Effects of combined exercises on shoulder mobility and strength of the upper extremities in breast cancer rehabilitation: a 3-week randomized controlled trial.

PURPOSE: The aim of this study was to analyze the effects of mobility training with FIVE® devices in combination with device-supported strength exercises for shoulder mobility and strength of the upper extremities in women with breast cancer. METHODS: We conducted a pretest-posttest intervention study with female breast cancer patients (n = 41) who were randomly assigned to two groups by lot during their stationary follow-up treatment at a rehabilitation clinic in the south of Germany between February and March 2020. As part of exercise therapy, the intervention group (n = 24) performed a mobility training with FIVE® devices combined with device-supported strength training, whereas the control group (n = 17) completed device-supported strength training. Before and after the 3-week intervention (3 training sessions/week), shoulder mobility and isokinetic maximal strength were tested. RESULTS: Both groups achieved significant improvements in shoulder mobility in the frontal and sagittal plane (between 3.8 and 15.35%; p < 0.05) and in strength performance (31.36% [IG] vs. 51.24% [CG]; p < 0.001). However, no robust evidence could be determined about potential interaction effects. CONCLUSION: A combined device-supported strength and mobility training (FIVE®) showed no advantages. Therefore, a variety of exercise methods is possible in exercise therapy of breast cancer patients. CLINICAL TRIAL REGISTRATION NUMBER: Since the University of Education Weingarten does not assign clinical trial registration numbers or ethical approval numbers, none could be assigned for this study.

Application of Dried Urine Spots for Non-Targeted Quadrupole Time-of-Flight Drug Screening.

The use of dried urine spots (DUS) can simplify sample handling, shipment and storage when compared to liquid urine samples. To prepare DUS, a small amount of urine is pipetted on a filter paper card. The subsequent drying of the specimen can prevent the post-sampling formation or degradation of substances (e.g., caused by bacteria). To evaluate the potential of DUS screening, 17 authentic urine samples, containing a broad range of substances, were extracted and analyzed on a Sciex TripleTOF® 5600+ System using a non-targeted screening and library searching approach. The screening results were compared to the analysis of the same urine sample in liquid form, using the same high-resolution liquid chromatography--quadrupole time-of-flight mass spectrometry method. More than 65 different legal and illegal drugs were successfully identified within the investigated 17 urine samples using the DUS screening approach. When compared to the analysis of liquid urine, the following compounds could not be identified: 1x ecgonine methyl ester, 1x nicotine, 1x promazine and 1x 11-nor-9-carboxy-∆9-tetrahydrocannabinol. Overall, 95.2% of the target substances that have been detected in liquid urine were identified correctly using the DUS approach. In conclusion, DUS screening offers a simple, cost-effective and easier sample handling alternative to the traditional use of liquid urine and provides the detection of the most important substances for forensic requirements. Furthermore, the DUS sample preparation can be fully automated (sample documentation, internal standard application and extraction).

SWATH data independent acquisition mass spectrometry for screening of xenobiotics in biological fluids: Opportunities and challenges for data processing.

SWATH data independent acquisition (DIA) mass spectrometry (MS) has become an established technique in MS-based 'omics' research and is increasingly used for the screening of xenobiotics (e.g. drugs, drug metabolites, pesticides, toxicants). Such xenobiotic screening methods are mostly applied for tentative compound identification purposes based on spectral library searching, while additional data processing techniques are scarcely used thereby leaving the full potential of these methods often unused. Here we present an analytical workflow for screening xenobiotics in human samples using SWATH/MS based on which we highlight opportunities for unlocking unused potential of these methods. The workflow was applied to urine samples from subjects who tested positive for THC and/or cocaine during roadside drug testing with the goal of confirming the positive roadside drug tests and identifying compounds that relate to illicit drug use (e.g. cutting agents, tobacco components) or associate with corresponding lifestyle choices (e.g. nasal decongestants, painkillers). These goals could only be reached by complementing spectral library search procedures with additional multivariate data analyses due to inherent incompleteness of the spectral library that was employed. Such incompleteness represents a common challenge for applications where limited or no metadata is available for study samples, for example in toxicology, doping control in sports, and workplace or roadside drug testing. It furthermore sets the stage for employing additional data processing techniques as is outlined in the presented work.

Quantitative determination of CBD and THC and their acid precursors in confiscated cannabis samples by HPLC-DAD.

Analysis of cannabis has gained new importance worldwide, mainly for quality control within the legalized recreational and medical cannabis industry, but also for forensic differentiation between drug-type cannabis and legal products such as fiber hemp and CBD-rich/THC-poor cannabis. We herein present an HPLC-DAD method for quantitative analysis of major neutral and acidic cannabinoids in herbal cannabis and hashish: Δ9-tetrahydrocannabinol (THC), Δ9-tetrahydrocannabinolic acid A (THCA), cannabidiol (CBD), cannabidiolic acid (CBDA), and cannabinol (CBN). Plant material was dried, homogenized and extracted with a mixture of methanol/hexane. Chromatographic separation of the analytes was achieved on a core-shell C8 column using gradient elution with water/acetonitrile containing 0.1% formic acid. The analytical run time was 13 min and analytes were detected at 210 nm. The method is selective, sensitive, accurate, and precise, as confirmed through validation according to ICH and AOAC guidelines. Linearity in herbal cannabis ranged from 0.04 to 4.00% for the neutral cannabinoids, and from 0.40 to 20% for the acids. Linear ranges in hashish samples were 0.13-13.33% and 1.33-66.66%, respectively. The presented method was successfully applied to characterize 110 cannabis samples seized by the Swiss police, demonstrating its applicability for routine cannabis potency testing in the forensic setting.

Investigating the ability of the microbial model Cunninghamella elegans for the metabolism of synthetic tryptamines.

Tryptamines can occur naturally in plants, mushrooms, microbes, and amphibians. Synthetic tryptamines are sold as new psychoactive substances (NPS) because of their hallucinogenic effects. When it comes to NPS, metabolism studies are of crucial importance, due to the lack of pharmacological and toxicological data. Different approaches can be taken to study in vitro and in vivo metabolism of xenobiotica. The zygomycete fungus Cunninghamella elegans (C. elegans) can be used as a microbial model for the study of drug metabolism. The current study investigated the biotransformation of four naturally occurring and synthetic tryptamines [N,N-Dimethyltryptamine (DMT), 4-hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET), N,N-di allyl-5-methoxy tryptamine (5-MeO-DALT) and 5-methoxy-N-methyl-N-isoporpoyltryptamine (5-MeO-MiPT)] in C. elegans after incubation for 72 hours. Metabolites were identified using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqTOF) instrument. Results were compared to already published data on these substances. C. elegans was capable of producing all major biotransformation steps: hydroxylation, N-oxide formation, carboxylation, deamination, and demethylation. On average 63% of phase I metabolites found in the literature could also be detected in C. elegans. Additionally, metabolites specific for C. elegans were identified. Therefore, C. elegans is a suitable complementary model to other in vitro or in vivo methods to study the metabolism of naturally occurring or synthetic tryptamines.

Determination of the formation rate of phosphatidylethanol by phospholipase D (PLD) in blood and test of two selective PLD inhibitors.

Phosphatidylethanol (PEth) is an alcohol biomarker formed from phosphatidylcholine (PC) by the enzyme phospholipase D (PLD) in the presence of ethanol. A drinking study revealed individual differences in maximum PEth levels after drinking to a targeted blood alcohol concentration (BAC) of 0.1%. This seemed to be due to different PLD activities in the tested persons. Furthermore, post-sampling formation of PEth occurred in blood samples, still containing alcohol. Therefore, a standardized in vitro test for measuring individual PEth formation rates was developed. Two PLD inhibitors were tested for their potency to inhibit post-sampling PEth formation. PEth-negative blood samples were collected from a volunteer. Ethanol was added in different concentrations (0.01-0.3% BAC) directly after blood sampling. The specimens were incubated at 37 °C. Aliquots were taken at the start of the incubation, and every hour until 8 h after start of incubation, and one sample was taken on subsequent days over 1 week. PEth 16:0/18:1 and PEth 16:0/18:2 were determined by online SPE-LC-MS/MS. Furthermore, this test system was applied to blood samples of 12 volunteers. For the inhibition tests, fresh blood (spiked with 0.1% ethanol) was spiked with 30, 300, 3000, or 30,000 nM of either halopemide or 5-fluoro-2-indolyl-deschlorohalopemide (FIPI), and incubated at 37 °C. PEth concentrations were determined hourly over 5 h on the first day and once on day 2 and day 3. PEth formation was linear in the first 7 h of incubation and dependent on the alcohol concentration. The formation rates of PEth 16:0/18:1 were 0.002 µmol L-1 h-1 (0.01% BAC), 0.016 µmol L-1 h-1 (0.1% BAC), 0.025 µmol L-1 h-1 (0.2% BAC), and 0.029 µmol L-1 h-1 (0.3% BAC). For PEth 16:0/18:2, the formation rates were 0.002 µmol L-1 h-1 (0.01% BAC), 0.019 µmol L-1 h-1 (0.1% BAC), 0.025 µmol L-1 h-1 (0.2% BAC), and 0.030 µmol L-1 h-1 (0.3% BAC). Maximum concentrations reached 431 ng/mL (PEth 16:0/18:1) and 496 ng/mL (PEth 16:0/18:2) at 0.3% BAC after 3 days. Maximum velocity (vmax) was not reached under these conditions. PEth formation in blood of the 12 volunteers ranged between 0.011 and 0.025 µmol L-1 h-1 for PEth 16:0/18:1 and between 0.014 and 0.021 µmol L-1 h-1 for PEth 16:0/18:2. PEth formation in human blood was inhibited by halopemide in a concentration-dependent manner. However, a complete inhibition was not achieved by the applied maximum concentration of 30,000 nM. FIPI showed a better inhibition of PEth formation. A complete inhibition could be achieved by a concentration of 30,000 nM for the first 24 h (for PEth 16:0/18:1) and for 48 h (for PEth 16:0/18:2). Formation of PEth was found to be dependent on the BAC. As a consequence, it is essential to inhibit PLD activity after blood collection to avoid post-sampling formation of PEth in blood samples with a positive BAC. Inhibition of PEth formation was more effective using FIPI, compared to halopemide.

In vitro phase I metabolism of three phenethylamines 25D-NBOMe, 25E-NBOMe and 25N-NBOMe using microsomal and microbial models.

Numerous 2,5-dimethoxy-N-benzylphenethylamines (NBOMe), carrying a variety of lipophilic substituents at the 4-position, are potent agonists at 5-hydroxytryptamine (5HT2A ) receptors and show hallucinogenic effects. The present study investigated the metabolism of 25D-NBOMe, 25E-NBOMe, and 25N-NBOMe using the microsomal model of pooled human liver microsomes (pHLM) and the microbial model of the fungi Cunninghamella elegans (C. elegans). Identification of metabolites was performed using liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS) with a quadrupole time-of-flight (QqToF) instrument. In total, 36 25D-NBOMe phase I metabolites, 26 25E-NBOMe phase I metabolites and 24 25N-NBOMe phase I metabolites were detected and identified in pHLM. Furthermore, 14 metabolites of 25D-NBOMe, 11 25E-NBOMe metabolites, and nine 25N-NBOMe metabolites could be found in C. elegans. The main biotransformation steps observed were oxidative deamination, oxidative N-dealkylation also in combination with hydroxylation, oxidative O-demethylation possibly combined with hydroxylation, oxidation of secondary alcohols, mono- and dihydroxylation, oxidation of primary alcohols, and carboxylation of primary alcohols. Additionally, oxidative di-O-demethylation for 25E-NBOMe and reduction of the aromatic nitro group and N-acetylation of the primary aromatic amine for 25N-NBOMe took place. The resulting 25N-NBOMe metabolites were unique for NBOMe compounds. For all NBOMes investigated, the corresponding 2,5-dimethoxyphenethylamine (2C-X) metabolite was detected. This study reports for the first time 25X-NBOMe N-oxide metabolites and hydroxylamine metabolites, which were identified for 25D-NBOMe and 25N-NBOMe and all three investigated NBOMes, respectively. C. elegans was capable of generating all main biotransformation steps observed in pHLM and might therefore be an interesting model for further studies of new psychoactive substances (NPS) metabolism.

Study of the in vitro and in vivo metabolism of 4-HO-MET.

4-Hydroxy-N-methyl-N-ethyltryptamine (4-HO-MET) is a new psychoactive substance (NPS) of the chemical class of tryptamines. It shows structural similarities to the endogenous neurotransmitter serotonin, and is a serotonergic hallucinogen, affecting emotional, motoric, and cognitive functions. The knowledge about its biotransformation is mandatory to confirm the abuse of the substance by urine analysis in forensic cases. Therefore, phase I metabolites were generated by the use of the pooled human liver microsomes (pHLM) in vitro model and analyzed by high-performance liquid chromatography high-resolution tandem mass spectrometry with information-dependent acquisition (HPLC-IDA-HR-MS/MS). Furthermore, three authentic urine samples was analyzed and results were compared: 12 different in vitro and 4 in vivo metabolites were found. The predominant biotransformation steps observed in vitro were mono- or dihydroxylation of 4-HO-MET, besides demethylation, demethylation in combination with monohydroxylation, formation of a carboxylic acid, deethylation, and oxidative deamination. In vivo, monohydroxylation, and glucuronidation were detected. A metabolic pathway based on these results was proposed. For the analysis of urine samples in forensic cases, the N-oxide metabolite and the HO-alkyl metabolite are recommended as target compounds, besides the glucuronides of 4-HO-MET and the parent compound 4-HO-MET itself.

Analysis of volatiles in fire debris by combination of activated charcoal strips (ACS) and automated thermal desorption-gas chromatography-mass spectrometry (ATD/GC-MS).

Adsorption of volatiles in gaseous phase to activated charcoal strip (ACS) is one possibility for the extraction and concentration of ignitable liquid residues (ILRs) from fire debris in arson investigations. Besides liquid extraction using carbon dioxide or hexane, automated thermo-desorption can be used to transfer adsorbed residues to direct analysis by gas chromatography-mass spectrometry (GC-MS). We present a fire debris analysis work-flow with headspace adsorption of volatiles onto ACS and subsequent automated thermo-desorption (ATD) GC-MS analysis. Only a small portion of the ACS is inserted in the ATD tube for thermal desorption coupled to GC-MS, allowing for subsequent confirmation analysis with another portion of the same ACS. This approach is a promising alternative to the routinely used ACS method with solvent extraction of retained volatiles, and the application to fire debris analysis is demonstrated.

Development and Validation of an LC-MS/MS Method and Comparison with a GC-MS Method to Measure Phenytoin in Human Brain Dialysate, Blood, and Saliva.

Phenytoin (PHT) is one of the most often used critical dose drugs, where insufficient or excessive dosing can have severe consequences such as seizures or toxicity. Thus, the monitoring and precise measuring of PHT concentrations in patients is crucial. This study develops and validates an LC-MS/MS method for the measurement of phenytoin concentrations in different body compartments (i.e., human brain dialysate, blood, and saliva) and compares it with a formerly developed GC-MS method that measures PHT in the same biological matrices. The two methods are evaluated and compared based on their analytical performance, appropriateness to analyze human biological samples, including corresponding extraction and cleanup procedures, and their validation according to ISO 17025/FDA Guidance for Industry. The LC-MS/MS method showed a higher performance compared with the GC-MS method. The LC-MS/MS was more sensitive, needed a smaller sample volume (25 µL) and less chemicals, was less time consuming (cleaning up, sample preparation, and analysis), and resulted in a better LOD (<1 ng/mL)/LOQ (10 ng/mL). The calibration curve of the LC-MS/MS method (10-2000 ng/mL) showed linearity over a larger range with correlation coefficients r2 > 0.995 for all tested matrices (blood, saliva, and dialysate). For larger sample numbers as in pharmacokinetic/pharmacodynamic studies and for bedside as well as routine analyses, the LC-MS/MS method offers significant advantages over the GC-MS method.

Study of the in vitro and in vivo metabolism of the tryptamine 5-MeO-MiPT using human liver microsomes and real case samples.

The synthetic tryptamine 5-methoxy-N-methyl-N-isopropyltryptamine (5-MeO-MiPT) has recently been abused as a hallucinogenic drug in Germany and Switzerland. This study presents a case of 5-MeO-MiPT intoxication and the structural elucidation of metabolites in pooled human liver microsomes (pHLM), blood, and urine. Microsomal incubation experiments were performed using pHLM to detect and identify in vitro metabolites. In August 2016, the police encountered a naked man, agitated and with aggressive behavior on the street. Blood and urine samples were taken at the hospital and his premises were searched. The obtained blood and urine samples were analyzed for in vivo metabolites of 5-MeO-MiPT using liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS). The confiscated pills and powder samples were qualitatively analyzed using Fourier transform infrared (FTIR), gas chromatography-mass spectrometry (GC-MS), LC-HRMS/MS, and nuclear magnetic resonance (NMR). 5-MeO-MiPT was identified in 2 of the seized powder samples. General unknown screening detected cocaine, cocaethylene, methylphenidate, ritalinic acid, and 5-MeO-MiPT in urine. Seven different in vitro phase I metabolites of 5-MeO-MiPT were identified. In the forensic case samples, 4 phase I metabolites could be identified in blood and 7 in urine. The 5 most abundant metabolites were formed by demethylation and hydroxylation of the parent compound. 5-MeO-MiPT concentrations in the blood and urine sample were found to be 160 ng/mL and 3380 ng/mL, respectively. Based on the results of this study we recommend metabolites 5-methoxy-N-isopropyltryptamine (5-MeO-NiPT), 5-hydroxy-N-methyl-N-isopropyltryptamine (5-OH-MiPT), 5-methoxy-N-methyl-N-isopropyltryptamine-N-oxide (5-MeO-MiPT-N-oxide), and hydroxy-5-methoxy-N-methyl-N-isopropyltryptamine (OH-5-MeO-MiPT) as biomarkers for the development of new methods for the detection of 5-MeO-MiPT consumption, as they have been present in both blood and urine samples.

Evaluation of N-acetyltaurine as an ethanol marker in human blood.

To investigate the potential of N-acetyltaurine (NAcT) in blood as a biomarker for alcohol uptake, a previously published LC-MS/MS method for urine was modified to simultaneously detect NAcT and ethyl glucuronide (EtG). The method was applied in a drinking study and by analyzing 147 forensic case samples. In the drinking study, contrary to EtG, NAcT proved to be an endogenous substance, which was present at 22 ± 7 ng/mL (13-31 ng/mL) in the blood after 2 weeks of abstinence. A moderate increase in NAcT to 40 ± 10 ng/mL (27-57 ng/mL) was observed after drinking. Within 24 h, the NAcT concentrations declined to starting concentrations in seven out of eight subjects. Peak EtG concentrations (c¯max) of 445 ± 101 ng/mL (278-662 ng/mL) were reached. While EtG in blood can be used to detect alcohol consumption even if ethanol is already eliminated, some of the maximum NAcT concentrations after a single ethanol dose were in the range of endogenous levels detected prior to the start of drinking in other subjects. In the 147 blood samples, the following concentrations were found: blood alcohol concentration (BAC): 1.22 ± 0.95 g/kg (0-3.46 g/kg); NAcT: 37.8 ± 18.4 ng/mL (12.1-109 ng/mL); EtG: 1149 ± 1121 ng/mL (0-5950 ng/mL). ROC curve analysis for BAC thresholds at 0.8 and 1.6 g/kg were performed for EtG and NAcT. Due to the presence of endogenous NAcT levels resulting in a lower sensitivity and selectivity when compared to EtG, and due to a minor increase in concentration after alcohol uptake, the usefulness of NAcT as an alcohol biomarker in blood is very limited.

Calypso's spell: accidental near-fatal thiacloprid intoxication.

Acute life-threatening intoxications with insecticides are rare. We report a case of accidental near-fatal thiacloprid intoxication with mass spectrometry-based analytical confirmation. The initial clinical presentation resembled imminent brain death and/or severe postanoxic encephalopathy. Prolonged supportive treatment resulted in full recovery underlining intoxication as an important differential diagnosis in unclear coma.

MEMS and FOG Technologies for Tactical and Navigation Grade Inertial Sensors-Recent Improvements and Comparison.

In the following paper, we present an industry perspective of inertial sensors for navigation purposes driven by applications and customer needs. Microelectromechanical system (MEMS) inertial sensors have revolutionized consumer, automotive, and industrial applications and they have started to fulfill the high end tactical grade performance requirements of hybrid navigation systems on a series production scale. The Fiber Optic Gyroscope (FOG) technology, on the other hand, is further pushed into the near navigation grade performance region and beyond. Each technology has its special pros and cons making it more or less suitable for specific applications. In our overview paper, we present latest improvements at NG LITEF in tactical and navigation grade MEMS accelerometers, MEMS gyroscopes, and Fiber Optic Gyroscopes, based on our long-term experience in the field. We demonstrate how accelerometer performance has improved by switching from wet etching to deep reactive ion etching (DRIE) technology. For MEMS gyroscopes, we show that better than 1°/h series production devices are within reach, and for FOGs we present how limitations in noise performance were overcome by signal processing. The paper also intends a comparison of the different technologies, emphasizing suitability for different navigation applications, thus providing guidance to system engineers.

Rapid quantification of free and glucuronidated THCCOOH in urine using coated well plates and LC-MS/MS analysis.

AIM: Generally, urine drug testing for cannabis abuse involves measuring total concentrations of 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) obtained by enzymatic and/or alkaline hydrolysis of THCCOOH-glucuronide. As hydrolysis can be inconsistent and incomplete, direct measurement of the two metabolites is preferable. Methodology & results: We developed a high-throughput LC-MS/MS method for simultaneous quantification of free and glucuronidated THCCOOH in urine using coated 96-well plates for analyte extraction and column-switching chromatography. Excellent separation of the two analytes was achieved within 2.5 min, with linear ranges from 5 to 2000 µg/l for THCCOOH and from 10 to 4000 µg/l for THCCOOH-glucuronide. CONCLUSION: The method was successfully validated and applied to authentic urine samples from cannabis consumers, demonstrating its applicability for routine cannabinoid testing.

Accelerated quantification of amphetamine enantiomers in human urine using chiral liquid chromatography and on-line column-switching coupled with tandem mass spectrometry.

Amphetamine (AM) is a powerful psychostimulant existing in two enantiomeric forms. Stereoselective analysis of AM in biosamples can assist clinicians and forensic experts in differentiating between abuse of illicitly synthesized racemic AM and ingestion of pharmaceutical AM formulations containing either S-AM or different proportions of the S- and R-enantiomers. Therefore, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantifying AM enantiomers in urine was newly developed. The method comprised dilution with water, followed by injection of the diluted sample onto an achiral C18 trapping column for purification and subsequent backflush elution to a chiral Lux 3 µm AMP LC column by means of a switching valve. An isocratic mobile phase of 25 % acetonitrile in 0.1 M aqueous ammonia was used for enantiomeric separation. Injection, cleanup, and backflush of the next sample were performed before the previous sample had eluted from the analytical column, thus enabling simultaneous enantioseparation of up to three samples within the analytical column. This novel chromatographic concept allowed for increased sample throughput by accelerating both the sample preparation and the LC analysis. Analyte detection was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was successfully validated through assessment of its linearity, lower limit of quantification, accuracy and precision, selectivity, matrix effect, carry-over, dilution integrity, and re-injection reproducibility. Linearity ranged from 0.05 to 25 mg/L for both enantiomers. Proof of the method included analysis of urine samples obtained from drug abusers and patients receiving an S-AM prodrug. Graphical Abstract Enantioselective determination of amphetamine in human urine using liquid chromatography with achiral-chiral column-switching and tandem mass spectrometry.

Assessing cannabis consumption frequency: Is the combined use of free and glucuronidated THCCOOH blood levels of diagnostic utility?

Heavy cannabis consumption is considered incompatible with safe driving. In Swiss traffic policy, drivers suspected of regular cannabis use are therefore required to undergo medical assessment of their long-term fitness to drive. A whole blood concentration of the cannabis metabolite 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol (THCCOOH) of 40 µg/L is currently used by Swiss forensic experts as the decision limit for regular cannabis consumption. The present study aimed to investigate the suitability of THCCOOH-glucuronide blood levels as an additional and/or better marker for the frequency of cannabis use. Whole blood samples collected from 23 heavy (≥10 joints/month) and 25 occasional smokers (≥1 joint/month, but ≤ 1 joint/week) enrolled in a placebo-controlled cannabis smoking study were analyzed for THCCOOH and THCCOOH-glucuronide. Based on receiver operating characteristic (ROC) curve analysis, concentration thresholds could be established for distinguishing between these two groups. Proposed thresholds for heavy use were THCCOOH-glucuronide > 52 µg/L (100% specificity; 41% sensitivity) and/or total THCCOOH > 58 µg/L (100% specificity; 43% sensitivity). Optimum thresholds for occasional use were THCCOOH-glucuronide < 5 µg/L (73% specificity; 97% sensitivity) and/or total THCCOOH < 5 µg/L (62% specificity; 98% sensitivity). Our results indicate that the THCCOOH-glucuronide whole blood concentration is a useful parameter that complements the free THCCOOH level to assess the frequency of cannabis consumption. The consideration of the blood concentrations of both free and glucuronidated THCCOOH improves the identification of heavy users whose fitness to drive has to be carefully assessed. Copyright © 2016 John Wiley & Sons, Ltd.

N-Acetyltaurine as a novel urinary ethanol marker in a drinking study.

The forensic utility of N-acetyltaurine (NAcT) in urine as a marker for ethanol intake was examined. A HILIC-based liquid chromatography method for the mass spectrometric determination of NAcT, taurine, and creatinine in urine was developed and validated to investigate NAcT formation and elimination in a drinking study. Thereby, eight subjects ingested 0.66 to 0.84 g/kg alcohol to reach a blood alcohol concentration (BAC) of 0.8 g/kg. Blood and urine were taken every 1.5-2 h, during the first 8 h. NAcT and taurine levels were measured and corrected for the urine's dilution by normalization to a creatinine concentration of 1 mg/mL. For the determination of NAcT and taurine, uncorrected lower limits of quantitation (LLOQs) were at 0.05 µg/mL of urine. In the drinking study, NAcT proved to be an endogenous compound, which is present at a range of 1.0 to 2.3 µg/mL in urine of alcohol-abstinent subjects. Maximum NAcT concentrations were reached in samples taken 3 to 6 h after the start of drinking, whereby an upregulation in N-acetyltaurine could be found for all the subjects. The mean peak concentrations (c̅ max) of 14 ± 2.6 µg/mL (range 9-17.5 µg/mL) were reached. Within 24 h, the NAcT levels declined to endogenous concentrations. The detectability of NAcT was found to be slightly shifted compared to BAC: When BAC was not detectable anymore, NAcT levels were still elevated. After 24 h, when ethyl glucuronide (EtG) and ethyl sulfate (EtS) were still detectable, NAcT concentrations showed endogenous levels again. Positive NAcT results can be used as an indicator for recent alcohol consumption. A direct relationship between NAcT and taurine concentrations could not be found. Graphical abstract N-acetyltaurine concentrations for eight subjects during the first 24 h after an alcohol consumption of 0.8 g/kg.

Application of phosphatidylethanol (PEth) in whole blood in comparison to ethyl glucuronide in hair (hEtG) in driving aptitude assessment (DAA).

For driving aptitude assessment (DAA), the analysis of several alcohol biomarkers is essential for the detection of alcohol intake besides psycho-medical exploration. In Switzerland, EtG in hair (hEtG) is often the only direct marker for abstinence monitoring in DAA. Therefore, the suitability of phosphatidylethanol (PEth) was investigated as additional biomarker. PEth 16:0/18:1 and 16:0/18:2 were determined by online-SPE-LC-MS/MS in 136 blood samples of persons undergoing DAA and compared to hEtG, determined in hair segments taken at the same time. With a PEth 16:0/18:1 threshold of 210 ng/mL for excessive alcohol consumption, all (n = 30) but one tested person also had hEtG values ≥30 pg/mg. In 54 cases, results are not in contradiction to an abstinence as neither PEth (<20 ng/mL) nor hEtG (<7 pg/mg) was detected. In eight cases, both markers showed moderate consumption. Altogether, PEth and hEtG were in accordance in 68 % of the samples, although covering different time periods of alcohol consumption. With receiver operating characteristic analysis, PEth was evaluated to differentiate abstinence, moderate, and excessive alcohol consumption in accordance with hEtG limits. A PEth 16:0/18:1 threshold of 150 ng/mL resulted in the best sensitivity (70.6 %) and specificity (98.8 %) for excessive consumption. Values between 20 and 150 ng/mL passed for moderate consumption, values <20 ng/mL passed for abstinence. As PEth mostly has a shorter detection window (2-4 weeks) than hEtG (up to 6 months depending on hair length), changes in drinking behavior can be detected earlier by PEth than by hEtG analysis alone. Therefore, PEth helps to improve the diagnostic information and is a valuable additional alcohol marker for DAA.