Olof Beck

Detection Times for Urinary Ethyl Glucuronide and Ethyl Sulfate in Heavy Drinkers during Alcohol Detoxification

AIMS Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are conjugated ethanol metabolites formed in low amounts after alcohol consumption. Compared with ethanol, EtG and EtS are excreted in urine for a prolonged time, making them useful as sensitive alcohol biomarkers. This study determined the detection times for EtG and EtS in alcoholic patients undergoing alcohol detoxification. METHODS Alcohol-dependent patients (n = 32) with an initial alcohol concentration >or=1 g/L based on breath testing were followed during detoxification. Urine samples for determination of EtG, EtS, ethanol and creatinine were collected on admission to the hospital and thereafter once daily for several days. EtG and EtS measurements were performed by liquid chromatography-mass spectrometry (LC-MS) and EtG also using an immunochemical assay (DRI-EtG EIA, ThermoFisher/Microgenics). RESULTS The detection time for urinary EtG was weakly correlated (r = 0.434, P = 0.013) with the initial alcohol concentration (range 1.0-3.4 g/L). For EtG, the individual time range until return to below the applied cut-off limit (<0.5 mg/L) was approximately 40-130 h (median 78) with a similar time course observed for EtS. After correction for urine dilution, the time until an EtG/creatinine ratio <0.5 mg/g was approximately 40- 90 h (median 65). The detection times after an estimated zero ethanol concentration were approximately 30-110 h (median 66) for EtG and approximately 30- 70 h (median 56) for EtG/creatinine. The EtG results by LC-MS and the immunoassay were in good agreement. CONCLUSIONS During alcohol detoxification, EtG and EtS remained detectable in urine for several days. The detection times showed wide inter-individual variations, also after adjusting values for urine dilution and to the estimated times for a completed ethanol elimination.

Naltrexone for the Treatment of Amphetamine Dependence: A Randomized, Placebo-Controlled Trial

OBJECTIVE Currently there is no approved pharmacotherapy for amphetamine dependence. Recent human laboratory studies have demonstrated that naltrexone modulates some of the reinforcing effects of amphetamine. The aim of this study was to investigate the efficacy of naltrexone in comparison with placebo in reducing relapse to amphetamine use in amphetamine-dependent patients. METHOD Eighty patients who met DSM-IV criteria for amphetamine dependence were randomized to 12 weeks of double-blind naltrexone (50 mg) or placebo treatment. Patients visited the clinic twice weekly to receive medication and relapse prevention therapy and leave urine samples, which were analyzed for drug toxicology and for assessing adherence to medication via detection of naltrexones metabolite (6-beta-naltrexol). The main outcome measure was abstinence from amphetamine use, as indicated by the total number of negative amphetamine urine samples during 12 weeks of treatment. All missing urine samples were defined for the analysis as positive for amphetamine. RESULTS Overall, 55 patients (68.8%) completed the trial. The intention-to-treat analysis showed that the naltrexone group had a significantly higher number of amphetamine-negative urine samples compared with the placebo group. Survival analyses showed that the treatment groups differed in rate of continuous abstinence, in both the intention-to-treat and completer samples, in favor of naltrexone treatment. There was a significant reduction in craving levels and self-reported consumption of amphetamine in the naltrexone group compared with the placebo group. Treatment with naltrexone was well tolerated in this sample. CONCLUSIONS This trial demonstrated the efficacy of naltrexone in reducing amphetamine use in amphetamine-dependent individuals.

Direct quantification of ethyl glucuronide in clinical urine samples by liquid chromatography-mass spectrometry.

Ethyl glucuronide is a minor metabolite of ethanol, and its presence in urine can be used as a laboratory test to detect recent alcohol intake, even for some time after the ethanol is no longer measurable. A simple analytical procedure was developed based on direct injection of urine diluted with a deuterated internal standard into an electrospray liquid chromatographic–mass spectrometric (LC-MS) system. A novel LC system using a porous graphite column (Hypercarb) enabled an isocratic elution with retention times of 5–6 minutes. The intra- and inter-assay coefficients of variation were 2–12%, and the measuring range was 0.1–1,500 mg/L (0.45–6,750 &mgr;mol/L). Ethyl glucuronide was found to be stable in urine for more than 4 days at room temperature, and no artifactual formation was observed on storage of urine samples fortified with 1% ethanol. Ethyl glucuronide was not detected in urine samples collected after abstinence from alcohol. Intake of a very low amount (7 g) of ethanol produced ethyl glucuronide values up to 8.4 mg/L after 4 hours and was still detectable at 6 hours. When the method was applied for routine screening of 252 clinical urine samples (range, 0–1,240 mg/L), it fulfilled the need for a simple and reliable assay to be used in the evaluation of urinary ethyl glucuronide as a routine test of recent alcohol intake.

Naltrexone Attenuates the Subjective Effects of Amphetamine in Patients with Amphetamine Dependence

Amphetamine abuse and dependence is a global health concern with a collateral increase in medical and social problems. Although some of the neurobiological mechanisms underlying amphetamine dependence and its devastating effects in humans are known, the development of rational and evidence-based treatment is lagging. There is evidence from preclinical studies suggesting that the endogenous opioid system plays a role in mediating some of the behavioral and neurochemical effects of amphetamine in a variety of controlled settings. In the present study we assessed the effects of naltrexone, an opioid antagonist (50 mg) on the subjective physiological and biochemical response to dexamphetamine (30 mg) in 20 amphetamine-dependent patients. Patients received naltrexone/amphetamine followed by placebo/amphetamine, 1 week apart in a randomized double-blind placebo-controlled design. The primary objective of the study was to evaluate the effect of pretreatment with naltrexone on the subjective response to amphetamine, using a Visual Analog Scale. The secondary objective was to investigate the effects of naltrexone on physiological and biochemical responses to amphetamine, as measured by changes in blood pressure, heart rate, skin conductance, and cortisol. Naltrexone significantly attenuated the subjective effects produced by dexamphetamine in dependent patients (p<0.001). Pretreatment with naltrexone also significantly blocked the craving for dexamphetamine (p<0.001). There was no difference between the groups on the physiological measures. The results suggest that the subjective effects of amphetamine could be modulated via the endogenous opioid system. The potential of naltrexone as an adjunct pharmaceutical for amphetamine dependence is promising.

Multicomponent LC–MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine—Experience from the Swedish population

The advent of new not yet legally regulated psychoactive substances sold over the Internet has created a challenge for clinical toxicology and drug testing laboratories. The routine use of immunoassay screening may no longer be the optimal solution in many instances since the number of analytes covered is becoming insufficient. The aim of this work was to design, validate and apply a multi-component LC-MS/MS method suitable for screening of a large number of target analytes belonging to the class of new psychoactive substances - legal highs. The analytical method was using a five-fold dilution of urine with internal standard (pethidine-d5) and injection of 2μL. The chromatographic system was using a 1.7-μm 100mm×2.1mm Ethylene Bridged Hybrid (BEH) C18 column and gradient elution with a flow rate of 600μL/min. Solvent A consisted of 0.1% formic acid and Solvent B was 100% acetonitrile. The gradient elution application was designed to have a wide polarity coverage with total run time of 4.0min. The tandem mass spectrometer was using an electrospray interface and operated in positive mode. Selected reaction monitoring of two ion transitions was used for each of 26 analytes. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (0.1-10μg/mL), acceptable imprecision in quantification (CV<15%). Some analytes were found not to be stable in urine upon storage. The method was successfully applied in routine drug testing. A total of 87 positive samples with 100 analytical findings were found to contain O-desmethyl-cis-tramadol (mostly without mitragynine), methylenedioxypyrovalerone, 4-fluoroamphetamine, methoxetamine, desoxypipradol, 4-fluoromethcathinone, 5,6-methylenedioxy-2-aminoindane, 4-methylmethcathinone, 3-fluoromethcathinone, 4-hydroxy-N-methyl-N-ethyltryptamine, α-methylamino-butyrophenone and 4-methoxymethcathinone.

Plasma concentrations of lamotrigine and its 2-N-glucuronide metabolite during pregnancy in women with epilepsy

Objective: To further characterize pregnancy‐induced alterations in the pharmacokinetics of lamotrigine (LTG).

Time course of ethanol-induced changes in serotonin metabolism.

The effect of acute ethanol consumption on serotonin metabolism was examined in healthy volunteers in the fasted and fed state by determination of plasma and urinary levels of the serotonin metabolites 5-hydroxyindole-3-acetic acid (5-HIAA) and 5-hydroxytryptophol (5-HTOL). The plasma and urinary levels of 5-HIAA were reduced by about 40% and 25%, while the 5-HTOL levels were increased on an average 7-fold and 50-fold, respectively, after oral intake of ethanol (0.8 g/kg) over 30 min in a fasted state. The maximal effect on both 5-HIAA and 5-HTOL levels was found 4-6 h after starting drinking. Urinary 5-HTOL and the 5-HTOL/5-HIAA ratio did not return to baseline until 19 h after the start of the administration (i.e., about 10 h after ethanol reached zero level). The mean 24-h excretion of 5-HTOL was increased 15-fold by the ethanol intake, while the 5-HIAA excretion was not significantly different. A clear dose dependent effect was observed in one individual who also ingested a lower amount of ethanol (0.5 g/kg). When ethanol (0.8 g/kg) was ingested over 3 h together with food, the urinary level of 5-HTOL and the 5-HTOL/5-HIAA ratio did not return to baseline until after 20-22 h. In other subjects who had unlimited access to ethanol and ingested between 1.3-2.3 g/kg together with food, the time to reach baseline 5-HTOL/5-HIAA ratio in urine ranged from 20 h to over 26 h.

5-Hydroxytryptophol in the cerebrospinal fluid and urine of alcoholics and healthy subjects

SummaryThe serotonin metabolite 5-hydroxytryptophol was determined in cerebrospinal fluid and urine of alcoholics and healthy subjects, by a glass capillary gas chromatographic-mass spectrometric method. The urinary excretion rate (14.6±2.9 pmoles/μmoles creatinine) and urine (109±20 pmoles/ml) and cerebrospinal fluid (4.12±0.21 pmoles/ml) concentrations in healthy subjects were established. Only 1% of the 5-hydroxytryptophol in urine occurred in free form. Ethanol ingestion (80, 120 g) by healthy subjects lead to a 20–100-fold increase in the urinary excretion rate of 5-hydroxytryptophol. In cerebrospinal fluid the increase was about 60%. Alcoholics had increased urinary excretion rates and cerebrospinal fluid levels during intoxication, which were in the same range as in intoxicated healthy subjects. During recovery from intoxication, the 5-hydroxytryptophol level in alcoholics decreased, but the CSF levels were still higher than in healthy subjects.

Method development for routine liquid chromatography-mass spectrometry measurement of the alcohol biomarker phosphatidylethanol (PEth) in blood.

BACKGROUND Phosphatidylethanol (PEth) is a group of phospholipids formed from ethanol and phosphatidylcholine by action of phospholipase D. Measurement of PEth in whole blood samples is employed as an alcohol biomarker. This work aimed to further develop an LC-MS method for PEth to make it practical for routine laboratory use. METHODS Blood samples were obtained from blood donors and from the clinical samples pool. A whole blood total lipid extract was separated on a C4 column, followed by ESI-MS detection of the deprotonated molecules in SIM mode or ESI-MS/MS detection of the major product ions (fatty acid fragments) by SRM. RESULTS Initial results indicated that individual calibration curves are required for MS quantitation of some PEth forms, and that deuterated analogs are preferable over phosphatidylpropanol as the internal standard. PEth-16:0/18:1 was the single most sensitive molecular form as alcohol biomarker, being detected in every of 211 blood specimens containing 0.1-20 μmol/L total PEth at reporting limits in the range 0.1-1.0 μmol/L. PEth-16:0/18:1 and 16:0/18:2, accounting for about 36% and 26%, respectively, of the total amount, correlated well with total PEth (R(2)=0.922-0.940), but the correlation was better for the sum of both forms (R(2)=0.994). Based on analysis of specimens from 200 blood donors, 95% reference intervals (CLSI C28-A3) were estimated to be <0.70 μmol/L for total PEth, <0.20 μmol/L for PEth-16:0/18:1, and <0.18 μmol/L for PEth-16:0/18:2. CONCLUSIONS The LC-ESI-MS(/MS) method allowed for simultaneous qualitative and quantitative measurements of PEth forms in whole blood samples. Related to the routine application of blood PEth as alcohol biomarker, reference intervals were suggested for total PEth and the major molecular forms PEth-16:0/18:1 and 16:0/18:2.

Identification of novel psychoactive drug use in Sweden based on laboratory analysis – initial experiences from the STRIDA project

Abstract Aim. The study aimed to collect information concerning the increasing use of new psychoactive substances, commonly sold through online shops as ‘Internet drugs’ or ‘legal highs’, or in terms of masked products such as ‘bath salts’ and ‘plant food’. Methods. The Karolinska Institutet and Karolinska University Laboratory and the Swedish Poisons Information Centre have initiated a project called ‘STRIDA’ aiming to monitor the occurrence and trends of new psychoactive substances in Sweden, and collect information about their clinical symptoms, toxicity and associated health risks. A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) multi-component method has been developed, currently allowing for the determination of > 80 novel psychoactive compounds or metabolites thereof. This study focused mainly on the particular drug substances identified and the population demographics of the initial STRIDA cases. Results. In urine and/or blood samples obtained from 103 consecutive cases of admitted or suspected recreational drug intoxications in mostly young subjects (78% were ≤ 25 years, and 81% were males) presenting at emergency departments all over the country, psychoactive substances were detected in 82%. The substances comprised synthetic cannabinoids (‘Spice’; JWH analogues), substituted cathinones (‘bath salts’; e.g. butylone, MDPV and methylone) and tryptamines (4-HO-MET), plant-based substances (mitragynine and psilocin), as well as conventional drugs-of-abuse. In 44% of the cases, more than one new psychoactive substance, or a mixture of new and/or conventional drugs were detected. Conclusion. The initial results of the STRIDA project have documented use of a broad variety of new psychoactive substances among mainly young people all over Sweden.