Forensic confirmatory analysis of ethyl sulfate—a new marker for alcohol consumption—by liquid-chromatography/electrospray ionization/tandem mass spectrometry

Ethyl sulfate (EtS)—a new direct marker for ethanol intake besides ethyl glucuronide (EtG) and others—was detected in urine samples by electrospray ionization tandem mass-spectrometry (LC-ESI-MS/MS). Ethyl sulfate sodium salt was used for method development, yielding a precursor [M − H]− m/z 125 and product ions m/z 97 [HSO4]− and m/z 80 [SO3]−. Pentadeuterated EtS (D5-EtS) was synthesized by esterification of sulfuric acid with anhydrous hexadeutero ethanol ([M − H]− m/z 130, product ions m/z 98 [DSO4]− and m/z 80 [SO3]−). After addition of D5-EtS and D5-EtG, urine samples were analyzed by direct injection into the gradient LC-MS/MS system. Analysis was performed in accordance with forensic guidelines for confirmatory analysis using one precursor and two product ions. EtS has been detected (in addition to EtG) in the urine samples of nine volunteers after drinking sparkling wine containing between 9 and 49 g of ethanol. Both EtS and EtG could be detected up to 36 h after consumption of alcohol. The excretion profile was found to be similar to that of EtG. No EtS was found in teetotalers’ urine samples. Method validation parameters are presented. EtS was stable in urine upon storage up to twenty days at room temperature. In addition to EtG, EtS can be used to detect recent alcohol consumption, thus providing a second marker for the time range of up to approximately one day after elimination of ethanol from urine samples. The determination of EtS can be used in addition to EtG as proof of ethanol consumption in workplace monitoring programs

Determination of ethyl glucuronide and ethyl sulfate from dried blood spots

Background: Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are non-oxidative minor metabolites of ethanol. They are detectable in various body fluids shortly after initial consumption of ethanol and have a longer detection time frame than the parent compound. They are regarded highly sensitive and specific markers of recent alcohol uptake. This study evaluates the determination of EtG and EtS from dried blood spots (DBS), a simple and cost-effective sampling method that would shorten the time gap between offense and blood sampling and lead to a better reflectance of the actual impairment. Methods: For method validation, EtG and EtS standard and quality control samples were prepared in fresh human heparinized blood and spotted on DBS cards, then extracted and measured by an LC-ESI-MS/MS method. Additionally, 76 heparinized blood samples from traffic offense cases were analyzed for EtG and EtS as whole blood and as DBS specimens. The results from these measurements were then compared by calculating the respective mean values, by a matched-paired t test, by a Wilcoxon test, and by Bland-Altman and Mountain plots. Results and discussion: Calibrations for EtG and EtS in DBS were linear over the studied calibration range. The precision and accuracy of the method met the requirements of the validation guidelines that were employed in the study. The stability of the biomarkers stored as DBS was demonstrated under different storage conditions. The t test showed no significant difference between whole blood and DBS in the determination of EtG and EtS. In addition, the Bland-Altman analysis and Mountain plot confirmed that the concentration differences that were measured in DBS specimens were not relevan

Creatinine in urine - a method comparison.

Drug screening in urine is widely applied in forensic toxicology. Contrary to blood analysis, excessive or reduced fluid intake can substantially alter the concentration of substances in urine. As a standard to detect urinary dilution, creatinine concentrations are analyzed. A sample with a concentration below 20 mg/dL is generally defined as too diluted to provide a valid result in abstinence control samples. This work investigates the potential of three different methods for the determination of creatinine concentrations in urine samples: A ZIC-HILIC based LC-MS/MS method, a spectrophotometric method on an AU 480 chemistry system, and a portable, immunoassay based point-of-care testing device was compared by measuring 200 urine samples. When comparing the two laboratory methods, LC-MS/MS and spectrophotometry, a mean difference of 3.7 ± 14 mg/dL was found, indicating that the spectrophotometric method is slightly overestimating the creatinine concentration. When comparing the LC-MS/MS method with the point-of-care testing device, a mean concentration difference within the calibration range for POCT (>20 mg/dL (excluding 16 samples) and <500 mg/dL (excluding 4 samples)) of 21 ± 37 mg/dL was found, indicating that the point-of-care testing device overestimates the measured creatinine concentration. A point-of-care testing device as used during this study can provide valuable information for on-site analysis. However, reported concentrations above 500 mg/dL should be further evaluated e.g. by dilution of the sample

On-line SPE LC-MS/MS for the quantification of Δ9-tetrahydrocannabinol (THC) and its two major metabolites in human peripheral blood by liquid chromatography tandem mass spectrometry

A universal and robust analytical method for the determination of Δ9-tetrahydrocannabinol (THC) and two of its metabolites Δ9-(11-OH)-tetrahydrocannabinol (11-OH-THC) and 11-nor-Δ9-carboxy-tetrahydrocannabinol (THC-COOH) in human whole blood was developed and validated for use in forensic toxicology. Protein precipitation, integrated solid phase extraction and on-line enrichment followed by high-performance liquid chromatography separation and detection with a triple quadrupole mass spectrometer were combined. The linear ranges used for the three cannabinoids were from 0.5 to 20ng/mL for THC and 11-OH-THC and from 2.5 to 100ng/mL for THC-COOH, therefore covering the requirements for forensic use. Correlation coefficients of 0.9980 or better were achieved for all three analytes. No relevant hydrolysis was observed for THC-COOH glucuronide with this procedure — in contrast to our previous GC-MS procedure, which obviously lead to an artificial increase of the THC-COOH concentration due to the hydrolysis of the glucuronide-conjugate occurring at high pH during the phase-transfer catalyzed methylation ste

Signatures of electron correlations in the transport properties of quantum dots

The transition matrix elements between the correlated $N$ and $N\!+\!1$ electron states of a quantum dot are calculated by numerical diagonalization. They are the central ingredient for the linear and non--linear transport properties which we compute using a rate equation. The experimentally observed variations in the heights of the linear conductance peaks can be explained. The knowledge of the matrix elements as well as the stationary populations of the states allows to assign the features observed in the non--linear transport spectroscopy to certain transition and contains valuable information about the correlated electron states.Comment: 4 pages (revtex,27kB) + 3 figures in one file ziped and uuencoded (postscript,33kB), to appear in Phys.Rev.B as Rapid Communicatio

No blue-yellow color vision impairment after acute ethanol ingestion

Several studies showed that a chronic ethanol exposure can cause color vision deficiencies. There is no agreement about the axis of color defects due to alcohol misuse since changes in the red-green and the blue-yellow axis are described in literature. The acute influence of alcohol on the blue-yellow color vision isn’t studied as well. The aim of this study was to determine the effect of acute alcohol ingestion on the blue-yellow color vision by using short wavelength automated perimetry (SWAP) and anomaloscopy with Moreland equation. This is the first study evaluating that question by using SWAP and anomaloscopy. 16 healthy subjects without history of alcohol-related and ophthalmological problems were examined by SWAP and anomaloscopy (Moreland equation) before and after alcohol ingestion. Mean sensitivity (MS), mean deviation (MD), loss of variance (LV), reliability factor (RF) and duration of examination were assessed for perimetry and match midpoint (MP), matching range (MR) and duration of examination for anomaloscopy. Blood alcohol concentrations (BAC) were determined by gas chromatography and phosphatidylethanol concentrations (marker of an alcohol misuse) by liquid-chromatography tandem-mass spectrometry in venous blood samples from a cubital vein. Mean blood BAC was 0.86 +/- 0.20 g/kg while performing perimetry and 0.84 +/- 0.20 g/kg while performing anomaloscopy. MS, MD, RF, MP, MR and duration of perimetry examination were not altered significantly after alcohol intake. LV showed a significant increase. The duration of anomaloscope testing was shortened significantly under influence of alcohol. The subjects also revealed a significant narrower matching range after alcohol intake. In the range of 0.8 g/kg BAC, no blue-yellow vision deficiencies could be demonstrated. In further studies, the effect of higher BAC on blue-yellow vision should be investigated by different methods

Inhibition of bacterial degradation of EtG by collection as dried urine spots (DUS)

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are direct alcohol consumption markers widely used nowadays for clinical and forensic applications. They are detectable in blood and urine even after consumption of trace amounts of ethanol and for a longer time frame, being detectable even when no more ethanol is present. The instability of EtG against bacterial degradation in contaminated urine samples and/or the possible postcollection synthesis of this metabolite in samples containing, e.g., Escherichia coli and ethanol, may cause false identification of alcohol uptake. Therefore, it is of paramount importance to constrict these error sources by inhibition of any bacterial growth causing hydrolization or synthesis of EtG. This study evaluates a new method of collecting urine samples on filter paper, dried urine spots (DUS), for simultaneous detection of EtG, EtS and creatinine, having the great advantage of inhibiting bacterial activity. In addition, a method validation for the determination of EtG and EtS in DUS was performed according to the FDA guidelines. Sterile-filtered urine was spiked with EtG and EtS, inoculated with E. coli and incubated. Liquid and dried urine samples were collected after various time intervals up to 96h. Liquid samples were frozen immediately after collection, whereas aliquots for DUS were pipetted onto filter paper, allowed to dry and stored at RT until analysis 1week after. The specimens were analyzed by LC-ESI-MS/MS. As expected, degradation of EtG, but not of EtS, was observed in contaminated liquid urine samples. However, the specimens collected on filter paper and stored at RT showed no degradation during storage. Therefore, collecting urine samples on filter paper for EtG and EtS analysis turns out to be a reliable method to avoid bacterial degradation of EtG and EtS, and consequently, stabilization of these ethanol metabolites is achieved. In addition, simultaneous measurement of creatinine content as an indicator of urine dilution helps to interpret the results. Method validation for EtG and EtS in DUS was satisfactory, showing the linearity of the calibration curves in the studied concentration range, good precision, accuracy and selectivit

Behavioural and skill-based early interventions in children with autism spectrum disorders

Introduction: Autism spectrum disorders (ASD) comprise typical or infantile autism (Kanner syndrome), Asperger’s disorder and atypical autism or pervasive developmental disorder - not otherwise specified. The syndrome is characterized by deficits in (1) verbal and nonverbal communication, (2) reciprocal social interaction and (3) repetitive patterns of behaviour, interests and activities. Early behavioural interventions are based on learning theory and behaviour therapy. They take into account specific deficits in perception, emotional reactions, social interaction and communication. In Germany, these comprehensive models are not widely evaluated and implemented. Research questions: * What are the clinical effectiveness and safety of early behavioural or skills-based early interventions in autism compared to other interventions or to treatment as usual? * What are specific factors responsible for the effectiveness? * What are the cost-effectiveness and cost consequences of different early interventions in autism? * Which legal, social and ethical aspects are relevant with regard to the implementation of the respective interventions in persons with autism? Methods: Following a systematic review of the literature, controlled studies on early behavioural or skills-based interventions published since 2000 in English or German with children until the age of twelve are included and critically appraised. Studies must have at least ten participants per intervention group. Results: In total, 15 publications based on 14 studies, eight systematic reviews and one health economic study are included. Most studies evaluate early interventions based upon the Lovaas model (Early intensive behavioural treatment (EIBT), Applied behavioural analysis (ABA)). Other evaluate pragmatic interventions or interventions based on other theoretical models like specific parent interventions, responsive education and prelinguistic milieu teaching, joint attention, symbolic play, and picture exchange communication system. Behaviour analytic interventions referring to the Lovaas model remain the most empirically evaluated early interventions in autism. Preschool children with autism can achieve improvements in cognitive and functional domains when treated within behavioural interventions with a frequency of at least 20 hours per week. It is not clear which is the minimum duration of effective interventions, and which active components are necessary for the effectiveness. There was no high quality evidence for other comprehensive early interventions. The identified health economic study is not suitable to evaluate the cost-effectiveness or cost consequences of early interventions. No publications concerning legal, ethical or social aspects were identified. The financial situation of persons with autisms and their families will be improved through the implementation of the “Pflege-Weiterententwicklungsgesetz” (Pf-WG). Further questions concern the organisation of care and the legal representation of autistic patients. Ethical questions arise mainly in the context of the equal supply of care to each individual patient in all regions of the country and the situation of the caregivers. Discussion: There are only a few studies with high methodology evaluating early interventions in children with autism. Most studies have a short duration with a lack of blinded outcome assessment in many cases. The lack of high quality comparative studies does not allow answering questions of comparative effectiveness of early interventions in autism. It can be concluded that interventions referring to the Lovaas model seem to have the highest effectiveness. This seems to be especially true when they are run clinic-based. However, there was no solid evidence with regard to factors responsible for the effectiveness of programms according to the ABA model. With regard to communication improvement, a systematic parent training seems to be superior to treatment as usual where a mixture of therapeutic elements is used. As well for clinical and health economic studies there is a substantial problem of generalisability into the German context. The identified health economic study is not suitable to evaluate the cost-effectiveness or cost consequences of early interventions. Conclusion: Based on the available studies, there is no sufficient evidence for any of the evaluated behavioural early intervention programmes. Studies suggest that preschool children with autism in behavioural intervention programmes with a frequency of at least 20 hours per week can achieve improvements in cognitive and functional domains. There is no evidence that in a substantial portion of the children a normal development can be achieved by early interventions. Most research evidence is available for ABA. A minimal necessary intensity of interventions to achieve positive outcomes cannot be derived from literature. There are no valid statements possible as to cost-effectiveness or consequences of these interventions. Effective early interventions may reduce total autism costs in the long run. This may be achieved when the initial high treatment expenditures are more than compensated later if persons with this disorder have better social functioning