Quantitative Determination of Ethyl Glucuronide and Ethyl Sulfate in Postmortem and Antemortem Whole Blood Using Phospholipid Removal 96-Well Plate and UHPLC-MS-MS.

Postmortem ethanol formation is a well-known problem in forensic toxicology. Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are ethanol metabolites that can be used to distinguish antemortem alcohol intake from postmortem formation of ethanol and in addition can be a helpful tool in assessment of the hip-flask defense. To an aliquot of 100 µL whole blood, internal standard (IS) and water was added before protein precipitation treatment (PPT) with ice-cold acetonitrile (ACN). The supernatants were filtered through a 96-well phospholipid removal plate, evaporated to dryness and reconstituted in 150 µL water/ACN/formic acid (FA). Identification of compounds was performed using multiple reaction monitoring (MRM) in negative mode. Gradient elution was performed on a C18 column with methanol (MeOH) and 0.1% FA. The run time was 4.5 min, and 0.5 µL was injected on an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS-MS) instrument. Linearity was achieved (coefficient of determination (R2) ≥ 0.999) for EtG in the range of 0.089 to 22 mg/L (0.40-100 µM) and EtS 0.025 to 6.3 mg/L (0.20-50 µM). The limit of quantification (LOQ) was 0.067 mg/L (0.30 µM) for EtG and 0.019 mg/L (0.15 µM) for EtS. Between assay accuracy was -15% to 8% and precision reported as relative standard deviation (RSD) was ≤ 4.5%. Precision, estimated as the RSD of the concentration difference between results from two independent analyses of authentic whole blood samples, was ≤ 6.7%. Recovery was ≥ 61% for EtG and ≥ 77% for EtS and matrix effects (ME) were 99% to 103%. Method comparison was carried out with a previously used UHPLC-MS-MS method, and satisfactory agreement was achieved, and external proficiency testing control samples had z-score < ± 1. The method has been used in routine work for more than 4 years analyzing about 6,000 antemortem and postmortem whole blood samples and has proven to be robust and reliable.

EtG and EtS in Autopsy Blood Samples With and Without Putrefaction Using UPLC-MS-MS.

Analytical challenges related to postmortem specimens are well known. The degree of putrefaction of the corpse will influence the quality of the blood samples, and both the efficiency of sample preparation and the subsequent chromatographic performance can be affected. An ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS) method was developed and validated for the determination of ethyl glucuronide (EtG) and ethyl sulfate (EtS) in postmortem whole blood. Sample preparation prior to UPLC-MS-MS analysis consisted of protein precipitation and filtration through a phospholipid removal plate. Chromatography was achieved using an HSS T3 column and gradient elution with formic acid in water in combination with methanol. The injection volume was 0.5 µL. Negative electrospray ionization was performed in the multiple reaction monitoring mode. Two transitions were monitored for the analytes and one for the internal standards. The between-assay relative standard deviations were in the range of 1.7-7.0% and the limits of quantification were 0.025 and 0.009 mg/L for EtG and EtS, respectively. Recovery was 51-55% and matrix effects ranged from 98% to 106% (corrected with internal standard). Blood samples from nine autopsy cases with various extents of putrefaction were analyzed. The sample preparation efficiently removed the phospholipids from the blood specimens. The samples were clean and the analytical quality of the chromatographic performance was satisfactory for both analytes irrespective of the degree of putrefaction.

Determination of safety margins for whole blood concentrations of alcohol and nineteen drugs in driving under the influence cases.

Legislative limits for driving under the influence of 20 non-alcohol drugs were introduced in Norway in February 2012. Per se limits corresponding to blood alcohol concentrations (BAC) of 0.2g/kg were established for 20 psychoactive drugs, and limits for graded sanctions corresponding to BACs of 0.5 and 1.2g/kg were determined for 13 of these drugs. This new legislation made it possible for the courts to make sentences based on the analytical results, similar to the situation for alcohol. To ensure that the reported concentration is as least as high as the true concentration, with a 99% safety level, safety margins had to be calculated for each of the substances. Diazepam, tetrahydrocannabinol (THC) and alcohol were used as model substances to establish a new model for estimating the safety margins. The model was compared with a previous used model established several years ago, by a similar yet much simpler model, and they were found to be in agreement. The measurement uncertainties depend on the standard batch used, the work list and the measurements' replicate. A Bayesian modelling approach was used to determine the parameters in the model, using a dataset of 4700 diazepam positive specimens and 5400 THC positive specimens. Different safety margins were considered for low and high concentration levels of diazepam (≤2µM (0.6mg/L) and >2µM) and THC (≤0.01µM (0.003mg/L) and >0.01µM). The safety margins were for diazepam 19.5% (≤2µM) and 34% (>2µM), for THC 19.5% (≤0.01µM) and 24.9% (>0.01µM). Concentration dependent safety margins for BAC were based on a dataset of 29500 alcohol positive specimens, and were in the range 10.4% (0.1g/kg) to 4.0% (4.0g/kg) at a 99% safety level. A simplified approach was used to establish safety margins for the compounds amphetamine, MDMA, methamphetamine, alprazolam, phenazepam, flunitrazepam, clonazepam, nitrazepam, oxazepam, buprenorphine, GHB, methadone, ketamine, cocaine, morphine, zolpidem and zopiclone. The safety margins for these drugs were in the range 34-41%.

Alcohol hangover as a cause of impairment in apprehended drivers.

OBJECTIVE: Previous studies have already shown the possibility of impairment during a hangover phase, after alcohol ingestion, when the blood alcohol concentration has returned to zero. The prevalence of drivers being in a hangover phase, in the driving population, and the relation to impairment relevant for traffic safety has, however, not been previously studied. The aim of this study was to investigate the prevalence and the concentrations of the 2 ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), in blood, indicating very recent alcohol intake, among apprehended drivers, in which no psychoactive substances, including alcohol, were detected. The aim was also to study these findings in relation to the impairment observed in these drivers. METHODS: Blood samples, drawn from suspected drunk or drugged drivers, were analyzed for a broad repertoire of psychoactive substances, with a clinical test for impairment (CTI) being performed at the same time. One hundred and forty-six cases, in which no psychoactive substances were detected and where a valid CTI was performed, were analyzed for EtG and EtS in blood. The prevalence and concentrations were related to the conclusions of the CTIs. RESULTS: EtS and EtG were detected in a total of 19 of the 146 cases (13%). Among the "impaired" drivers, EtG and EtS were detected in 16 cases (18%), whereas among "not impaired" drivers they were detected in 3 cases (5%). There were significantly more detections of EtS (and EtG) among the impaired group of drivers compared to the nonimpaired drivers (P =.030), and the concentrations of both EtG (P =.027) and EtS (P =.026) were significantly higher in the group of impaired drivers compared to the nonimpaired drivers. There was a statistically significant positive correlation between the concentrations of EtG (Spearman's rho = 0.170, P =.041) and EtS (Spearman's rho = 0.189, P =.022) and the degree of impairment. CONCLUSIONS: EtG and EtS were prevalent findings in blood collected from the apprehended drivers, testing negative for all psychoactive substances. The higher rates of detections of EtG and EtS in impaired compared to nonimpaired drivers, and also the positive correlation between concentrations of EtG and EtS and the degree of impairment, indicate that hangover symptoms may be relevant for traffic safety.

Sex, BMI and age in addition to dietary intakes influence blood concentrations and congener profiles of dioxins and PCBs.

SCOPE: The aim of this study was to i) characterize dietary polychlorinated biphenyls (PCBs) and dioxin exposure in consumers of fish from the PCB contaminated Lake Mjøsa in Norway ii) examine the influence of demographic factors on blood concentrations and congener composition of dioxins and PCBs, iii) characterize dietary sources and possible exposures above tolerable intake. METHODS AND RESULTS: Blood samples were analysed for dioxin-like (dl) compounds (PCDD/Fs and dl-PCBs) and non-dl-PCBs (ndl-PCBs). Dietary exposures were calculated using food frequency questionnaires (n=64). Men had higher median intake of dl-compounds than women (1.2 and 0.85 pg TEQ/kg bw/day), but similar blood concentrations (23.3 and 25.8, pg TEQ/g lipid weight (lw)). For non-dl-PCBs, intakes (6.5 and 4.5 ng/kg bw/day) and blood concentrations (381 and 224 ng/g lw) were higher in men than in women. Blood concentrations correlated with dietary intakes in men only. Increasing BMI and age elevated blood concentrations mainly in women. Men and women had different blood congener profiles, with a higher share of PCB-126 in women, despite similar dietary congener profiles. Eleven participants exceeded the tolerable intake for dl-compounds. Fish from Lake Mjøsa was the main dietary source. CONCLUSION: The higher influence of BMI and age for women than for men may have implications for risk assessment.

Consumption of fish from a contaminated lake strongly affects the concentrations of polybrominated diphenyl ethers and hexabromocyclododecane in serum.

Very high concentrations of polybrominated diphenyl ethers (PBDE) have been reported in fish from Lake Mjøsa in Norway. This study was performed to examine the serum concentrations of PBDE and hexabromocyclododecane (HBCD) in consumers of fish from this lake and to investigate possible relationships between serum concentrations, self-reported fish intake and calculated total dietary PBDE exposure. Serum concentrations of the sum of the seven PBDE (BDE-28, 47, 99, 100, 153, 154 and 183) were significantly higher than those of a reference group of Norwegians eating only food with background levels of contamination (medians: 18 ng/g lipids men, 8.4 ng/g lipids women). The median dietary intake of Sum 7 PBDE was 2549 ng/day (30 ng/kg body weight/day), the highest dietary intake of PBDE reported. The contribution from fish caught from the contaminated lake comprised 98.7% of the total dietary exposure. For men, serum levels of PBDE were strongly correlated with the calculated dietary exposure, except for BDE-209. This suggests that sources other than the diet are important for human BDE-209 exposure. The median serum HBCD concentration was 4.1 and 2.6 ng/g lipids for men and women, respectively, and was also found to be associated with consumption of fish from Lake Mjøsa.

Comparison of GC and LC determinations of hexabromocyclododecane in biological samples - results from two interlaboratory comparison studies.

In order to assess the quality and comparability of results from determinations of 1,2,5,6,9,10-hexabromocyclododecane (HBCD) in biological samples, two interlaboratory comparison studies have been organized. Up to 13 laboratories determined either the total HBCD concentration, or concentrations of alpha-, beta- and gamma-HBCD, or both in cod liver oil, herring filet, salmon filet, butter and chicken meat. The laboratories were able to determine total HBCD concentrations in the marine samples with satisfying quality (RSD <35%). However, the analysis of samples with low HBCD contamination (

Occupational exposure to hexabromocyclododecane at an industrial plant.

Occupational exposure to hexabromocyclododecane (HBCD) among workers at an industrial plant producing expandable polystyrene (PS) added HBCD as flame retardant has been assessed in the present study. Airborne dust samples were collected near the breathing zone of 10 male workers during three 8-h work shifts. The HBCD concentrations in the airborne dust varied from 0.2 to 150 microg/m3 (mean 12.2 and median 2.1 microg/m3). Two serum samples were obtained from each of the workers. The mean serum concentration was 190 ng/g lipids; the median was 101 ng/g lipids (range 6 to 856 ng/g lipids). HBCD was not detected above 1 ng/g lipids (LOD) in any samples from persons in a reference group with no occupational exposure to HBCD. The contribution of gamma-HBCD to the total HBCD serum concentration was notably high (39%) compared to what has usually been observed in biological samples. There was no clear correlation of serum levels with average HBCD concentrations in the airborne dust samples collected near the subjects' breathing zone. The elevated exposure levels reported in this study compared to urban air and serum levels in general populations suggest that further and more detailed exposure assessment studies should be initiated in industries where HBCD is applied.

Automated solid-phase extraction for the determination of polybrominated diphenyl ethers and polychlorinated biphenyls in serum--application on archived Norwegian samples from 1977 to 2003.

An analytical method comprised of automated solid-phase extraction and determination using gas chromatography mass spectrometry (single quadrupole) has been developed for the determination of 12 polybrominated diphenyl ethers (PBDEs), 26 polychlorinated biphenyls (PCBs), two organochlorine compounds (OCs) (hexachlorobenzene and octachlorostyrene) and two brominated phenols (pentabromophenol, and tetrabromobisphenol-A (TBBP-A)). The analytes were extracted using a sorbent of polystyrene-divinylbenzene and an additional clean-up was performed on a sulphuric acid-silica column to remove lipids. The method has been validated by spiking horse serum at five levels. The mean accuracy given as recovery relative to internal standards was 95%, 99%, 93% and 109% for the PBDEs PCBs, OCs and brominated phenols, respectively. The mean repeatability given as RSDs was respectively 6.9%, 8.7%, 7.5% and 15%. Estimated limits of detection (S/N=3) were in the range 0.2-1.8 pg/g serum for the PBDEs and phenols, and from 0.1 pg/g to 56 pg/g serum for the PCBs and OCs. The validated method has been used to investigate the levels of PBDEs and PCBs in 21 pooled serum samples from the general Norwegian population. In serum from men (age 40-50 years) the sum of seven PBDE congeners (IUPAC No. 28, 47, 99, 100, 153, 154 and 183) increased from 1977 (0.5 ng/g lipids) to 1998 (4.8 ng/g lipids). From 1999 to 2003 the concentration of PBDEs seems to have stabilised. On the other hand, the sum of five PCBs (IUPAC No. 101, 118, 138, 153 and 180) in these samples decreased steadily from 1977 (666 ng/g lipids) to 2003 (176 ng/g lipids). Tetrabromobisphenol-A and BDE-209 were detected in almost all samples, but no similar temporal trends to that seen for the PBDEs were observed for these compounds, which might be due to the short half-lives of these brominated flame retardants (FR) in humans.