[Direct metabolites of ethanol as biological markers of alcohol use: basic aspects and applications]. / Direkte ethanolmetaboliten als biomarker für alkoholkonsum: grundlagen und anwendungen.

In addition to self reports and questionnaires, biomarkers are of relevance in the diagnosis of and therapy for alcohol use disorders. Traditional biomarkers such as gamma-glutamyl transpeptidase or mean corpuscular volume are indirect biomarkers and are subject to the influence of age, gender and non-alcohol related diseases, among others. Direct metabolites of ethanol such as ethyl glucuronide (EtG), ethyl sulphate (EtS) and phosphatidylethanol (PEth) are direct metabolites of ethanol, that are positive after intake of ethyl alcohol. They represent useful diagnostic tools for identifying alcohol use even more accurately than traditional biomarkers. Each of these drinking indicators remains positive in serum and urine for a characteristic time spectrum after the cessation of ethanol intake - EtG and EtS in urine up to 7 days, EtG in hair for months after ethanol has left the body. Applications include clinical routine use, emergency room settings, proof of abstinence in alcohol rehabilitation programmes, driving under influence offenders, workplace testing, assessment of alcohol intake in the context of liver transplantation and foetal alcohol syndrome. Due to their properties, they open up new perspectives for prevention, interdisciplinary cooperation, diagnosis of and therapy for alcohol-related problems.

Comparison of direct and indirect alcohol markers with PEth in blood and urine in alcohol dependent inpatients during detoxication.

The importance of direct and indirect alcohol markers to evaluate alcohol consumption in clinical and forensic settings is increasingly recognized. While some markers are used to prove abstinence from ethanol, other markers are suitable for detection of alcohol misuse. Phosphatidyl ethanol (PEth) is ranked among the latter. There is only little information about the correlation between PEth and other currently used markers (ethyl glucuronide, ethyl sulfate, carbohydrate deficient transferrin, gamma-glutamyl transpeptidase, and methanol) and about their decline during detoxification. To get more information, 18 alcohol-dependent patients in withdrawal therapy were monitored for these parameters in blood and urine for up to 19 days. There was no correlation between the different markers. PEth showed a rapid decrease at the beginning of the intervention, a slow decline after the first few days, and could still be detected after 19 days of abstinence from ethanol.

Detection of ethyl glucuronide in blood spotted on different surfaces.

This study aims to show that sensitive detection of ethyl glucuronide in dried blood spotted onto various surfaces after a period of 24h is feasible. At present, there is insufficient information how tightly ethyl glucuronide (EtG) binds to various materials and how easily it can be eluted. 4ml aliquots of blood samples obtained from seven volunteers after consumption of alcoholic beverages were applied to six different surfaces. After drying and a 24h-storage at 20±2°C the samples were re-dissolved in water, and EtG was subsequently analyzed by a LC-MS Paul-type ion trap. A comparison was made between dried and corresponding fluid samples. EtG was detectable in all subjects' samples following consumption of alcohol. EtG was also detectable after a storage time of four weeks at 4°C in whole blood that had been preserved with EDTA. EtG was detectable in all samples dried on different surfaces and its concentration remained relatively constant irrespective of the particular condition of the material. Detection of EtG in blood spots from the scene may indicate recent alcohol consumption in cases where collection of blood remained undone or could not be performed.

Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS.

Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues--improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid-liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm x 2 mm, 3 microm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).

Detection and identification of 700 drugs by multi-target screening with a 3200 Q TRAP LC-MS/MS system and library searching.

The multi-target screening method described in this work allows the simultaneous detection and identification of 700 drugs and metabolites in biological fluids using a hybrid triple-quadrupole linear ion trap mass spectrometer in a single analytical run. After standardization of the method, the retention times of 700 compounds were determined and transitions for each compound were selected by a "scheduled" survey MRM scan, followed by an information-dependent acquisition using the sensitive enhanced product ion scan of a Q TRAP hybrid instrument. The identification of the compounds in the samples analyzed was accomplished by searching the tandem mass spectrometry (MS/MS) spectra against the library we developed, which contains electrospray ionization-MS/MS spectra of over 1,250 compounds. The multi-target screening method together with the library was included in a software program for routine screening and quantitation to achieve automated acquisition and library searching. With the help of this software application, the time for evaluation and interpretation of the results could be drastically reduced. This new multi-target screening method has been successfully applied for the analysis of postmortem and traffic offense samples as well as proficiency testing, and complements screening with immunoassays, gas chromatography-mass spectrometry, and liquid chromatography-diode-array detection. Other possible applications are analysis in clinical toxicology (for intoxication cases), in psychiatry (antidepressants and other psychoactive drugs), and in forensic toxicology (drugs and driving, workplace drug testing, oral fluid analysis, drug-facilitated sexual assault).

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC-ESI-MS/MS.

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)--a biomarker for ethanol uptake--has been developed by LC-ESI-MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents-PEth 18:1/18:1, PEth 16:0/16:0-were determined in post-mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g-dose of ethanol. Furthermore, PEth 18:1/16:0 or its isobaric isomer PEth-16:0/18:1 was detected. In comparison to previous high-performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC-MS/MS-method is more sensitive--with a limit of detection below 20 ng/ml--and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC-MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC-MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate.

Suicidal poisoning with mercaptodimethur-morphological findings and toxicological analysis.

In the western countries, the number of fatal intoxications with plant protecting agents has decreased to some extent due to laws restricting the use of highly toxic pesticides like halogenated hydrocarbons. Nevertheless, in consideration of the easy availability of most plant protectants, the small fraction of such fatalities among suicides and intoxications is astonishing. An 80-year-old woman died of an intoxication with methiocarb (mercaptodimethur), a carbamate type pesticide and as such a reversible inhibitor of the acetylcholinesterase. The case is presented because it is the first explicit report on a fatal poisoning of a human with methiocarb. The methiocarb concentrations detected were 6,100 microg/g in stomach content, 4.0 microg/ml in heart blood, 11 microg/g in kidney, 1.9 microg/ml in urine, 25 microg/g in liver, 2 microg/g in bile and 2.5 microg/g in brain tissue.

Toxicological analysis after asphyxial suicide with helium and a plastic bag.

A 23-year-old man was found on a raised hide in lying position, the head wrapped in a plastic bag connected with a helium gas cylinder by a polypropylene tube. The autopsy did not show any specific findings nor did the routine toxicological analysis reveal significant information regarding the cause of death (BAC 0.9 mg/g, diphenhydramine 0.81 microg/ml in heart serum). For the detection of helium in the lungs, gas samples from both lungs were collected by a method ensuring minimal dilution. Gas analyses were performed using a GC-MS with a split-splitless injector and a headspace syringe. As carrier gas the commonly used helium was replaced by nitrogen. Helium was found in clearly elevated concentrations in gas samples from both lungs. Therefore, suffocation by breathing helium enriched, and thus oxygen deficient atmosphere, can strongly be assumed as the cause of death.

Prevalence of gamma-hydroxybutyrate (GHB) in serum samples of amphetamine, metamphetamine and ecstasy impaired drivers.

Two hundred and forty-seven serum samples which have been collected by police during roadside testing and have been found positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDE) were analyzed for gamma-hydroxybutyrate (GHB). Serum samples were spiked with deuterated GHB as internal standard and acetonitrile was added to achieve dilution and protein precipitation. Samples were analyzed with a LC-MS/MS system operated in the multiple reaction monitoring mode (MRM) using a TurboIonSpray source. Chromatographic separation was achieved using a Synergi Polar RP column applying a gradient elution with a runtime of 15 min. To differentiate between endogenous and exogenously administered GHB a cut-off concentration of 10 microg/mL was applied. Five samples exceeded this concentration and were found positive for GHB. These samples were only found positive for amphetamine but no other amphetamine derivatives were detected, while in three samples THC and in one sample cocaine, benzoylecgonine and ethanol were found.

A case of Munchausen syndrome by proxy with subsequent suicide of the mother.

Munchausen syndrome by proxy is a subtle and difficult to diagnose form of child abuse in which the carer (usually the mother) simulates, manipulates or produces symptoms of illness in the victim. In most cases the detrimental effect is caused by applying foreign substances or by airway obstruction. In the presented case a 20-month-old girl developed a spreading soft-tissue infection resistant to treatment on the left upper arm after vaccination, which required a number of surgical interventions. Repeatedly, microorganisms from the intestinal flora were isolated from the wound secretion. After the girl suffered respiratory and circulatory arrest, which required resuscitation measures, chemical toxicological tests revealed not medically prescribed benzodiazepines in serum and urine. When the mother, a trained nurse, was confronted with the allegation to have manipulated the symptoms of the illness she committed suicide. The forensic autopsy of the suicide produced numerous hints suggesting chronic self-damaging behaviour described as Munchausen syndrome. This case shows a number of manipulation forms with the maintenance of a chronic skin and soft tissue infection belonging to the rarer forms of inflicting damage to the child. It also illustrates that confrontation with the allegation of Munchausen syndrome by proxy creates a very stressful emotional situation that may lead to a suicidal act.

Development of a multi-target screening analysis for 301 drugs using a QTrap liquid chromatography/tandem mass spectrometry system and automated library searching.

A new multi-target screening (MTS) procedure for drugs in blood and urine for toxicological analysis has been developed using a hybrid triple-quadrupole linear ion trap mass spectrometer (QTrap) for the fast detection and identification of 301 forensically important drugs, e.g. tranquilizers (benzodiazepines), hypnotics, drugs of abuse (opiates, cocaine, amphetamines, cannabinoids), antidepressants, neuroleptics, and some cardiac drugs, in one single liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Samples were extracted either with liquid-liquid extraction or solid-phase extraction. A multiple reaction monitoring (MRM) as survey scan and an enhanced product ion (EPI) scan as dependent scan were performed in an information-dependent acquisition (IDA) experiment. Finally, drug identification was carried out by library search with a newly developed MS/MS library based on EPI spectra at three different collision energies in positive mode. The advantage of this newly developed method is the possibility to detect and identify 301 drugs in one single LC/MS/MS run.

[gamma-Hydroxybutyrate intoxication]. / Intoxikation mit gamma-Hydroxybutyrat.

The drug gamma-Hydroxybutyrate (GHB), also known as liquid ecstasy, has now reached Europe. Estimating the dosage of liquid GHB is especially difficult leading to unintentional intoxication because the exact concentration is not known. We repeatedly had to treat young patients intoxicated by GHB in our intensive care unit. We describe the course and treatment of three patients with GHB intoxication. If alcohol or mixed intoxication with drugs detected in traditional hospital toxicological assays has been excluded as the cause of unconsciousness in young patients from disco's, an intoxication with GHB should be considered. The therapy is mainly symptomatic and supportive but monitoring in an intensive care unit with the option of short term respirator therapy is necessary. Serum and urine samples taken on arrival should be conserved for further investigation in a forensic institute.

The induction of gene mutations and micronuclei by oxiranes and siloranes in mammalian cells in vitro.

Oxiranes and siloranes are candidate molecules for the development of composite materials with low shrinkage. Since some of these molecules are highly reactive, they could lead to adverse biological effects from underlying genetic mechanisms. Therefore, we analyzed the formation of micronuclei (chromosomal aberrations) and the induction of gene mutations (HPRT assay) in mammalian cells. The numbers of micronuclei induced by the oxirane di(cyclohexene-epoxidemethyl)ether (Eth-Ep) at low concentrations (10 micro M) were about five-fold higher than controls. The related compound epoxy cyclohexyl methyl-epoxy cyclo-hexane carboxylate (Est-Ep) was less effective. The activity of diglycidylether of bisphenol A (BADGE) was even lower but similar to the most reactive silorane, di-3,4-epoxy cyclohexylmethyl-dimethyl-silane (DiMe-Sil). No induction of micronuclei was detected in the presence of a rat liver homogenate (S9). Est-Ep and Eth-Ep also induced gene mutations. Our analyses indicated low mutagenic potentials of siloranes; however, some oxiranes induced strong effects at two genetic endpoints.

Kinetics of kavain and its metabolites after oral application.

Kavain metabolism in humans was the target of this current investigation. In the present study a high-performance liquid chromatographic (HPLC-DAD) assay method for the simultaneous determination of kavain and its main metabolites (p-hydroxykavain, p-hydroxy-5,6-dehydrokavain and p-hydroxy-7,8-dihydrokavain) in serum and urine was developed and validated. The metabolites were mainly excreted in the form of their conjugates. All kavain metabolites were detectable in serum and urine, except for p-hydroxy-7,8-dihydrokavain, which was found in urine only. Confirmation of the results and identification of the metabolites were performed by LC-MS or LC-MS-MS. Kinetics of kavain and its metabolites in serum were investigated after administration of a single oral dose (800 mg kavain). Within 1 and 4 h after uptake, the serum concentrations ranged between 40 and 10 ng/ml for kavain, 300 and 125 ng/ml for p-hydroxykavain, 90 and 40 ng/ml for o-desmethyl-hydroxy-5,6-dehydrokavain, and 50 and 30 ng/ml for 5,6-dehydrokavain.

Tuning compounds for electrospray ionization/in-source collision-induced dissociation and mass spectra library searching.

Tuning compounds for positive and negative electrospray ionization (ESI) were tested for the tuning of in-source collision-induced dissociation (ESI/CID) with three types of SCIEX API instruments (API 365, 2000 and 3000) in the single-quadrupole mode. The vacuum interfaces of these instruments differ slightly in geometry, but the principles of ionization and solvent evaporation by nebulizer and curtain gases, orifice and skimmer are identical. For comparison of in-source CID, breakdown curves of haloperidol, paracetamol, metronidazole and metamizole were acquired by increasing the orifice voltages. The API 2000 and 3000 required higher orifice voltages than did the API 365 to induce a similar degree of fragmentation of the protonated or deprotonated molecules to characteristic fragment ions. This increase of orifice voltage could be demonstrated with each of the four compounds tested by a shift of the maxima of the breakdown curves to higher orifice voltages. A procedure with three collision energy (CE) levels for drug identification with a mass spectra library set up with an API 365 therefore required an adjustment of the orifice voltages to higher values when being transferred to an API 2000 or API 3000. The corresponding orifice voltages for the three instruments were 20/50/80 V (API 365), 30/90/130 V (API 2000) and 40/80/120 V (API 3000). However, a change in orifice voltage of +/-10 V (with the API 2000 and 3000) hardly influenced the fit values of a library search for each single CE level. For adjusting orifice voltages with different instruments, a tuning procedure with haloperidol and paracetamol is presented. With this tuning procedure an ESI/CID mass spectra library set up for API 365 and API 150 could also be used for drug identification with an API 2000 and an API 3000 with good library search results.

Tune compounds for electrospray ionisation/in-source collision-induced dissociation with mass spectral library searching.

Haloperidol, paracetamol, metronidazole and metamizole have been tested as tune compounds for electrospray ionisation in-source collision-induced dissociation MS (ESI-CID-MS) with two different mass spectrometers (Sciex API 365 and Agilent 1100 MSD SL). The different electrospray sources of API 365 and MSD 1100 SL consist of an orifice with nitrogen curtain gas and a capillary interface, respectively. In-source CID occurs in both interfaces in front of the skimmers, which separate a region with a vacuum of approximately 300 Pa and the high vacuum (<10(-3) Pa). Comparison of the breakdown curves of selected tune compounds, depending on collision energy (orifice or fragmentor voltage), showed, that very similar fragmentation can be obtained with both instruments, when adjusting the fragmentor voltage of the MSD 1100 SL to higher values than the orifice voltage of the API 365. For three energy levels--low, medium and high--the corresponding voltages were 20, 50 and 80 V for the API 365 and 110, 190, 230 V for the MSD 1100 SL. These voltages resulted in the most similar spectra for haloperidol and paracetamol with both instruments. The comparison of ESI-CID-MS of all tune compounds at three energy levels showed, that - despite variations in relative ion abundances - all significant ions were present in one of the three CID spectra. Therefore, mass spectral library searching of an ESI-CID-MS library set-up with one of the two instruments should be possible with the other instrument after adjusting the CID energies by means of at least two tune compounds such as haloperidol and paracetamol, metronidazole or metamizole.

Fast confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine by LC/MS/MS using negative atmospheric-pressure chemical ionisation (APCI).

A fast method using automated solid-phase extraction (SPE) and short-column liquid-chromatography coupled to tandem mass-spectrometry (LC/MS/MS) with negative atmospheric-pressure chemical ionisation (APCI) has been developed for the confirmation of 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH) in urine samples. This highly specific method which combines chromatographic separation and MS/MS-analysis can be used for the confirmation of positive immunoassay results with a NIDA cut-off of 15ng/ml. The conjugates of THC-COOH were hydrolysed prior to SPE, and a standard SPE was performed using C18-SPE columns. No derivatisation of the extracts was needed as in GC/MS analysis, and the LC run-time was 6.5min by gradient elution with a retention time of 2.4min. Linearity of calibration was obtained in the range between 0 and 500ng/ml (correlation coefficient R(2)=0.998). Using linear regression (0-50ng/ml) the limit of detection (LOD) was 2.0ng/ml and the limit of quantitation (LOQ) was 5.1ng/ml; day-to-day reproducibility and precision were tested at 15 and 250ng/ml and were 13.4ng/ml+/-3.3% and 255.8ng/ml+/-4.5%, respectively.

Prevalence of nicotine consumption in drug deaths.

One hundred consecutive drug death victims autopsied at the Institute of Forensic Medicine, University of Freiburg, between 1995 and 1997 were studied retrospectively as to whether the drug users had also consumed nicotine. The study included histological examination of the lung tissue for smoker cells and radioimmunological as well as GC-MS assays of the urine for cotinine, the primary metabolite of nicotine. It was found that 98 out of 100 drug victims had consumed nicotine in addition to illicit drugs or replacements. Yellowish-brown discolorations on the middle and index fingers were discernible in 44 drug victims, whereas fresh or scarred burns due to glowing cigarettes were found in six deceased drug consumers. Diseases of the bronchial system typical of heavy smokers were seen in 35 cases. Siderophages could be demonstrated in 17 of the 100 drug deaths.

Post-mortem detection and identification of sildenafil (Viagra) and its metabolites by LC/MS and LC/MS/MS.

The highly putrefied corpse of an 80-year-old man was found in the apartment which he had rented to a prostitute. A package of Viagra 25 was found beside the corpse and three tablets were missing. Autopsy revealed severe coronary artery sclerosis as well as signs of previous myocardial infarctions. For the detection and identification of sildenafil and three metabolites in urine and tissue samples, solid-phase extraction, LC/MS and MS/MS methods were developed. Blood was not available for toxicological analysis due to the putrefaction. For method development, urine from a volunteer who had ingested 25 mg sildenafil was collected over 8 h, and three metabolites were identified by MS/MS. These metabolites were also found in the victim's urine. These findings prove that sildenafil was taken some time prior to death, but the causality of sildenafil intake and fatal cardiac failure could not be proven, since no blood was available for analysis. However, the administration of sildenafil was contraindicated due to several previous myocardial infarctions.