Forensic Science International

Editorial Board

2012-05-10

Editorial

Pascal Kintz — 2011-10-24

In modern clinical and forensic toxicology, analytical procedures must not only be sensitive but also highly specific, because in most cases the analytes are not known in advance and many other xenobiotics or endogenous biomolecules may interfere with their detection. The particular task of analytical toxicology is to analyze complex biological matrices, such as hair. Appropriate sample preparation prior to instrumental analysis is therefore a key step.

Consensus of the Society of Hair Testing on hair testing for chronic excessive alcohol consumption 2011

Pascal Kintz — 2011-10-24

Can ethyl glucuronide in hair be determined only in 3cm hair strands?

Ronald Agius, Liliane Martins Ferreira, Michel Yegles — 2011-10-24

Abstract: This paper addresses the suitability of ethyl glucuronide in hair (EtGH) strands other than 3cm for alcohol consumption. This issue will be addressed (a) by statistically comparing the distribution of EtGH results for 3cm hair strands to other hair strands analysed from 4126 cases and (b) by examining the stability of EtGH in an 8cm hair strand and two 12cm hair samples of two volunteers and a post-mortem case using 1cm segmental analysis. For 3464 driving license re-granting Medical and Psychological Assessment (MPA) cases, the detection of alcohol consumption using hair lengths longer than 3cm was never significantly less than for 3cm hair lengths, even up to 12cm hair lengths analysed non-segmented. For 662 non-MPA cases, where, in contrast to MPA cases, generally no abstinence was required, an increase in the EtGH positivity rate was observed with increasing hair length analysed up to 9cm, indicating that EtG-washout effects seem to play a minor role if any. For both MPA and non-MPA hair samples less than 3cm, a drastic, significant increase in the number of positive EtGH samples were observed, compared to 3cm hair lengths, strongly supportive of EtGH incorporation from sweat after a recent alcohol consumption. Segmental studies indicated that EtG is stable in the hair matrix up to 12cm long, hence supporting the above results. Even though both the statistical and the stability studies are preliminary results which need to be confirmed by other studies, they both provide evidence for the determination of alcohol consumption using EtGH in hair lengths longer than 3cm. Amendments to the Consensus of the Society of Hair Testing, the German driving license re-granting guidelines and EWDTS hair guidelines with respect to testing for abstinence and/or alcoholism are proposed for the benefit of the donors.

Ethyl glucuronide in hair – A highly effective test for the monitoring of alcohol consumption

Ronald Agius, Thomas Nadulski, Hans-Gerhard Kahl, Bertin Dufaux — 2011-10-24

Abstract: In Germany drink driving offenders lose their license and must prove abstinence for one year in order to regain it. In this paper we assess the newly introduced ethyl glucuronide (EtG) tests in urine and hair in this alcohol abstinence monitoring. 20% (80 out of 386) of the 3cm long hair samples were tested positive for EtG in hair, compared to only 2% (92 out of 4248 samples) in urine in the same time period. Additionally 50% of the samples positive for EtG in hair had EtG values greater than 30pg/mg hair, indicating chronic alcohol consumption in the last three months. This study shows that four EtG tests in 3cm hair lengths reveal a significantly higher percentage of drink driving offenders who fail to be sober in the rehabilitation period, than do six random EtG tests in urine. Presumably, the hair test is more adequate to monitor long term alcohol abstinence than the urine test as defined by the new driving license re-granting medical and psychological assessment (MPA) in Germany.

Automated fast procedure for the simultaneous extraction of hair sample performed with an automated workstation

I. Angeli, M. Minoli, A. Ravelli, F. Gigli, F. Lodi — 2011-10-31

Abstract: Introduction: Hair testing has a leading role in toxicology practice and even more in those aspects tightly linked to the assessment of psychoactive drug use and abuse in social life.Aim: The objective of the present study was to develop and validate an automated SPE sample-preparation step, suited for GC/MS confirmation analysis of basic drugs in hair drug control. The method was studied and optimized for quantitative determination and in a second time it was extended to real hair samples.The purpose of method validation was to ensure good reliability, reproducibility and quickness.Methods: Janus Automated Workstation (PerkinElmer) was employed to perform SPE hair extraction, using 96-well plate SPEC MP1 acquired from Varian (Agilent Technologies). After derivatization of dried extracts, screening confirmations were performed using gas chromatography (GC) followed by mass spectrometry (MS). GC/MS data were validated following standard guidelines, but our attention was focused on three headings: samples cross-contamination, “memory effect” and extraction recovery.Results: Validation requests were fully accomplished and we always obtained best results with the automated procedure. For instance, analytes mean recovery was between 70 and 90% and data analysis proved that no contamination between samples occurred.Conclusions: The automated workstation has shown good reliability (cross contamination and “memory effect” were tested and excluded), effectiveness (no false negative was detected), solvent saving (500μL/sample vs traditionally LLE 4mL/sample) and quickness (50min for 96 tests cycle).

Society of Hair Testing guidelines for drug testing in hair

Gail A.A. Cooper, Robert Kronstrand, Pascal Kintz — 2011-11-16

Abstract: The Society of Hair Testing (SoHT) Guidelines for Drug Testing in Hair provide laboratories with recommended best practice guidelines whether they are currently offering drug testing in hair, or plan to offer a hair testing service in the future. The guidelines include reference to recommended sample collection and storage procedures, through sample preparation, pre-treatment and analysis and the use of cut-offs.

LSD in pubic hair in a fatality

2011-10-24

Abstract: Lysergic acid diethylamide (LSD) is a potent hallucinogen, active at very low dosage and its determination in body fluids in a forensic context may present some difficulties, even more so in hair. A dedicated liquid chromatography–electrospray-tandem mass spectrometry (LC–ES-MS/MS) assay in hair was used to document the case of a 24-year-old man found dead after a party. Briefly, after a decontamination step, a 50mg sample of the victim's pubic hair was cut into small pieces (<1mm length), and incubated overnight in 3mL of phosphate buffer pH 5 at room temperature. After a liquid–liquid extraction (dichloromethane/ether), the extract was analyzed using a LC–ES-MS/MS method exhibiting a limit of quantification of 0.5pg/mg for LSD. A LSD concentration of 0.66pg/mg of pubic hair was observed. However, this result remains difficult to interpret owing to the concomitant LSD presence in the victim's post mortem blood and urine, the lack of previously reported LSD concentrations in hair, and the absence of data about LSD incorporation and stability in pubic hair.

Value of the concept of minimal detectable dosage in human hair

Pascal Kintz — 2011-10-24

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.

Simultaneous detection of seventeen drugs of abuse and metabolites in hair using solid phase micro extraction (SPME) with GC/MS

2011-11-03

Abstract: Introduction: The analysis of pediatric and adult hair is a useful non-invasive biomarker to effectively detect long term exposure to various xenobiotics, specifically drugs of abuse such as cocaine, opiates and amphetamines. Very often individuals are using, or are exposed to multiple drugs simultaneously and therefore it is important to be able to detect them in the same analysis. We have developed a sensitive and specific solid phase micro extraction (SPME) coupled with gas chromatography mass spectrometry (GC/MS) to detect 17 different analytes in hair using a single extraction method.Method: Five milligrams of hair is extracted overnight, subjected to solid phase extraction (SPE) and then to SPME-GC/MS. The aimed analytes include amphetamine, methamphetamine, MDA, MDMA, cocaine, benzoylecognine, norcocaine, cocaethylene, methadone, codeine, morphine, 6-AM, oxycodone, oxymorphone, hydrocodone, hydromorphone and meperidone.Results: The following are the LOD of the various drugs: 0.2ng/mg hair for amphetamine, methamphetamine, MDA, MDMA, morphine, codeine, 6-AM, oxycodone, oxymorphone, hydromorphone, hydrocodone, meperidine and 0.13ng/mg hair for cocaine, benzoylecognine, cocaethylene, norcocaine and methadone.Conclusion: This GC/MS method is sensitive and specific to detect the presence of these 17 analytes in as little as 5mg of hair and is especially useful for newborn and child hair analysis where the amount of hair is often very limited.

Detection of polybrominated biphenyl ethers (PBDEs) in pediatric hair as a tool for determining in utero exposure

Katarina Aleksa, Amanda Carnevale, Cynthia Goodyer, Gideon Koren — 2011-11-14

Abstract: Introduction: Cryptorchidism, or undescended/maldescended testis, is the most common birth defect of male genitalia. Its prevalence has been increasing over the past few decades. This may be due to an increase in the prevalence of anti-androgenic chemicals such as polychlorinated biphenyls, organochloride pesticides, plasticizers and fungicides. A newer group of chemicals, brominated flame retardants (BFRs), are being implicated as endocrine-disrupting chemicals. These chemicals are used worldwide in polymers that are incorporated into a variety of consumer products (e.g., textile, computers and televisions, insulating foam, electrical equipment and kitchen appliances). In order to quantify BFRs we introduce the use of hair levels of polybrominated diphenyl esters (PBDEs) as biomarkers of systemic exposure. This approach will allow for the estimation of in utero BFR exposure, in the process of evaluating the potential link between the incidence of cryptorchidism in newborn males and level of exposure of the pregnant mother to environmentally relevant BFRs. For that end we have developed a GC/MS assay in which children's hair is analyzed for the presence of polybrominated biphenyl ethers (PBDEs).Methods: In this pilot, 10–40mg of hair from 24 children (12 newborn and 12 from children 1 to 15 years) was extracted overnight at 40°C with 4N HCl and hexane (4:1). The samples were eluted from 2g NaSO4:2g Florisil SPE columns with 8mL hexane. Dried samples are reconstituted with anhydrous isooctane and injected onto a GC/MS and analyzed for BDE-28, BDE-47, BDE-99, BDE-100, BDE-153, BDE-154, BDE-183 and BDE-209.Results: PBDEs were detected in all of the newborn and child hair. The ΣPBDE ranged from 0.038 to 1.01pg/mg newborn hair and from 0.208 to 2.695ng/mg child hair. The most abundant PBDE in newborn hair was BDE-153 while in child hair the variable PBDEs were BDE-47 and BDE-99. The highest molecular weight congener BDE-209 was detected in 10/24 pediatric hair samples. The LOQ is 0.0625pg/mg (BDE-209 0.625pg/mg) and the efficiency of extraction was between 70 and 90%.Conclusion: This GC/MS method is sufficiently sensitive to detect the presence of all 8 PBDE congeners tested in as little as 10mg of pediatric hair. The results show that PBDEs are present in newborn hair, making this matrix useful in examining in utero exposure to PBDEs and linking it to cryptorchidism.

Evidence of mephedrone chronic abuse through hair analysis using GC/MS

M. Martin, J.F. Muller, K. Turner, M. Duez, V. Cirimele — 2011-11-01

Abstract: Mephedrone is a synthetic derivative of cathinone which is becoming more common on the recreational drug market. Several intoxications following mephedrone abuse have been reported though published papers have focused essentially on analytical approaches for biological fluids and only one has involved a hair sample. After the development and validation of a new method, the first series of positive results for mephedrone in hair specimens is reported here.After decontamination of the hair strand in methylene chloride, hair segments were cut into small pieces with scissors, weighed and incubated overnight in Soerensen buffer pH 7.0 in the presence of deuterated 3,4-methylenedioxymethamphetamine (MDMA) at 40°C. The incubation medium was extracted using ethyl acetate after alkalinisation with 1N sodium hydroxide (NaOH). Before injection, the dry extract was derivatized using a mixture of heptafluorobutyric anhydride/ethyl acetate (100:50, v/v), evaporated and dissolved in ethyl acetate (25μl). After introduction of 1μl of the extract onto a splitless injector, chromatographic separation was achieved on a HP 6890 gas chromatograph equipped with a 5-MS capillary column. Detection was achieved in single ion monitoring mode (m/z 254–119–210 for mephedrone, m/z 258–213 for MDMA-d5) using a 5973 MSD operating in electron impact mode.Sixty-seven hair specimens were tested for mephedrone. Thirteen of them were found positive for mephedrone with concentrations ranging from 0.2 to 313.2ng/mg with a mean concentration of 26.8ng/mg. It was difficult to compare our findings due to a lack of reference data, nevertheless mephedrone seems well incorporated into hair (concentrations in the ng/mg range) like other stimulant drugs such as amphetamines or cocaine.The aim of this work was to develop a specific and accurate method for mephedrone analysis in hair specimens and its application to a large number of samples (n=67). The developed analytical method appears sensitive enough to reveal occasional to regular use of mephedrone.

Detection and quantification of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair by GC/MS/MS in Negative Chemical Ionization mode (NCI) with a simple and rapid liquid/liquid extraction

M. Minoli, I. Angeli, A. Ravelli, F. Gigli, F. Lodi — 2011-10-31

Abstract: Introduction: The fine detection of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in hair matrix remains one of the most important topics in hair analysis. This relevance lies in the necessity to obtain evidence of effective drug consumption and dispel any doubt of environmental contamination. THCCOOH is the highest and mainly represented Δ9-THC metabolite, but its concentration in hair is very low. A sensitive method for quantitative determination of THCCOOH in hair was developed. As first step, the method was tested with different SPE/LLE conditions, but the best results were obtained with a simple ad hoc LLE extraction. The final method was fully validated, evaluating parameters like extraction recovery, linearity, specificity and sensitivity. More than one hundred hair samples were then analyzed with the validated method. Data analysis was performed so as to determine respective concentrations of the metabolite and active molecule.Methods: Hair was washed and cut into small pieces (2–4mm). Samples (20–50mg) were spiked with deuterated internal standard (THC-d3 and THCCOOH-d3) and then hydrolyzed at 90°C in 1mL of 1M NaOH for 15min. THC was isolated by a LLE basic extraction with n-hexane:ethyl acetate (9:1). Next the aqueous solution was acidified (pH 4) adding concentrated acetic acid. THCCOOH was extracted with the same mixture. Dried extracts were derivatized with pentafluoropropionic anhydride and hexafluoroisopropanol and analyzed by GC/MS/MS (Agilent 7000B triple quadrupole) in NCI mode.Results: The linear range of THCCOOH is 0.1–5pg/mg, with good correlation coefficients (r2>0.9993). This method has great sensitivity (LOD 0.01pg/mg to LOQ 0.04pg/mg), high recovery, reproducibility and robustness.Conclusions: Based on these results, the method proved to be effective for the rapid determination of THC and THCCOOH in hair specimens.

Comparison of extraction procedures for benzodiazepines determination in hair by LC–MS/MS

Luca Morini, Claudia Vignali, Marina Polla, Andrea Sponta, Angelo Groppi — 2011-11-09

Abstract: Introduction: The use of a LC–MS/MS system for benzodiazepines detection remarkably increased the analytical sensitivity of these drugs in biological matrices, in particular in non-conventional ones such as hair. Since the amount of hair sample available for the analysis is frequently limited and, moreover, it needs to be checked for many other drugs and compounds of forensic interest, it is important to develop a sample preparation procedure able to detect either benzodiazepines and as many as possible other substances. The aim of this study was to compare the sensitivity of two different hair sample preparation procedures for benzodiazepines detection in hair.Methods: About 20mg hair, previously washed with organic solvent and cut into small pieces, were ultrasonicated with a phosphate buffer (pH 8.4) up to 1h and then extracted with dichloromethane/diethyl ether. The organic solvent was then dried under nitrogen flow and samples were reconstituted with 60μl methanol. Finally a 5μl aliquot was injected in the LC–MS/MS system. The second procedure consisted of an ultrasonication of hair samples in 700μl of methanol. Samples were then directly analyzed. Both the methods were fully validated.Results: Thirty-five compounds among benzodiazepines and their metabolites were screened using both the procedures. The methods fulfilled all the validation parameters and were applied on either spiked blank hair and real positive samples. While phosphate extraction allowed to reach a LOQ for almost all the substances ranging from 0.1 to 5pg/mg, thus guaranteeing to evaluate even a single dose administration (as confirmed by real positive cases) the sensitivity of the methanol extraction showed a LOQ ranging from 1 to 20pg/mg, still enough to assess a therapeutic use of almost all the benzodiazepines; yet the methanolic incubation allows a simple and rapid analytical procedure due to the direct injection of the extraction solvent.Conclusion: Even though a methanol extraction procedure for benzodiazepines determination is useful for forensic toxicological purposes also when a wider range of substances is needed and in case of a small amount of hair available, it is advisable to prefer a phosphate extraction when detection of a single dose administration is required.

Hair and urine testing to assess drugs of abuse consumption in couples undergoing assisted reproductive technology (ART)

2011-10-24

Abstract: For the first time in Europe hair and urine testing have been applied to assess drugs of abuse consumption in couples undergoing assisted reproductive technology and the eventual association of toxic habits with other lifestyle, health status and sociodemographic factors was also investigated.Couples attending five assisted reproduction centers in Rome were invited to join the study. When they presented at the Centre for the visit, they were asked to answer a structured questionnaire concerning sociodemographic characteristics and lifestyle habits, and at the same time to provide hair and urine samples.Hair and urine testing for drugs of abuse, urinary profile of principal endogenous steroids involved in fertility process (testosterone, epitestosterone, androsterone, etiocholanolone and dehydroepiandrosterone) and of alcohol and tobacco smoke biomarkers were performed with validated methodologies. Of the 594 enrolled individuals (297 couples), 352 (164 couples and 24 single individuals from the couple) completed the questionnaire and gave both hair and urine samples, apart from 3 bald men, who only gave urine samples. Urine testing showed an overall 4.8% (17 individuals) positivity to drugs of abuse: 4.2% to cannabinoids, 1.4% to cocaine and 0.85% to both drugs. Results of 4cm segment hair samples testing matched those from urine samples. Thus, taking together, results of urine and hair testing confirmed repeated use of cannabis, cocaine and both drugs in 3.7, 0.85 and 0.57% examined individuals, respectively. Drug consumers were in a statistically higher percentage active smokers and alcohol drinkers, less prone to physical activity and with a trend towards higher weight than non consumers. Finally, repeated drug consumption was associated with significant lower concentration of urinary testosterone in males and of urinary dehydroepiandrosterone in females. The findings of the present study confirm the suitability of urine testing to disclose recent drugs of abuse consumption and of hair analysis to verify repeated consumption. Association between different toxic habits and sedentary lifestyle is also substantiated by the obtained results in our cohort of couples attending assisted reproduction centers.

Development and validation of a liquid chromatography–tandem mass spectrometry assay for hair analysis of atomoxetine and its metabolites: Application in clinical practice

2011-10-24

Abstract: Atomoxetine (ATX) is a potent inhibitor of the noradrenaline reuptake transporter approved since 2002 for the treatment of attention-deficit/hyperactivity disorder (ADHD) in children, adolescents, and adults as alternative treatment to methylphenidate.A procedure based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed for the determination of ATX and its main metabolites (4-hydroxyatomoxetine – 4 hydroxyATX – and N-desmethylatomoxetine – des-methylATX) in hair of one treated child and five treated adolescents. Since hair samples can be easily collected without the need for specials skills and exposing a patient to discomfort, hair testing of ATX and eventually of its metabolites should be useful, especially in case of pediatric patients, to check compliance in a wider time-window. After addition of duloxetine as internal standard, hair samples were overnight digested with 2ml 1M NaOH at 45°C. Then, analytes were extracted from alkaline solution with two different 2ml aliquots of tert-butyl methyl ether. Chromatographic separation was achieved at ambient temperature using a reverse-phase column and a mobile phase of 40% of water–60% 5mM ammonium acetate, 50mM formic acid, 4mM trifluoroacetic acid in acetonitrile–water (85:15, v/v). The mass spectrometer was operated in positive ion mode using multiple reaction monitoring.The method was linear over the concentration range 0.2–50ng/mg hair for the all analytes under investigation, with an intra- and inter-assay imprecision and inaccuracy always less than 20% and an analytical recovery between 33.1% and 76.1%, depending on the considered analyte. Only ATX and 4-hydroxyATX were detected in hair samples with concentrations varying from 0.2 to 2.0ng/mg hair and from 0.3 to 1.0ng/mg, respectively.Notwithstanding the absence of any dose–hair concentration relationship, hair monitoring of ATX and concomitant medications commonly administrated in ADHD children and adolescents can be crucial in verifying long-term compliance to prescribed medication in individuals displaying a non negligible tendency to refuse drugs and to lie on the adherence to therapy as a specific symptom of the disease.

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

Sebastian Broecker, Sieglinde Herre, Fritz Pragst — 2011-10-31

Abstract: The retrospective investigation of the exposure to toxic substances by general unknown screening of hair is still a difficult task because of the large number of possible poisons, the low sample amount and the difficult sample matrix. In this study the use of liquid chromatography–hybrid quadrupole time-of-flight mass spectrometry (LC–QTOF-MS) was tested as a promising technique for this purpose. In the optimized procedure, 20mg hair were decontaminated with water and acetone and two times extracted by 18h incubation with 0.5ml of a mixture of methanol/acetonitrile/H2O/ammonium formate at 37°C. A mixture of deuterated standards from different drug groups was added for quantification and method control. The united extracts were evaporated to a residue of 0.5ml and 5μl were injected without clean-up for LC–QTOF-MS measurement (instrument Agilent 6530) with positive electrospray ionization and in data dependent acquisition mode. For peak identification the accurate mass data base and spectral library of the authors was used which contains accurate mass CID spectra of more than 2500 and theoretically calculated accurate mass data of more than 7500 toxicologically relevant substances. Validation at the example of 24 illegal drugs, their metabolites and benzodiazepines resulted in limits of detection of 0.003–0.015ng/mg, and limits of quantification of 0.006–0.021ng/mg with good accuracy and intra- and interday reproducibility. The matrix effect by ion suppression/enhancement was 72–107% for basic drugs and 42–75% for benzodiazepines. Yields of the hair extraction above 90% were determined for 59 drugs or metabolites. The method was applied to hair samples from 30 drug fatalities and from 60 death cases with known therapeutic drug intake at life time. Altogether 212 substances were identified with a frequency per drug of 1–40 (mean 4.2) and per case of 2–33 (mean 10.2), between them 35 illegal drug related substances and 154 therapeutic drugs. Comparison with the data known from case histories and from the analysis of blood, urine and gastric content showed only a low agreement, with many unexpected drugs detected and many reported drugs not detected in hair. Basic drugs and metabolites such as opioides, cocaine, amphetamines, several groups of antidepressants, neuroleptics, beta-blockers or the metamizole metabolite noramidopyrine were found with high frequency whereas acidic and several neutral drugs such as cannabinoids, salicylic acid, furosemide, barbiturates, phenprocoumone or cardiac glycosides could not be detected with sufficient sensitivity, mainly because of the low ion yield of positive ESI for these compounds. The advantage of a comprehensive acquisition of all substances is paid by a lower sensitivity in comparison to targeted screening LC–MS/MS procedures. In conclusion, the procedure of sample preparation and LC–QTOF-MS analysis proved to be a robust and sensitive routine method in which the qualitative screening for a wide variety of toxic substances in hair is combined with the quantitative determination of selected illegal drugs.

Practical experiences in application of hair fatty acid ethyl esters and ethyl glucuronide for detection of chronic alcohol abuse in forensic cases

2011-10-31

Abstract: This article presents results from 1872 hair samples, which were analyzed for fatty acid ethyl esters (FAEEs) and ethyl glucuronide (EtG). The results were evaluated in the context of self-reported drinking behavior, the use of hair cosmetics, the gender of the sample donors and hair sample length. For comparison, CDT and GGT in serum were available in 477 and 454 cases, respectively. A number of alcohol abstainers or low moderate drinkers and excessive drinkers were selected for assessment of cut-offs for FAEEs in the proximal 6cm hair segments and for EtG in the proximal 3cm hair segments. Cut-off values were assessed by ROC analysis. It was found that the cut-offs of 1.0ng/mg FAEE and 30pg/mg EtG presently used for excessive drinking lead to a low portion of false positives (4% and 3% respectively) but to a higher portion of false negatives (23% and 25% respectively). Comparison of the mean and medium concentrations in samples without any reported hair cosmetics (N=1079) and in samples with reported use of hair spray (N=79) showed an increase by the factor of about two for FAEE but no significant difference for EtG. Mean values of EtG were decreased by 80% in bleached samples (N=164) and by 63% in dyed samples (N=96). There was no significant effect of bleaching and dyeing on FAEE. Hair gel and hair wax, oil or grease showed no significant effect on both FAEE and EtG.With respect to gender and investigated hair length ambiguous results were obtained because of major differences in the compared subpopulations of male with higher alcohol consumption and mainly shorter hair, and less drinking female with longer hair. For excessive drinkers FAEEs in the 0–6cm hair segment and EtG in the 0–3cm segment decreased with increasing time of reported abstinence before sample collection. These drinkers attain the level of teetotalers only after more than 10 months of abstinence. In comparison to scalp hair, FAEEs recovered from armpit hair and leg hair were lower and from chest hair were higher. EtG in armpit hair was lower and in leg hair higher than in scalp hair. It is concluded that the combined use of FAEE and EtG essentially increases the accuracy of interpretation since both markers complement each other by a different sensitivity to sources of error.

Maternal hair testing for the assessment of fetal exposure to drug of abuse during early pregnancy: Comparison with testing in placental and fetal remains

2011-10-31

Abstract: Drug use by pregnant women in the first trimester of pregnancy and subsequent fetal exposure during early gestation can be assessed only by repetitive/systematic maternal blood/urine analysis or segmental hair analysis. No evidence of any relationship between maternal/fetal exposure during this specific period of gestation has been demonstrated to date in a human model.Methods: To clarify drugs toxicokinetics and transplacental passage during early pregnancy, the presence of the most widely used recreational drugs of abuse and metabolites was investigated in the proximal 4cm hair segments of women undergoing voluntary termination of pregnancy (n=280) during the 12th week of gestation and the results were compared to those from placenta and fetal tissue samples in order to verify whether maternal hair testing can reflect fetal exposure and, if so, to what extent. Hair, placenta and fetal remains were analyzed by validated gas chromatography mass spectrometry assays.Results: Eighty one positive hair samples were identified: 60 were positive for cannabis (74.1%), 28 for cocaine (34.6%), 7 for opiates (8.6%), 3 for MDMA (3.7%) and 18.5% were positive for more than one drug. The positive hair test results were confirmed in placenta/fetal tissues in 10 cases out of 60 for cannabis (16. 7%); in 7 out of 28 for cocaine (25%); and none for the 6 opiates positive cases and 3 MDMA cases, respectively.Conclusion: Drugs/metabolites in hair of pregnant women can be used as biomarkers of past drug use (repetitive or sporadic), although the use is not always reflected in fetal/placental tissues. There are several possible hypotheses to explain the results: (1) the use occurred before the start of pregnancy, (2) past sporadic consumption which could be measured in hair but not in fetal and placental remains because of the narrow window of drug detection in placental/fetal tissues; (3) the sensitivity of the analytical methods was not high enough for the detection of the minute amount of drugs of abuse and metabolites which reached these tissues (4) there is a large variability in the transplacental passage of drugs of abuse and in the placenta's metabolizing capacity.

Positive EtG findings in hair as a result of a cosmetic treatment

2011-10-24

Abstract: In a case of a driving ability assessment, hair analysis for ethyl glucuronide (EtG) was requested by the authorities. The person concerned denied alcohol consumption and did not present any clinical sign of alcoholism. However, EtG was found in concentrations of up to 910pg/mg in hair from different sampling dates suggesting an excessive drinking behavior. The person declared to use a hair lotion on a regularly base. To evaluate a possible effect of the hair lotion, prospective blood and urine controls as well as hair sampling of scalp and pubic hair were performed. The traditional clinical biomarkers of ethanol consumption, CDT and GGT, were inconspicuous in three blood samples taken. EtG was not detected in all collected urine samples.The hair lotion was transmitted to our laboratory. The ethanol concentration in this lotion was determined with 35g/L. The EtG immunoassay gave a positive result indicating EtG, which could be confirmed by GC–MS/MS-NCI. In a follow-up experiment the lotion was applied to the hair of a volunteer over a period of six weeks. After this treatment, EtG could be measured in the hair at a concentration of 72pg/mg suggesting chronic and excessive alcohol consumption. Overnight incubation of EtG free hair in the lotion yielded an EtG concentration of 140pg/mg.In the present case, the positive EtG hair findings could be interpreted as the result of an EtG containing hair care product. To our knowledge, the existence of such a product has not yet been reported, and it is exceptionally unusual to find EtG in cosmetics. Therefore, external sources for hair contamination should always be taken into account when unusual cosmetic treatment is mentioned. In those cases, it is recommended to analyze the hair product for a possible contamination with EtG. The analysis of body hair can help to reveal problems occurring from cosmetic treatment of head hair. As a consequence, the assessment of drinking behavior should be based on more than one diagnostic parameter.

The standardization of results on hair testing for drugs of abuse: An interlaboratory exercise in Lombardy Region, Italy

C. Stramesi, C. Vignali, A. Groppi, M. Caligara, F. Lodi, S. Pichini, C. Jurado — 2011-10-24

Abstract: Hair testing for drugs of abuse is performed in Lombardy by eleven analytical laboratories accredited for forensic purposes, the most frequent purposes being driving license regranting and workplace drug testing. Individuals undergoing hair testing for these purposes can choose the laboratory in which the analyses have to be carried out. The aim of our study was to perform an interlaboratory exercise in order to verify the level of standardization of hair testing for drugs of abuse in these accredited laboratories; nine out of the eleven laboratories participated in this exercise. Sixteen hair strands coming from different subjects were longitudinally divided in 3–4 aliquots and distributed to participating laboratories, which were requested to apply their routine methods. All the participants analyzed opiates (morphine and 6-acetylmorphine) and cocainics (cocaine and benzoylecgonine) while only six analyzed methadone and amphetamines (amphetamine, methamphetamine, MDMA, MDA and MDEA) and five Δ9-tetrahydrocannabinol (THC). The majority of the participants (seven labs) performed acidic hydrolysis to extract the drugs from the hair and analysis by GC–MS, while two labs used LC–MS/MS. Eight laboratories performed initial screening tests by Enzyme Multiplied Immunoassay Technique (EMIT), Enzyme-linked Immunosorbent Assay (ELISA) or Cloned Enzyme Donor Immunoassay (CEDIA). Results demonstrated a good qualitative performance for all the participants, since no false positive results were reported by any of them. Quantitative data were quite scattered, but less in samples with low concentrations of analytes than in those with higher concentrations. Results from this first regional interlaboratory exercise show that, on the one hand, individuals undergoing hair testing would have obtained the same qualitative results in any of the nine laboratories. On the other hand, the scatter in quantitative results could cause some inequalities if any interpretation of the data is required.

Diethyl phosphates accumulation in rabbits’ hair as an indicator of long term exposure to diazinon and chlorpyrifos

2011-10-24

Abstract: Long term exposure to organophosphate pesticides can be evaluated by quantitative analysis of their non-specific metabolites in hair matrix. The aim of this study was to determine whether these metabolites can be internally incorporated into the hair of rabbits exposed to diazinon and chlorpyrifos. The influence of dose and dose duration of each pesticide dosage were investigated. Three groups of rabbits were exposed to different dosages of diazinon (3.0 and 6.0mg/kg/day) and chlorpyrifos (18.0mg/kg/day) via drinking water. Hair samples were collected every month and analyzed for diethyl phosphate (DEP) and diethyl thiophosphate (DETP) by gas chromatography–mass spectrometry (GC–MS). The mean concentrations of the low-dose treated group, ranged from 112 to 257pg/mg for DEP and from 295 to 515pg/mg for DETP in hair. The high-dose treated group demonstrated a range of mean concentrations from 142 to 585pg/mg for DEP and from 406 to 988pg/mg for DETP in hair. For the chlorpyrifos treated group, the concentrations ranged from 138 to 1070 for DEP and from 554 to 886pg/mg for DETP. Analysis revealed the incorporation of these metabolites into the rabbit hair in a dosage and dose duration-dependent manner. These data confirms the ability of using hair analysis for diethyl phosphates to assess long-term OP exposure.

The atlas of dialkylphosphates; assessment of cumulative human organophosphorus pesticides’ exposure

Matthaios P. Kavvalakis, Aristidis M. Tsatsakis — 2011-10-24

Abstract: Organophosphorus pesticides (OPs) are a group of chemicals with significant health interest, due to their wide spectrum of action and their excessive use both indoors (household) and outdoors (occupationally). The non-specific metabolites of OPs, dialkylphosphates (DAPs), are the most commonly used indicators for the assessment of cumulative OP exposure in humans. This review presents studies on human biomonitoring of OPs in the general population and in occupationally exposed humans. Furthermore, cases of OP intoxication determined by the measurement of DAP metabolites in various biological samples are included. In many studies, urine samples from both the general population and exposed populations have been analyzed mainly in Europe and America, while other matrices such as amniotic fluid, meconium, hair and blood have been less studied. A variety of analytical techniques were used for the determination of DAPs in these matrices. In studies measuring DAPs in urine samples, the detected concentrations ranged from 18 to 830ppb for the general population, while the corresponding values for exposed populations ranged from 29 to 1370ppb. Studies on amniotic fluid indicated DAP levels of 0.3–2.8ppb. Studies on meconium samples showed a concentration range of 0.5–16,000ppb. DAP levels in hair samples ranged from 40 to 165ppb for the general population and from 181.7 to 812.9ppb for the exposed population. Each matrix provides specific information on OP exposure, namely acute, long-term, chronic or prenatal. Meconium and hair can indicate cumulative exposure, while amniotic fluid is an indicator of fetal exposure to xenobiotics. Thus, various biological samples provide a more comprehensive view of OP exposure. In general, dimethylphosphate (DMP) and diethylphosphate (DEP) levels were higher in mainly urine samples, than other methyl and ethyl phosphates. In addition, results in the existing literature are sufficient to demonstrate the difference in levels of DAPs in general and occupationally exposed populations, mainly in urine and hair samples. However, more studies are needed to measure DAP levels in matrices such as amniotic fluid, meconium and hair to add to the literature and confirm existing data.

The influence of ethanol containing cosmetics on ethyl glucuronide concentration in hair

Liliane Martins Ferreira, Tina Binz, Michel Yegles — 2011-11-03

Abstract: Ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE), non-volatile, direct metabolites of ethanol have been shown to be suitable markers for the evaluation of social and chronic excessive alcohol consumption. Previous investigations have shown that the regular use of hair-care products with high alcohol content lead to an increase of FAEE concentration and consequently gave false-positive results for the determination of FAEE in hair. In this study we investigated the influence of a long-term hair treatment with EtOH containing lotion, on the EtG concentrations in hair.In this study 7 volunteer subjects (classified as either rare, social or heavy drinkers) treated the right side of their scalp every day during a one or two month period with a commercial hair tonic (Seborin), which contains 44.0% ethanol (vol%). Collection of hair specimens from both sides of the scalp was done one day before hair treatment, one week and one month after treatment (for 5 subjects also after two months of treatment). A hair segment of 3 centimeters (cm) was cut and then washed with water and acetone, and then pulverized. EtG was quantified by GC/MS after pulverization and 2h of ultrasonication in water, extraction by solid phase extraction using Oasis MAX columns and derivatization with HFBA. Measurements were done in negative chemical ionization mode using EtG-D5 as internal standard. Comparison of EtG concentration in the treated and in the non-treated hair specimens did not show any increase at the different dates of collection for the 7 subjects.In conclusion, these results show that there is no indication for an increase of EtG after use of ethanol containing hair cosmetics.

Accidental potassium dichromate poisoning. Toxicokinetics of chromium by ICP-MS-CRC in biological fluids and in hair [Forensic Sci. Int. 217 (2012) e8–e12]

2012-04-18