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As gamma-hydroxybutyric acid (GHB) underlies fast metabolization, its determination from hair may presumably offer a detection window superior to that of body fluids. Due to the wide range of endogenous concentration levels, the evidence of an exogenous ingestion is challenging. As already shown for other drugs, the temporal resolution obtained by applying single hair microanalysis provides further information. Therefore, a method for the extraction and quantification of GHB in 2-mm hair segments (seg) was optimized and validated (limit of detection [LOD]: 2.5 pg/seg, lower limit of quantification [LLOQ]: 5 pg/seg), and five single hairs were examined, each for three non-users and for three (alleged) users. A major challenge was the choice of appropriate extraction tubes without remains of GHB. In two samples from non-users, GHB could not or could only be detected in trace amounts. In the third sample, concentrations between the LOD and 31.1 pg/seg (mean: 9.5, median: 8.4; each pg/seg) were detected with decreasing values towards the tips. In two samples of persons with assumed GHB intake, maximum concentrations of 6.8 and 30.7 pg/seg were measured, but no significant concentration peaks indicating a single ingestion could be observed. The third sample showed concentrations of 7.6-55.2 pg/seg (mean: 28.8, median: 29.6; each pg/seg). In this case, the obtained profiles showing at least two reproducible concentration maxima between 20 and 40 mm point to an ingestion of GHB. The concentration profiles from single hairs were reproducible in each case, reflecting the concentration course of routine 1-cm segmental analysis. These are the first results published on GHB testing in segmented single hairs, and the results must be verified further., (© 2024 John Wiley & Sons Ltd.)

In September 2019, a 22-year-old man with a history of drug abuse presented to the hospital with altered mental status. Due to a suspected drug overdose, a blood sample taken on admission and a urine sample collected 30 h thereafter were submitted to our laboratory to test for illegal drugs, pharmaceutical substances, and designer drugs. During the routine toxicological analysis of the serum sample, morphine and phenobarbital were identified by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Additionally, two compounds showing identical accurate masses and isotope ratios as the designer benzodiazepine diclazepam and the benzodiazepine lormetazepam were found. However, retention times differed significantly from the expected values, and the acquired MS/MS spectra did not match the library entries of the two compounds, indicating the presence of two previously unknown substances. After further investigation, SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone), a methaqualone analog, which has recently emerged on the research chemical market, and its hydroxy metabolite were tentatively identified by accurate mass, isotope matching, and plausible fragmentation. However, for unequivocal confirmation and quantification, a reference standard is required. As no reference material was available by the end of 2019, SL-164 was obtained from an online shop, and its identity and purity (97.8%) were confirmed by nuclear magnetic resonance spectroscopy. The subsequent quantitative analysis revealed a concentration of 390 ng/mL SL-164 in serum. In the urine sample, the parent compound was not detected, but three suspected monohydroxylated metabolites were found. This example shows that LC-QTOF-MS is a powerful approach for the (tentative) identification of unknown compounds in biological matrices., (© 2024 John Wiley & Sons Ltd.)

Phosphatidylethanol (PEth) has recently become a popular direct alcohol marker for evaluating drinking behavior. This study aimed at gaining further information on the long-term stability of five PEth homologues (16:0/18:1, 16:0/18:2, 16:0/20:4, 18:0/18:1, 18:0/18:2) in whole blood (WB) and dried blood spots (DBS) stored at -80°C, 4°C, and room temperature (18°C) over a period of 60 days. Venous blood was taken from 10 volunteers (five females and five males, aged 21-40 years) with a moderate drinking behavior and a negative breath alcohol test at the time of collection. 100 μL aliquots of WB were prepared in addition to 20 μL DBS samples. The initial PEth concentrations were determined on the day of the blood collection. On days 1, 3, 5, 7, 11, 17, 40, and 60, DBS were analyzed in triplicate by means of LC-MS/MS. On these days, WB aliquots having been stored until that time were used to create further DBS in triplicate, which were subsequently stored at 18°C and analyzed in a single batch after day 60. All homologues, except PEth 16:0/20:4, were stable at -80°C in DBS and WB for 60 days. The initial PEth 16:0/18:1 concentrations remained stable in both DBS and WB in all but one volunteer's specimen at 4 and 18°C. Apart from this exception, simultaneously detected PEth homologues 16:0/18:2, 18:0/18:1, and 18:0/18:2 remained stable over at least 40 days in DBS. Nevertheless, the storage time between sample collection and analysis should be kept as short as possible if an ethanol-free sample cannot be ensured., (© 2023 John Wiley & Sons, Ltd.)

The aim of this study was to monitor seven phosphatidylethanol (PEth) homologues in dried blood spots (DBS) and ethyl glucuronide in hair (EtGH) over a 6-month period of drinking while documenting the daily drinks (amount and type) of alcohol via app. A total of 23 volunteers (12 males and 11 females) aged 19-54 years were enrolled. At four-weekly intervals, capillary blood to create DBS and after 3 and 6 months, respectively, a strand of hair (proximal, 3 cm) was collected. Analyses of EtGH and PEth homologues were performed using liquid chromatography-tandem mass spectrometry. All participants consumed alcohol during the 6 months. Only one participant tested negative for both PEth and EtGH. Eight participants had PEth 16:0/18:1 concentrations between 20 and <210 ng/mL (mean: 45.6 ng/mL) but EtGH concentrations below 5 pg/mg. PEth 16:0/18:1 concentrations between 20 and <210 ng/mL and EtGH concentrations between 5 and <30 pg/mg were assigned to eight subjects, uniformly matching them in the category of socially accepted drinking behavior. Four test subjects exceeded the cutoff for social drinking behavior in both PEth 16:0/18:1 (mean: 528 ng/mL) and EtGH (mean: 84.5 pg/mg). Two participants exceeded the threshold for PEth 16:0/18:1 of 210 ng/mL in blood but remained below 30 pg EtG/mg hair. PEth showed a higher detection rate for alcohol consumption than EtGH did. Moreover, PEth concentrations reacted quickly to changes in drinking behavior, whereas EtGH concentrations remained similar over time., (© 2023 John Wiley & Sons, Ltd.)

A previously published method for single hair analysis has been applied to a doping case for further clarification. Amphetamine could be detected in multiple micro segments resulting in two distinct concentration peaks in several hairs. The consumption of a contaminated food supplement as possible source for the amphetamine is discussed., (© 2022 John Wiley & Sons, Ltd.)

A high volume of fluid can strongly reduce a drug's concentration in urine. Therefore, to detect diluted samples, the concentration of creatinine in urine is determined during testing drugs of abuse. If the concentration is below 20 mg creatinine/dl urine, the urine sample is usually rejected for drug testing. It should be examined whether creatine or creatinine ingestion can mask urine dilution by increasing the creatinine concentration. A total of 18 subjects drank 1.3 L of water and 0.2 L of orange juice on each of the three testing days: (1) without creatine, (2) with 20 g of creatine, and (3) with 20 g of creatine following incubation for 4 days in orange juice at room temperature; an acidic environment should promote conversion of creatine to creatinine. The lowest creatinine concentrations in urine were observed on average 2 h after fluid intake. At that time, ingestion of fluid without creatine, with creatine, and with creatine(ine)-orange juice mixture resulted in mean values of 11.6, 22.5, and 28.3 mg creatinine/dl urine, respectively. It can be concluded that ingestion of creatine or creatinine can increase the concentration of creatinine in urine and thus mask dilution of a sample. The conversion of creatine in orange juice further increases availability of creatinine as it is obvious from urine creatinine concentration. Therefore, creatine ingestion during drug testing will give rise to negative results due to matrix adulteration. In a case of suspected creatine supplementation, the creatine content of the sample should be determined in addition to creatinine., (© 2021 John Wiley & Sons, Ltd.)

One of the major challenges of testing drugs of abuse is the detection of highly diluted urine samples. The ingestion of a large amount of fluid can considerably reduce the concentration of substances, possibly resulting in inaccurate drug testing. For detection, determination of urinary creatinine is a widely established procedure. In this study, results from the most popular methods, including photospectrometry (Jaffe) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), have been compared regarding 327 urine abstinence control samples. Because samples with creatinine concentrations close to the cutoff of 20 mg/dL are of particular interest, only samples below 50 mg/dL were considered. Results revealed a close correlation of creatinine concentrations by both analytical methods with an R 2 value of 0.9005. A mean concentration difference of 3.30 ± 3.45 mg/dL was observed, indicating a moderate underestimation by the Jaffe reaction. Graphical analyses showed high accordance between both methods with only a few outliers. Due to easy handling and for economic reasons, the spectrometric method is often preferred over LC-MS/MS. For urine samples with creatinine concentrations close to the cutoff, confirmation through a second method should be performed to avoid a possible disadvantage or even severe consequences for the respective individual. It is recommended that each laboratory establishes a reliable verification method., (© 2021 John Wiley & Sons, Ltd.)

Hair is the matrix of choice in forensic toxicology when retrospective analysis is needed. Nonetheless, due to misalignment, different growth stages and segmentation lengths of 0.5-1 cm, resolution of time is limited. By segmental analysis of single hairs, most of these factors can be compensated and resolution of time is enhanced. A method for manually segmenting single hairs in 2-mm sections and screening for 156 analytes by liquid chromatography coupled to tandem mass spectrometry has been developed and validated. The method was applied to 15 single-dose cases concerning different pharmaceuticals by analyzing 10 hairs each, sampled 1 and 2 months after ingestion in most cases. The validation showed a lower limit of quantification of ≤1.25 pg/segment for ~90% of analytes and good accuracy. Many substances could be detected in the presented cases, whereas detection of benzodiazepines and low dosed opioids remains challenging. In positive cases, characteristic peak-shaped concentration profiles across the hairs were obtained. The segment with most coinciding peak maxima can be allocated to the time of ingestion. A method for the determination of individual hair growth rate was applied and revealed a gap between expected and actual position of peak maxima. Additionally, different localization of simultaneously administered substances was observed. These findings were tried to be explained by different routes of incorporation and may contribute to current knowledge. The presented method may directly be applied to similar questions in hair analysis, and the findings are considered important for interpreting further results in single hair analysis., (© 2021 John Wiley & Sons, Ltd.)

Driving under the influence of drugs (DUID) is a serious global problem and poses a public health risk. With new psychoactive substances (NPS) entering the illicit drug market several years ago, a significant number of highly potent and harmful drugs have become easily available and the use of these substances may impair a person's ability to drive a vehicle safely. Since NPS are not usually covered in routine toxicological analyses used in DUID investigations, only little is known about their prevalence. To gather more information on the prevalence of NPS in cases of impaired driving, a retrospective study was conducted to determine the prevalence of these drugs in blood samples of DUID suspects in southern Germany. A total of 837 blood samples, which were collected in the German federal states Baden-Württemberg and Bavaria in 2017 and 2018, were reanalyzed for designer stimulants and synthetic cannabinoids by liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). For the analysis of synthetic cannabinoids, a more sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) screening method was additionally used. A total of 14 cases (1.6%) tested positive for NPS. Designer stimulants were detected in two cases (0.2%) and synthetic cannabinoids were found in 12 cases (1.4%). The rather low prevalence rate of 1.6% estimated in this study suggests that driving under the influence of NPS does not play a large role in southern Germany. Nonetheless, in all cases in which the psychophysical impairment cannot be explained by routine toxicological findings, a screening for NPS should additionally be performed., (© 2020 John Wiley & Sons, Ltd.)

Highly diluted urine is among the most commonly observed factors affecting the validity of urine testing for drug abuse. A minimum creatinine concentration of 20 mg/dL urine has been proposed as a marker for dilution of a urine sample. This study investigates the effect of water consumption on creatinine concentration, as well as its effect on specific gravity and osmolality. In this study, 22 subjects (17 women and 5 men) were included to determine the influence of sex and weight on the impact of excessive water consumption on these markers of urine dilution. The subjects consumed 0.5 L, 1.0 L, and 1.5 L of water, respectively, within 15 minutes. The mean minimum creatinine concentrations (Jaffé reaction) for the void 2 hours after fluid intake were 60.4 mg/dL, 15.8 mg/dL, and 10.9 mg/dL for the respective ingested volumes of water. Mean creatinine concentrations excreted by men were significantly higher than those excreted by women. Participants with a weight below 60 kg tended to excrete lower urine creatinine concentrations. 50% of the volunteers with a BMI < 20 kg/m 2 and 20% of the volunteers with a BMI > 20 kg/m 2 exhibited creatinine concentrations below the threshold value of 20 mg/dL. A similar pattern was established for gravity and osmolality. Due to its simple determination, creatinine may be preferred over specific gravity or osmolality. In order to evaluate the internal dilution of a urine sample for legally defensible drug testing, it may be necessary to account for sex and body weight., (© 2018 John Wiley & Sons, Ltd.)

A 100 μg/L or higher concentration of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in blood serum is generally assumed to be associated with regular and/or heavy use of cannabis. At present, determination of the extent of cannabis use by means of the concentration of THC-COOH in hair has not been assessed. Therefore, we aimed at establishing a threshold for THC-COOH concentrations in hair to prove frequent consumption by comparing THC-COOH concentrations in 129 corresponding serum and hair samples, respectively. The concentration of THC-COOH in the serum was analyzed by gas chromatography-mass spectrometry and in the hair by liquid chromatography-tandem mass spectrometry. Data were statistically evaluated using receiver operating characteristic curves and contingency tables. Our results suggest that a THC-COOH concentration of ≥4.2 pg/mg in hair was always accompanied by a THC-COOH concentration of at least 100 μg/L in blood serum. Should this be confirmed by further studies of a larger study population, a hair concentration of 4.2 pg/mg THC-COOH can be set as a threshold to predict regular and/or heavy consumption of cannabis even if no corresponding blood sample is available for analysis., (© 2018 John Wiley & Sons, Ltd.)

Testing hair for cannabis use has increasingly been scrutinised due to exposure to second-hand smoke or environmental contamination. Confirmation of drug use involving detection of metabolites such as 11-nor-9-carboxy-delta-9-tetrahydrocannabinol (THC-COOH) and 11-hydroxy-delta-9-tetrahydrocannabinol (THC-OH) having very rarely been considered. We developed a new, simplified procedure with regard to expenditure of time and material to determine delta-9-tetrahydrocannabinol (THC, qualitatively), as well as THC-OH and THC-COOH (quantitatively) from 587 hair samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was compared to hitherto established methods (n = 3). Compared to conventional methanolic extraction alkaline dissolution resulted in higher concentrations for THC-OH. Concentrations determined from specimens ranged from 0.01 to 18.7 ng THC/mg hair, 0.05-37.6 pg THC-OH/mg hair, and from 0.1 to 54.3 pg THC-COOH/mg hair. THC was detectable in 70.4% samples along with both metabolites from more than half of these samples. In 12.9% of THC-positive cases, neither THC-OH nor THC-COOH were present. In 8.9% of THC-negative cases, it was possible to detect metabolites either alone or in combination. THC-OH could more frequently be detected than THC-COOH and appeared to be less susceptible to cosmetic treatment. In summary, THC-OH turned out to be a further suitable marker to prove cannabis use. Determination of both metabolites is recommended to unequivocally differentiate consumption from external exposure or contamination., (© 2018 John Wiley & Sons, Ltd.)

Although hair is widely used to identify drug use, there is a risk of false positives due to environmental contamination. This especially applies to cocaine (COC). Several strategies such as detection of norcocaine (NCOC) or cocaethylene, metabolite concentration ratios or intricate washing procedures have been proposed to differentiate actual use from contamination. The aim of the present study was to identify hydroxy metabolites of COC in hair specimens, thus enabling unambiguous prove of ingestion. A suspect screening of 41 COC-positive samples for these compounds was performed by liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS). Once identified, mass transitions for o-, p- and m-isomers of hydroxy COC as well as p- and m-isomers of hydroxy benzoylecgonine (BE) and hydroxy NCOC were introduced into a routine procedure for testing drugs of abuse in hair by liquid chromatography-tandem mass spectrometry (LC-MS/MS) which was applied to 576 hair samples. Hydroxy metabolites were present in 92.2% of COC-positive hair samples; their detection rate exceeded that of cocaethylene and NCOC. Moreover, p-OH-BE, m-OH-BE as well as p-OH-NCOC and m-OH-NCOC have been identified for the first time in COC-positive hair specimens. Hydroxy cocainics could be detected in samples having a negative conclusion on drug use applying hitherto established criteria. We suggest a more conclusive interpretation outcome including detection of hydroxy metabolites into the evaluation of COC-positive hair samples., (Copyright © 2017 John Wiley & Sons, Ltd.)