Correlation of the incidence of cocaine and cocaethylene in hair and postmortem biologic samples

Hair samples are useful as a matrix for drug testing because drugs can be detected in hair for longer periods than in blood or urine. The authors report a prospective comparison of the detection of cocaine and cocaethylene in routine postmortem biologic specimens to the detection of cocaine and cocaethylene in hair. The authors collected hair samples from various areas of the head in 53 autopsy cases, prepared them, and analyzed them by gas chromatography/mass spectrometry (GC/MS) for cocaine and cocaethylene. The authors compared the results of hair analysis with the results of toxicologic analysis performed on routine postmortem samples by enzyme multiplied immunoassay technique and GC/MS. Cocaine was found in either biologic fluids or in hair in 16 of 53 samples tested. Nine samples were positive for cocaine in both biologic fluids and hair. Five samples contained cocaine only in biologic fluids, and two contained cocaine only in hair. Cocaethylene was present in two cases. Drug screening of hair provides additional information in some autopsy cases, but the authors have not made hair analysis a routine practice. It may prove useful to save hair samples in all cases for later analysis if warranted by additional history or autopsy findings.     

Review of analytical methods for screening and quantification of fentanyl analogs and novel synthetic opioids in biological specimens

Fentanyl, fentanyl analogs, and other novel synthetic opioids (NSO), including nitazene analogs, prevail in forensic toxicology casework. Analytical methods for identifying these drugs in biological specimens need to be robust, sensitive, and specific. Isomers, new analogs, and slight differences in structural modifications necessitate the use of high-resolution mass spectrometry (HRMS), especially as a non-targeted screening method designed to detect newly emerging drugs. Traditional forensic toxicology workflows, such as immunoassay and gas chromatography mass spectrometry (GC–MS), are generally not sensitive enough for detection of NSOs due to observed low (sub-μg/L) concentrations. For this review, the authors tabulated, reviewed, and summarized analytical methods from 2010–2022 for screening and quantification of fentanyl analogs and other NSOs in biological specimens using a variety of different instruments and sample preparation approaches. Limits of detection or quantification for 105 methods were included and compared to published standards and guidelines for suggested scope and sensitivity in forensic toxicology casework. Methods were summarized by instrument for screening and quantitative methods for fentanyl analogs and for nitazenes and other NSO. Toxicological testing for fentanyl analogs and NSOs is increasingly and most commonly being conducted using a variety of liquid chromatography mass spectrometry (LC–MS)-based techniques. Most of the recent analytical methods reviewed exhibited limits of detection well below 1 μg/L to detect low concentrations of increasingly potent drugs. In addition, it was observed that most newly developed methods are now using smaller sample volumes which is achievable due to the sensitivity increase gained by new technology and new instrumentation.     

Improved gas chromatography-negative ion chemical ionization tandem mass spectrometric method for determination of 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid in hair using mechanical pulverization and bead-assisted liquid-liquid extraction

A gas chromatography-negative ion chemical ionization tandem mass spectrometric (GC-NCI-MS/MS) method was developed and validated for the determination of 11-nor-Δ(9)-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) in human hair. After decontamination, hair samples were weighed (25mg), mechanically pulverized with a bead mill, and incubated in 0.7 mL of 1.0M sodium hydroxide at 95 °C for 30 min. Bead-assisted liquid-liquid extraction was performed with n-hexane:ethyl acetate (9:1, v/v), a method developed in our laboratory. The extract was evaporated to dryness, derivatized with pentafluoropropanol and pentafluoropropionic anhydride, and analyzed by GC-MS/MS in the negative ion chemical ionization mode using methane as the reagent gas. The linear ranges were 0.05-10.0 pg/mg for THC-COOH with the coefficient of determination (r(2) = 0.9976). The intra-day and inter-day precisions were within 1.7 and 13.8%, respectively. The intra-day and inter-day accuracies were -4.8 to 10.0% and -3.9 to 3.8%, respectively. The limit of detection and quantification were 0.015 and 0.05 pg/mg, respectively. The recoveries were in the range of 79.4-87.1%. The results indicate that the proposed method is simple, rapid, accurate, and precise for determination of THC-COOH in hair. The method identified THC-COOH in hair specimens from suspected marijuana abusers.     

Evaluation of the One-Step ELISA kit for the detection of buprenorphine in urine, blood, and hair specimens

A solid-phase enzyme immunoassay involving microtiter plates was recently proposed by International Diagnostic Systems corporation (IDS) to screen for buprenorphine in human serum. The performance of the kit led us to investigate its applicability in other biological matrices such as urine or blood, and also hair specimens. Low concentrations of buprenorphine were detected with the ELISA test and confirmed by HPLC/MS (buprenorphine concentrations measured by HPLC/MS: 0.3 ng/mL in urine, 0.2 ng/mL in blood, and 40 pg/mg in hair). The intra-assay precision values were 8.7% at 1 ng/mL of urine (n = 8), 11.5% at 2 ng/mL in serum (n = 8), and 11.5% at 250 pg/mg of hair (n = 8), respectively. The immunoassay had no cross-reactivity with dihydrocodeine, ethylmorphine, 6-monoacetylmorphine, pholcodine, propoxyphene, dextromoramide, dextrometorphan at 1 and 10 mg/L, or codeine, morphine, methadone, and its metabolite EDDP. A 1% cross-reactivity was measured for a norbuprenorphine concentration of 50 ng/mL. Finally, the immunoassay was validated by comparing authentic specimens results with those of a validated HPLC/MS method. From the 136 urine samples tested, 93 were positive (68.4%) after the ELISA screening test (cutoff: 0.5 ng/mL) and confirmed by HPLC/MS (buprenorphine concentrations: 0.3-2036 ng/mL). From the 108 blood or serum samples screened, 27 were positive (25%) after the ELISA test with a cutoff value of 0.5 ng/mL (buprenorphine concentrations: 0.2-13.3 ng/mL). Eighteen hair specimens were positive (72%) after the screening (cutoff: 10 pg/mg) and confirmed by LC/MS (buprenorphine concentrations: 40-360 pg/mg). The ELISA method produced false positive results in less than 21% of the cases, but no false negative results were observed with the immunological test. Four potential adulterants (hypochloride 50 mL/L, sodium nitrite 50 g/L, liquid soap 50 mL/L, and sodium chloride 50 g/L) that were added to 10 positive urine specimens (buprenorphine concentrations in the range 5.3-15.6 ng/mL), did not cause a false negative response by the immunoassay.     

Hair analysis of seven bodybuilders for anabolic steroids,ephedrine, and clenbuterol

Several bodybuilders, all winners of international competitions, were arrested for trafficking of a number of doping agents including anabolic steroids, ephedrine, beta-adrenergics, human chorionic gonadotropin, antidepressants, and diuretics. In accordance with the recent French law against doping, the judge asked to test seven bodybuilders to identify doping practices. Hair and urine specimens were collected for analysis. After decontamination, a 100 mg hair strand was pulverized in a ball mill, hydrolyzed, extracted, and derivatized to be tested by GC/MS for anabolic steroids, beta-adrenergic compounds, ephedrine, and other doping agents. Urine was analyzed for anabolic steroids and metabolites, beta-adrenergic compounds, ephedrine, and human chorionic gonadotropin, in addition to a broad spectrum screening with GC/MS. The following compounds were detected in urine: ephedrine (29 and 36 ng/mL, n = 2), clenbuterol (0.2 to 0.3 ng/mL, n = 3), norandrosterone (4.7 to 100.7 ng/mL, n = 7), norethiocholanolone (0.9 to 161.8 ng/mL, n = 6), stanozolol (1 to 25.8 ng/mL, n = 4), methenolone (2.5 to 29.7 ng/mL, n = 4), testosterone (3 to 59.6 ng/mL, n = 7), epitestosterone (1 to 20.4 ng/mL, n = 7) and ratio testosterone/epitestosterone >6 for four subjects (18.5 to 59.6). The following drugs were detected in hair: ephedrine (0.67 and 10.70 ng/mg, n = 2), salbutamol (15 to 31 pg/mg, n = 3), clenbuterol (15 to 122 pg/mg, n = 6), nandrolone (1 to 7.5 pg/mg, n = 3), stanozolol (2 to 84 pg/mg, n = 4), methenolone (17 and 34 ng/ml, n = 2), testosterone enanthate (0.6 to 18.8 ng/mg, n = 5), and testosterone cypionate (3.3 to 4.8 ng/mg, n = 2). These results document the doping practice and demonstrate repetitive exposure to anabolic compounds and confirm the value of hair analysis as a complement to urinalysis in the control of doping practice.     

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.     

A Fatal Case of Poisoning with Fentanyl Transdermal Patches in Japan

Fentanyl transdermal patches have been used to treat cancer‐ and noncancer‐related chronic pain. However, its inappropriate or illegal application may cause fatal poisoning. We herein present the case of a Japanese woman in her 40s who was found dead with seven 25‐μg/h fentanyl transdermal patches on her body. We established a detailed toxicological analysis procedure to quantify fentanyl, and its metabolite norfentanyl, and other drugs (acetaminophen, allylisopropylacetylurea, celecoxib, estazolam, promethazine, and sertraline) in human whole blood by ultra‐high‐performance liquid chromatography–tandem mass spectrometry. The measured fentanyl and norfentanyl concentrations in the femoral and cardiac blood were 0.051 and 0.072 μg/mL and 0.033 and 0.076 μg/mL, respectively. The decedent's fentanyl concentrations were consistent with previously reported postmortem blood levels for fatal cases of poisoning by fentanyl transdermal patches. Based on the decedent's case history, autopsy findings, and toxicological analyses, the cause of death was identified as intoxication with transdermal fentanyl.     

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.     

Amphetamine, cocaine and cannabinoids use among truck drivers on the roads in the State of Sao Paulo, Brazil

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.     

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.     

Time-resolved hair analysis of MDMA enantiomers by GC/MS-NCI

The aim of the study was to determine the enantioselective disposition of 3,4-methylenedioxymethamphetamine (MDMA) and other amphetamine-type stimulants (ATS) in segmented hair specimens of self-declared ecstasy abusers, who took part in a double-blind placebo-controlled six-way crossover study during approximately 7 weeks, during which they received a 75 and a 100 mg dose of racemic MDMA twice. Hair specimens were washed and cut into pieces of 2 cm length. After digestion and solid phase extraction, the enantiomers were derivatized with a chiral agent (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride, developed at the authors laboratory and quantified by gas chromatography coupled to mass spectrometry operating in the negative chemical ionization mode. Most of the hair specimens that were tested positive for MDMA showed a predominance of the (R)-enantiomer. The R/S ratios of MDMA varied between 1.02 and 2.75 and total concentrations ranged from 0.1 to 20.1 ng/mg. The enantiomers of its metabolite 3,4-methylenedioxyamphetamine (MDA) were also quantified in most hair segments. The R/S ratios of MDA varied between 0.60 and 1.60, while the concentrations of the enantiomers ranged from 10 to 160 pg/mg hair. When segmental analysis was performed on single hair specimens, no inversion of the R versus S ratios of MDMA and MDA was observed. The predominance of (R)-MDMA in hair was in accordance with those already published for other matrices. Furthermore, both enantiomers of amphetamine (AM) were also detected in hair segments of four volunteers and the R/S ratios ranged from 1.00 to 1.47.     

An unusual fatality in a child due to oxycodone

An unusual fatality secondary to oxycodone in a child is reported. A 2-year-old female child was conveyed to a local hospital after exhibiting signs of rubbing of the mouth and staggering. A hospital toxicological immunoassay screen for drugs of abuse and tricyclic antidepressants was performed on a urine sample and reported as negative. She was discharged and found unresponsive the next morning. She was conveyed to a second hospital in full cardiopulmonary arrest and despite resuscitative efforts, was pronounced dead upon arrival. An autopsy was performed and postmortem specimens were submitted and screened for drugs using mainly chromatographic techniques. Quantitation was achieved by gas chromatography with nitrogen phosphorus detection. Confirmation was performed by gas chromatography/mass spectrometry. Oxycodone was the only drug detected in the following concentrations: heart blood, 1.36 mg/L; gastric contents, 7.33 mg in 33 mL (222.34 mg/L); liver, 0.2 mg/kg; and urine, 47.23 mg/L (47,230 ng/mL). In addition, immunoassay testing of the urine was positive for the opiate class of drugs. This case report demonstrates an unusual cause of death in a young child with emphasis on potential limitation in hospital urine screening tests and the importance of complete forensic toxicological testing in all child deaths.     

Ethyl glucuronide: unusual distribution between head hair and pubic hair

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

Monitoring compliance to therapy during addiction treatments by means of hair analysis for drugs and drug metabolites using capillary zone electrophoresis coupled to time-of-flight mass spectrometry

Capillary electrophoresis coupled to time-of-flight mass spectrometry was used in the present work for the determination of therapeutic and abused drugs and their metabolites in the hair of subjects undergoing addiction treatments, in order to monitor their compliance to therapy. For this purpose a rapid, qualitative drug screening method was adopted based on capillary electrophoresis hyphenated with time-of-flight mass spectrometry, which had earlier been developed and validated for the forensic-toxicological analysis of hair, limitedly to illicit/abused drugs [1]. Sampling of hair was carried out in order to refer to a time window of about two months from the date of sampling (i.e. 2cm ca. from cortex). A single extraction procedure was applied, allowing the determination in the hair matrix of "drugs of abuse" referred to the past abuses, and therapeutic drugs prescribed in the detoxification program as well as their metabolites. Analyte identification was based on accurate mass measurements and comparison of isotope patterns, providing the most likely matching between accurate mass value and elemental formula. Small molecules (<500Da) of forensic and toxicological interest could be identified unambiguously using mass spectrometric conditions tailored to meet a mass accuracy ≤5ppm. In the present study, the proposed approach proved suitable for the rapid broad spectrum screening of hair samples, although needing further confirmation of results by using fragmentation mass spectrometry.     

Target screening and confirmation of 35 licit and illicit drugs and metabolites in hair by LC-MSMS

A liquid chromatography-tandem mass spectrometry (LC-MSMS) target screening in 50mg hair was developed and fully validated for 35 analytes (Δ9-tetrahidrocannabinol (THC), morphine, 6-acetylmorphine, codeine, methadone, fentanyl, amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, benzoylecgonine, cocaine, lysergic acid diethylamide, ketamine, scopolamine, alprazolam, bromazepam, clonazepam, diazepam, flunitrazepam, 7-aminoflunitrazepam, lorazepam, lormetazepam, nordiazepam, oxazepam, tetrazepam, triazolam, zolpidem, zopiclone, amitriptyline, citalopram, clomipramine, fluoxetine, paroxetine and venlafaxine). Hair decontamination was performed with dichloromethane, and incubation in 2 mL of acetonitrile at 50°C overnight. Extraction procedure was performed in 2 steps, first liquid-liquid extraction, hexane:ethyl acetate (55:45, v:v) at pH 9, followed by solid-phase extraction (Strata-X cartridges). Chromatographic separation was performed in AtlantisT3 (2.1 mm × 100 mm, 3 μm) column, acetonitrile and ammonium formate pH 3 as mobile phase, and 32 min total run time. One transition per analyte was monitored in MRM mode. To confirm a positive result, a second injection monitoring 2 transitions was performed. The method was specific (no endogenous interferences, n=9); LOD was 0.2-50 pg/mg and LOQ 0.5-100 pg/mg; linearity ranged from 0.5-100 to 2000-20,000 pg/mg; imprecision <15%; analytical recovery 85-115%; extraction efficiency 4.1-85.6%; and process efficiency 2.5-207.7%; 27 analytes showed ion suppression (up to -86.2%), 4 ion enhancement (up to 647.1%), and 4 no matrix effect; compounds showed good stability 24-48 h in autosampler. The method was applied to 17 forensic cases. In conclusion, a sensitive and specific target screening of 35 analytes in 50mg hair, including drugs of abuse (THC, cocaine, opiates, amphetamines) and medicines (benzodiazepines, antidepressants) was developed and validated, achieving lower cut-offs than Society of Hair Testing recommendations.     

Blood, brain, and hair GHB concentrations following fatal ingestion

Despite the increasing incidence of illicit use of gamma-hydroxybutyrate (GHB), little information is available documenting levels of the drug in GHB fatalities. We measured GHB levels in postmortem blood, brain and hair specimens from a suspected overdose case by gas chromatography/mass spectrometry (GC/MS) following solid phase extraction (SPE) and derivatization with bis(trimethyl-silyl) trifluoroacetamide (BSTFA). Examination found 330 microg/mL GHB in femoral blood and 221 ng/mg GHB in frontal cortex brain tissue, values higher than those typically reported in the literature. The hair shaft was negative for GHB whereas the plucked root bulbs with outer root sheath attached (2,221 ng/mg) and root bulbs after washing and removal of the outer root sheath (47 ng/mg) contained the drug. Our results are consistent with an acute single dose of GHB and, as the toxicology screen was negative for other drugs of abuse, emphasize the significant danger of this drug.     

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

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/H(2)O/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.     

Testing for anabolic steroids in hair from two bodybuilders.

Two male bodybuilders were recently arrested by the French customs in Strasbourg (France) in possession of 2050 tablets and 251 ampoules of various anabolic steroids. It was claimed that the steroids were for personal use and not for trafficing as suggested by the police. Urine and hair specimens were collected from both suspects to clarify the claims. Nandrolone, stanozolol, testosterone and their corresponding metabolites were identified in the urine of both subjects. After decontamination, the hair was hydrolyzed by sodium hydroxide in presence of deuterated internal standards. After extraction with ethyl acetate and silylation, the drugs were identified by GC-MS in the electron impact mode. Hair from both males were positive for nandrolone (196 and 260 pg/mg), testosterone (46 and 71 pg/mg) and stanozolol (135 and 156 pg/mg), clearly indicating steroids abuse. Although not yet recognized by the International Olympic Committee, hair analysis may be a useful adjunct to conventional drug testing in urine from athletes.     

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.