Confirmation of the full conversion of ethylene oxide to 2-chloroethanol in fumigated foodstuffs: possible implications for risk assessment.

This study describes the extension of a gas chromatography mass spectrometry (GC-MS) method, initially devoted to the analysis of ethylene oxide (EO) in ice cream, to a larger range of food items including herbs, spices, vegetables, inorganic salts, food supplements, thickeners, etc. Results are reported as EOTotal according to EC 2015/868 definition (expressed as EO equivalents as the sum of native EO and 2-chloroethanol (2-CE) after acidic hydrolysis) with a limit of quantification at 0.01 mg/kg regardless of the food item. Its ruggedness was demonstrated through fortification experiments on hundreds of samples. Re-analysis of 146 positive food samples without hydrolysis demonstrated that not EO but 2-CE is the predominant analyte detected in the different processed ingredients suspected to have been previously treated with EO. A series of eight contaminated dried herbs and spices were also re-analysed by four ISO 17025 accredited commercial laboratories making use of different analytical strategies for EO determination in foods. Each laboratory reported EOTotal levels within the same concentration range, but the resulting reproducibility ranged from 23% to 41% depending on the sample. Additionally, we show that results of free EO from methods based on conversion to 2-iodoethanol may lead to artefactual detection of native EO (false positive). An official method of analysis applicable for different food matrices would be useful to avoid discrepancies of results. Altogether, these data re-enforce the fact that in absence of native EO in food items, risk assessment of EO in foodstuffs should consider the predominance of 2-CE. A toxicological risk assessment using the food additive xanthan gum as a case study is discussed.

Trace-level quantification of ethylene oxide and 2-chloroethanol in low-viscosity hydroxypropyl methylcellulose with solid phase microextraction and GC-MS.

Ethylene oxide (EtO) is naturally present in numerous food products but recently EtO and its degradation product of 2-chloroethanol (2-CE) have been reported in amounts exceeding the maximum residue limit in Europe. The reports include hard capsules for dietary supplements made from low viscous hydroxypropyl methylcellulose (HPMC). The European council (EC) has proposed a generalized method for spices, seeds, and capsules utilizing QuEChERS, solid phase extraction (SPE), and GC-MS/MS to accommodate the need for analyte specificity, trace-level analysis, and higher throughput. HPMC has unique solvation properties and, without care, can potentially be transferred to the instrument. The current work presents the development of two methods specific for EtO and 2-CE in low viscous HPMC using solid phase microextraction (SPME) and GC-MS. Method optimization for solvation, SPME settings, and GC-MS settings are presented along with validation based on standard addition. Both methods present a high degree of specificity and limits of detection (EtO 6.7 µg/kg and 2-CE 12 µg/kg), comparable to those obtained with the EC method. Apart from sampling, the methods were fully automated and rely on low cost GC-MS instrumentation, widely available. Analyzed samples did not contain EtO or 2-CE, and method development was done with spiked samples. Contamination from plastic containers and analytical carry-over are shown as possible sources of EtO and 2-CE.

Analysis of ethylene oxide in ice creams manufactured with contaminated carob bean gum (E410).

Residues of ethylene oxide (EO), a banned fumigant in the EU, were found at amounts above the maximum residue limit (MRL) in carob (locust) bean gum (additive E410). The pesticide entered the food chain via stabiliser blends that are used as minor ingredients in the manufacture of ice cream. Consequently, all products that contained the non-compliant ingredient were withdrawn or recalled in several countries across the EU, in most cases irrespective of whether the pesticide residue was detectable or not in the final product. This is the first report of a reliable method to determine EO and its metabolite/marker compound 2-chloroethanol (2-CE), either together or independently in ice cream, with a limit of quantification at 0.01 mg EO/kg and recovery in the range of 87-104% across the levels investigated (0.01, 0.02 and 0.06 mg EO/kg). The method applies QuEChERS extraction and isotope dilution gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). High resolution mass spectrometry (HRMS) confirmed the specificity of low mass ions. Data on the stability of EO and 2-CE under thermal conditions revealed that 2-CE is relatively stable in an ice cream matrix (ca. 80% recovery of spiked material). Importantly, this study also demonstrates that not EO, but 2-CE is the predominant analyte detected in the contaminated samples, which is new information of significance in terms of the overall risk assessment of EO in foodstuffs.

An unusual trichloroethanol fatality attributed to sniffing trichloroethylene.

We report the death of a 28-year-old man due to sniffing a contact cement containing trichloroethylene. Initial testing revealed the presence of 80 mg/L trichloroethanol in cardiac blood, and the death was ruled as being due to trichloroethanol toxicity resulting from chloral hydrate ingestion. However, further investigation of the case revealed that the trichloroethanol resulted from trichloroethylene abuse. Subsequent targeted analysis for trichloroethylene, four months after the death, confirmed its presence in cardiac blood (1.1 mg/L), bile (4.5 mg/L), and liver (2.5 mg/kg). Trichloroethanol was initially detected during routine drug screening that employed gas chromatography (GC) using an HP-5 column with electron capture detection and subsequently quantitated by GC using the same column as for the initial screen, but with flame-ionization detection (FID); ethchlorvynol was the internal standard. Trichloroethylene was quantitated by headspace GC with a Restek Rtx-BAC1 column and FID; 1,1,1-trichloroethane was the internal standard.

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.

Assessment of cancer risk from ethylene oxide residues in spices imported into New Zealand.

Quantitative estimates of cancer risks from ethylene oxide (ETO) residues were constructed based on 200 retail samples of various spices in New Zealand. Two samples of cinnamon contained detectable ETO. The highest value encountered was 15 ppm. ETO was not detected in the remaining 198 samples. However, 31 samples had detectable levels of ethylene chlorohydrin (ECH) and/or ethylene bromohydrin (EBH). A conservative estimate of ETO intake, based on average spice consumption, was 3.4 x 10(-6) mg/kg/day. Cancer potency factors for ETO ranging from 0.29 to 0.55 (mg/kg/day)(-1) were used to form cancer risk estimates. The resulting estimates of average lifetime excess cancer risk was 0.8 x 10(-6) to 1.7 x 10(-6). The US 97.5 percentile value for spice consumption (2.8 kg spices per year), gave an extreme upper-end estimate of lifetime cancer risk of approximately 1.4 x 10(-5). These risks are practically negligible considering the conservative assumptions used in estimating exposure to ETO. The exposures to ECH and EBH are 200-300-fold higher than to ETO. These compounds are of lesser potency to ETO in terms of mutagenicity or carcinogenicity in studies to date. However, the precise contribution of these compounds to the cancer risk estimate is uncertain due to large toxicological data gaps, including the absence of a 2-year cancer bioassay by the oral route.

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.

Perimortem fixation of the gastric and duodenal mucosa: a diagnostic indication for oral poisoning.

Two cases of fatal oral poisoning are presented. In the first case, a 40-year-old man died due to a lethal dose of mercury (blood concentration 113.8 microg/ml) and in the second, a 34-year-old man died of chloralhydrate overdose with a lethal blood concentration of trichloroethanol (52 microg/ml), the active metabolite of chloralhydrate. In both cases gross examination and histology showed an unusually well preserved gastrointestinal mucosa in addition to unspecific signs of intoxication. The two cases demonstrate that the phenomenon of perimortal fixation is a useful indication for the forensic pathologist and should direct the suspicion to oral poisoning. The detection of fixation facilitates toxicology screening by indicating that the relevant substance must have the capability to precipitate proteins.

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.

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.

Determination of chlorobutanol in mouse serum, urine and embryos by capillary gas chromatography with electron capture detection.

A sensitive and specific method for the determination of chlorobutanol (1,1,1-trichloro-2-methyl-2-propanol) in mouse serum, urine, and embryos by capillary gas chromatography with electron capture detection is described. For sample preparation n-hexane was used to extract chlorobutanol and the internal standard 2,2,2-trichloroethanol (TCE) from each matrix. Following extraction chromatographic separation of the samples was achieved with a fused-silica capillary column (30 m x 0.25 mm I.D., 0.25 micron film thickness). The method as described has the required sensitivity to quantitate chlorobutanol in individual embryos following administration of a single oral dose of the drug to a pregnant mouse. The limit of detection was 1 pg on column and the detector response was linear from 1 to 100 micrograms/ml for serum, 0.2 to 20 micrograms/ml for urine, and 1 to 10 ng/embryo.

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%.

Chloral hydrate overdose: trichloroethanol detection by gas chromatography/mass spectrometry.

A case is presented where an individual ingested a fatal dose of chloral hydrate. Trichloroethanol (TCE), the metabolite of chloral hydrate, was initially identified by the Fujiwara reaction and quantified by gas chromatography/mass spectrometry in blood )127 mg/l), urine (128 mg/l) and stomach contents (25 mg total).

Determination of ethylene oxide residues in processed food products by gas-liquid chromatography after derivatization.

A simple, sensitive and fast method (taking only 4-5 h) for the determination of residues of ethylene oxide (EO) and its reaction product, ethylene chlorohydrin (ECH), is described. For the analysis sodium hydroxide is added to the sample where ECH is transformed to EO. This is followed by the distillation of EO into dilute sulphuric acid containing sodium iodide, whereby EO is converted into ethylene iodohydrin (EIH). The EIH content is determined by gas chromatography using electron capture detection. The method has proved to be applicable to the analysis of low residue levels (less than 0.05 mg/kg, calculated as EO) in various foods, including processed foods. The collaborative studies carried out with four food products in six laboratories were remarkably successful with regard to repeatability of the method and reproducibility of the results. The recoveries of ECH were 50%-60%. In the 204 food products examined EO residues were found in 96 samples at concentration levels between 0.05 mg/kg and 1800 mg/kg.

Simple and accurate method for determination of ethylene chlorohydrin in dried spices and condiments.

A simple and accurate method is described for the determination of ethylene chlorohydrin (ECH) by using capillary gas chromatography (GC) and flame ionization detection. Acetonitrile-methanol was chosen as the extraction solvent in preference to other solvents because its use reduced the number of compounds detected by the GC system, thus enabling easier identification and quantitation of ECH. The coefficient of variation for the method is 2.7% at 5 ppm, and recovery is good, even for the standard addition of 1 ppm. Fifteen different spices and condiments were analyzed using this method; 20% were identified as positive for ECH. The method also identifies the related compound ethylene bromohydrin (EBH).

2-chloroethyl fatty acid esters as indicators of 2-chloroethanol in black walnuts, seasoning mixes, and spices.

Residues of 2-chloroethyl fatty acid esters (CEEs) and 2-chloroethanol (ECH), by-products of ethylene oxide fumigation, were determined in black walnuts, seasoning mixes, and spices. Extracts containing ECH and CEE were cleaned up by previously described procedures, and residue levels were quantitatively determined using a gas chromatograph equipped with a halogen-selective electrolytic conductivity detector. All food products that contained CEE residues also contained ECH. ECH residues ranged from less than 0.2 to 880 ppm and were less than 0.2-7 times the CEE levels found.

High performance liquid chromatography determination of ethylene glycol and ethylene chlorohydrin in tissues.

A simultaneous determination of ethylene glycol (EG) and ethylene chlorohydrin (EC), which are formed during sterilization of bone and dura mater with ethylene oxide, has been reported. The EG and EC residues in tissues were extracted with HPLC grade water and analyzed by reversed-phase high-performance liquid chromatography (HPLC) with a RefractoMonitor detector. The isocratic elution was employed with HPLC grade water as the solvent. Since the reference chamber of the detector was filled with solvent and was not changed during the run, the use of gradient programming was not employed. With spherisorb ODS (5 microns) C18 column and 20 microL sample injection, the retention times for EG and EC were (104 +/- 2) sec and (202 +/- 4) sec, respectively. The unknown concentration was calculated from the calibration curve and the weight of the sample tissue. The HPLC analysis provided a simple, accurate and reproducible method of determination of EG and EC residues in ethylene-oxide sterilized tissues.