Development, validation, and application of an LC-MS/MS method for mitragynine and 7-hydroxymitragynine analysis in hair.

The entire scalp hair of a self-declared Kratom consumer of 3 grams per day was acquired during an ethical committee approved study. As no values of the concentration in hair of the two Kratom alkaloids mitragynine or 7-hydroxymitragynine were found in the literature, an already established method for the analysis of benzodiazepines/z-substances was extended for the detection of mitragynine and 7-hydroxymitragynine with LC-MS/MS, and successfully validated. The limits of detection and quantification for mitragynine were 2 pg/mg and 4 pg/mg, respectively. Those of 7-hydroxymitragynine were 20 pg/mg and 30 pg/mg, respectively. The method was applied to the entire scalp hair, divided in 91 regions, of the study participant. A narrow mitragynine concentration distribution with values between 1054 pg/mg and 2244 ng/mg (mean 1517 ng/mg) and no clear scalp region associated distribution pattern was obtained. 7-Hydroxymitragynine was not detected in any hair sample. After validation, the method was established as routine and subsequently 300 samples (mainly abstinence controls for drugs of abuse) were analyzed, allowing the investigation of the prevalence of Kratom consumption in our population. None of the analyzed routine hair samples were positive for mitragynine or 7-hydroxymitragynine, providing no evidence that Kratom consumption is prevalent in the investigated population.

Distribution pattern of common drugs of abuse, ethyl glucuronide, and benzodiazepines in hair across the scalp.

While hair analysis is important and accepted in forensic applications, fundamental knowledge gaps still exist, exacerbated by a lack of knowledge of the incorporation mechanisms of substances into hair. The influence of the hair sampling location on the head on ethyl glucuronide (EtG) and cocaine concentrations was investigated by measuring the complete scalp hair of 14 (2 EtG, 4 cocaine, 8 both EtG and cocaine) study participants in a grid pattern for EtG, drugs of abuse, and benzodiazepines. Head skin perfusion and sweating rates were investigated to rationalize the concentration differences. For EtG, ratios between maximum and minimum concentrations on the scalp ranged from 2.5 to 7.1 (mean 4.4). For cocaine, the ratios ranged from 2.8 to 105 (mean 17.6). EtG concentrations were often highest at the vertex, but the distribution was strongly participant dependent. Cocaine and its metabolites showed the lowest concentrations at the vertex and the highest on the periphery, especially at the forehead. These differences led to hair from some head parts being clearly above conventional cut-offs and others clearly below. In addition to EtG and cocaine, the distributions of 24 other drugs of abuse and benzodiazepines/z-substances and metabolites are described. No clear pattern was observed for the head skin perfusion. Sweating rate measurements revealed higher sweating rates on the periphery of the haircut. Therefore, sweat could be a main incorporation route for cocaine. Concentration differences can lead to different interpretations depending on the sampling site. Therefore, the results are highly relevant for routine forensic hair analysis.

Cannabinoid concentrations in blood and urine after smoking cannabidiol joints.

In Switzerland, the sale of cannabis with tetrahydrocannabinol (THC) content less than 1% has recently been legalized. As a consequence, cannabis with low THC and high cannabidiol (CBD) values up to approximately 25% is legally available on the market. In this study, we investigated cannabinoid blood and urine concentrations of a naive user and of a modeled chronic user after smoking a single CBD joint. Chronic use was modeled as smoking 2 joints per day for 10 days. Joints contained 200mg of cannabis with THC concentrations of 0.94% and 0.8% and CBD concentrations of 23.5% and 17% in the naive-smoker and chronic-smoker experiment, respectively. After smoking, blood and urine samples were collected for 4 and 20h after smoking start, respectively. THC blood concentrations reached 2.7 and 4.5ng/mL in the naive and chronic user, respectively. In both cases, the blood THC concentration is significantly above the Swiss road traffic threshold of 1.5ng/mL. Consequently, the user was legally unfit to drive directly after smoking. CBD blood concentrations of 45.7 and 82.6ng/mL were reached for the naive and chronic user, respectively. During the 10-day smoking period, blood and urine samples were regularly collected. No accumulation of any cannabinoid was found in the blood during this time. Urinary 11-nor-9-carboxy-THC concentrations seemed to increase during the 10-day period, which is important in abstinence testing.

Sample preparation method for the combined extraction of ethyl glucuronide and drugs of abuse in hair.

Often in hair analysis, a small hair sample is available while the analysis of a multitude of structurally diverse substances with different concentration ranges is demanded. The analysis of the different substances often requires different sample preparation methods, increasing the amount of required hair sample. When segmental hair analysis is necessary, the amount of hair sample needed is further increased. Therefore, the required sample amount for a full analysis can quickly exceed what is available. To combat this problem, a method for the combined hair sample preparation using a single extraction procedure for analysis of ethyl glucuronide with liquid chromatography-multistage fragmentation mass spectrometry/multiple reaction monitoring (LC-MS3 /MRM) and common drugs of abuse with LC-MRM was developed. The combined sample preparation is achieved by separating ethyl glucuronide from the drugs of abuse into separate extracts by fractionation in the solid-phase extraction step during sample clean-up. A full validation for all substances for the parameters selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, matrix effects, and recovery was successfully completed. The following drugs of abuse were included in the method: Amphetamine; methamphetamine; 3,4-methylenedioxy-N-methylamphetamine (MDMA); 3,4-methylenedioxyamphetamine (MDA); 3,4-methylenedioxy-N-ethylamphetamine (MDE); morphine; 6-monoacetylmorphine; codeine; acetylcodeine; cocaine; benzoylecgonine; norcocaine; cocaethylene; methadone; 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) and methylphenidate. In conclusion, as only 1 sample preparation is needed with 1 aliquot of hair, the presented sample preparation allows an optimal analysis of both ethyl glucuronide and of the drugs of abuse, even when the sample amount is a limiting factor.

Distribution pattern of ethyl glucuronide and caffeine concentrations over the scalp of a single person in a forensic context.

The distribution of analyte concentrations in hair across the scalp has not been thoroughly investigated. Differences in concentrations depending on sampling location are problematic, especially when measuring a second strand to confirm the result of the first measurement. Aiming at a better understanding of the concentration differences, the distribution of ethyl glucuronide (EtG) and caffeine concentrations in hair across the entire head of one test subject was investigated by dividing the scalp completely into regions of ca 2 cm × 2 cm area, yielding a total of 104 samples. For the quantification of EtG, a novel LC-MS3 /MRM method was developed and validated with a limit of detection and limit of quantification of 2 and 4 pg/mg, respectively. Large variations of the concentration across the head were found, with factors of ca 3.0 and 10.6 for EtG and caffeine, respectively. These differences could not be attributed to measurement error alone. The concentrations were projected onto the subject's head, and concentration patterns were identified for EtG and caffeine. When examining multiple strands from within one 2 cm × 2 cm sampling area, the strands showed similar concentrations. Segmental analysis of selected 3 cm strands showed decreasing concentrations of EtG and caffeine from proximal to distal end, possibly due to wash-out of the analytes. Copyright © 2017 John Wiley & Sons, Ltd.

Systematic Exploration of Biotransformation Reactions of Amine-Containing Micropollutants in Activated Sludge.

The main removal process for polar organic micropollutants during activated sludge treatment is biotransformation, which often leads to the formation of stable transformation products (TPs). Because the analysis of TPs is challenging, the use of pathway prediction systems can help by generating a list of suspected TPs. To complete and refine pathway prediction, comprehensive biotransformation studies for compounds exhibiting pertinent functional groups under environmentally relevant conditions are needed. Because many polar organic micropollutants present in wastewater contain one or several amine functional groups, we systematically explored amine biotransformation by conducting experiments with 19 compounds that contained 25 structurally diverse primary, secondary, and tertiary amine moieties. The identification of 144 TP candidates and the structure elucidation of 101 of these resulted in a comprehensive view on initial amine biotransformation reactions. The reactions with the highest relevance were N-oxidation, N-dealkylation, N-acetylation, and N-succinylation. Whereas many of the observed reactions were similar to those known for the mammalian metabolism of amine-containing xenobiotics, some N-acylation reactions were not previously described. In general, different reactions at the amine functional group occurred in parallel. Finally, recommendations on how these findings can be implemented to improve microbial pathway prediction of amine-containing micropollutants are given.