Forensic Science International

Editorial Board

2010-03-20

Editorial

Pascal Kintz — 2010-01-08

Given the limitations of self-reports on drug use, testing for drugs of abuse is important for most clinical and forensic toxicological situations, both for assessing the reality of the intoxication and for evaluation of the level of drug impairment.

Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption 2009

Pascal Kintz — 2010-01-08

Alcohol is a legal compound in many countries and is consumed in much higher amounts in comparison to other drugs of abuse and by a much higher portion of the population. Compared to other substances, the detection of chronic excessive alcohol consumption by hair analysis has some specific characteristics.

Validation of a headspace solid-phase microextraction–GC–MS/MS for the determination of ethyl glucuronide in hair according to forensic guidelines

2010-01-11

Abstract: The analysis of ethyl glucuronide (EtG) in hair is a powerful tool for chronic alcohol abuse control because of the typical wide detection window of the hair matrix and due to the possibility of segmentation, allowing evaluation of alcohol consumption in different periods. Additionally, EtG in hair is often the only diagnostic parameter of choice for alcohol abuse when other clinical parameters such as ALT, AST, γGT and CDT (asialotransferrin and disialotransferrin) are in the normal range and EtG in urine negative. In this paper, we describe the development, optimization and validation of a new method based on hair extraction with water, clean-up by solid phase extraction (SPE), derivatization with heptafluorobutyric anhydride and headspace solid-phase microextraction (HS-SPME) in combination with GC–MS/MS according to forensic guidelines. The assay linearity of EtG was confirmed over the range from 2.8 to 1000pg/mg hair, with a coefficient of determination (r2) above 0.999. The LLOQ was 2.8pg/mg and the LLOD was 0.6pg/mg. An error profile calculated according to the “Guide to the Expression of Uncertainty in Measurement” (GUM) at 99% confidence intervals for the range 5–750pg/mg hair did not exceed 10%. This range corresponds to more than 98% of the positive samples analysed.

Hair analysis for Δ9-tetrahydrocannabinolic acid A—New insights into the mechanism of drug incorporation of cannabinoids into hair

2010-02-03

Abstract: Differentiation between external contamination and incorporation of drugs or their metabolites from inside the body via blood, sweat or sebum is a general issue in hair analysis and of high concern when interpreting analytical results. In hair analysis for cannabinoids the most common target is Δ9-tetrahydrocannabinol (THC), sometimes cannabidiol (CBD) and cannabinol (CBN) are determined additionally. After repeated external contamination by cannabis smoke these analytes are known to be found in hair even after performing multiple washing steps. A widely accepted strategy to unequivocally prove active cannabis consumption is the analysis of hair extracts for the oxidative metabolite 11-nor-9-carboxy-THC (THC-COOH). Although the acidic nature of this metabolite suggests a lower rate of incorporation into the hair matrix compared to THC, it is not fully understood up to now why hair concentrations of THC-COOH are generally found to be much lower (mostly <10pg/mg) than the corresponding THC concentrations.Δ9-Tetrahydrocannabinolic acid A (THCA A) is the preliminary end product of the THC biosynthesis in the cannabis plant. Unlike THC it is non-psychoactive and can be regarded as a ‘precursor’ of THC being largely decarboxylated when heated or smoked. The presented work shows for the first time that THCA A is not only detectable in blood and urine of cannabis consumers but also in THC positive hair samples. A pilot experiment performed within this study showed that after oral intake of THCA A on a regular basis no relevant incorporation into hair occurred. It can be concluded that THCA A in hair almost exclusively derives from external contamination e.g. by side stream smoke. Elevated temperatures during the analytical procedure, particularly under alkaline conditions, can lead to decarboxylation of THCA A and accordingly increase THC concentrations in hair. Additionally, it has to be kept in mind that in hair samples tested positive for THCA A at least a part of the ‘non-artefact’ THC probably derives from external contamination as well, because in condensate of cannabis smoke both THC and THCA A are present in relevant amounts. External contamination by side stream smoke could therefore explain the great differences in THC and THC-COOH hair concentrations commonly found in cannabis users.

A tendency for re-offending in drug-facilitated crime

2010-02-01

Abstract: The authors present 3 cases that demonstrate a return to DFC following periods of inactivity. The offences occurred in Paris and its suburbs and in each of the cases there were two distinct periods of activity by the offenders with 2, 8 and 22 victims attributed to each of the perpetrators.To 20mg of decontaminated and cut hair, 100pg/mg of clonazepam-d4 was added as internal standard. Hair specimens were extracted with CH2Cl2/ether after incubation overnight at 56°C in pH 7.6 buffer. Extractions were performed on blood and urine using Toxi-tube A® with 5ng/mL of clonazepam-d4. The residues were analyzed by LC–ESI-MS/MS. Calibration curves in blood and urine (0.5–500ng/mL) were prepared by spiking aliquots of blank fluids (r2>0.9816 for all drugs). LOD in body fluids ranged 0.5–10ng/mL. Calibration curves in hair (0.5–100pg/mg) were prepared by spiking aliquots of blank hair (r2>0.9877 for all drugs). LOD in hair ranged 0.5–5pg/mg.Case #1: Two young women were raped with an interval of approximately 1 year between the incidents. Lorazepam (present, <2pg/mg) was detected in hair obtained from the first victim, and zolpidem (19pg/mg) in hair of the second one. The offender was in jail between the two offences. Case #2: The offender approached a total of 8 men and women who were aged over 50 years. The offender was in jail between the two series of respectively 3 and 5 victims. Zopiclone was detected in victims’ hair (n=7) at concentrations 13–42pg/mg. Case #3: The offender stole thousands of Euros using credit cards obtained from 22 different wealthy victims. He employed a cocktail of up to 6 drugs made up of: flunitrazepam, clonazepam, doxylamine, cyamemazine, zolpidem and lorazepam. Drugs were detected in all victims’ hair (n=18) at concentrations in the range 1–81pg/mg for all drugs. Between the two series (of respectively 4 and 16 victims) the offender spent 6 months in jail, and then police spent 6 months looking for him while he was under judiciary control prior to his judgment.Segmental hair analysis permits retrospective information on drug exposure and should be considered in the investigation of drug-facilitated crimes not only to prove single exposure but also when there has been any appreciable delay in samples being obtained for analysis. Indeed, in 56% cases reported in this paper, due to the long time that elapsed between offences and the opportunity to obtain samples for analysis hair analysis was considered the only viable matrix to investigate the possibility of drug involvement in the crimes.Our experience demonstrates that the incidence of re-offending in DFC after a period of inactivity (often due to imprisonment) may be of concern, notably in big cities.

High throughput analysis of drugs of abuse in hair by combining purposely designed sample extraction compatible with immunometric methods used for drug testing in urine

R. de la Torre, E. Civit, F. Svaizer, A. Lotti, M. Gottardi, M. Miozzo — 2010-01-18

Abstract: Drug testing in hair usually requires a rather complex sample treatment before drugs are amenable to analysis by either immunological and/or chromatographic coupled to mass spectrometry methods. Immunological methods applied are usually dedicated to hair analysis as analytes present in this matrix are not always the same present in urine. Comedical s.a.s. laboratories recently commercialized reagents (VMA-T) purposely designed for hair sample treatment which are compatible with current immunometric methods used for urine drug testing. This is possible as some analytes (6-MAM and cocaine) present in hair after sample treatment are converted to those detected in urine (morphine and benzoylecgonine). A correlation study for several drug classes performed in two laboratories with 32 clinical and 12 spiked drug free (controls) hair samples shows that implementation of the method on clinical chemistry analyzers is easy and that results obtained by different operators and instruments are comparable and reproducible. The main advantage of VMA-T method is the possibility to simultaneously extract from hair main drug classes, in a period of time lower than 2h and its compatibility with immunological methods applied in urine drug testing.

Exposure to psychoactive substances in women who request voluntary termination of pregnancy assessed by serum and hair testing

2010-01-08

Abstract: Drug abuse is a worldwide phenomenon with significant health and socioeconomic impact and it is of particular concern in women of reproductive age and in pregnant women. We aimed to investigate the prevalence of drug use by serum and hair testing in a cohort of pregnant women at 12th week gestation who decided voluntarily to interrupt their pregnancy and to investigate the relationship between drug exposure and induced abortions (IA), repeated IA and contraception. The study was conducted in an obstetrics clinic authorised to perform IA in Murcia, Spain during an 18 months period (2007–2009).Apart from serum and/or hair testing, the 142 women enrolled in the study completed a detailed questionnaire regarding drug, alcohol and tobacco use in the previous 3 months. Serum and hair samples were analyzed by gas chromatography mass spectrometry assays. Hair and serum samples showed a 30% overall positivity to drugs of abuse. Of these samples, 20.4, 14.1, 4.2 and 1.4% were positive for cannabinoids, cocaine, opiates, and MDMA, respectively, with polydrug use in 5.6% cases. In this cohort, a positive association was found between drug use and repeated IA. The results highlight the need for promoting pregnancy planning for young women in general, especially when consuming psychoactive substances as well as promote safe and accessible contraception in women of reproductive age. In women requesting IA, specific drug abuse counselling should be implemented.

Prenatal hair development: Implications for drug exposure determination

Joey Gareri, Gideon Koren — 2010-01-14

Abstract: Neonatal hair is a clinically important toxicological matrix, as it allows determination of in utero drug exposure. This paper serves to review the physiological development of the hair follicle and hair production during fetal life. An understanding of the mechanisms and timing of hair development in the prenatal period is critical to effectively assessing the time window of exposure determination associated with toxicological analysis of neonatal hair.

An assessment of cortisol analysis in hair and its clinical applications

R. Gow, S. Thomson, M. Rieder, S. Van Uum, G. Koren — 2010-01-22

Abstract: Hair analyses for exogenous compounds, specifically drugs of abuse, have been a useful tool in detecting long-term drug exposure. More recently, studies have delved into the exposure of endogenous compounds in hair.Cortisol is synthesized in the adrenal cortex in response to stress-induced activation of the hypothalamic–pituitary–adrenal (HPA) axis. While catecholamines generally indicate acute stress, cortisol can be used as an indicator for sub-acute and chronic stress.Studies on the effects of chronic stress are most often subjective in nature, relying on questionnaires asking the participant to recall on past stressors. This can lead to the issue of recall and reporting bias. A new objective measure of chronic stress is needed for a more accurate understanding of the effects of chronic stress on the body. This review uses emerging evidence to describe the usefulness of hair analysis for cortisol and discusses the current methods used.

Gas chromatography–mass spectrometry assay for the simultaneous quantification of drugs of abuse in human placenta at 12th week of gestation

2010-01-07

Abstract: We describe the development and validation of a method for the quantification of drugs of abuse, using gas chromatography–mass spectrometry (GC/MS), in human placenta. Concentration ranges covered were 5–500ng/g for amphetamine, methamphetamine, MDMA, methadone, cocaine, benzoylecgonine, cocaethylene, morphine, 11-nor-9-carboxy-delta-9-tetrahydrocannabinol, nicotine, and cotinine. Intra-assay and inter-assay imprecisions were less than 15.7% for lower quality control samples and less than 14.9% for medium and high quality control samples. Recovery range was 36.2–83.7%. Placenta samples were kept at −80°C until analysis; analytes were stable after three freeze–thaw cycles (samples stored at −20°C). This accurate and precise assay has sufficient sensitivity and specificity for the analysis of specimens collected from women who voluntarily terminated their pregnancy at 12th week of gestation. The method has proven to be robust and accurate for the quantification of the principal recreational drugs of abuse in this period of the prenatal life. This is the first report that highlights the presence of drugs of abuse during the first trimester of gestation.

Rapid and simple determination of psychotropic phenylalkylamine derivatives in human hair by gas chromatography–mass spectrometry using micro-pulverized extraction

Jin Young Kim, Soon Ho Shin, Jae Il Lee, Moon Kyo In — 2010-01-08

Abstract: A gas chromatography–mass spectrometric (GC–MS) method was developed and validated for the determination of five psychotropic phenylalkylamine derivatives (amphetamine, AP; methamphetamine, MA; 3,4-methylenedioxyamphetamine, MDA; 3,4-methylenedioxymethamphetamine, MDMA; norketamine, NKT) in human hair. Hair samples (10mg) were washed with distilled water and acetone, mechanically pulverized for 1.5min with a bead mill, and then incubated in 1mL of methanol under ultrasonication at 50°C for 1h. The resulting solutions were evaporated to dryness, derivatized using heptafluorobutyric anhydride (HFBA) at 50°C for 30min, and analyzed by GC–MS. The linear ranges were 0.1–20.0ng/mg for AP and MA and 0.05–20.0ng/mg for MDA, MDMA, and NKT, with the coefficients of determination (r2>0.9982). The intra-day and inter-day precisions were within 11.5% and 12.8%, respectively. The intra-day and inter-day accuracies were −4.1% to 5.8% and −6.6% to 4.2%, respectively. The limits of detections (LODs) for each compound were lower than 0.028ng/mg. The recoveries were in the range of 78.9–101.2%. Based on these results, the method proved to be effective for the rapid and simple determination of phenylalkylamine derivatives in hair specimens.

Interpretation of hair findings in children after methadone poisoning

Pascal Kintz, Julie Evans, Marion Villain, Vincent Cirimele — 2010-01-08

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°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:The following concentrations were obtained from the children who had died following a methadone overdose: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.

Hair analysis for drugs in driver's license regranting. A Swedish pilot study

Robert Kronstrand, Ingrid Nyström, Malin Forsman, Kerstin Käll — 2010-01-08

Abstract: When being convicted for petty drug offence or driving under the influence of drugs in Sweden, the driving license may be suspended. To regain the license, the person has to prove that he or she has been drug free during an observation period. This is controlled by urine samples taken at several occasions. However, the risk of manipulation and the risk of false negative urine samples are high. In addition, many people find it difficult or embarrassing to urinate when observed. Hair sampling might therefore be a welcome option to this procedure, with its easy sampling and minimal risk of manipulation. The longer detection window may also provide better information to the physician. The aim of this work was to evaluate if clients preferred hair samples to urine and to investigate practical and interpretive problems or advantages with hair samples. Ninety-nine hair samples and 198 urine samples were collected from 84 clients during the 12 month study period. Hair samples were divided into either one segment (0–3cm) or two segments (0–3 and 3–6cm) depending on the length. The hair samples were screened with LC–MS–MS for 20 drugs and confirmation of positive results were performed with GC–MS or LC–MS–MS. The results were compared to urine samples taken at two occasions during the observation period. To cover the timeframe of the urine samples hair was collected 2 weeks after the second sample. The urine samples were analysed with immunochemical screening and positive results confirmed with GC–MS or LC–MS–MS. Seventy-four clients presented with negative results in both urine and hair. Hair analysis identified illegal drugs at seven different occasions whereas urine failed to identify any illegal drugs. However the thresholds used may still be too high to find sporadic use as clients that admitted to use drugs sporadically presented with drug concentrations lower than the agreed thresholds but above the limit of detection. This implicates that the physician must have an understanding and knowledge of the limitations of the screening methods used. Another important outcome was that the clients approved of hair sampling considering it a better means to prove their drug abstinence. In addition, both the clients and the clinicians thought hair sampling easier than urine sampling. We believe that hair analysis can offer several advantages compared to urine analysis for clinicians working with driving license regranting.

Pharmacokinetics of methylphenidate in oral fluid and sweat of a pediatric subject

2010-01-22

Abstract: Methylphenidate (MPH) is a stimulant medication widely used for treating attention-deficit hyperactivity disorder (ADHD) in children and adolescents. Therapeutic monitoring for this drug is essentially lacking and alternative biological matrices, such as oral fluid and sweat, should be investigated for noninvasive assessment of short- and long-term history of drug use. We report the excretion profile of MHP and its metabolite ritalinic acid (RA) in oral fluid and sweat from a 12-year-old boy treated with the extended release drug formulation.Concentrations of MPH and RA in oral fluid, sweat and plasma were measured by liquid chromatography–mass spectrometry. Oral fluid-to-plasma ratio at each time interval was calculated at the start of the treatment and correlated with salivary pH. Excretion of MPH in sweat patches, collected up to 24h with PharmChek patches was also investigated.MPH and RA were both detected in oral fluid with a pharmacokinetic profile similar to that in plasma. Oral fluid peak concentrations of MPH ranged between 13.5 and 30.9ng/mL at 3.0h after drug intake. Oral fluid-to-plasma MPH ratio between 13.1 and 3.2 demonstrated an accumulation of the drug in oral fluid. Conversely, RA was found in oral fluid at peak concentration (23.4–62.9ng/mL) equivalent to one-tenth of those found in plasma. Concentration profiles of MPH and RA in oral fluid were quite constant during the four weeks of drug administration.In sweat, MPH was detected for the first time at 5h after drug administration (range: 9.3–11.2ng/patch) up to 24h (range: 29.8–38.7ng/patch). RA was not detected in the sweat patches during the 24h time of collection.The results suggest that measurement of MPH in oral fluid can be used as a potential alternative to drug monitoring in plasma. Moreover, MPH measurement in sweat patches can be used for noninvasive monitoring of MPH consumption and misuse in situations where detection of recent abuse is of interest.

Solid-phase microextraction for the detection of codeine, morphine and 6-monoacetylmorphine in human hair by gas chromatography–mass spectrometry

M. Moller, K. Aleksa, P. Walasek, T. Karaskov, G. Koren — 2010-01-18

Abstract: Introduction: Opiate hair analysis continues to prove difficult due to the scarcity of hair sample and low drug concentrations. For this reason, we developed a sensitive method utilizing headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography–mass spectrometry (GC–MS) for the detection of three principle opiates; codeine, morphine, and 6-acetylmorphine.Methods: Experimental conditions for HS-SPME and GC–MS were systematically optimized to produce the sensitive analytical method reported. Briefly, opiates were extracted from adult hair with methanol under agitation. The methanolic extract was then decanted into SPME autosampler vials, where deuterated standards of each of the 3 opiates were added at a concentration of 2ng/mg. Samples were dried under N2, derivatized, and subjected to HS-SPME coupled with GC/MS for analysis.Results: Preliminary datum for this study indicates detection limits for these 3 opiates are superior to that reported in the literature; an LOQ of 0.01ng/mg for morphine and 6-acetylmorphine and 0.005ng/mg for codeine. Linearity was evident between 0.01ng/mg and 5ng/mg for each opiate, with R2 above 0.992. The robustness of the method was demonstrated to be acceptable as inter-day and intra-day precision fell below 15% for each opiate analyzed.Conclusion: Compared with conventional methods, this method of detection for opiates is fast, simple, and accurate, with the sensitivity and specificity required in forensic and clinical toxicology.

Semi-quantitative analysis of drugs of abuse, including tetrahydrocannabinol in hair using aqueous extraction and immunoassay

Cynthia Coulter, James Tuyay, Margaux Taruc, Christine Moore — 2010-01-18

Abstract: A semi-quantitative analytical screening procedure for the determination of cocaine, amphetamines, opiates, and delta-9-tetrahydrocannabinol in hair has been developed. The procedure employs an aqueous extraction buffer, uses only 10mg of hair, requires 2h of incubation for the extraction to occur, and multiple drug classes can be screened using enzyme linked immunosorbent assays. Hair calibration standards were prepared around the recommended cut-off concentrations of the Society of Hair Testing. All drug classes showed excellent linearity over the concentration range tested, indicating that immunochemical screening can be used in a semi-quantitative mode for hair analysis using an aqueous buffer, rapid extraction and a small amount of hair.

Ethyl glucuronide and ethyl sulfate in meconium and hair-potential biomarkers of intrauterine exposure to ethanol

2010-01-08

Abstract: This study investigated ethyl glucuronide (EtG) and ethyl sulfate (EtS) concentration in meconium and in maternal and neonatal hair (HEtG and HFAEEs, respectively) as potential markers of intrauterine exposure to ethanol together with meconium fatty acid ethyl esters (FAEEs) in a cohort of 99 mother–infant dyads, 49 coming from the Arcispedale of Reggio Emilia (Italy) and 50 from the Hospital del Mar of Barcelona (Spain). FAEEs, EtG and EtS were measured in meconium samples using liquid chromatography–tandem mass spectrometry. A head space-solid phase microextraction-gas chromatography–mass spectrometry was used to test HEtG and HFAEEs in hair samples from mothers and their newborns. Eighty-two meconium samples (82.8%) tested positive for EtG, 19 (19.2%) for EtS while 22 (22.2%) showed FAEEs levels higher than 2nmol/g, the cut-off used to differentiate daily maternal ethanol consumption during pregnancy from occasional or no use. Although EtG and EtS in meconium did not correlate with total FAEEs concentration, a good correlation between EtG, EtS and ethyl stearate was observed. Moreover, EtG correlated well with ethyl palmitoleate, while EtS with ethyl laurate, myristate and linolenate. Neither maternal nor neonatal hair appears as good predictors of gestational ethanol consumption and subsequent fetal exposure in these mother–infant dyads. In conclusion, these data show that meconium is so far the best matrix in evaluating intrauterine exposure to ethanol, with EtG and EtS being potentially good alternative biomarkers to FAEEs.

11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic acid ethyl ester (THC-COOEt): Unsuccessful search for a marker of combined cannabis and alcohol consumption

2010-01-14

Abstract: 11-Nor-Δ9-tetrahydrocannabinol-9-carboxylic acid ethyl ester (THC-COOEt) can be presumed to be a mixed metabolite formed during combined consumption of cannabinoids and alcohol. In order to examine this hypothesis, THC-COOEt and its deuterated analogue D3-THC-COOEt were synthesized as reference substance and internal standard from the corresponding carboxylic acids and diazoethane and methods were developed for the sensitive detection of THC-COOEt in plasma and hair based on gas chromatography–electron impact mass spectrometry after silylation with N-methyl-N-tert-butyldimethylsilyl-trifluoroacetamide and gas chromatography–negative chemical ionization mass spectrometry (GC-NCI-MS) as well as tandem mass spectrometry (GC-NCI-MS-MS) after derivatization with pentafluoropropionyl anhydride. The methods were applied for THC-COOEt determination to plasma samples from 22 drunk driving cases which contained both ethanol (0.30–2.16mg/g) and THC-COOH (15–252ng/mL) as well as to 12 hair samples from drug fatalities which were both positive for THC (0.09–2.04ng/mg) and fatty acid ethyl esters as markers of chronic alcohol abuse (0.70–6.3ng/mg). In none of these samples THC-COOEt could be found with limits of detection of 0.3ng/mL in plasma and 2pg/mg in hair in 11 samples using GC-NCI-MS and 0.2pg/mg in one sample using GC-NCI-MS. Therefore, the use of this compound as a marker for combined cannabis and alcohol consumption could not be achieved.

Simultaneous screening and quantification of 52 common pharmaceuticals and drugs of abuse in hair using UPLC–TOF-MS

2010-01-11

Abstract: An UPLC–TOF-MS method for simultaneous screening and quantification of 52 drugs in hair was developed and validated. The selected drugs represent the most common classes of pharmaceuticals and drugs of abuse such as amphetamines, analgesics, antidepressants, antipsychotics, benzodiazepines, cocaine, ketamine and opioids. Hair samples were extracted with methanol:acetonitrile:ammonium formate (2mM, 8% acetonitrile, pH 5.3) overnight at 37°C. The target drugs were separated and quantified using a Waters ACQUITY UPLC coupled to a Waters Micromass LCT Premier XE Time-of-Flight mass spectrometer. Total chromatographic run time was 17min. The data were treated with the MassLynx software ChromaLynx XS and QuanLynx for automated identification and quantification, respectively. The limits of detection ranged from 0.01 to 0.10ng/mg using a 10-mg hair sample and the limit of quantification was 0.05ng/mg for 87% of the analytes. A good linear behaviour was achieved for most of the analytes in the range from LOQ to 10 or 25ng/mg except for the amphetamines. The method showed an acceptable precision and trueness, since the obtained CV and BIAS values were ≤25% for 81% of the analytes. The extraction recoveries for 92% of the analytes ranged between 84 and 106% and the extraction recoveries for all analytes were better than 60%. The method was applied to 15 autopsy hair samples from forensic investigations showing a wide abuse pattern of many pharmaceuticals and drugs of abuse within a period of less than three months. The present study demonstrated that the combination of accurate mass and retention time can provide good selectivity, which demonstrates that the TOF instrument is adequate for both screening and quantification purposes. Furthermore, it was shown that screening with the ChromaLynx XS software is less sensitive and selective for some analytes than the QuanLynx software, especially in low concentrations.

Changes in antidepressant metabolism in pregnancy evidenced by metabolic ratios in hair: A novel approach

Lisa O’Brien, Carina Baumer, Detlef Thieme, Hans Sachs, Gideon Koren — 2010-01-11

Abstract: Background: Depression and other psychiatric illnesses are common during pregnancy and are often treated with antidepressants. Physiological changes of pregnancy may alter the pharmacokinetics of medications and ultimately affect the dose required to maintain effective therapy. Human hair offers a safe, non-invasive way to monitor long term systemic exposures to medications.Objective: To determine whether the ratio of hair antidepressant: major metabolite differed when early and late pregnancy was compared to the postpartum period.Methods: Segmental analyses using liquid chromatography–mass spectrometry–mass spectrometry were performed on hair samples. The mean concentration of parent compound and metabolite was found for each trimester and the postpartum period.Results: Twelve women provided hair samples of which nine samples were long enough to analyze the first and third trimesters along with the postpartum period. Citalopram, venlafaxine, fluoxetine and sertraline were the antidepressants studied. In the citalopram group, a statistically significant difference existed between the citalopram:norcitalopram ratio when the first trimester was compared to the postpartum period (0.89±0.26 versus 1.4±0.24 respectively, p=0.022). A statistically significant difference also existed between the third trimester and the postpartum period for the citalopram group (0.9±0.14 and 1.4±0.24 respectively, p=0.048). No other statistically significant differences were found.Conclusion: It is important that variations in drug metabolism during pregnancy be considered as these changes may necessitate a dosage adjustment to ensure that therapeutic failure does not occur during pregnancy.

Assessment of exposure to environmental tobacco smoke in young adolescents following implementation of smoke-free policy in Italy

2010-01-08

Abstract: We investigated acute and chronic exposure to environmental tobacco smoke (ETS) in a cohort of young adolescents using urinary cotinine and hair nicotine testing after recent implementation of Italian smoke free legislation.Study subjects were 372 Italian young adolescents, between 10 and 16 years of age from the principal city of Sicily, Palermo. Urine and hair samples were collected between November 2005 and May 2006, when the legislation to ban smoking in all the enclosed places of employment (including bars, restaurants, pubs) was completely enforced. An exhaustive questionnaire including sociodemographic characteristics and active and passive exposure to cigarette smoking was completed. Urinary cotinine was analyzed by radioimmunoassay and hair nicotine by a validated GC/MS method.Based on urinary cotinine results, 2.1% and 89% of the study participants, respectively, showed non-exposure and low acute exposure to ETS, whereas only 1.6% presented very high exposure or a hidden active smoking habit in the recent past. Hair nicotine disclosed non-exposure and low exposure to ETS in 11.8% and 65.6% of the young adolescents, respectively, taking into consideration a larger time-window. High repeated exposure, suggesting active smoking in some cases was observed in 8.6% of the study subjects. Hair nicotine was inversely related to educational level of the adolescents’ parents.Overall, due to the implementation of smoke-free legislation and information campaign against smoking, a significant trend toward low exposure to ETS was observed in this study cohort with no association between exposure to ETS and respiratory illnesses.

Combined use of fatty acid ethyl esters and ethyl glucuronide in hair for diagnosis of alcohol abuse: Interpretation and advantages

F. Pragst, M. Rothe, B. Moench, M. Hastedt, S. Herre, D. Simmert — 2010-01-11

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 AUCEtOH, despite large inter-individual variations. It is demonstrated by calculation of AUCEtOH on monthly basis for moderate, risky and heavy drinking that AUCEtOH 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.

Fatty acid ethyl ester concentrations in hair and self-reported alcohol consumption in 644 cases from different origin

Silke Süße, Carl M. Selavka, Tom Mieczkowski, Fritz Pragst — 2010-01-11

Abstract: For diagnosis of chronic alcohol abuse, fatty acid ethyl esters (FAEE) were determined in hair samples from 644 individuals, mainly parents from child protection cases. The analysis for ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate was performed according to a validated procedure consisting of external degreasing by two times washing with n-heptane, extraction with a mixture of dimethylsulfoxide and n-heptane, separation and evaporation of the n-heptane layer, headspace solid phase microextraction of the residue after addition of phosphate buffer pH 7.6 and gas chromatography–mass spectrometry using deuterated internal standards. For interpretation, the sum of the concentrations of the four esters CFAEE was used with the cut-off's 0.5ng/mg for the proximal scalp hair segment 0–3cm or less and 1.0ng/mg for scalp hair samples with a length between 3 and 6cm and for body hair.CFAEE ranged from 0.11 to 31ng/mg (mean 1.77ng/mg, median 0.82ng/mg). The mean concentration ratio between the 4 esters was 8:45:38:9. 298 cases had CFAEE above the cut-off's. Self-reported drinking data were obtained in 553 of the cases in the categories abstinent (156 cases), moderate drinking (252 cases) and excessive drinking (145 cases). Median and box-plot data clearly demonstrate differentiation of these ingestor sub-populations by CFAEE. However, in the abstinent and moderate groups the consumption was frequently underreported (37 and 110 cases positive) whereas in the group self-reported excessive drinking 32 cases were negative. Comparison of CFAEE with carbohydrate-deficient transferrin (CDT) in 139 cases and gamma-glutamyltransferase (GGT) in 136 cases showed a good agreement in CDT- and GGT positive cases (27/28 and 32/41) but a large portion of the negative CDT- and GGT-results with positive hair test (44/100 and 48/95) which is explained mainly by the much shorter time window of CDT and GGT.No significant correlation was found between persons weight and CFAEE showing that the test is not biased against physical fitness or obesity. Furthermore, there was no statistically significant difference between scalp hair (541 samples) and hair from other body sites (84 samples).In conclusion, FAEE in hair appeared to be suitable markers for the detection of excessive drinking. However, as there is no proportionality between drinking amount and CFAEE, the additional use of other markers can increase the reliability of the interpretation.

Buprenorphine detection in hair samples by immunometric screening test: Preliminary experience

Fiorenza Svaizer, Andrea Lotti, Massimo Gottardi, Maria Pia Miozzo — 2010-01-18

Abstract: The recent introduction of buprenorphine use by the Drug Addiction Services has induced toxicology laboratories to develop new qualitative or semiquantitative screening assay for its determination in hair samples. The aim of this preliminary study was to verify the correlation between the buprenorphine intake and the immunometric screening test results (VMA-T Comedical and buprenorphine CEDIA/Thermo-Fisher/Microgenics reagents) and therefore their comparison with the liquid chromatography coupled with mass spectrometry (LC/MS) results. Hair samples were obtained from 32 subjects without buprenorphine-therapy reported and 17 in treatment. In glass test tube with hermetic cap were weighed 33mg of 49 finely cut hair samples, washed with 1mL of SLV-VMA-T washing solution, which is then completely sucked and eliminated. The samples were extracted with 400μL of VMA-T reagent for an hour at 100°C. The extracts were analysed by immunometric screening test on ILab 650 chemistry analyser, using buprenorphine CEDIA reagent assay. From the 32 non-takers of drug, 30 semiquantitative results were less than 10pg/mg and 2 were over 10pg/mg; from the 17 subjects with therapy, all were over 10pg/mg (range 13–50pg/mg); no samples were false-negative. Results suggest that exist a good relationship between the administration of buprenorphine and its concentration in hair, detectable through this method and reagents line.

Ethyl glucuronide determination in meconium and hair by hydrophilic interaction liquid chromatography–tandem mass spectrometry

2010-01-11

Abstract: Ethyl glucuronide (EtG) detection in non-conventional matrices, such as hair and meconium, can provide useful information on alcohol abuse over a long time frame, for example during pregnancy or after a withdrawal treatment. This study reports on the development, validation and application of a new hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method for the analysis of EtG in meconium and hair. For each matrix, the sample preparation and the chromatographic separation were thoroughly optimised. Additionally, experiments with reversed-phase liquid chromatography were also performed in the development stages. Analyses were carried out using a Phenomenex Luna HILIC column (150mm×3mm, 5μm) and a mobile phase composed by ammonium acetate 2mM and acetonitrile, in gradient. Different SPE cartridges (Oasis MAX, Oasis WAX, aminopropyl silica) and solvents were tested in order to obtain the highest recoveries and cleanest extracts. Optimal results were obtained for meconium with aminopropyl cartridges, while for hair an incubation of 16h with 2mL of water and acetonitrile (50/50, v/v) provided good results. The analytical method was validated for both matrices (meconium and hair) by assessing linearity, precision, accuracy, recovery and limit of quantification. The calibration curve concentrations ranged from 50 to 1200pg/mg for meconium and from 20 to 1000pg/mg for hair. Real meconium and hair samples were analyzed and results were consistent with literature.

Heroin markers in hair of a narcotic police officer: Active or passive exposure?

Marion Villain, Jean-François Muller, Pascal Kintz — 2010-01-22

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.