F. Musshoff

A High-Performance Liquid Chromatographic–Tandem Mass Spectrometric Method for the Determination of Ethyl Glucuronide and Ethyl Sulfate in Urine Validated According to Forensic Guidelines

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are powerful markers for alcohol intake and abuse. Several analytical procedures for the quantification of EtG and EtG in serum and urine have been developed so far. Many of the published methods show limits of detections (LODs) or limits of quantifications (LOQs) for EtG in urine within the range of 0.1 mg/L or higher. Since this is the actual cutoff value for proving abstinence in Germany, problems may occur if urine samples are highly diluted. In this paper, the validation of a highly sensitive, fast and simple LC-MS-MS for the determination of EtG and EtS in urine is described. The calibration curves for EtG and EtS is linear over the whole range (0.025-2.0 mg/L). Very low detection limits can be achieved (LOD: EtG 0.005 mg/L, EtS 0.005 mg/L; and LOQ: EtG 0.019 mg/L, EtS 0.015 mg/L). All data for selectivity, precision and accuracy, recovery, as well as for the processed sample and the freeze/thaw stability, comply with the guidelines of the German Society of Toxicological and Forensic Chemistry. Strong matrix-related effects can be compensated for by using an internal standard. Finally, the applicability of the procedure is proven by analysis of 87 human urine samples and by successful participation in interlaboratory comparison tests.

Cocaine and benzoylecgonine concentrations in fluorinated plasma samples of drivers under suspicion of driving under influence

Due to an in vitro decomposition of cocaine (COC), usually an analytical detection in unstabilized blood taking systems is impossible and for this reason the main metabolite benzoylecgonine (BZE) is determined. In a few regions in Germany the local authorities use systems containing sodium fluoride for taking a blood sample. Caused by inhibition of esterases in the taken sample COC is still detectable in blood samples. Cases of driving under the influence (DUI) with positive findings for COC and/or BZE were evaluated and substance concentrations in plasma were correlated with documented deficiencies in the psycho-physical performance. In 51.5% of all cases positive for cocaine-like substances besides BZE a positive result was also given for COC. If both substances were found (mean COC concentration 0.836 mg/L) the concentration of BZE was significantly higher (mean 0.669 mg/L) compared to cases with a single detection of BE (mean 0.209 mg/L) (p=0.001). In 72 cases without any detection of further drugs cocaine users seemed rather excited and stimulated towards intervening police officers, in particular when COC was present in the blood (17.8%). Also in the medical investigation reports a rather stimulative effect (25% vs. 3.6% sedated) was to be registered. Whereas with a sole determination of BZE, a stimulated (19%) as well as a sedated impression (14.9%) was described. Definite concentration-effect relations could not be recognized. Indeed, more peculiarities were to be registered with the simultaneous detection of COC than with a sole BZE determination. The determination of COC and the differences in the BZE concentration can be explained by the fact that the simultaneous detection of both substances is indicative for of a consumption shortly before the blood sampling. A sole detection of BZE is more likely indicative for a consumption already some time ago. Therefore, in the first case one would rather suggest an acute intoxication phase. A determination of BZE without COC is more likely indicative for a transition to the drug-induced exhaustion phase which is also to be expected after the consumption of COC. The absence of COC can be seen within the scope of a validity check as an indication of a possible exhaustion reaction. A use of fluoride stabilized blood sampling systems is advised. This makes it easier to investigate the state of intoxication or to appraise the temporal connection between COC consumption and incident or blood sampling.

Determination of hypoglycaemia induced by insulin or its synthetic analogues post mortem

The determination of human insulin or its synthetic analogues in post-mortem specimens represents a challenge for forensic toxicologists due to its proven instability in post-mortem blood. We present two cases of an insulin-induced hypoglycaemia. In the first case, ante-mortem material was available for the detection of an injection with human insulin. Human insulin was detected by immunopurification with magnetic beads and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses at a concentration of 5180 μU/ml. The molar ratio human insulin:C-peptide was 111. The second case describes a suicide by self-injection of Insulin lispro and determination of the drug after pre-extraction with methanol and immunopurification by LC-MS/MS at the injection site, in vitreous humour and organs. Apart from the well-known matrices--femoral blood and urine--the specimen vitreous humour and the injection site promise the best possibilities for a proof of insulin at autopsy. In addition to insulin analyses, the parameters C-peptide, proinsulin, glucose, lactate, and sulfonylureas should be measured in case of suspected fatal hypoglycaemia.

Estimation of gamma-hydroxybutyrate (GHB) co-consumption in serum samples of drivers positive for amphetamine or ecstasy.

There is no toxicological analysis of gamma-hydroxybutyrate (GHB) applied routinely in cases of driving under influence (DUI); therefore the extent of consumption of this drug might be underestimated. Its consumption is described as occurring often concurrently with amphetamine or ecstasy. This study examines 196 serum samples which were collected by police during road side testing for GHB. The samples subject to this study have already been found to be positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDEA). Analysis has been performed by LC/MS/MS in the multiple reaction monitoring (MRM) mode. Due to its polarity, chromatographic separation of GHB was achieved by a HILIC column. To differentiate endogenous and exogenous levels of GHB, a cut-off concentration of 4μg/ml was applied. Of the 196 samples, two have been found to be positive for GHB. Of these samples, one sample was also positive for amphetamine and one for MDMA. Whilst other amphetamine derivates were not detected in these samples, both samples were found to be positive for cannabinoids. These results suggest that co-consumption of GHB with amphetamine or ecstasy is relatively low (1%) for the collective of this study.

A SPME-GC/MS Procedure for the Determination of Fatty Acid Ethyl Esters in Hair for Confirmation of Abstinence Test Results

Fatty acid ethyl esters (FAEE), direct metabolites of ethanol, are suitable alcohol markers that can be detected in different tissues. The determination of FAEE in hair can help to evaluate social and excessive alcohol consumption. Due to the presence of FAEE in the hair of teetotalers, proving alcohol abstinence seems to be impossible. To verify these results, an solid phase micro extraction-gas chromatography/mass spectrometry procedure for the determination of the four FAEE: ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate in hair was validated with special focus on low concentration levels. Besides very high sensitivity (limits of detection between 0.005 and 0.009 ng/mg), good results for linearity, precision and accuracy, recovery and stability were achieved. In addition, 73 hair samples with measured ethyl glucuronide (EtG) concentrations between 4 and 10 pg/mg were analyzed for FAEE. By using the following cut-offs: EtG: 7 pg/mg, FAEE: 0.2 ng/mg a satisfying matching rate of 72.6% was found. This shows that FAEE can be determined to verify borderline EtG concentrations even in the context of abstinence tests. However, the diversified influencing factors on analyte concentrations in hair, which may explain the large deviations between EtG and FAEE results observed in some cases, have to be mentioned when interpret ambiguous results.

Determination of glycyrrhetic acid after consumption of liquorice and application to a fatality

Besides alcohol and drugs of abuse, several popular foods contain potentially toxic substances and cases of intoxication after consumption of these foods attract notice of forensic toxicology. This is also true for the case of a 34-year-old woman who was suspected to have suffered lethal acute intoxication from eating nothing but liquorice over a period of several months. The liquorice ingredient glycyrrhizin and its metabolite glycyrrhetic acid, which elicits a mineralocorticoid effect, were determined in the sort of liquorice the woman had consumed by using LC-MS/MS. In addition, a fast and sensitive procedure for the quantification of glycyrrhetic acid including a simple sample preparation was developed. The method was proven to be accurate and precise. In a liquorice ingestion experiment, 200 g of liquorice had to be eaten. Afterwards, concentrations of glycyrrhetic acid in the blood of up to 434 ng/ml were measured. Since only traces of glycyrrhetic acid had been found in the blood and stomach content of the deceased woman, the possibility of acute lethal glycyrrhetic acid intoxication could be eliminated. Excluding other causes of death, the woman is believed to have died from a lethal hyperglycemic coma. Nonetheless, the influence of harmful and toxic substances in food should be taken into consideration in special cases.

Klinisch-toxikologische Untersuchungen in forensischen Laboratorien

ZusammenfassungAn einigen medizinischen Fakultäten werden aufgrund der apparativen Ausstattung und Expertise forensisch-toxikologische Laboratorien der Institute für Rechtsmedizin auch in die klinisch-toxikologische Analytik eingebunden, insbesondere bei Fragestellungen, die über automatisierte Laborparameterbestimmungen hinausgehen. Ein Situationsbericht aus dem Institut für Rechtsmedizin der Universität Bonn untermauert die Bedeutung solcher klinisch-toxikologischer Analysen. Als Fazit kann festgehalten werden, dass klinisch-toxikologische Untersuchungen durch Institute für Rechtsmedizin nicht nur wichtige Dienstleistungen für das eigene Universitätsklinikum darstellen, sie dienen darüber hinaus der fachlichen und wissenschaftlichen Vernetzung.AbstractIn some medical faculties institutes of Forensic Medicine also carry out clinical toxicological investigations due to their expertise in forensic toxicology and the methods available, especially in cases where specific chromatographic methods are necessary. Experiences with clinical toxicological investigations for our own university hospital and surrounding hospitals will be reported. Clinical toxicology is not only a service for the hospital but promotes scientific and routine cooperation.

Positive Befunde in Haaren und Knochenmark

ZusammenfasungNach 7½-jähriger Lagerung in einem Erdgrab wurden forensisch-toxikologische Analysen an verschiedenen Proben vorgenommen, die bei einer Sektion des teilskelettierten Körpers gewonnen worden waren. Für Pipamperon und Haloperidol wurden in Restgeweben positive Befunde im Spurenbereich ermittelt; die Medikation mit beiden Arzneimittelwirkstoffen war bekannt. In Kopf- und Schamhaaren sowie Knochenmark wurden die Substanzen in höheren Konzentrationen und mit Signalrauschverhältnissen aufgefunden, die eine sichere Identifizierung und Quantifizierung erlaubten. Haare und Knochenmark können als geeignete Matrizes für Post-mortem-Analysen angesehen werden, selbst nach jahrelanger Lagerung in einem Erdgrab. Auch in solchen Fällen kann die forensisch-toxikologische Untersuchung ggf. wichtige Informationen liefern.AbstractAfter 7½ years in an earth grave a forensic toxicological analysis was performed on several samples obtained at autopsy of a partly skeletonized body. Positive findings at trace levels were achieved for pipamperon and haloperidol in residual tissue. Medication with both drugs was known. In head hair, pubic hair and bone marrow the drugs were found in higher concentrations and with signal-to-noise ratios allowing reliable identification and quantification. Hair and bone marrow have to be considered as useful matrices for post-mortem analyses even after many years in an earth grave. Even in such cases a forensic toxicological analysis can lead to important information.