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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.forsciint.2009.12.033 Byline: Pascal Kintz (a), Julie Evans (b), Marion Villain (a), Vincent Cirimele (a) Keywords: Hair; Contamination; Decontamination; Methadone; Child; Administration Abstract: Methadone is not licensed for use in children though it can be employed for the management of neonatal opiate withdrawal syndrome. During the last 2 years, our laboratory has been asked to test for methadone and EDDP, its major metabolite, in hair from children that were admitted to hospital unconscious and where methadone had already been identified in a body fluid (4 cases) or where the children were deceased and evidence of methadone overdosage having already been established (2 cases). In all of these cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention. After decontamination with dichloromethane and segmentation the hair was cut into small pieces, incubated overnight at 40[degrees]C, liquid-liquid extracted and analysed with LC-MS/MS, using 2 transitions per compound. The LOQ for both methadone and EDDP was 10pg/mg. In the first series involving children admitted to hospital, the following results were obtained: acents case 1: 4x1cm section, methadone at 0.05-0.08ng/mg, no EDDP detected, acents case 2: 4x1cm section, methadone at 0.13-0.15ng/mg, EDDP at 0.02ng/mg, acents case 3: 3x1.5cm section, methadone at 0.07-0.09ng/mg, EDDP at 0.01-0.03ng/mg, acents case 4: 6x2cm section, methadone at 0.06-0.13ng/mg, EDDP at 0.02-0.03ng/mg. The following concentrations were obtained from the children who had died following a methadone overdose: acents case 5: 2x2cm section, methadone at 0.53-0.58ng/mg, no EDDP detected, acents case 6: 4x1cm section, methadone at 0.44-0.77ng/mg, EDDP at 0.04-0.06ng/mg. The first observation is that all these concentrations are low by comparison with those observed in adults on methadone maintenance therapy. However, the more surprising observation is the relative homogenous concentrations along the hair locks in each specific case. This raises concerns around the possibility that contamination could have occurred prior to sampling and makes it hard to reach a conclusion regarding the possibility of repeated methadone exposure in the months prior the incidents. In these cases it was impossible to conclude that the children were deliberately administered methadone. The results of the analysis of hair could indicate that they were in an environment where methadone was being used and where the drug was not being handled and stored with appropriate care. The homogenous concentrations found on segmental analyses could be indicative of external contamination that may have arisen not only from direct contamination with the drug but also via contamination with body fluids at the post mortem or from sweat produced close to the time of the incident. In view of these results we concluded that a single determination should not be used firmly to discriminate long-term exposure to a drug. Author Affiliation: (a) Laboratoire ChemTox, 3 rue Gruninger, 67400 Illkirch, France (b) ROAR Forensics Ltd, Malvern, UK Article History: Received 10 June 2009; Accepted 24 August 2009

The major problem after testing the hair of a child for drugs is the interpretation of the findings. In 2016, the laboratory received several hair specimens with the request to verify if there was any evidence of previous methadone exposure by the donor of the sample. Case 1 was a child admitted to the Emergency Unit for intense sedation and breathing difficulties. Cases 2-4 involved children found dead at home. In all cases, methadone and EDDP, its metabolite, were found in blood. After decontamination, the hair was analysed with LC-MS/MS for methadone and EDDP. The LOQ for both drugs was 10pg/mg. Concentrations were in the range 60-1590 and <10-220pg/mg for methadone and EDDP, respectively. In all of the cases, segmental analysis revealed approximately the same amount of drug along the hair lock. As a consequence, contamination was considered as an issue and interpretation of the results was a challenge that deserves particular attention. It must be considered that the amount of hair from children, available for analysis, can be low, particularly when several drugs have to be tested. This has consequences on the limit of quantitation and the identification of the metabolite(s). It must be also noted that hair from children is finer and more porous in comparison with adult (risk of higher contamination by sweat versus adults). It is very difficult to put any window of detection when testing for drugs in young children as hair growing is asynchronous. It is even more complicated as it has been demonstrated that drugs can be incorporated during pregnancy in the hair of the foetus, which will contribute to the positive findings after delivery. Several weeks or months after delivery, identification of a drug in hair can indicate: 1, in-utero exposure, or 2, exposure after delivery, or 3, a mix of both situations. Whereas the detection of drugs in a child's hair unambiguously shows drug handling in the environment of the child, it is difficult to distinguish between systemic incorporation into hair after ingestion or inhalation and external deposition into hair from smoke, dust, or contaminated surfaces. However, the interpretation of hair results with respect to systemic or only external exposure is particularly important in case of children for a realistic assessment of the toxic health risk. Practising scientists have the responsibility to inform the child protection authorities, courts, etc about these limitations. [ABSTRACT FROM AUTHOR]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.forsciint.2009.12.032 Byline: Marion Villain, Jean-Francois Muller, Pascal Kintz Keywords: Contamination; Drugs of abuse; Hair; Trafficking Abstract: On March 2007, a police officer (46-year-old man) and a clerk (37-year-old woman) were arrested and subjected to investigation on the charges of drugs of abuse trafficking. The loving couple was exploiting their administrative positions to make money with the resale of seized drugs. The laboratory was requested to analyse their hair for drugs of abuse. Hair of the 2 subjects tested positive for heroin by GC-MS. A few days later, analysis of hair obtained from 11 other police officers of the same unit was requested, in order to compare the results, as external contamination was proposed to account for the positive results. The aim of the investigations was to demonstrate that passive contamination could not occur for persons dealing every day with drugs of abuse with minimal caution and hygiene, and that the measured concentrations in the arrested subjects correspond to personal abuse. All the narcotic team tested negative, irrespective of the compound. Author Affiliation: Laboratoire ChemTox, 3 rue Gruninger, 67400 Illkirch, France Article History: Received 10 June 2009; Accepted 3 August 2009

Morphine sulfate misuse is essentially observed among regular heroin injectors. To our knowledge, primary addiction to morphine sulfate is exceptional, especially among young adolescents. A 13-year-old girl, with no history of addiction, was found dead with three empty blisters of Skenan(®) LP 30 mg at her side. Opiates were detected in biological fluids and hair by chromatographic methods. Blood analyses confirmed morphine overdose (free morphine: 428 ng/mL; total morphine: 584 ng/mL) and segmental hair analysis confirmed regular exposure over several months (maximum morphine concentration 250 pg/mg). Suspecting the victim's mother of recreational use of Skenan(®), the magistrate ordered analysis of her hair, with negative results. From an epidemiological viewpoint, this case of oral morphine sulfate abuse in an adolescent with no previous history suggests the emergence of a new trend of morphine sulfate consumption. From a toxicological viewpoint, it demonstrates the value of hair testing, which documented the victim's regular exposure and made an important contribution to the police investigation. [ABSTRACT FROM AUTHOR]

Abstract: Amitriptyline, temazepam, tramadol and dihydrocodeine are prescription-only-medications that are rarely prescribed to children. Each of these drugs has a sedative effect on the central nervous system; their combined use could cause an exacerbation of the sedative effects. Amitriptyline (a tricyclic antidepressant) can be prescribed to treat nocturnal enuresis; temazepam (a hypnotic) can be used as a premedicant in inpatient and day-case surgery; tramadol (a synthetic opioid analgesic) is used to treat moderate or severe pain, though it is not recommended for children under the age of 12 years and dihydrocodeine (opioid analgesic), which is available in combination with acetaminophen (Co-dydramol®), is not recommended for children under the age of 4 years; in children over 4 years, a reduced dose is necessary. The North West Forensic Science Service Laboratory, Euxton, Lancashire, was asked by a British police force to analyze three separate hair samples, which had been collected from a young child following their discovery as a result of a large scale kidnap and false imprisonment investigation. After decontamination and segmentation (20 x 1-cm section), two of the three hair specimens were analyzed by liquid chromatography coupled with tandem mass spectrometry after alkaline (pH 9.5) extraction using methylene chloride/isopropanol/n-heptane (25:10:65, v/v/v). The entire length of each hair specimen tested positive for amitriptyline and nortriptyline (7–314 pg/mg amitriptyline; 7–318 pg/mg nortriptyline), temazepam (2–29 pg/mg), tramadol (60–2000 pg/mg) and dihydrocodeine (10–90 pg/mg) demonstrating that the child had ingested these drugs on more than one occasion prior to the kidnap. In this case, the child's mother and the mothers' partner were found guilty of kidnap, false imprisonment and perverting the course of justice. There are very few studies citing the concentrations of these drugs in children – especially children's hair samples. This case demonstrates the added value of hair testing and emphasizes the importance of using hair samples to complement conventional analysis. [Copyright &y& Elsevier]

Abstract: Excluding laboratory mistakes, a false positive hair result can be observed in case of contamination from environmental pollution (external contamination) or after drug incorporation into the hair from the individual body fluids, such as sweat or putrefactive fluid (post mortem artifact). From our 20 years experience of hair testing, it appears that artifact(s) cannot be excluded in some post mortem cases, despite a decontamination procedure. As a consequence, interpretation of the results is a challenge that deserves particular attention. Our strategy will be reviewed in this paper, based on six cases. In all cases, a decontamination procedure with two washes of 5ml of dichloromethane for 5min was performed and the last dichloromethane wash was negative for each target drug. From the histories, there was no suspicion of chronic drug use. In all six cases, the concentrations detected were similar along the hair shaft, irrespective of the tested segment. We have considered this as indicative of external contamination and suggested to the forces or the judges that it is not possible to indicate exposure before death. In contrast to smoke, it seems that contamination due to aqueous matrices (sweat, putrefactive fluid, blood) is much more difficult to remove. To explain potential incorporation of 7-aminoflunitrazepam via putrefactive material, the author incubated negative hair strands in blood spiked at 100ng/ml and stored at +4°C, room temperature and +40°C for 7, 14 and 28 days. After routine decontamination, 7-aminoflunitrazepam tested positive in hair, irrespective of the incubation temperature, as early as after 7 days (233–401pg/mg). In all periods, maximum concentrations were observed after incubation at room temperature. The highest concentration (742pg/mg) was observed after 28 days incubation at room temperature. It is concluded that a standard decontamination procedure is not able to completely remove external contamination in case of post mortem specimens. Homogenous segmental analyses can be probably indicative of external contamination and therefore a single hair result should not be used to discriminate long-term exposure to a drug. Nor should the presence of a metabolite be considered as a discrimination tool, as it can also be present in putrefactive material. [Copyright &y& Elsevier]

Abstract: Ethyl glucuronide (EtG) is a minor metabolite of ethanol that can be detected in hair. In some specific situations, head hair can be missing, and therefore, alternative anatomical locations of hair are of interest. In this study, paired hair specimens (head hair and pubic hair) from eight social drinkers were analyzed for EtG. Each sample was decontaminated by two dichloromethane bathes (5ml) for 2min. After cutting into small pieces, about 50mg of hair was incubated in 2ml water in the presence of 10ng of EtG-d5, used as internal standard and submitted to ultra-sonication for 2h. The aqueous phase was extracted by SPE using Oasis MAX columns. The hair extract was separated on an ACQUITY BEH HILIC column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 221–85 and 75 and m/z 226–75 for EtG and the IS, respectively. This laboratory is using a positive cut-off at 50pg/mg. All eight head hair specimens were negative for EtG at a limit of quantitation fixed at 10pg/mg. Surprisingly, EtG was identified at high concentrations in pubic hair, in the range 12–1370pg/mg. It appears, therefore, that it is not possible to document the drinking status of a subject by simply switching from head hair to pubic hair. [Copyright &y& Elsevier]

Abstract: Diphenhydramine is one of the first effective antihistamine agents to have been discovered. The compound is also used for its sedative and antiemetic effects. The first case involving repetitive sedation linked to the use of diphenhydramine as a drug-facilitated crime and subsequent impairment of a 9-year-old female victim is reported. Due to the long delay between the alleged crime and clinical examination, collection of blood or urine was of little value. Hence, the laboratory developed an original approach based on hair testing by LC-MS/MS. A single strand of hair from the victim was sampled about 7 weeks after the last suspected administration and was cut into small segments. After cutting into small pieces, about 20mg of hair per segment was incubated overnight in a phosphate buffer (pH 8.4). The aqueous phase was extracted with 5ml of a mixture of methylene chloride/diethyl ether (80/20), in presence of diazepam-d 5, used as internal standard (IS). The hair extract was separated on an XTerra MS C18 column using a gradient of acetonitrile and formate buffer. Detection was based on two daughter ions: transitions m/z 256.2–152.1 and 167.1 and m/z 289.9–154.0 for diphenhydramine and the IS, respectively. In the hair of the child, diphenhydramine was detected at concentrations in the range 33–39pg/mg, depending on the segment. [Copyright &y& Elsevier]

Drug facilitated sexual assaults (DFSA) have been increasingly reported in the medical literature since the 1980s but their legal recognition is more recent, at least in Europe. From a case treated in south-eastern France, whose judicial consequences were known, it seemed of interest to carry out an international study of jurisprudence concerning this type of rape. While from the medical viewpoint the drugs used are well-known and their presence can be clinically verified, the legal consequences of their use in subsequent criminal prosecution is less clear-cut. Some European countries have no jurisprudence in this area, while others consider the use of drugs as an aggravating circumstance. In France, it was only in 2003 that the first case of DFSA was truly punished by the judicial system, with considerable media attention. By contrast, in English-speaking countries, particularly the United States, the use of drugs to facilitate sexual assault has frequently been recognized in legislation and in criminal prosecutions. [ABSTRACT FROM AUTHOR]

Abstract: A procedure is presented for the screening of 17 benzodiazepines and hypnotics in oral fluid after collection with the Intercept® device by LC-MS/MS (alprazolam, 7-aminoclonazepam, 7-aminoflunitrazepam, bromazepam, clobazam, diazepam, lorazepam, lormetazepam, midazolam, nordiazepam, oxazepam, temazepam, tetrazepam, triazolam, zaleplon, zopiclone and zolpidem). The method involves extraction of 0.5mL of oral fluid (previously stored in the Intercept blue buffer) treated with 0.5mL of phosphate buffer (pH 8.4) in the presence of 5ng diazepam-d5 used as internal standard, with 3mL of diethyl ether/methylene chloride (50/50) and separation using liquid chromatography–tandem mass spectrometry. The limits of quantification for all benzodiazepines and hypnotics range from 0.1 to 0.2ng/mL. Linearity is observed from the limit of quantification of each compound to 20ng/mL (r 2 >0.99). Coefficients of variation at 2ng/mL, measured on 6 points range from 4 to 8% for all drugs, except zopiclone (34%). Extraction recovery, measured at the same concentration was higher than 90%. Ion suppression was evaluated for each compound and was lower than 10% for all drugs except zopiclone (93%). These results were found suitable to screen for 17 benzodiazepines in oral fluid and detect them at very low concentrations, making this method suitable for monitoring subjects under the influence. [Copyright &y& Elsevier]

The purported lowering of sex opposition, coupled with a possible abrupt unconsciousness-inducing effect and ease of administration in spiked drinks have resulted in the use of hypnotics in cases of drug-facilitated offense. Among these compounds, lorazepam possesses amnesic properties and can impair an individual rapidly.The chances to detect this substance increase if the most sensitive methods are used and if the biological fluid which allows the longest possible detection time is available.In order to document the window of detection of lorazepam, we have orally administered 2.5 mg of the drug to three volunteers and collected oral fluid (n=l) over 8 h, urine (n=2) over 144 h and hair (n=3) 4 weeks after exposure. Lorazepam was analyzed by LC-MS/MS after alkalinisation (to pH 8.4 with phosphate buffer) and extraction by dichloromethane/diethyl ether in presence of diazepam-d5, used as internal standard. Reversed-phase separation on a XTerra C18 column was achieved in 12 min, under gradient conditions. Molecular ions (m/z 321 and 290 for lorazepam and the IS, respectively) were selected in Ql and the corresponding daughter ions (m/z 303 and 275 for lorazepam and m/z 154 and 198 for the IS) were detected in Q3 after collision with argon.Urine tested positive for lorazepam over 144 h (2–4 ng/ml), with a peak detected after 24 h exposure (411–880 ng/ml). Oral fluid tested positive for lorazepam over 8 h (0.7 ng/ml). Despite a limit of quantitation at 1 pg/mg, we were unable to detect a single lorazepam dose in hair, contrarily to most other benzodiazepines that are detectable.Therefore, in case of drug-facilitated crimes involving lorazepam, urine appears as the best specimen to document exposure, particularly if LC-MS/MS is used. [Copyright &y& Elsevier]

Since gamma-hydroxybutyrate (GHB) is present in both blood and urine of the general population as an endogenous compound, toxicologists must be able to discriminate between these endogenous levels and a concentration resulting from exogenous exposure. The implementation of a cut-off concentration must be done cautiously, due to the wide distribution of endogenous concentrations.To verify the accuracy of a proposed 50 mg/l postmortem blood cut-off, we tested 71 autopsy cases of subjects where the cause of death could exclude GHB exposure. The delay between death and autopsy ranged from 12 to 72 h. GHB was tested by gas chromatography–mass spectrometry (GC–MS) after precipitation. Briefly, 20 μl of blood, bile, or vitreous humor was pipetted in a glass tube, followed by 20 μl of GHB-d6 and 45 μl of acetonitrile. After vortexing and centrifugation, the supernatant was collected and evaporated to dryness. The residue was derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) for 20 min at 70 °C. After injection on a 30 m HP5 MS capillary column, GHB (m/z 233, 204, and 147) and GHB-d6 (m/z 239) were identified by MS.GHB tested positive in all the 71 whole blood (cardiac) specimens, with concentrations in the range 0.4–409 mg/l, with a major distribution in the range 10–40 mg/l. A concentration >50 mg/l was observed in 14 cases. As there was no data to support GHB exposure, this was considered as postmortem formation. In order to discriminate this contamination, when available, femoral blood, bile or/and, vitreous humor were tested. The following results were obtained: cardiac blood (55–409 mg/l) versus bile (6.1–238 mg/l) in seven cases; cardiac blood (51–409 mg/l) versus femoral blood (17–44 mg/l) in five cases, and cardiac blood (51–409 mg/l) versus vitreous humor (3.9–2 mg/l) in six cases. It is obvious that bile does not fit the requirements for discrimination and that femoral blood and mostly vitreous humor can be of particular interest. These results demonstrate that a positive (>50 mg/l) postmortem blood GHB concentration cannot support alone drug exposure and that it is essential to document the case with other specimens, including peripheral blood and vitreous humor. [Copyright &y& Elsevier]

It is generally accepted that chemical testing of biological fluids is the most objective means of diagnosis of drug use. The presence of a drug analyte in a biological specimen can be used to document exposure. The standard in postmortem drug testing is a general unknown screening, followed by the gas chromatographic/mass spectrometric confirmation conducted on a whole blood sample. In recent years, remarkable advances in sensitive analytical techniques have enabled the analysis of drugs in unconventional biological specimens such as hair. The advantages of this sample over traditional media, like urine and blood, are obvious: collection is almost non-invasive, relatively easy to perform, and in forensic situations it may be achieved under close supervision of law enforcement officers to prevent adulteration or substitution. Moreover, the window of drug detection is dramatically extended to weeks, months or even years. The aim of this review is to document the current status of hair analysis in postmortem toxicology. [Copyright &y& Elsevier]

Objectives: Buprenorphine at high dosage became available in France in 1996, as a substitution treatment for heroin addicts. Since this date, numerous deaths were attributed to this drug. This paper reports a new series of 13 fatalities involving buprenorphine observed at the Institute of Legal Medicine of Strasbourg, between August 2000 to October 2001.Design and methods: During the mentioned period, about 800 forensic cases were screened at the laboratory. Buprenorphine and its primary metabolite norbuprenophine were assayed in postmortem specimens by HPLC/MS. From these 13 subjects, 11 were male. Blood levels ranged from 0.3 to 7.7 ng/mL (mean 3.5 ng/mL) and 0.3 to 16.2 ng/mL (mean 2.9 ng/mL) for buprenorphine and norbuprenorphine, respectively. The mean values appear to be within the therapeutic range.Conclusions: IV injection of crushed tablets, a concomitant intake of psychotropics (especially benzodiazepines and neuroleptics) and the high dosage of the buprenorphine formulation available in France appear as the major risk factors for such fatalities. [Copyright &y& Elsevier]

Abstract: The influence on drug incorporation of melanin affinity, lipophilicity, and membrane permeability is of paramount importance. Despite their high lipophilicity, some drugs have quite low incorporation rate into hair, suggesting that the higher incorporation rates of basic drugs (cocaine, amphetamines.) than neutral (steroids, benzodiazepines, cannabinoids…) or acidic ones are strongly related to the penetrating ability of the drug to break through the membrane based on the pH gradient between blood and the acidic hair matrix. When using hair analysis as a matrix during investigative analysis, e.g. workplace drug testing, doping, driving under the influence, drug-facilitated crime, the question of importance is to know whether the analytical procedure was sensitive enough to identify traces of drugs; this is particularly important when the urine sample(s) of the subject was positive and the hair sample(s) was negative. It has been accepted in the forensic community that a negative hair result cannot exclude the administration of a particular drug, or one of its precursors and the negative findings should not overrule a positive urine result. Nevertheless, the negative hair findings can, on occasion, cast doubt on the positive urine analysis, resulting in substantial legal debate and various consequences for the subject. The concept of minimal detectable dosage in hair is of interest to document the negative findings, but limited data is currently available in the scientific literature. Such data includes cocaine, codeine, ketamine, some benzodiazepines and some unusual compounds. Until laboratories will have sensitive enough methodologies to detect a single use of drug, care should be taken to compare urine and hair findings. [Copyright &y& Elsevier]

Abstract: Since 1960, it has been demonstrated by various analytical procedures that high concentrations of arsenic were present in Napoleon''s hair. Various authors, indicating that the detected arsenic levels are a consequence of external contamination, have challenged the results of these examinations. We have tested two strands of hair, referenced as Noverraz and Grand Maréchal Bertrand. Samples were incubated 6h in water at 90°C, and arsenic speciation was carried out by HPLC–ICP/MS. The residue was injected on a cation-exchange PRP-X200 column that allows the detection of arsenobetaine and on an anion-exchange PRP-X100 column to test for the mineral forms. In these conditions, the inorganic species As(III), As(V) and their metabolites (DMA and MMA) were separated. Analysis of hair samples highlighted massive amounts of total arsenic (42.1 and 37.4ng/mg). Arsenical species found in the two samples of analyzed hair are distributed in the following: As(III) 31.1 and 44.7%; As(V) 66.3 and 53.2%; DMA 0.42 and 0.15%. Traces of MMA were detected, and 2% of the species could not be identified. These results prove that more than 97% of the arsenic found in the hair of Napoleon is in inorganic form, which is consistent with a chronic intoxication to arsenic. [Copyright &y& Elsevier]