Determination of psychoactive substances in hair for forensic purposes

Hair analysis for abstinence testing has been gaining ever more traction as an alternative to classic matrices such as blood or urine due to the many benefits it offers, the most important being a long window of detection. However, hair analysis still suffers from a lack of knowledge concerning the factors that can influence the concentrations ultimately found in the hair. One such influencing factor, and the topic of this thesis, is the sampling location on the head. This topic had been examined by Dussy et al. 2014 in a small study. The study had a small sample size of three test subjects and did not investigate the entire head systematically. Additionally, only ethyl glucuronide (EtG) and caffeine were investigated. Therefore, little could be said about the patterns of distribution or the possible extent of differences, especially about any other substance than EtG and caffeine.
In a first step, the entire scalp hair of a single person was investigated. For this, the scalp hair was divided into individual strands of roughly 3x3 cm area yielding a total of 104 strands across the head. Each strand was analyzed for EtG and caffeine. For this, a method for the analysis of EtG using LC-MS3 was developed, as the previous employed GC-MS/MS method was not suitable for large series of samples. The complete analysis yielded EtG concentrations between 6.8 and 20.2 pg/mg. For caffeine, values between 1.1 and 12.0 ng/mg were obtained. The distribution patterns of EtG and caffeine were markedly different. While caffeine clearly showed higher concentrations towards the edges of the haircut, EtG was not as clearly distributed except for lower concentration at the neck.
As these results were very promising, the distribution was investigated more thoroughly. Cocaine is the second most important substance for hair analysis after EtG at our lab, as it is the most prevalent in the routine population. Therefore, the study focused on alcohol and cocaine consuming individuals. For each individual, all hair was sampled in the same way as in the previous single person study. As the sample preparation for EtG and drugs of abuse (DoA) was normally done with different methods at our lab, the analysis of the many hundreds to thousands of hair samples would have exceeded the manageable workload. Therefore, a combined sample preparation method for EtG, caffeine, DoA and benzodiazepines/z-substances (BZD-Z) was developed. The method is based on the different retention characteristics of the substances to an Oasis Max SPE cartridge. Using this combined sample preparation, a large workload reduction was possible. Also in cases with little available hair, a full analysis was still possible.
In addition to determining the shape and extent of the substance distributions, finding explanations for the observed differences was of interest. Therefore, in cooperation with the clinic for angiology and the department for sport, exercise and health, the head skin perfusion and sweating rates across the scalp were investigated, as the bloodstream and the sweat are thought to be important incorporation pathways. The head skin perfusion was measured using a laser Doppler anemometer. Each sample area was scanned allowing the same cartography of the perfusion rates as with the substance concentrations. The head skin sweating rates were measured using a cycling cap which was lined with pads of strongly water absorbing material. The test subjects were asked to cycle for 10 minutes on an ergometer after which the cycling cap was placed on their head and another 10 minutes of cycling were commenced. The weight of the pads was measured before and after cycling to obtain the weight of the excreted sweat.
Thirteen alcohol and/or drug consuming persons, 12 of which were cocaine consumers and 9 of which were alcohol consumers could be enlisted. The distribution patterns for 29 different substances were obtained. For many substances (almost all DoA and BZD-Z) the distribution showed a clear pattern of higher concentrations on the periphery of the head and especially on the forehead. EtG in general showed a reverse behavior to this, with higher concentrations towards the vertex posterior region. The extent of the found differences across the head was depended strongly on substance and on the individual. Cocaine showed factors between 2.8 and 105 (median 8.4) between lowest and highest concentrations, while e.g. methadone showed factors of only 1.6 to 4.2 (median 2.0). EtG showed factors between 2.5 and 7.1 (median 4.0). These differences are very relevant as typically cut-off thresholds are applied when evaluating hair results. This leads to situations in which results of samples from different parts of the head lead to a different evaluation of the consumption habits. In the case of EtG, two thresholds (7 pg/mg [5 pg/mg according to the new SoHT consensus of 06.08.2019] and 30 pg/mg for abstinence and chronic excessive consumption, respectively) are applied. One participant showed concentrations between 6.2 pg/mg and 30.4 pg/mg. For this participant, depending on sampling location, the whole range of interpretations is possible if applying the former cut-offs. For seven of nine study participants, the EtG concentrations fell into more than one category. This is problematic and should be considered when sampling and documenting sampling in routine work. The perfusion measurements did not show a clear pattern of perfusion rates, with only the back of the neck showing significantly lower perfusion rates across participants. The sweat rates showed large differences with sweating being highest on the forehead and generally decreasing towards the vertex posterior. The sweat rates showed a similar behavior to the concentrations of most substances, meaning the concentration differences could be caused by sweating. EtG showed a reverse distribution to the sweat rates, so might instead be washed out by the sweat.
From these results, sweat rate differences across the scalp are believed to be a major source of the observed concentration differences. For sweat to be a major incorporation pathway, substances must on the one hand be able to cross from the bloodstream into the sweat, and on the other be able to go from the sweat into the hair. Most research studying the incorporation of substances from the sweat into the hair were conducted within the context of external contamination. Therefore, these studies usually focused on cocaine and employed high concentrations and unrealistic conditions for modeling the daily incorporation from sweat of a drug user. On this basis, the incorporation of the routine panel of substances into the hair from artificial sweat solution was investigated to test the plausibility of the hypothesis that sweat is a major contributor to the concentration differences found across the head. This project is currently ongoing and definite results are not yet available.
One study participant reported consuming Kratom every day. As mitragynine and 7-hydroxymitragynine (7-Hy-Mitra) have recently been listed as scheduled substances in Switzerland, a method was developed for the measurement of mitragynine and 7-Hy-Mitra in hair and applied to the hair of the study participant. 7-Hy-Mitra could not be detected in any sample. Mitragynine showed a narrow concentration distribution with concentrations between 1.1 ng/mg and 2.2 ng/mg with no clear pattern. The method was applied to routine drug of abuse hair samples to estimate the prevalence of Kratom consumption. After measurement of 300 routine samples, not a single sample was positive, showing a low prevalence of Kratom consumption.
Recently, hemp material containing less than 1 % THC and high amounts of cannabidiol (CBD) have become available for legal purchase in Switzerland. No information was available if smoking this CBD rich hemp could yield THC concentrations above the legal cut-off of 1.5 ng/mL in blood (2.2 ng/mL with the confidence interval; VRV Art. 2 § 2). Therefore, a self-experiment was conducted to determine if concentrations above this legal limit could be reached. In this experiment, an employee of our institute smoked a CBD joint while blood was collected at regular intervals. Urine was collected from every spontaneous void. To see if an accumulation of THC could occur, the same person smoked two CBD joints per day for ten day. While smoking the last joint, blood and urine were again collected. The experiment showed that blood concentrations above the legal threshold of THC could be achieved with THC concentration of 2.7 ng/mg and 4.5 ng/mg after the single and chronic smoking experiments, respectively. No accumulation of THC in blood or urine could be detected during the 10-day smoking period. A slight accumulation of THC-COOH in urine is possible, but could not be definitely confirmed.