the adult population, but in the less mature subjects it was approximately three (group 2: 27.8 h) to four times (group 1: 39.8 h) greater. Trichloroacetic acid had a remarkably long residence time in the study population after a single dose of CH. The concentration of this metabolite failed to decline even 6 days after dose. These issues should be carefully considered when CH administration is contemplated for clinical use in neonates, infants and children.

Death after chloral hydrate sedation: report of case.

A young healthy female died after taking chloral hydrate syrup before surgery to extract third molars. Various aspects of the use of chloral hydrate are discussed, including the metabolism, active moiety, reported side effects, and effects on the heart. Recommendations are made concerning patient supervision, dosage limitations, and degree of sedation.

Extrahepatic metabolism of chloral hydrate, trichloroethanol and trichloroacetic acid in dogs.

To examine the details concerning that part of TRI metabolism which was carried out by the extrahepatic organs, we studied the extrahepatic metabolism of chloral hydrate (CH), free-trichloroethanol (F-TCE) and trichloroacetic acid (TCA) using a method developed in our laboratory. Bypass and non-bypass dogs were given CH, F-TCE and TCA, and we compared the concentrations these substances and their metabolites in the serum and urine of the two groups of animals. In the bypass dogs, F-TCE, TCA and conjugated-trichloroethanol (Conj-TCE) appeared in the blood and urine 30 min. after the CH administration, and TCA and Conj-TCE appeared 30 min. after the F-TCE. All levels of administered substance were higher in bypass dogs than in non-bypass dogs, and the compounds were metabolized in small amounts in the extrahepatic organs compared with the liver. Therefore, administered substances remained at high levels in the serum and were excreted in large amounts in the urine in the form of unchanged substances. The metabolized percentage volumes of CH to TCA in the bypass dogs were 10-20%, and those of F-TCE to TCA were very small, while these percentage values of CH to F-TCE were the same or slightly smaller, respectively. Moreover, trichloroethylene (TRI) acts to decrease the leukocyte count in the blood, but the TRI metabolites described above do not have this function.