Post-mortem evidence of doxylamine in toxicological analyses.

BACKGROUND: Doxylamine (DA) is widely available in pharmacies without prescription and can be used in suicidal intention because of its sedative and anticholinergic properties. Research of literature shows that only a few publications deal with post-mortem evidence of DA and its interpretation during toxicological examination. MATERIAL AND METHODS: In this study, all cases with a positive detection of DA during toxicological analyses with high-performance liquid chromatography in the time period 2000 to 2010 at the Institute of Legal Medicine and Forensic Sciences in Berlin, Germany were retrospectively analysed and interpreted, taking into account police investigations, autopsy results and toxicological analyses. RESULTS: In total, 22 cases with DA intoxications were discovered (♂=16/♀=6, age-at-death range 17 to 90years). Maximum blood concentration was measured at 77.5µg/mL. Cause of death was due to DA intoxication in eight suicide cases; seven of those were combined intoxications (DA and other substances, particularly diphenhydramine). During the evaluated time period no monointoxications with DA were discovered. CONCLUSION: Benchmarks published in past literature are meant as orientation during evaluation of post-mortem DA evidence. These should not be used as absolute values and need to be interpreted individually in each case. Post-mortem redistribution needs to be considered as a main factor in alteration of DA concentration measurement. Furthermore, proof of DA ingestion found in gastric content should only be interpreted quantitatively due to unreliable calculation of the ingested amount. In conclusion, a variety of factors, such as the time period between time of death and the time of the first toxicological analysis, the condition of the body and the findings at autopsy, must also be critically considered.

Hair analysis in the detection of long-term use of non-steroidal anti-inflammatory drugs and its relation to gastrointestinal hemorrhage: an examination of 268 hair and blood samples from autopsy cases.

Non-steroidal anti-inflammatory drugs (NSAIDs) are commonly used as analgesics and antipyretics in Western countries. Gastrointestinal (GI) disorders are common side effects of NSAIDs and other drugs. This study investigated the correlation between chronic use of these substances and GI lesions by analyzing postmortem blood and hair samples from autopsy cases. This study included 268 hair and blood samples from autopsy cases. Deceased individuals with GI lesions were selected for the case group (n = 132) and those without any GI lesions were placed in the control group (n = 136). Collection of the samples took place from 2008 until 2010 at the Institute of Legal Medicine and Forensic Sciences, Charité-Universitätsmedizin Berlin, Germany. HPLC-DAD was used to analyze the blood samples while hair samples were analyzed using LC-quadrupole-time-of-flight-MS. The proximal 0-6 cm hair segment was analyzed. The full length of shorter hair samples was analyzed when longer segments were unavailable. Method validation was performed according to the guidelines of the German Society of Toxicological and Forensic Chemistry (GTFCh). Twenty-three per cent of the case group blood samples included one or more NSAIDs while 19 % of the control group blood samples included one or more NSAIDs. In contrast, the hair analysis results demonstrated that samples from the control and case group differed significantly; 67 % of the case group tested positive for one or more NSAIDs while 38 % of the control group tested positive for one or more NSAIDs. Hair analysis results provided a strong indication of a relationship between frequent NSAID consumption and GI lesions.

Methadone and illegal drugs in hair from children with parents in maintenance treatment or suspected for drug abuse in a German community.

BACKGROUND: Children living in homes with drug-addicted parents are in a steady danger of poisoning and may suffer from neglect, maltreatment, and lagging behind in development. Hair analysis could be a suitable way to examine this endangering exposure to drugs. METHODS: Hair samples from 149 children (aged 1-14 years) living with parents substituted by methadone and/or suspected for abuse of illegal drugs, and from 124 of the parents in a German community were investigated by liquid chromatography-hybrid quadrupole time-of flight mass spectrometry and by headspace solid phase microextraction gas chromatography-mass spectrometry for methadone, heroin, cocaine, amphetamines, ecstasy, cannabinoids and benzodiazepines and their metabolites or degradation products (32 compounds). RESULTS: From the children's hair, only in 35 samples, no drugs were detected. Cannabinoids were found in 56 samples, in 20 of them as the only drug. In the remaining 95 samples, methadone was identified 35 times with additional use of illegal drugs in 28 cases. Drug use in the children's environment was obvious for heroin in 44 cases, cocaine in 73 cases, amphetamine or ecstasy in 6 cases, and diazepam in 8 cases. The concentrations varied from limit of quantification to 2.16 ng/mg of methadone, 11.1 ng/mg of 6-acetylmorphine, 17.8 ng/mg of cocaine, 3.29 ng/mg of amphetamine, and 0.72 ng/mg of Δ-tetrahydrocannabinol. In general, hair from younger children contained higher concentrations than from their elder siblings. Systemic incorporation of methadone, cocaine, or cannabinoids appeared likely from detection of the nonhydrolytic metabolites 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine in 11 cases, norcocaine in 16 cases, and 11-nor-9-carboxy-Δ-tetrahydrocannabinol in 9 cases. Within the families, hair samples of children and parents provided often the same drug pattern. External deposition from smoke and by contact with contaminated surfaces or parent's hands and systemic deposition after passive smoking, administration, or oral intake by hand-to-mouth transfer were discussed as alternative incorporation mechanisms into hair. CONCLUSIONS: Altogether, investigation of children's hair proved to be a useful way to detect endangering drug use in their environment and lead to a more thorough inspection and measures to improve their situation in many of the cases.

Fatty acid ethyl esters in hair as alcohol markers: estimating a reliable cut-off point by evaluation of 1,057 autopsy cases.

Alcohol abuse is a widespread problem, especially in Western countries. Therefore, it is important to have markers of alcohol consumption with validated cut-off points. For many years research has focused on analysis of hair for alcohol markers, but data on the performance and reliability of cut-off values are still lacking. Evaluating 1,057 cases from 2005 to 2011, included a large sample group for the estimation of an applicable cut-off value when compared to earlier studies on fatty acid ethyl esters (FAEEs) in hair. The FAEEs concentrations in hair, police investigation reports, medical history, and the macroscopic and microscopic alcohol-typical results from autopsy, such as liver, pancreas, and cardiac findings, were taken into account in this study. In 80.2 % of all 1,057 cases pathologic findings that may be related to alcohol abuse were reported. The cases were divided into social drinkers (n = 168), alcohol abusers (n = 502), and cases without information on alcohol use. The median FAEEs concentration in the group of social drinkers was 0.302 ng/mg (range 0.008-14.3 ng/mg). In the group of alcohol abusers a median of 1.346 ng/mg (range 0.010-83.7 ng/mg) was found. Before June 2009 the hair FAEEs test was routinely applied to a proximal hair segment of 0-6 cm, changing to a routinely investigated hair length of 3 cm after 2009, as proposed by the Society of Hair Testing (SoHT). The method showed significant differences between the groups of social drinkers and alcoholics, leading to an improvement in the postmortem detection of alcohol abuse. Nevertheless, the performance of the method was rather poor, with an area under the curve calculated from receiver operating characteristic (ROC curve AUC) of 0.745. The optimum cut-off value for differentiation between social and chronic excessive drinking calculated for hair FAEEs was 1.08 ng/mg, with a sensitivity of 56 % and a specificity of 80 %. In relation to the "Consensus on Alcohol Markers 2012" by the SoHT, an increase in the cut-off value for FAEEs in the proximal hair segment 0-3 cm from 0.5 to 1 ng/mg may be advisable to avoid excessive numbers of false positive results.

Detecting alcohol abuse: traditional blood alcohol markers compared to ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs) measurement in hair.

Alcohol abuse is a common problem in society; however, the technical capabilities of evaluating individual alcohol consumption using objective biomarkers are rather limited at present. In recent years research has focused on alcohol markers using hair analysis but data on performance and reliable cut-off values are still lacking. In this study 169 candidates were tested to compare traditional biomarkers, such as carbohydrate-deficient-transferrin (CDT), gamma glutamyl transferase (GGT), aspartate amino transferase, alanine amino transferase and the mean corpuscular volume of the erythrocytes, with alcohol markers detectable in hair such as ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEEs). This study revealed that EtG, GGT and CDT showed the best results, demonstrating areas under the curve calculated from receiver operating characteristics of 0.941, 0.943 and 0.899 respectively. The lowest false-negative and false-positive rates were obtained by using a combined interpretation system for hair EtG and FAEEs. All markers demonstrated only low to moderate correlations. Optimum cut-off values for differentiation between social and chronic excessive drinking calculated for hair EtG and FAEEs were 28 pg/mg and 0.675 ng/mg, respectively. The critical values published in the "Consensus on Alcohol Markers 2012" by the Society of Hair Testing were confirmed.

Toxicological identification of diphenhydramine (DPH) in suicide.

Diphenhydramine (DPH), an H1-antihistamine, is identified during postmortem toxicological analyses on a relatively rare but still regular basis. This study examines suicidal intoxications with DPH by analyzing blood and gastric content concentration levels. Twenty cases of DPH intoxications within a 10-year period (2000-2010) were discovered by screening the autopsy records of the Institute of Legal Medicine and Forensic Sciences (ILMFS) in Berlin, Germany. In four cases, DPH levels were lower than 1 µg/mL and hence were not considered likely to be responsible for causing death. In 11 cases, DPH played a role in the fatal episode, and five of these cases were monointoxications. Considering that more than 8,000 autopsies were performed by the ILMFS within the time period under examination, there is only one monointoxication case every 2 years, which makes it a rare occurrence. In two of these intoxications, DPH was only measured in toxic but not "lethal" concentrations in blood, with a concentration of 5 µg/mL being generally used as the cut off between categories according to forensic literature. This raises the question as to whether a strict boundary for a "lethal" blood concentration, as suggested in some literature, can be set and applied in any of these cases. This study shows that an individual interpretation of each case is of utmost importance for correct classification. A thorough toxicological analysis of peripheral venous blood and gastric content, as well as a detailed work-up of the death circumstances, are the basis of an exact interpretation of intoxications with DPH.

General unknown screening in hair by liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS).

The retrospective investigation of the exposure to toxic substances by general unknown screening of hair is still a difficult task because of the large number of possible poisons, the low sample amount and the difficult sample matrix. In this study the use of liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) was tested as a promising technique for this purpose. In the optimized procedure, 20mg hair were decontaminated with water and acetone and two times extracted by 18h incubation with 0.5ml of a mixture of methanol/acetonitrile/H(2)O/ammonium formate at 37°C. A mixture of deuterated standards from different drug groups was added for quantification and method control. The united extracts were evaporated to a residue of 0.5ml and 5µl were injected without clean-up for LC-QTOF-MS measurement (instrument Agilent 6530) with positive electrospray ionization and in data dependent acquisition mode. For peak identification the accurate mass data base and spectral library of the authors was used which contains accurate mass CID spectra of more than 2500 and theoretically calculated accurate mass data of more than 7500 toxicologically relevant substances. Validation at the example of 24 illegal drugs, their metabolites and benzodiazepines resulted in limits of detection of 0.003-0.015ng/mg, and limits of quantification of 0.006-0.021ng/mg with good accuracy and intra- and interday reproducibility. The matrix effect by ion suppression/enhancement was 72-107% for basic drugs and 42-75% for benzodiazepines. Yields of the hair extraction above 90% were determined for 59 drugs or metabolites. The method was applied to hair samples from 30 drug fatalities and from 60 death cases with known therapeutic drug intake at life time. Altogether 212 substances were identified with a frequency per drug of 1-40 (mean 4.2) and per case of 2-33 (mean 10.2), between them 35 illegal drug related substances and 154 therapeutic drugs. Comparison with the data known from case histories and from the analysis of blood, urine and gastric content showed only a low agreement, with many unexpected drugs detected and many reported drugs not detected in hair. Basic drugs and metabolites such as opioides, cocaine, amphetamines, several groups of antidepressants, neuroleptics, beta-blockers or the metamizole metabolite noramidopyrine were found with high frequency whereas acidic and several neutral drugs such as cannabinoids, salicylic acid, furosemide, barbiturates, phenprocoumone or cardiac glycosides could not be detected with sufficient sensitivity, mainly because of the low ion yield of positive ESI for these compounds. The advantage of a comprehensive acquisition of all substances is paid by a lower sensitivity in comparison to targeted screening LC-MS/MS procedures. In conclusion, the procedure of sample preparation and LC-QTOF-MS analysis proved to be a robust and sensitive routine method in which the qualitative screening for a wide variety of toxic substances in hair is combined with the quantitative determination of selected illegal drugs.

Combined use of liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and high performance liquid chromatography with photodiode array detector (HPLC-DAD) in systematic toxicological analysis.

Time of flight mass spectrometry provides new possibilities of substance identification by determination of the molecular formula from accurate molecular mass and isotope pattern. However, the huge number of possible isomers requires additional evidence. As a suitable way for routine performance of systematic toxicological analysis, a method for combined use of liquid chromatography-hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) and high performance liquid chromatography with diode array detector (HPLC-DAD) was developed and applied to blood samples from 77 death cases. The blood samples were prepared by extraction with CH(2)Cl(2) and by protein precipitation with acetonitrile (1:4 (v/v)). The evaporated extracts were reconstituted in 35% acetonitril/0.1% formic acid/H(2)O and aliquots were injected for analysis by LC-QTOF-MS (Agilent 6530) and HPLC-DAD (Agilent 1200). A valve switching system enabled simultaneous operation of both separated chromatographic lines under their respective optimal conditions using the same autosampler. The ESI-QTOF-MS instrument was run in data dependent acquisition mode with switching between MS and MS/MS (cycle time 1.1s) and measuring the full mass spectra and the collision induced dissociation (CID) fragment spectra of all essential [M+H](+) ions. Libraries of accurate mass CID spectra (~2500 substances) and of DAD-UV spectra (~3300 substances) of the authors were used for substance identification. The application of this procedure is demonstrated in detail at four examples with multiple drug intake or administration. In the 77 cases altogether 198 substances were identified (87 by DAD and 195 by QTOF-MS) with a frequency between 1 and 20. In practical application, the sample preparation proved to be suitable for both techniques and for a wide variety of substances with different polarity. The automatic performance of the measurements was efficient and robust. Mutual confirmation, decrease of false positive and false negative identifications, and the semi-quantitative estimation of the concentrations by HPLC-DAD for a first assessment of the toxicological relevance of the qualitative result were shown to be the main advantages of the method combination.

Development and practical application of a library of CID accurate mass spectra of more than 2,500 toxic compounds for systematic toxicological analysis by LC-QTOF-MS with data-dependent acquisition.

A library of collision-induced dissociation (CID) accurate mass spectra has been developed for efficient use of liquid chromatography in combination with hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) as a tool in systematic toxicological analysis. The mass spectra (Δm < 3 ppm) of more than 2,500 illegal and therapeutic drugs, pesticides, alkaloids, other toxic chemicals and metabolites were measured, by use of an Agilent 6530 instrument, by flow-injection of 1 ng of the pure substances in aqueous ammonium formate-formic acid-methanol, with positive and negative electrospray-ionization (ESI), selection of the protonated or deprotonated molecules [M+H](+) or [M-H](-) by the quadrupole, and collision induced dissociation (CID) with nitrogen as collision gas at CID energies of 10, 20, and 40 eV. The fragment mass spectra were controlled for structural plausibility, corrected by recalculation to the theoretical fragment masses and added to a database of accurate mass data and molecular formulas of more than 7,500 toxicologically relevant substances to form the "database and library of toxic compounds". For practical evaluation, blood and urine samples were spiked with a mixture of 33 drugs at seven concentrations between 0.5 and 500 ng mL(-1), prepared by dichloromethane extraction or protein precipitation, and analyzed by LC-QTOF-MS in data-dependent acquisition mode. Unambiguous identification by library search was possible for typical basic drugs down to 0.5-2 ng mL(-1) and for benzodiazepines down to 2-20 ng mL(-1). The efficiency of the method was also demonstrated by re-analysis of venous blood samples from 50 death cases and comparison with previous results. In conclusion, LC-QTOF-MS in data-dependent acquisition mode combined with an accurate mass database and CID spectra library seemed to be one of the most efficient tools for systematic toxicological analysis.

A sudden death with lung embolism after inadvertent infusion of zinc oxide shake lotion.

A 74-year old woman in postoperative treatment after a colonic surgery died immediately after perfusion of about 1.5 mL of a white emulsion which was believed to contain 1% propofol via cardiac catheter into the right atrium. It was strongly suspected that a syringe with a zinc oxide shake lotion (consisting of 20% ZnO, 20% talc, 25% glycerol and 35% water) which was intended for external treatment had been mistaken for the propofol syringe. During autopsy, an anatomic cause of death could not be found. In order to exclude an intoxication and to determine the significance of the perfusion fluid in this context, toxicological and histological investigations were performed. Propofol and other drugs applied to the patient were found in therapeutic or sub-therapeutic range. However, in comparison to a control case, the zinc concentrations determined by AAS were about 200 times higher in lung tissue, 10 times higher in heart blood and 3-4 times higher in kidney and liver tissue. No increase was seen in venous blood. Histology showed a strong embolism of the lung tissue with birefingent sharp-edged crystals, which were identified as the talcum, and an amorphous component (ZnO). The same embolism was seen to a medium extent also in the brain sections and to a low extent in heart, liver, pancreas and kidney. Pulmonary embolism by talcum and zinc oxide was established as the cause of death which occurred by syringe swap due to insufficient security precautions in the drug administration. The results are discussed in context of pulmonary microembolism cases frequently described for drug addicts after injection of crashed talcum containing tablets.

Poisonings with diphenhydramine--a survey of 68 clinical and 55 death cases.

The antihistaminic drug diphenhydramine (DPH) is mainly used as a sedative, hypnotic and antiemetic. In many countries it is over-the-counter available, very common, and generally regarded as a harmless drug. Sixty-eight non-fatal and 55 fatal poisonings with DPH alone or in combination with other drugs were investigated in the Institute of Legal Medicine of the University Hospital Charité between 1992 and 2004. The analytical investigations were performed by HPLC with photodiode array detector (HPLC-DAD). The DPH concentrations ranged from 0.5 to 8.9 microg/mL in the non-fatal cases and from 0.3 to 119 microg/mL in fatal cases. The intoxication symptoms stated during emergency admission were inconsistent, with somnolence, sedation and retardation on one hand and tachycardia, anticholinergic syndrome, agitation, hallucinations, confusion, tremor, convulsions, delirium and coma on the other. In three cases rhabdomyolysis occurred. A concentration above 5 microg/mL can be regarded as potentially lethal. In many of the survivors the time course of the concentrations of DPH and the metabolites desmethyldiphenhydramine (DM-DPH) and diphenylmethoxyacetic acid (DPMA) were investigated. Whereas DM-DPH is present in blood from the very beginning because of the high first pass metabolism, DPMA is slowly formed over several metabolic steps. For this reason, the concentration ratio DPMA/DPH can be used for an approximate estimation of the time between drug intake and sampling in clinical cases or of the survival time after drug ingestion in death cases. In some of the deaths the concentrations in heart blood were much higher than in venous blood. This is explained mainly by agonal aspiration of the vomited gastric content. Besides the majority of suicidal cases also a case of child maltreatment and a case, in which the drug was forcibly administrated in a drug facilitated crime, were investigated. From the results it follows that diphenhydramine is not less poisonous than other prescribed hypnotics. However, despite the hallucinogenic effects, an abuse for recreational purposes was not observed until now.

Selectivity of substance identification by HPLC-DAD in toxicological analysis using a UV spectra library of 2682 compounds.

The UV spectra and relative retention times (RRT) of 2682 toxicologically relevant substances were measured by high-performance liquid chromatography with diode array detection (HPLC-DAD) in an acetonitrile phospate buffer (pH 2.3) mixture on an RP8 column and were arranged in a database. A complete survey of the molecular structures of all database entries showed the presence of 1650 different chromophores or chromophore combinations. The specificity of the UV spectrum for substance identification was determined by calculation of the similarity indices (SI) of all possible substance pairs within the database with an SI > 0.9990, which indicated spectral identity. In a similar way, the RRT was evaluated for all possible pairs: two compounds were declared indistinguishable because the RRT of at least one of them fell into the RRT error window of the other. Although the use of the RRT alone produced unsatisfactory identification results, 1619 substances (60.4%) were unambiguously identified by their UV spectrum only. This rate was increased to 84.2% by the combination of spectrum and RRT. The selectivity parameters discrimination power (DP) and mean list length (MLL) were calculated (DP = 0.9999, MLL = 1.253) and compared with literature data, which proved HPLC-DAD to be one of the most reliable methods for substance identification in toxicological analysis. The practical relevance of the results for systematic toxicological analysis is demonstrated by the example of a multidrug intoxication and in the context of sample preparation methods routinely used.