Will tetrahydrocannabinol be formed from cannabidiol in gastric fluid? An in vivo experiment.

Cannabidiol (CBD) products have ascribed an uprising trend for their health-promoting effects worldwide. In contrast to Δ9-tetrahydrocannabinol (THC), CBD exhibits no state of euphoria. Since conversion of CBD into THC in an acidic environment has been reported, it has not been proved whether this degradation will also occur in human gastric fluid. A total of 9 subjects ingested 400 mg CBD as a water-soluble liquid together with lecithin as an emulsifier and ethanol as a solubilizer. Blood samples were taken up to 4 h, and urine samples were submitted up to 48 h. THC, 11-hydroxy-Δ9-THC (THC-OH), 11-nor-9-carboxy-Δ9-THC (THC-COOH), CBD, 7-hydroxy cannabidiol (7-OH-CBD), and 7-carboxy cannabidiol (7-CBD-COOH) were determined in blood and THC-COOH and 7-CBD-COOH in urine by LC-MS/MS. Neither THC, THC-OH, nor THC-COOH were detectable in any serum specimen. Only two urine samples revealed THC-COOH values slightly above the threshold of 10 ng/ml, which could also be caused by trace amounts of THC being present in the CBD liquid. It can be concluded that negative consequences for participants of a drug testing program due to a conversion of CBD into THC in human gastric fluid appear unlikely, especially considering a single intake of dosages of less than 400 mg. Nevertheless, there is a reasonable risk for consumers of CBD products being tested positive for THC or THC metabolites. However, this is probably not caused by CBD cyclization into THC in human gastric fluid but is most likely due to THC being present as an impurity of CBD products.

Formation of phosphatidylethanol and ethylglucuronide after low to moderate alcohol consumption in volunteers with a previous three-week alcohol abstinence.

AIMS: Phosphatidylethanol (PEth) is only formed when ethanol is present in blood. This direct alcohol marker has been widely discussed, including the minimum amount of ethanol being necessary to form as much PEth as to exceed the threshold of 20 ng/mL in previously PEth negative subjects. In order to corroborate hitherto existing results, a drinking study including 18 participants after a 3-week alcohol abstinence was performed. METHODS: They consumed a pre-calculated amount of ethanol to reach a blood alcohol concentration (BAC) of at least 0.6 g/kg. Blood was drawn before and periodically seven times after alcohol administration on day 1. Blood and urine were also collected the next morning. Dried blood spots (DBS) were prepared immediately from collected venous blood. BAC was determined by head space gas chromatography and the concentrations of both PEth (16:0/18:1, 16:0/18:2 and five additional homologues) and ethyl glucuronide (EtG) were analysed using liquid chromatography-tandem mass spectrometry. RESULTS: Out of 18, 5 participants had concentrations of PEth 16:0/18:1 above the threshold of 20 ng/mL, and 11 out of the 18 subjects had concentrations between 10 and 20 ng/mL. In addition, four persons had PEth 16:0/18:2 concentrations above 20 ng/mL the following morning. All test subjects tested positive for EtG in DBS (≥ 3 ng/mL) and urine (≥100 ng/mL) upon 20-21 h after alcohol administration. CONCLUSION: By combining both a lower cutoff of 10 ng/mL and the homologue PEth 16:0/18:2, the sensitivity to detect a single alcohol intake after a 3-week abstinence increases to 72.2%.

Return of the Quaaludes? Prolonged agitated delirium after intentional ingestion of the methaqualone analog SL-164 - a case report.

Background: A 22-year-old male with a known history of drug abuse presented to our department with prolonged agitated delirium, myocloni, tachycardia and subfebrile temperature after the deliberate ingestion of opium poppy tea (Papaver somniferum L.) together with the methaqualone analog SL-164 (5-chloro-3-(4-chloro-2-methylphenyl)-2-methyl-4(3H)-quinazolinone) which is sold online as a designer drug. Methods: SL-164 and its hydroxy metabolites were detected in serum and urine via liquid chromatography-quadrupole-time-of-flight mass spectrometry (LC-QTOF-MS). Results: The pronounced delirium was treated with benzodiazepines and neuroleptics; temporary medical restraint had to be applied. Symptoms completely resolved over the next 72 h and the patient was discharged on day three able to give consent. Conclusions: Although methaqualone was a popular and widespread sedative in the 1950s and 60 s before its discontinuation in the USA in 1985, derivatives of the methaqualone class have not previously played a large role as drugs of abuse in the rapidly growing market of new psychoactive substances. To our knowledge, this is the first case of agitated delirium with detection of SL-164 and hydroxylated metabolites in a patient's serum and urine.

Comparison of concentrations of drugs between blood samples with and without fluoride additive-important findings for Δ9-tetrahydrocannabinol and amphetamine.

Fluoride is a common stabilizing agent in forensic toxicology to avoid the frequent problem of degradation of drugs in blood samples especially described for cocaine. In cases only samples with addition of fluoride are available, it is a crucial question if also concentrations of common drugs other than cocaine (amphetamines, opiates and cannabinoids) are affected by fluoride. So far, there are only rare literature data available on discrepant results especially for Δ9-tetrahydrocannabinol (THC). In this study, comparative analysis of positive tested paired routine plasma/serum samples (n = 375), collected at the same time point (one device with and one without fluoride), was carried out with special focus on cannabinoids. Samples were measured with validated routine liquid chromatography-tandem mass spectrometry methods for THC, 11-hydroxy-THC (THC-OH), 11-nor-9-carboxy-THC (THC-COOH), cocaine, benzoylecgonine, ecgonine methyl ester, morphine, codeine, amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyamphetamine, and 3,4-methylenedioxyethylamphetamine, and results were statistically evaluated. Beside the expected stabilization effect on cocaine and the consequently reduced concentration of ecgonine methyl ester in fluoride samples, benzoylecgonine was elevated compared to respective samples without fluoride. Most importantly, new findings were significantly reduced mean concentrations of THC (- 17%), THC-OH (- 17%), and THC-COOH (- 22%) in fluoride samples. Mean amphetamine concentration was significantly higher in samples with the additive (+ 6%). For the other amphetamine type of drugs as well as for morphine and codeine, no significant differences could be seen. Whenever specified thresholds have been set, such as in most European countries, the use of different blood sample systems may result in a motorist being differently charged or prosecuted. The findings will support forensic toxicologists at the interpretation of results derived from fluoride-stabilized blood samples.

Is it possible to detect PEth 16:0/18:1 and PEth 18:1/18:1 in red blood cells after 20 years of storage in liquid nitrogen?

INTRODUCTION: Phosphatidylethanol (PEth), as measured in freshly drawn blood samples, could be a promising new biomarker for habitual alcohol consumption, but it is still unknown whether PEth can also be determined from blood samples having been stored frozen for a longer period. MATERIALS AND METHODS: PEth 16:0/18:1 and PEth 18:1/18:1 were determined by LC-MS/MS from red blood cells (RBC) derived from blood samples of (I) 20 healthy volunteers (after 1 month of storage at -80 °C) and (II) 232 participants of the population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Heidelberg study (after 20 years of storage at -196 °C). Analyses involved liquid-liquid extraction from 100 µl aliquots with phosphatidylpropanol (PProp 18:1/18:1) as the internal standard. Extracts were subjected to a 10-min LC gradient separation, using multiple reaction monitoring of deprotonated molecules for quantification. RESULTS: After 1 month of storage at -80 °C, PEth was detectable in all samples at mean concentrations of 393.6 ± 12.4 ng/ml (PEth 16:0/18:1) and 43.3 ± 1.1 ng/ml (PEth 18:1/18:1). In samples stored for 20 years at -196 °C, PEth was detectable in 23.7% of all samples at mean concentrations of 412.2 ± 655.5 ng/ml (PEth 16:0/18:1) and 38.0 ± 74.8 ng/ml (PEth 18:1/18:1). DISCUSSION AND CONCLUSION: PEth can be determined reliably from samples of moderate habitual alcohol consumers after 1 month of storage at -80 °C. Our data suggest that PEth is generally also detectable in samples after 20 years of storage at -196 °C. Further studies are needed to assess the still unknown impact of storage duration and temperature on different PEth specimen concentrations.

Improved detection of alcohol consumption using the novel marker phosphatidylethanol in the transplant setting: results of a prospective study.

Phosphatidylethanol (PEth) is a new, highly specific alcohol marker. The aim of this study was to assess its diagnostic value in the liver transplant setting. In 51 pre- and 61 post-transplant patients with underlying alcoholic liver disease PEth, ethanol, methanol, carbohydrate-deficient transferrin (CDT), and ethyl glucuronide in urine (uEtG) and hair (hEtG) were tested and compared with patients' questionnaire reports. Twenty-eight (25%) patients tested positive for at least one alcohol marker. PEth alone revealed alcohol consumption in 18% of patients. With respect to detection of alcohol intake in the preceding week, PEth showed a 100% sensitivity. PEth testing was more sensitive than the determination of ethanol, methanol, CDT or uEtG alone [sensitivity 25% (confidence interval (CI) 95%, 7-52%), 25% (7-52%), 21% (6-45%) and 71% (41-91%), respectively], or ethanol, methanol and uEtG taken in combination with 73% (45-92%). Specificity of all markers was 92% or higher. Additional testing of hEtG revealed alcohol consumption in seven patients, not being positive for any other marker. Phosphatidylethanol was a highly specific and sensitive marker for detection of recent alcohol consumption in pre- and post-transplant patients. The additional determination of hEtG was useful in disclosing alcohol consumption 3-6 months retrospectively.

Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia.

INTRODUCTION: Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options. CASE REPORT: We report the case of a suicidal yew ingestion admitted to our hospital under prolonged cardiopulmonary resuscitation due to pulseless electrical activity. Extra-corporeal life support (ECLS) was established to maintain adequate organ perfusion. Repeated administration of digoxin-specific Fab antibody fragments, which cross-react with taxine, was associated with an immediate conversion from asystole to broad-complex bradycardia and a gradual normalization of the electrocardiogram (ECG). This was paralleled by a recovery of the cardiac function and weaning from the ECLS. The taxine metabolite 3,5-dimethoxyphenol could be detected by mass spectrometry before but not after the first Fab-fragment treatment. In contrast, the total amount of taxine (including the neutralized, Fab fragment-bound fraction) was increased after each Fab fragment administration, suggesting an accumulation of neutralized, since antibody-bound taxine in the blood by anti-digoxin Fab fragments. DISCUSSION: In conclusion, the successful clinical course of this case suggests a benefit of an early anti-digoxin Fab-fragment administration for the treatment of yew intoxication.

Identification and characterization of sulfonyltransferases catalyzing ethyl sulfate formation and their inhibition by polyphenols.

Ethyl sulfate (EtS) is a minor metabolite of ethanol, usually being present along with ethyl glucuronide in both blood and urine. At present, there have been few studies on sulfotransferases (SULTs) catalyzing EtS formation. Moreover, inhibition by nutritional components on EtS formation, e.g., polyphenols that are extensively sulfonated, has not been addressed at all. Firstly, the incubation procedure was optimized with regard to buffer, substrate concentration, and incubation time. Recombinant SULT enzymes including SULT1A1, 1A3, 1B1, 1E1, and 2A1 were screened for their activity towards ethanol; subsequently, respective kinetics was investigated. The inhibitory potential of resveratrol, quercetin, and kaempferol being abundant in beer and wine was studied thereafter. Analysis was performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) using deuterated EtS as the internal standard. All enzymes are involved in the sulfonation of ethanol; respective kinetics followed the Michaelis-Menten model. Among the five SULTs under investigation, SULT1A1 displayed the highest activity towards ethanol followed by SULT2A1. Polyphenols significantly reduced the formation of EtS. Results revealed multiple SULT isoforms being capable of catalyzing the transfer of a sulfo group to ethanol; nevertheless, the relevance of SULTs' polymorphism on the sulfonation of ethanol needs further appraisal. Nutritional components such as polyphenols effectively inhibit formation of EtS; this observation may partly serve as an explanation of the highly inter-individual variability of EtS findings in both blood and urine.

A potential new metabolite of gamma-hydroxybutyrate: sulfonated gamma-hydroxybutyric acid.

Detection of gamma-hydroxybutyric acid (GHB) became crucial in many clinical and forensic settings due to its increasing use for recreational purposes and drug-facilitated sexual assault. Its narrow window of detection of about 3-12 h in urine represents a major problem. Analogous to ethyl glucuronide, the recently identified GHB-glucuronide exhibits a longer window of detection than the parent drug. It appeared reasonable that a sulfonated metabolite of GHB (GHB-SUL) will also be formed. Due to the lack of an appropriate standard, GHB was incubated with a human liver cytosolic fraction to produce GHB-SUL. Following development of a liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure GHB and GHB-SUL, authentic urine samples (n = 5) were tested for GHB-SUL. These investigations revealed detectable signals of both GHB and GHB-SUL, strongly indicating that GHB is not only glucuronidated but also sulfonated. Given that sulfonated metabolites generally have longer half-life times than the corresponding free drugs, GHB-SUL may serve as a biomarker of GHB misuse along with its glucuronide.

A methoxydiphenidine-impaired driver.

Methoxydiphenidine (MXP) was first reported in 1989 as a dissociative anesthetic but did not enter the market for pharmaceuticals. The substance re-appeared in 2013 as a new psychoactive substance. A case of driving under the influence of MXP is reported. The concentration of MXP has been determined from a serum sample (57 ng/mL) by liquid chromatography tandem mass spectrometry following liquid-liquid extraction. In addition, amphetamine, methylenedioxymethamphetamine, and its major metabolite were present in concentrations of 111, 28, and 3 ng/mL, respectively. The subject presented with amnesia, out-of-body experiences, bizarre behavior, and decreased motor abilities. At present, information on human toxicity of MXP is not available. MXP is comparable in structure as well as in action at the N-methyl-D-aspartate (NMDA) receptor to phencyclidine or ketamine. Therefore, it is likely that MXP exerts similar severe psychotropic action in man. However, there is no information on the duration and intensity of MXP's impairing effects, the interpretation of a particular concentration in the blood or serum, and its detectability in routine drug screenings. Confirmation analysis may be confined to cases where the police has specific intelligence that points to MXP use.

An unnatural death by propan-1-ol and propan-2-ol.

A fatality of an inpatient ingesting a disinfectant containing ethanol, propan-1-ol, and propan-2-ol is reported. The alleged survival time was about 1 h. Major findings at autopsy were an extended hemorrhagic lung edema, an edematous brain, and shock kidneys. Concentrations of alcohols and acetone, a major metabolite of propan-2-ol, were determined from body fluids (blood from the heart and the femoral vein, urine, gastric contents) and tissues (brain, muscle, liver, kidneys, lungs) by headspace/gas chromatography using 2-methylpropan-2-ol as the internal standard. All samples investigated were positive for propan-1-ol, propan-2-ol, ethanol, and acetone except stomach contents, where acetone was not detectable. The low concentration of acetone compared to propan-2-ol likely supports the short survival time. The concentration ratios estimated from the results are in accordance with the physico-chemical properties of the particular alcohols, their different affinities towards alcohol dehydrogenase as well as their interdependence during biotransformation. Autopsy did not reveal the cause of death. According to the few published data, blood concentrations of 1.44 and 1.70 mg/g of propan-2-ol and propan-1-ol, respectively, are considered sufficient to have caused the death. This case also points to the need to restrict access to antiseptic solutions containing alcohols in wards with patients at risk.

Simultaneous quantification of tobacco alkaloids and major phase I metabolites by LC-MS/MS in human tissue.

INTRODUCTION: Insurance agencies might request laboratories to differentiate whether a deceased has been a smoker or not to decide about refunding of his nonsmoker rate. In this context, the question on a solid proof of tobacco alkaloids and major metabolites in tissues came up. Currently, an appropriate assay is still lacking to analyze tissue distribution in smokers or nonsmokers. Nicotine (NIC), nornicotine (NNIC), anatabine (ATB), anabasine (ABS), and myosmine (MYO) are naturally occurring alkaloids of the tobacco plant; most important phase I metabolites of NIC are cotinine (COT), norcotinine (NCOT), trans-3'-hydroxycotinine (HCOT), nicotine-N'-oxide (NNO), and cotinine-N-oxide (CNO). An analytical assay for their determination was developed and applied to five randomly selected autopsy cases. METHODS: Homogenates using 500 mg aliquots of tissue samples were analyzed by liquid chromatography/tandem mass spectrometry following solid phase extraction. The method was validated according to current international guidelines. RESULTS: NIC, COT, NCOT, ABS, ATB, and HCOT could be detected in all tissues under investigation. Highest NIC concentrations were observed in the lungs, whereas highest COT concentrations have been found in the liver. MYO was not detectable in any of the tissues under investigation. CONCLUSIONS: The assay is able to adequately separate isobaric analyte pairs such as NIC/ABS/NCOT and HCOT/CNO thus being suitable for the determination of tobacco alkaloids and their phase I metabolites from tissue. More autopsy cases as well as corresponding body fluids and hair samples will be investigated to differentiate smokers from nonsmokers.

[In vitro characterization of glucuronosyl- and sulfotransferases involved in the conjugation of ethanol]. / In vitro Charakterisierung der Glucuronidierung und Sulfatierung von Ethanol.

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol; for some years, both compounds have been used as direct biomarkers of alcohol consumption in forensic and clinical settings as well as in traffic medicine. Drinking experiments showed individual variations of the formation of EtG and EtS. At present, our knowledge on enzymes involved in the conjugation of ethanol is incomplete and partly inconsistent. The purpose of the present study was to characterize those enzymes that are capable of catalyzing glucuronidation and sulfation of ethanol including some potential inhibitors. Following optimization of incubation conditions, the formation rates of EtG and EtS from ethanol via recombinant glucuronosyltransferases (UGTs, hepatic) and sulfotransferases (SULTs, hepatic, intestinal), the kinetics and the inhibitory potential of polyphenols such as quercetin, kaempferol and resveratrol were determined. Analysis was performed following either solid phase extraction due to severe ion suppression of EtG or direct injection of the EtS-containing incubation mixture by high-pressure liquid chromatography/tandem mass spectrometry. Deuterated analogues were used as internal standards. All UGTs were capable of metabolizing ethanol through glucuronidation; UGT1A9 and UGT2B7 exhibited the highest formation rates. All SULTs showed ethanol-sulfating activity with SULT1A1 being most active. Data for all enzymes could best be described by Michaelis-Menten kinetics. All polyphenols inhibited the conjugation of ethanol except UGT2B 15. Inhibition was reversible and competitive for most enzymes; mechanism-based inhibition was evident for UGT2B7 and SULT2A1 with regard to quercetin and for SULT1E1 with regard to kaempferol. These results suggest an influence on the formation rates of EtG and EtS by common food ingredients beside known polymorphisms of UGT and SULT family members. Further studies should be conducted to achieve a better understanding of the extent and significance of this influence.

[Diuretics and their potential effect on breath-alcohol concentration--a case report]. / Diuretika und ihre Auswirkung auf die Atemalkoholkonzentration--eine Fallvorstellung.

Many objections were raised to breath-alcohol analysis upon its introduction in the field of traffic law enforcement in Germany, but in the meantime this issue has become less relevant in forensic routine work. In the present case, the defending lawyer claimed that the ethanol concentration in the blood and hence in the breath of his client, which was 0.35 mg/l according to the Dräger Alcotest 7110® Evidential and thus above the legal limit of 0.25 mg/l, had been changed by diuretics taken 4 hours before the breath alcohol test, viz. 10 mg of torasemide, a loop diuretic, and 50 mg of spironolactone, a competitive aldosterone antagonist. According to the literature, the maximum urinary output in healthy subjects within the first 4 hours after 10 mg torasemide was 1450 ml. In patients suffering from heart failure, the urinary volume was reduced by a factor of 2.5-3; after chronic intake of torasemide, water loss did not differ from placebo. Spironolactone, which acts on the distal tubule, has little effect on urinary output. In a publication, the loss of water in excess within 24 hours was 90 ml. Co-administration of 100 mg spironolactone and 20 mg furosemide, which roughly compares to 10 mg torasemide, resulted in a mean urinary volume of 1566 ml within the first 4 hours. In terms of the reported case and provided that no compensatory fluid had been taken, a purely theoretical maximum shift of 0.007 mg/ may occur in the breath-alcohol concentration due to the smaller distribution volume even considering maximum urinary excretion values. On the other hand, already mild levels of dehydration may be associated with negative symptoms affecting driving ability.

Quantitative low-volume assay for simultaneous determination of fentanyl, norfentanyl, and minor metabolites in human plasma and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

A rapid and sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for simultaneous quantification of fentanyl (F), norfentanyl (NF), despropionylfentanyl (DPF), and hydroxynorfentanyl (OHNF) in human plasma and urine specimens has been developed and validated according to international guidelines. Analytes were extracted from 250-µL plasma or urine by liquid-liquid extraction. OHNF in urine affords a second extraction step and analysis with a different column. Calibration curves in plasma were linear from 0.05-10 ng/mL for F, 0.07-0.5 ng/mL for NF, 0.02-1.0 ng/ml for DPF, and 0.67-3.0 ng/mL for OHNF; in urine, from 0.09-10.0, 0.17-50, 0.08-1.0, and 1.0-5.0 ng/mL for F, NF, DPF, and OHNF, respectively. Analytical bias and intra- and inter-assay imprecision were within ± 15 % of target, except for OHNF in plasma and DPF in urine at the respective lower quality control level. All analytes were stable in processed samples when stored for 24 h at room temperature. Recoveries and process efficiencies were above 82.9 and 75.1 % for all analytes in plasma and urine. The low level of DPF in plasma indicated with a matrix effect of 71.3 % moderate ion suppression, all other analytes in plasma and urine showed no matrix effects. The lower limit of quantification (LOQ) in plasma was 0.05, 0.07, 0.02 and 0.67 ng/mL for F, NF, DPF, and OHNF, respectively. In urine, the LOQ of F, NF, DPF, and OHNF were 0.09, 0.17, 0.08, and 1.28 ng/mL, respectively. This assay has been applied to human specimens collected during a clinical drug-drug interaction study.

Identification and preliminary characterization of UDP-glucuronosyltransferases catalyzing formation of ethyl glucuronide.

Ethyl glucuronide (EtG), a minor metabolite of ethanol, is used as a marker of alcohol consumption in a variety of clinical and forensic settings. At present there are very few studies of UDP-glucuronosyltransferases (UGT), responsible for catalyzing EtG formation, and the possible effect of nutritional components, e.g. flavonoids, which are extensively glucuronidated, on EtG formation has not been addressed at all. The following incubation conditions were optimized with regard to previously published conditions: buffer, substrate concentration, and incubation time. Isolation of EtG from the incubation mixture was also optimized. Recombinant UGT enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, 2B10, 2B15) were screened for their activity towards ethanol, and kinetic data were then established for all enzymes. It was decided to study the effect of the flavonoids quercetin and kaempferol on glucuronidation of ethanol. Isolation was by solid-phase extraction (SPE) to minimize matrix effects. Analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS-MS), with EtG-d5 as the internal standard. SPE was vital to avoid severe ion suppression after direct injection of the incubation solution. EtG formation was observed for all enzymes under investigation; their kinetics followed the Michaelis-Menten model, meaning the maximum reaction rate achieved at saturating substrate concentrations (V(max)) and the substrate concentration at which the reaction rate is half of V(max) (Michaelis-Menten constant, K(m)) could be calculated. The highest rate of glucuronidation was observed with UGT1A9 and 2B7. After co-incubation with both flavonoids, formation of EtG was significantly reduced for all enzymes except for UGT2B15, whose activity did not seem to be affected. Results reveal that multiple UGT isoforms are capable of catalyzing glucuronidation of ethanol; nevertheless, the effect of UGT polymorphism on glucuronidation of ethanol needs further study. Formation of EtG is inhibited by the flavonoids under investigation. Obviously, nutritional components affect conversion of ethanol to EtG. This observation may serve as a partial explanation of its variable formation in man.

Monitoring of phosphatidylethanol in dried blood spots and of ethyl glucuronide in hair over 6 months of alcohol consumption.

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.

Single hair analysis for gamma-hydroxybutyric acid-Method optimization, validation, and application.

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.

Degradation of zopiclone during storage of spiked and authentic whole blood and matching dried blood spots.

Z-drugs such as zopiclone are increasingly involved in forensic cases. Its degradation occurs in solvents and biological fluids. It is assumed that hydrolysis largely accounts for the breakdown of zopiclone in aqueous media. Therefore, a stability study in blood at different storage conditions (-20, 4, 20, and 40°C) was performed to establish changes of the drug's concentration with time, also including its degradation product 2-amino-5-chloropyridine (ACP). As removal of the aqueous phase may stabilize molecules that are prone to hydrolysis, it was assessed whether the use of dried blood spots (DBS) may be an alternative for storing and analyzing zopiclone and ACP. Spiked and authentic blood samples and corresponding DBS were analyzed using fully validated LC-MS/MS assays. There was agreement between the measurement of zopiclone from either blood or matching DBS in freshly prepared samples. Results showed that zopiclone was unstable in blood at all storage temperatures except at -20°C. Stability of zopiclone in spiked and authentic blood was increased in DBS compared to matching blood samples stored at the same condition. About 85 % of the initial concentration of zopiclone was still intact in DBS on day 8 at 20°C. ACP was formed from zopiclone in equimolar amounts in both media. Therefore, determination of both zopiclone and ACP may be helpful to estimate the initial concentration in both media. Pre-analytical conditions have a major impact on the recovery of zopiclone from blood. With respect to its known advantages, DBS can be recommended as a valuable alternative for the determination of zopiclone from blood.

Stability of phosphatidylethanol species in spiked and authentic whole blood and matching dried blood spots.

BACKGROUND: Phosphatidylethanol (PEth) is currently under investigation as a highly sensitive and specific marker of alcohol misuse. As its stability in blood samples has not systematically been investigated, a study was performed to determine the stability of major PEth species in spiked and authentic whole blood and also in matching dried blood spots (DBS) at different conditions. METHODS: To PEth-free blood from teetotalers, low and high concentrations of two major PEth (18:1/18:1 and 16:0/18:1) species were added chosen on the basis of concentrations determined from authentic samples which were collected from the subjects undergoing alcohol detoxification treatment. Effects of sampling (EDTA or heparinized tubes), temperature, and time (≤30 days) were investigated. Processed samples (two at each condition, respectively) were subjected to LC gradient separation using multiple reaction monitoring. Stability was assessed using the critical difference or a periodic analysis result that was within 15 % of the initial concentration. Reaction kinetics of degradation was investigated with rate constants being checked for an Arrhenius relationship. RESULTS: PEth was stable in dried blood spot (DBS) stored either at room temperature or frozen, whereas it was not stable in whole blood except in samples stored at -80 °C. Activation energies increased in the following order: spiked heparinized blood < spiked EDTA blood < authentic EDTA blood. CONCLUSIONS: PEth is a labile analyte which is predominantly degraded by hydrolysis. Only at -80 °C, stability in whole blood can be ascertained, and analysis should be performed within 30 days. EDTA should be preferred over heparin as an additive. DBS is able to stabilize PEth thus partly resolving pre-analytical difficulties of PEth measurement.