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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.forsciint.2009.12.028 Byline: F. Pragst (a), M. Rothe (b), B. Moench (b), M. Hastedt (a), S. Herre (a), D. Simmert (a) Keywords: Alcohol marker; Ethyl glucuronide; Fatty acid ethyl ester; Hair analysis Abstract: In this study the combined use of fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) for diagnoses of chronically excessive alcohol abuse is investigated at 174 hair samples from driving ability examination, workplace testing and child custody cases for family courts and evaluated with respect to the basics of interpretation. Using the cut-off values of 0.50ng/mg for FAEE and 25pg/mg for EtG, both markers were in agreement in 75% of the cases with 103 negative and 28 positive results and there were 30 cases with FAEE positive and EtG negative and 13 cases with FAEE negative and EtG positive. As the theoretical basis of interpretation, the pharmacokinetics of FAEE and EtG is reviewed for all steps between drinking of ethanol to incorporation in hair with particular attention to relationships between alcohol dose and concentrations in hair. It is shown that the concentrations of both markers are essentially determined by the area under the ethanol concentration in blood vs. time curve AUC.sub.EtOH, despite large inter-individual variations. It is demonstrated by calculation of AUC.sub.EtOH on monthly basis for moderate, risky and heavy drinking that AUC.sub.EtOH increases very strongly in the range between 60 and 120g ethanol per day. This specific feature which is caused by the zero-order elimination of ethanol is a favorable prerequisite for a high discrimination power of the hair testing for alcohol abuse. From the consideration of the different profiles of FAEE and EtG along the hair and in agreement with the literature survey, a standardized hair segment 0-3cm is proposed with cut-off values of 0.5ng/mg for FAEE and 30pg/mg for EtG. This improves also the agreement between FAEE and EtG results in the cases of the present study. A scheme for combined interpretation of FAEE and EtG is proposed which uses the levels of abstinence and the double of the cut-off values as criteria in addition to the cut-off's. Considering the large variations in the relationship between ethanol dose and FAEE and EtG concentrations in hair, the combined use of both parameters strongly increases the accuracy of the diagnosis by mutual confirmation and identification of false positive or false negative results due to biological variations or analytical errors. Author Affiliation: (a) Institute of Legal Medicine, University Hospital Charite, Hittorfstr. 18, 14195 Berlin, Germany (b) Lipidomix GmbH, Berliner Allee 261-269, 13088 Berlin, Germany Article History: Received 12 June 2009; Accepted 22 July 2009

Purpose: This article describes the validation of multi-target methods for the determination of 76 different analytes in hair and nail samples. Segmented hair and nail samples taken from autopsy cases were included in this study., Method: Drugs of abuse, psychotropic drugs, and other drugs were included for the validation of this method. Hair and nail samples were ground using a ball mill and extracted for 18 h. Extracts were measured using a UHPLC-triple quadrupole-mass analyzer. Analytes were separated on a RP 18 column under gradient elution of the mobile phases, water with 0.1 % formic acid and acetonitrile. Whole nail and hair samples from seven autopsy cases were split into segments and analyzed., Results: The entire method was validated according to the German Society of Forensic Toxicology guidelines. In addition, the concentration ratios of selected substances and their metabolites were calculated. Similar concentration ratios in hair and nails were detected for 3,4-methylendioxyamphetamine (MDA)/3,4-methylenedioxy-N-methylamphetamine (MDMA), 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP)/methadone, and bisnortilidine/nortilidine in some of the cases. Reduced 6-monoacetylmorphine (6-MAM) and cocaine concentrations were observed as a result of sample preparation using the ball mill. Previous heroin intake could be assumed from the detection of 6-MAM and morphine in only one nail segment in one case., Conclusions: Nail samples may serve as an alternative matrix for the detection of long-term consumption of a wide range of drugs. Based on our results, drug concentrations in nails are not comparable to those in hair. The main mechanisms for drug incorporation into the nails may be during the formation of the nail plate by the germinal matrix. However, external contamination can also affect the analysis of nail clippings.

Hair analysis for drugs and drugs of abuse is increasingly applied in child protection cases. To determine the potential risk to a child living in a household where drugs are consumed, not only can the hair of the parents be analyzed but also the hair of the child. In the case of hair analysis for cannabinoids, the differentiation between external contamination and systemic uptake is particularly difficult, since the drug is quite often handled extensively prior to consumption (e.g. when preparing a joint) and smoke causes a further risk for an external contamination. Δ9-tetrahydrocannabinolic acid A (THCA-A), the non-psychoactive biogenetic precursor of Δ9-tetrahydrocannabinol (THC), is a suitable marker for external contamination since it is not incorporated into the hair matrix through the bloodstream in relevant amounts. In the presented study, hair samples from 41 children, 4 teenagers, and 34 drug-consuming parents were analyzed for THCA-A, THC and cannabinol (CBN) applying methanolic extraction and a fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (Method 1). For comparison, a part of the samples was also analyzed applying alkaline hydrolysis followed by liquid/liquid extraction and gas chromatography-mass spectrometry (GC-M)S (Method 2), or by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) (Method 3). Furthermore, 458 seized marihuana samples and 180 seized hashish samples were analyzed for the same cannabinoids by gas-chromatography-flame ionization detector (GC-FID). In all but one of the hair samples, the concentration of THCA-A was higher than the concentration of THC and in 14 cases no THC could be detected despite the presence of THCA-A, suggesting that in almost all cases a significant external contamination had occurred. Within-family comparison showed a higher THCA-A/THC ratio in hair of children than of their consuming caregivers. Mean and median of this ratio of all hair samples (6.7 and 4.2) were between those of marihuana (11.0 and 8.3) and hashish (2.8 and 2.1) with a large variation in all samples. Comparison of the Methods 1 to 3 showed clearly that the choice of the analytical procedure has a strong influence on the quantitative results, mainly because of decarboxylation of THCA-A during hair hydrolysis by NaOH and other analytical steps, which lead to artifactually elevated THC concentrations. In conclusion, these findings suggest that the major part of the cannabinoids detected in the hair samples from children arose from an external contamination through 'passive' transfer by e.g. contaminated hands or surfaces and not from inhalation or deposition of side stream smoke., (Copyright © 2014 John Wiley & Sons, Ltd.)

Contrary to the illegal use of any form of manufactured cocaine, chewing of coca leaves and drinking of coca tea are allowed and are very common and socially integrated in several South American countries. Because of this different legal state, an analytical method for discrimination between use of coca leaves and abuse of processed cocaine preparations is required. In this study, the applicability of hair analysis for this purpose was examined. Hair samples from 26 Argentinean coca chewers and 22 German cocaine users were analysed for cocaine (COC), norcocaine (NC), benzoylecgonine (BE), ecgonine methyl ester (EME), cocaethylene (CE), cinnamoylcocaine (CIN), tropacocaine (TRO), cuscohygrine (CUS) and hygrine (HYG) by hydrophilic interaction liquid chromatography (HILIC) in combination with triplequad mass spectrometry (MS/MS) and hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS). The following concentrations (range, median, ng/mg) were determined in hair of the coca chewers: COC 0.085-75.5, 17.0; NC 0.03-1.15, 0.12; BE 0.046-35.5, 6.1; EME 0.014-6.0, 0.66; CE 0.00-13.8, 0.38; CIN 0.005-16.8, 0.79; TRO 0.02-0.16, 0.023; CUS 0.026-26.7, 0.31. In lack of a reference substance, only qualitative data were obtained for HYG, and two metabolites of CUS were detected which were not found in hair of the cocaine users. For interpretation, the concentrations of the metabolites and of the coca alkaloids in relation to cocaine were statistically compared between coca chewers and cocaine users. By analysis of variance (ANOVA) significant differences were found for all analytes (α = 0.000 to 0.030) with the exception of TRO (α = 0.218). The ratios CUS/COC, CIN/COC and EME/COC appeared to be the most suitable criteria for discrimination between both groups with the means and medians 5-fold to 10-fold higher for coca chewers and a low overlap of the ranges between both groups. The same was qualitatively found for HYG. However, these criteria cannot exclude cocaine use in addition to coca chewing. In this regard screening for typical cutting agents can be helpful and led to the detection of levamisole (21×), lidocaine (6×) and paracetamol (3×) in the 22 samples from German cocaine users, whereas no levamisole, lidocaine (3×) and paracetamol (1×) were found in hair from the Argentinean coca chewers. These criteria have to be confirmed for South American cocaine consumers including smokers of coca paste and may be different because of different composition of the drug and other use habits.

Purpose: In this study, the usefulness of nail samples instead of hair for a general unknown screening (GUS) for drugs was tested. An alternative matrix for long term detection is still needed in cases where no hair is available for analysis., Methods: Hair and nail samples from 70 postmortem cases were analyzed by liquid-chromatography quadrupole time-of-flight mass spectrometry. Hair and nail samples were ground by a ball mill and extracted twice for 18 h. Extracts were measured in Auto MS/MS mode (data dependent mode acquisition)., Results: Only 10% of the cases showed a disagreement of results in hair and nail analysis where hair samples were tested positive and corresponding nail samples were tested negative in a general unknown screening for drugs. In most of the cases investigated the analysis of the nail clippings and whole nail samples led to results comparable to those obtained from hair analysis. The incorporation of a large number of substances into the nail matrix was proven by the detection of 89 different analytes (e.g. antidepressants, drugs of abuse or antihypertonics) in our tests., Conclusion: In cases where the amount of hair available is not sufficient for a general unknown screening for drugs, nails appear to be a useful comparable matrix for the detection of long-term drug consumption due to the comparison of the qualitative GUS results from the hair and nail samples in this study.

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.

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.

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.

Alcohol consumption during pregnancy is a widespread problem and can cause severe fetal damage. As the diagnosis of fetal alcohol syndrome is difficult, the implementation of a reliable marker for alcohol consumption during pregnancy into meconium drug screening programs would be invaluable. A previously published gas chromatography mass spectrometry method for the detection of fatty acid ethyl esters (FAEEs) as alcohol markers in meconium was optimized and newly validated for a sample size of 50 mg. This method was applied to 122 cases from a drug-using population. The meconium samples were also tested for common drugs of abuse. In 73 % of the cases, one or more drugs were found. Twenty percent of the samples tested positive for FAEEs at levels indicating significant alcohol exposure. Consequently, alcohol was found to be the third most frequently abused substance within the study group. This re-validated method provides an increase in testing sensitivity, is reliable and easily applicable as part of a drug screening program. It can be used as a non-invasive tool to detect high alcohol consumption in the last trimester of pregnancy. The introduction of FAEEs testing in meconium screening was found to be of particular use in a drug-using population.

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.

Fatty acid ethyl esters (FAEE) and ethyl glucuronide (EtG) were determined in 602 meconium samples in a maternal health evaluation study for detection of gestational alcohol consumption. A validated headspace solid phase microextraction method in combination with GC-MS was used for FAEE and the cumulative concentration of ethyl palmitate, ethyl linoleate, ethyl oleate, and ethyl stearate with a cut-off of 500 ng/g was applied for interpretation. A new and simple method was developed and validated for quantification of EtG from 10-20 mg meconium with D(5)-EtG as internal standard consisting of 30 min. extraction with methanol/water (1:1, v/v), evaporation of methanol, filtration of the aqueous solution through a cellulose filter and injection into LC-MS-MS. The limits of detection and quantification for EtG were 10 and 30 ng/g, the recovery 86.6 to 106.4% and the standard deviation of the concentrations ranged from 13% at 37 ng/g to 5% at 46,700 ng/g (N = 6). FAEE above the cut-off were found in 43 cases (7.1%) with cumulative concentrations between 507 and 22,580 ng/g and with one outlier of about 150,000 ng/g (EtG not detected). EtG was detected in 97 cases (16.3%) and concentrations between LOD and 10,200 ng/g with another outlier of 82,000 ng/g (FAEE 10,500 ng/g). Optimal agreement between the two markers was obtained with a cut-off for EtG of 274 ng/g and 547 cases with both FAEE- and EtG-negative, 33 cases with both FAEE- and EtG-positive, nine cases with FAEE-positive and EtG-negative, and seven cases with FAEE-negative and EtG-positive. Differences in physical, chemical, and biochemical properties and in the pharmacokinetic behavior are discussed as reasons for the deviating cases. In none of the 602 cases, serious alcohol consumption was reported by the mothers and no evidence for gestational ethanol exposure was observed in the medical investigation of the newborns. It is concluded that the combined use of FAEE and EtG in meconium as markers for fetal alcohol exposure essentially increases the accuracy of the interpretation and helps to avoid false positive and false-negative results.