Potential problems with the interpretation of hair analysis results

Due to differences in hair growth rate depending on anatomical region, age, gender, ethnicity and interindividual variability, interpretation of parent drug or/and metabolite concentrations in hair is not easy. Furthermore, as drug incorporation mechanisms into hair matrix is not yet fully understood, it is rather difficult to extrapolate details on time and dose from hair segment analysis. If incorporation sources other than from bloodstream (skin secretions and/or external/environmental contamination) are considered, interpretation becomes even more complicated. For evaluating possible passive contamination, it is essential to consider specific identification of metabolites, use of metabolite-to-parent drug ratios, assays of decontamination washes and analysis of specimens collected from other body parts. Cosmetic hair treatment, natural and artificial hair colour, differences in hair structure and specificity of analytical methodology may represent other bias sources affecting concentrations of drugs in hair. A suitable cut-off level related to the LOD will allow correct identification of drugs or metabolites in hair. Regarding the performance of different hair testing laboratories, little information is available at this time to what extent test results are comparable and their interpretation is consistent. Frequency of drug consumption and time intervals between multiple consumption or lag time between consumption and appearance in the hair has not been fully investigated and needs further research.     

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.     

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.     

Influence of bleaching on the enantiomeric disposition of amphetamine-type stimulants in hair

The aim of this work was to study the influence of hair bleaching on the enantiomeric ratios of amphetamine-type stimulants (ATS), including amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine and 3,4-methylenedioxyethylamphetamine. Hair specimens from 14 STA users were treated with a commercial bleaching product during 40 min. After alkaline digestion and solid-phase extraction of bleached and non-bleached hair, the STA enantiomers were derivatised with an in-house synthesised chiral reagent, the (2S,4R)-N-heptafluorobutyryl-4-heptafluorobutoyloxy-prolyl chloride. The diastereoisomers were quantified by GC/MS-NCI. The results showed that the concentrations of all enantiomers decreased in bleached hair in comparison with the non-treated hair (median values between 20 and 39%). The enantiomeric ratios of the STA in bleached hair were not significantly different from those determined in non-treated hair. Our findings pointed out that bleaching treatments decrease concentrations of STA in hair without influencing their enantiomeric ratios.     

Threshold values in toxicology - useful or not?

In many fields of toxicology, numbers are used as threshold values, e.g. as "acceptable daily intake values" resulting in maximum permissible concentrations in food or in animal feed by using "safety factors"; maximal admissible concentrations of toxic substances in the air at the workplace; cut-off values in analytical toxicology; limit values for biological specimens in the case of driving under the influence of drugs, guidance values for environmental specimens, etc. The philosophy behind these values must be well understood and they should only be applied to real cases by persons with enough toxicological background. The bad use of these numbers in toxicology can have dramatic consequences. Especially in regulatory toxicology their use should be made with great care. Moreover, tremendous improvements in analytical methodology, e.g. the decreasing of the limits of detection for many potentially toxic substances in recent years, should not end up in an overestimation of risks to humans. To avoid these abuses careful interpretations of analytical findings by qualified toxicologists are of paramount importance. The use and abuse of some of these threshold values will be outlined in several applications from analytical toxicology, risk assessment issues, forensic toxicology in post-mortem cases, as well as from the drugs and driving cases. Generally, if threshold values are considered as guidance values and not as the "absolute truth" in toxicology, they may be very useful in the interpretation of toxicology data.     

Influence of bleaching on stability of benzodiazepines in hair

In order to study the influence of hair bleaching on benzodiazepines concentrations, hair was treated with a bleaching product (Poly Blonde, Schwarzkopf & Henkel) for 20 min. The treated hair specimen was obtained from a person who died after an overdose of several illicit drugs associated with benzodiazepines. Bleached and non bleached hair were washed (acetone and water), pulverised and then incubated for 2 h in a thioglycolic solution. In the extracts obtained by solid-phase extraction on C18 columns, the different drugs with the corresponding deuterated standards were derivatized and determined by GC-MS in a SIM mode. These results show that the concentrations of all the drug detected decreased in bleached hair in comparison with non treated hair. Whereas the diminution was less important for cocaine and benzoylecgonine (decrease of 24.6 and 36.4%, respectively), concentrations for codeine, 6-monoacetylmorphine and morphine decreased more significantly (decrease of 57.5, 88.6 and 67.4%, respectively) as well as those of diazepam, nordazepam and 7-aminoflunitrazepam (decrease of 39.7, 67.7 and 61.8%, respectively). The results in this study agree with those of other authors that bleaching affects the stability of cocaine and opiates incorporated in hair. These findings also point out that bleaching influences the stability of entrapped benzodiazepines in hair. Finally, these results reconfirm that it is very important to consider the cosmetic history of a hair sample in the interpretation of hair analysis results.     

Drugs and chronic alcohol abuse in drivers

Blood specimens from 210 drivers (179 male and 31 female) apprehended in Luxembourg from autumn 2001 to spring 2002 and requested for the determination of their blood alcohol concentration (BAC) were tested for medicinal drugs, illicit drugs, and chronic alcohol abuse (by quantification of the carbohydrate-deficient transferrin: CDT). These additional analyses were performed anonymously and with permission of state prosecutor. The 22.8% had consumed medicinal drugs, with benzodiazepines and antidepressants (10.9 and 7.6%, respectively) as main psychoactive classes. Cannabis was the most detected illicit drug (9.5%) but only one in three had THC detectable in their blood. Association of two or more psychoactive substances (poly-drug use) was observed in 27.6% of drivers (90.6% of drug consumers). On the basis of CDT values, 29.5% of drivers investigated were assumed to be chronic alcohol abusers. Statistical analysis revealed that chronic alcohol abuse and medicinal psychoactive drugs were associated with significantly higher BAC. Medicinal psychoactive drugs were clearly associated with poly-drug use, and were furthermore detected at supra-therapeutic levels in 34.9%.