Determination of ethyl glucuronide in human hair by SPE and LC-MS/MS

A method for the sensitive and selective determination of ethyl glucuronide (EtG) in hair has been developed using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Washed and cut hair segments were extracted by ultrasonication (3h, 50 degrees C) and the extracts were cleaned-up with aminopropyl SPE columns. LC-MS/MS analysis was performed using a polar-endcapped phenyl-hexyl-RP-phase with negative mode electrospray ionisation (ESI) using a triple quadrupole mass spectrometer (Sciex API 365) with a turboionspray source and post-column addition of acetonitrile for enhanced sensitivity. The MS/MS transitions monitored were m/z 221 -->75 for EtG and 226 -->75 for D(5)-EtG as an internal standard. The method was selective and sensitive, with a detection limit of 51 pg/mg hair at a signal-to-noise ratio of 3:1. The mean recovery was 96%, with an intra- and inter-day precision of less than 11.7% at a concentration of 200 pg/mg. The linearity was assessed in the range of 25-2000 pg/mg hair, with a correlation coefficient of 0.997. The method was successfully applied to 97 human hair samples which were taken at autopsies from persons with known alcoholism or were obtained from alcoholics who were hospitalized for ethanol withdrawal, from social drinkers and from children having not consumed any alcohol. Although, approximately two-third of the alcoholics showed EtG concentrations in hair of higher than 51 pg/mg (up to >4000 pg/mg), in one-third the EtG concentration was below the detection limit. However, only in one of five hair samples of "social drinkers", the EtG concentration was above the detection limit (51 pg/mg). No EtG has been detected in the hair of children. These investigations demonstrate that heavy alcohol consumption may be but not necessarily has to be detectable by EtG analysis in hair.     

A study of distribution of ethyl glucuronide in different keratin matrices

Ethyl glucuronide (EtG) is a direct metabolite of ethanol, frequently used as a biomarker of alcohol abuse. To this purpose, EtG is preferentially determined in hair samples, using a cut-off value of 30pg/mg to discriminate between social and heavy drinkers, as recently fixed by an international consensus conference. Although this cut-off value is assumed for head hair, alternative matrices, such as pubic, axillary and chest hair, are often analyzed when head hair is not available. Previous studies suggested that determination of EtG in various keratin matrices may lead to different results; growth cycle and rate, urine contamination, distribution of sebum glands and other environmental factors are likely to contribute to these differences. We analyzed more than 2700 samples (head, pubic, chest and axillary hair) to evaluate the inter- and intra-individual distribution of the EtG concentration in the different keratin matrices. The data were interpreted on a statistical basis, on the assumption that large population data-sets will level off the average alcohol consumption of each group. From both inter- and intra-individual distribution data, significant differences were observed in EtG concentrations recorded in head, axillary and pubic hair samples. It is concluded that pubic hair cannot be utilized alternatively to head hair to prove chronic alcohol abuse, nor is axillary hair, since positive and negative biases respectively affect these determinations. In contrast, for chest hair, EtG distributions similar to head hair were found, although the large discrepancy between the examined population dimensions presently prevents any definitive conclusion. Thus, chest hair represents a promising alternative to head hair for EtG determinations, deserving further investigation on samples collected from the same individuals, in order to establish a clear correlation between their respective EtG concentrations.     

Sexual abuse and anti-wrinkle cream: evidence from octocrylene

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.     

Ethyl glucuronide in human hair after daily consumption of 16 or 32 g of ethanol for 3 months

The overall objectives of the study were to develop a sensitive method for ethyl glucuronide (EtG) determination in hair and then investigate if a low or moderate intake of ethanol could be differentiated from total abstinence. Forty-four subjects were included in the study, 12 males (7 drinkers and 5 abstinent) and 32 females (14 drinkers and 18 abstinent). The study lasted 3 months and the female drinkers consumed one glass (16 g of ethanol) and the males consumed two glasses (32 g of ethanol) of wine (13.5-14%) daily. Hair samples were collected as close as possible above the skin and the proximal 2 cm were analyzed for EtG. Hair was cut into pieces of about 0.5 cm length and washed before incubation overnight in water and then extracted on Clean Screen EtG Carbon columns. The LC/MS/MS system consisted of a Waters ACQUITY UPLC connected to an API 4000 triple quadrupole instrument. Two transitions for EtG and one for the internal standard EtG-D(5) were measured. The method was linear from 60 to 10,000 pg/sample. Imprecision studies were performed at three levels as well as with an authentic sample. Total imprecision was 16% at 200 pg/sample, 8% at 1000 pg/sample, 6% at 8000 pg/sample and 13% at 29 pg/mg in the authentic sample. Of those who drank two glasses of wine every day, four had measurable amounts of EtG in their hair (5-11 pg/mg), and in only one of the females drinking one glass of wine EtG was quantified (3 pg/mg). Among the 23 abstinent subjects two had traces of EtG in the hair. We conclude that persons who ingested 16 or 32 g of ethanol daily for 3 months presented with low concentrations of EtG in hair, well below the proposed threshold for overconsumption set at 30 pg/mg. In addition, none of those who ingested 16 g/day had concentrations over the proposed abstinence threshold of 7 pg/mg.     

Segmental determination of ethyl glucuronide in hair: a pilot study

Ethyl glucuronide (EtG) is a minor metabolite of ethanol. Its detection in hair is more and more studied in both clinical and forensic context for the purpose of alcohol abuse monitoring. In this pilot study, hair specimens from 15 patients included in a treatment program after alcohol abuse cessation, were segmented and analyzed for EtG. The results were then compared to their self-reported past alcohol consumption and to their blood biomarkers values (GGT, MCV, ASAT, ALAT). EtG concentrations measured in hair varied from 8 to 261 pg/mg. The pattern of EtG concentration detected in the different hair segments matched with the drinking history of patients, displaying variations (increase and decrease) in alcohol consumption and also time of cessation. Results also demonstrated the existence of a significant correlation (r(p)=0.5357; p=0.0390) between EtG concentration in hair and the amount of alcohol intake. Variations in the EtG concentrations with respect to hair segments may provide an overview of the drinking history of patients. Moreover, EtG concentration in hair may help to estimate the daily alcohol intake.     

Comparison of ethyl glucuronide and fatty acid ethyl ester concentrations in hair of alcoholics, social drinkers and teetotallers

In previous investigations hair analysis for ethyl glucuronide (EtG) and fatty acid ethyl esters (FAEE) proved to be suitable for the detection of excessive alcohol consumption. The aim of this study was to compare EtG and FAEE concentrations in hair of alcoholics, social drinkers and teetotallers. Hair samples from 10 alcoholics in withdrawal treatment, 11 fatalities with documented excessive alcohol consumption, four moderate social drinkers who consumed up to 20 g ethanol per day, and three strict teetotallers were analysed. After external degreasing with n-heptane, extraction with a dimethyl sulfoxide/n-heptane mixture and headspace solid-phase microextraction of the extracts, four fatty acid ethyl esters (FAEEs) (ethyl myristate, ethyl palmitate, ethyl oleate and ethyl stearate) were analysed by gas chromatography-mass spectrometry (GC-MS) with deuterated internal standards. EtG was determined by GC-MS/NCI after ultrasonication of the samples with H2O, cleanup by SPE with aminopropyl columns and PFP derivatisation. The following concentrations were measured for the four groups: teetotallers EtG < 0.002 ng/mg, FAEE 0.05-0.37 ng/mg, moderate social drinkers EtG < 0.002 ng/mg, FAEE 0.26-0.50 ng/mg, alcoholic patients EtG 0.030-0.415 ng/mg, FAEE 0.65-20.50 ng/mg and the fatalities with alcohol history EtG 0.072-3.380 ng/mg, FAEE 1.30-30.60 ng/mg. The results confirm that by using a cut-off value of the sum of FAEE > 1 ng/mg and/or a positive EtG result in hair, excessive alcohol consumption can be identified using hair analysis. However, no significant correlation between the EtG and FAEE concentrations in the positive cases could be shown. Segmental analysis of some of the specimens did not reveal the same distribution for EtG compared to FAEE in hair, and no chronological accordance compared to the self-reported alcohol consumption could be observed for both parameters. These different results of both methods are discussed in terms of differences between EtG and FAEE in mechanism of formation and incorporation into hair and elimination from hair.     

Comparison of ethyl glucuronide in hair with phosphatidylethanol in whole blood as post-mortem markers of alcohol abuse

Ethyl glucuronide (EtG) is a direct metabolite of ethanol and has been used as a marker of alcohol abuse in both urine and hair. This study investigated the value of EtG testing in post-mortem hair for diagnostic improvement of alcohol abuse in forensic medicine. Material from 70 consecutive medico-legal autopsies was collected in accordance with the recommendations on ethics by the Swedish National Board of Forensic Medicine. A method for determination of EtG in hair samples was developed using ultra performance liquid chromatography/electrospray tandem mass spectrometry (UPLC/ESI-MS/MS; LOQ, 2.5 pg/mg). The result of the EtG analysis was compared with the findings of phosphatidylethanol (PEth) in femoral whole blood, as measured by high performance liquid chromatography with an evaporative light-scattering detector (HPLC-ELSD; LOQ, 0.22 micromol/l). Evaluation of liver histology and anamnestic evidence of alcohol abuse of the deceased were taken in consideration for the interpretation. Measurable levels of EtG were present in 49 of the 70 autopsy cases whereas PEth was present in 36. Thirty-nine cases had EtG levels above the cutoff limit (> or = 30 pg/mg) compared with 29 for PEth (> or = 0.7 micromol/l). Fifteen cases had EtG as exclusive indicator for alcohol abuse compared with four cases for PEth. These findings suggest that measurements of EtG in hair may provide improved diagnostic information on alcohol abuse, due to a long retrospective time-window for detection and stability of EtG in hair in the decaying cadaver. However, an EtG level below the cutoff does not completely exclude previous alcohol abuse.     

Cannabinoid concentrations in hair from documented cannabis users

Fifty-three head hair specimens were collected from 38 males with a history of cannabis use documented by questionnaire, urinalysis and controlled, double blind administration of delta9-tetrahydrocannabinol (THC) in an institutional review board approved protocol. The subjects completed a questionnaire indicating daily cannabis use (N=18) or non-daily use, i.e. one to five cannabis cigarettes per week (N=20). Drug use was also documented by a positive cannabinoid urinalysis, a hair specimen was collected from each subject and they were admitted to a closed research unit. Additional hair specimens were collected following smoking of two 2.7% THC cigarettes (N=13) or multiple oral doses totaling 116 mg THC (N=2). Cannabinoid concentrations in all hair specimens were determined by ELISA and GCMSMS. Pre- and post-dose detection rates did not differ statistically, therefore, all 53 specimens were considered as one group for further comparisons. Nineteen specimens (36%) had no detectable THC or 11-nor-9-carboxy-THC (THCCOOH) at the GCMSMS limits of quantification (LOQ) of 1.0 and 0.1 pg/mg hair, respectively. Two specimens (3.8%) had measurable THC only, 14 (26%) THCCOOH only, and 18 (34%) both cannabinoids. Detection rates were significantly different (p<0.05, Fishers' exact test) between daily cannabis users (85%) and non-daily users (52%). There was no difference in detection rates between African-American and Caucasian subjects (p>0.3, Fisher's exact test). For specimens with detectable cannabinoids, concentrations ranged from 3.4 to >100 pg THC/mg and 0.10 to 7.3 pg THCCOOH/mg hair. THC and THCCOOH concentrations were positively correlated (r=0.38, p<0.01, Pearson's product moment correlation). Using an immunoassay cutoff concentration of 5 pg THC equiv./mg hair, 83% of specimens that screened positive were confirmed by GCMSMS at a cutoff concentration of 0.1 pg THCCOOH/mg hair.     

Detection of amitriptyline, nortriptyline and bromazepam in liver, CSF and hair in the homicidal poisoning of a one-month-old girl autopsied 8 months after death

We reported on the death by poisoning of a one-month-old baby that had followed the death of one of her sister (due to cyamemazine overdose). Exhumation of the corpse was done 8 months after burial and revealed the presence of amitriptyline. Parent drug and its metabolite were analysed by HPLC-MS/MS in positive ionisation mode on a C(18) analytical column using a gradient of acetonitrile and 2mM formate buffer at pH=3. Quantification is based on the main ion m/z=233, the common product ion of nortriptyline (MH(+), m/z 264), amitriptyline (MH(+), m/z 278) and nortriptyline D3 used as internal standard (MH(+), m/z 267). Amitriptyline and nortriptyline in the liver were measured at a concentration of 29.8 and 3.6 μg/g, respectively. Hair analyses revealed the presence of amitriptyline and nortriptyline at concentrations of 1811 and 43 pg/mg, respectively, while complementary analyses showed the presence of bromazepam in the hair at a concentration of 740 pg/mg, thus documenting previous administrations. The mother confessed later having used the drinkable form of the pharmaceutical LAROXYL(?) by pouring the content of a 20 ml bottle (at 40 mg/ml) into the feeding-bottle of her child. The milk was sweet but still bitter and following the testimony of a close relative, the whole family helped to feed the crying baby.     

Markers of chronic alcohol use in hair: comparison of ethyl glucuronide and cocaethylene in cocaine users

Two direct ethanol metabolites, namely ethyl glucuronide (EtG) and cocaethylene (CE), in the hair of cocaine (COC) users were compared in this study. Hair samples (n=68) were submitted to the determination of EtG (by liquid chromatography-electrospray-tandem mass spectrometry) and of COC and metabolites, including CE (by gas chromatography-mass spectrometry). Quantitative and qualitative results were compared. No quantitative correlation was found between EtG and CE, as well as between EtG and the cocaethylene concentration divided by the concentration of COC and its metabolites (benzoylecgonine and ecgonine methylester, as COC equivalents). Nevertheless, many factors are supposed to affect the amount of the two substances incorporated in the hair matrix, such as the subject's habits in ethanol and COC use, genetic variability in the metabolism of both substances, and the different chemical and physical properties of EtG and CE. When establishing a cut-off of 4 pg/mg for EtG and of 200 pg/mg for CE, 47 samples tested positive for EtG and 41 samples tested positive for CE; 12 samples out of the 47 EtG-positives tested negative for CE (25%), whereas 6 samples out of the 41 CE-positives tested negative for EtG (15%). According to these data, EtG appears to be a more sensitive and specific marker of non-moderate alcohol users than CE.     

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

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.     

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

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.     

Interpretation problems in a forensic case of abstinence determination using alcohol markers in hair

In a child custody case a mother with a longstanding history of alcohol misuse had to show absolute abstinence for one year. She entered a residential rehabilitation for six months and was tested two months later by way of a hair test for ethyl glucuronide (EtG) with the result of 22 pg/mg in the proximal 0-1cm segment and the segments 1-2 cm and 2-3 cm being negative. This was interpreted as a minimum alcohol intake of 20-50 units per week in the month before sampling. Since the mother denied any alcohol intake a second hair sample was collected seven weeks after the first and analyzed for fatty acid ethyl esters (FAEEs) by a second laboratory. A low concentration of 0.03 ng/mg was measured within the 0-6 cm segment of recently bleached hair and was interpreted as showing no evidence of alcohol use during the last six months. Three further hair samples were analyzed during the next nine months with low EtG values (<2.4-3.3 pg/mg, 0-3 cm segment) and low FAEE values (0.27-0.53 ng/mg, 0-6 cm segment). These findings were summarized as indicating continued low alcohol consumption over the past one year period. As a consequence of the conflicting results, the case was dealt with in a hearing before the Family Division of the High Court of London. It was concluded in the judgment that the evidence did not indicate that the mother had consumed alcohol in the period tested by the hair samples. It was stated that the evidence in this case highlighted the need for the exercise of considerable caution when hair tests for alcohol are being interpreted and relied upon, both generally and particularly in isolation, and that this case is a proper reminder of the need for expert evidence to be given in a manner according to the Practice Direction.     

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

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.     

Testing for zopiclone in hair application to drug-facilitated crimes

The use of a drug to modify a person's behaviour for criminal gain is not a recent phenomenon. However, the recent increase in reports of drug-facilitated crimes (sexual assault, robbery) has caused alarm in the general public. The drugs involved can be difficult to detect due to low dosages or chemical instability. They possess amnesic properties and can be quickly cleared from the body fluids. In these situations, blood or even urine can be of poor interest. This is the reason why this laboratory developed an original approach based on hair testing by LC-MS/MS. Zopiclone (Imovane), due to its short half-life associated with rapid hypnotic activity, is considered as a compound of choice to sedate victims. To document the detection of zopiclone in hair, we first tested specimens obtained from two volunteers who had ingested a single 7.5 mg Imovane tablet, and from repetitive consumers of zopiclone. After pH 8.4 buffer incubation and extraction with methylene chloride/diethyl ether (80/20 (v/v)), hair extracts were separated on a Xterra MS C18 column using a gradient of acetonitrile and formate buffer. Zopiclone and diazepam-d5, used as internal standard, were detected by tandem mass spectrometry. A single exposure to zopiclone was detectable in the first hair segment of two volunteers at concentration of 5.4 and 9.0 pg/mg, respectively. Hair from repetitive consumers tested positive for zopiclone at concentrations of 37 and 66 pg/mg. Hair analysis was applied to two authentic criminal cases. In the first one, zopiclone tested positive in the corresponding hair segment at 4.2 pg/mg, in accordance with a single exposure to the drug. In the other expertise, zopiclone was detected in the two segments analyzed, at 21.3 and 21.5 pg/mg, making unlikely the hypothetical single exposure to zopiclone.     

Testing human hair for cannabis

To validate information on cannabis use, we investigated human hair and pubic hair for cannabinoids (THC and THC-COOH) by gas chromatography/mass spectrometry. Samples (100 mg approximately) were decontaminated with methylene chloride, then pulverized and dissolved in 1 ml 1 N NaOH for 10 min at 95 °C in the presence of 200 ng of deuterated standards. After cooling, samples were extracted by n-hexane/ethyl acetate after acidification with acetic acid. After derivatization of the dry extract by PFPA/PFP-OH, the drugs were separated on a 30-m capillary column and detected using selected-ion monitoring (m/z 377 and 459 for THC and THC-COOH, respectively). Forty-three hair samples were obtained from fatal heroin overdose cases. Among them, 35% tested positive for cannabinoids. Hair concentrations ranged from 0.26 to 2.17 ng/mg (mean, 0.74 ng/mg) and 0.07 to 0.33 ng/mg (mean, 0.16 ng/mg) of THC and THC-COOH, respectively. As is generally the case for other drugs detected in hair, metabolite concentration was always lower when compared to the parent drug concentration. In pubic hair, THC concentrations ranged from 0.34 to 3.91 ng/mg (mean, 1.35 ng/mg) and THC-COOH concentrations from 0.07 to 0.83 ng/mg (mean, 0.28 ng/mg). In most cases, the highest cannabinoid concentration was found in pubic hair, suggesting that this sample may be the more suitable for cannabis testing.     

Incorporation of propyphenazone in beard hair of a migraine patient

The incorporation of propyphenazone in beard hair after consumption of this substance present in the analgesic Migraine-Kranit (Codali) was investigated. Because of a migraine attack a volunteer took four tablets of Migraine-Kranit (one tablet contains 150 mg propyphenazone) the first day and two tablets the second day. Shaved beard hair was collected 48, 72, 96 and 120 h after the first consumption of the analgesic drug. These hair specimens were washed (acetone and water), pulverized and then incubated during 2 h in a thioglycolic solution. After solid-phase extraction on C18 columns, propyphenazone was assayed in these extracts by GC/MS operating in selected ion monitoring mode (m/z 230, 215). Diazepam-d5 was used as an internal standard. In hair specimen 1 (48 h after consumption) the highest concentration was found (170 pg/mg hair). In hair specimen 2 (72 h) and 3 (96 h) the concentration were significantly lower (44 and 18 pg/mg, respectively). After 120 h no propyphenazone could be detected (limit of detection: 5 pg/mg hair). These results show that propyphenazone was already in beard hear 2 days after consumption, whereas no more presence could be shown after 120 h. As the time period of 2 days is too short to allow entrapment into the hair matrix from bloodstream and growing of hair out of the follicle, our results suggest that incorporation of propyphenazone may be mainly due to excretion in sweat and subsequent incorporation into the hair.     

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

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.     

Analysis of pain management drugs, specifically fentanyl, in hair: application to forensic specimens

This article discusses the immunoassay screening of pain management drugs, and the mass spectrometric confirmation of fentanyl in human hair. Hair specimens were screened for fentanyl, opiates (including oxycodone), tramadol, propoxyphene, carisoprodol, methadone, and benzodiazepines and any positive results were confirmed using gas chromatography or liquid chromatography with mass spectral detection. The specific focus of the work was the determination of fentanyl in hair, since autopsy specimens were also available for comparison with hair concentrations. Using two-dimensional gas chromatography with electron impact mass spectrometric detection, fentanyl was confirmed in four of nine hair specimens collected at autopsy. The accuracy of the assay at 10 pg/mg was 95.17% and the inter-day and intra-day precision was 5.04 and 13.24%, respectively (n=5). The assay was linear over the range 5-200 pg/mg with a correlation of r(2)>0.99. The equation of the calibration curve forced through the origin was y=0.0053x and the limit of quantitation of the assay was 5 pg/mg. The fentanyl concentrations detected were 12, 17, 490, and 1930 pg/mg and the results were compared with toxicology from routine post-mortem analysis. The screening of pain management drugs in hair is useful in cases where other matrices may not be available, and in routine testing of hair for abused drugs.     

Evaluation of nicotine and cotinine in human hair.

To validate data on tobacco use, the authors investigated the use of hair samples for quantifying nicotine and cotinine by gas chromatography/mass spectrometry. Hair was taken from 22 nonsmokers and 42 smokers, cut close to the scalp at the back of the head. The hair (about 100 mg from each subject) was incubated in 3 mL of 1N NaOH at 100 degrees C for 1 h. After this, the samples were extracted by diethyl ether. The drugs were separated on a 12-m BP-5 capillary column and detected using selected ion monitoring (nicotine, m/z 84; cotinine, m/z 98). Hair from nonsmokers and smokers contains nicotine and cotinine. Although it is difficult to determine an absolute cutoff level, an amount greater than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from nonsmokers. In the population of nonsmokers, the influence of environmental smoke exposure was noted.