Gisela Skopp

Ethyl Glucuronide Concentration in Serum of Human Volunteers, Teetotalers, and Suspected Drinking Drivers

The kinetic profile of ethanol and ethyl glucuronide (EtG) in serum was investigated in three subject groups: 1) Healthy, moderately drinking volunteers (daily intake less than 30 g ethanol) who ingested a single dose of ethanol. In this group the maximum of serum ethyl glucuronide concentration (SEtGC) and of serum ethanol concentration (SEC) did not exceed 3.7 mg/L and 1.5 g/L respectively. EtG peaked 2 to 3.5 h later than ethanol. EtG was eliminated with a terminal half-life of 2 to 3 h. EtG decreased slower than ethanol--the metabolite could still be determined in serum up to 8 h after complete ethanol elimination. 2) In serum samples of teetotalers neither ethanol nor EtG could be found. 3) In 37 of 50 serum samples of drivers suspected of driving under the influence of ethanol, SEtGC was found between the limit of detection (0.1 mg/L) and 20 mg/L. If the SEC is less than 1 g/L and the SEtGC is significantly higher than 5 mg/L, we assume alcohol misuse.

Dorsolateral Prefrontal Cortex N-Acetylaspartate/Total Creatine (NAA/tCr) Loss in Male Recreational Cannabis Users

BACKGROUND Cannabinoids present neurotoxic and neuroprotective properties in in vitro studies, inconsistent alterations in human neuroimaging studies, neuropsychological deficits, and an increased risk for psychotic episodes. METHODS Proton magnetic resonance spectroscopy ((1)H-MRS), neuropsychological testing, and hair analysis for cannabinoids was performed in 13 male nontreatment-seeking recreational cannabis users and 13 male control subjects. RESULTS A significantly diminished N-acetylaspartate/total creatine (NAA/tCr) ratio in the dorsolateral prefrontal cortex (DLPFC) was observed in cannabis users (p = .0003). The NAA/tCr in the putamen/globus pallidum region correlated significantly with cannabidiol (R(2) = .66, p = .004). Results of the Wisconsin Card Sorting test, Trail making Test, and D2 test for attention were influenced by cannabinoids. CONCLUSIONS Chronic recreational cannabis use is associated with an indication of diminished neuronal and axonal integrity in the DLPFC in this study. As chronic cannabis use is a risk factor for psychosis, these results are interesting because diminished NAA/tCr ratios in the DLPFC and neuropsychological deficits were also reported in schizophrenia. The strong positive correlation of NAA/tCr and cannabidiol in the putamen/globus pallidum is in line with neuroprotective properties of cannabidiol, which were also observed in in vitro model studies of Parkinsons disease.

On cosmetically treated hair — aspects and pitfalls of interpretation

Popular hair cosmetic treatments like bleaching or permanent waving were found to affect the stability of incorporated drugs and to cause alterations of the fibers at an ultrastructural level. This may result in a partial or complete loss of drug substances, depending on the particular drug molecule and on its concentration prior to the cosmetic treatment. Moreover, from literature, there is some evidence that drug molecules are not only incorporated into the growing fiber by passive diffusion from blood into the matrix cells and melanocytes, but that the substances enter the hair also via perspiration such as sweat and sebum. Since permed and bleached hair shows an enhanced sorption capacity, the risk of false positives or an unusually high drug concentration in cosmetically treated hair was under investigation. Virgin, permed, mildly as well as severely bleached tresses were exposed to artificial sweat or sebum containing cocaine, benzoylecgonine, 6-acetylmorphine, morphine and codeine (500 ng/g). Except codeine, the concentrations measured by GC/MS were very small and quite close to the detection limit indicating a minor importance of drug uptake into hair fiber from the endogenous-exogenous shunt via sebum or sweat. From the results it is concluded that an increased risk of false positive results in hair analysis on bleached and permanent waved hair fibers does exist, but is not particularly severe.

Diminished gray matter in the hippocampus of cannabis users: possible protective effects of cannabidiol.

BACKGROUND Chronic cannabis use has been associated with memory deficits and a volume reduction of the hippocampus, but none of the studies accounted for different effects of tetrahydrocannabinol (THC) and cannabidiol (CBD). METHODS Using a voxel based morphometry approach optimized for small subcortical structures (DARTEL) gray matter (GM) concentration and volume of the hippocampus were measured in 11 chronic recreational cannabis users and 13 healthy controls, and correlated with THC and CBD from hair analyses. GM volume was calculated by modulating VBM using Jacobian determinants derived from the spatial normalization. RESULTS Cannabis users showed lower GM volume located in a cluster of the right anterior hippocampus (P(uncorr)=0.002; effect size Cohens d=1.34). In a regression analysis an inverse correlation of the ratio THC/CBD with the volume of the right hippocampus (P(uncorr) p<0.001, Cohens d=3.43) was observed. Furthermore Cannabidiol correlated positively with GM concentration (unmodulated VBM data), but not with GM volume (modulated VBM) in the bilateral hippocampus (P=0.03 after correction for hippocampal volume; left hippocampus Cohens d=4.37 and right hippocampus 4.65). CONCLUSIONS Lower volume in the right hippocampus in chronic cannabis users was corroborated. Higher THC and lower CBD was associated with this volume reduction indicating neurotoxic effects of THC and neuroprotective effects of CBD. This confirms existing preclinical and clinical results. As a possible mechanism the influence of cannabinoids on hippocampal neurogenesis is suggested.

Determination of Morphine and 6-Acetylmorphine in Blood With Use of Dried Blood Spots

The use of dried blood spots (DBS) which has successfully been introduced in neonatal metabolic screening is an appropriate method to reduce virus infection risk to a minimum, facilitating regular mailing and handling of samples in the laboratory. Injection diacetylmorphine use is notably associated with a prevalence of infection and a risk of transmission of blood-borne viruses. The aim of the present study was to establish a method to determine morphine and 6-acetylmorphine (6-AM) as accurately and sensitively from DBS as from whole blood. Analysis by liquid chromatography/tandem mass spectrometry was checked for carryover, ion suppression/enhancement, linearity of response, lower limits of detection and quantification, and the within-run and between-run assay imprecision for both whole blood and DBS after liquid/liquid extraction. DBS drying time and elution were optimized, and extraction efficiency from DBS was compared with that of blood and of a solution of the pure compounds. Short-term stability of morphine and 6-AM was determined at −20°C, 4°C, and 40°C up to 7 days from both whole blood and DBS. Furthermore, it was tested whether analysis of DBS may be as reliable as that of whole blood investigating 50 authentic samples. The lower limit of detection was 0.4 ng of morphine per spot and 0.8 ng of 6-AM per spot using a DBS blood volume of 100 μL and was 0.3 and 0.7 ng/100 μL whole blood for morphine and 6-AM, respectively. Recovery rates of the analytes from DBS did not differ from those from whole blood and were ≥55% for 6-AM and ≥25% for morphine, and the within- and between-run coefficients of variation were always ≤9%. 6-AM degraded rapidly at both 4°C and 40°C in whole blood; however, it seemed to be much more stable in DBS. Significant correlations between real whole-blood samples and DBS were obtained. The DBS assay has potential as a precise and inexpensive option for the determination of morphine and 6-AM in small blood samples. Further, the DBS matrix proved to excellently stabilize 6-AM.

Biochemical approach on the conservation of drug molecules during hair fiber formation

A biochemical concept for the endogenous incorporation of drug molecules into growing hair is presented. It is based on the principles of transport across biomembranes, on the principles of biotransformation and drug melanin affinity. The approach gives explanations for current observations in hair analysis, which up to date have not been understood sufficiently. Phenomena such as the ratio of parent drug to metabolite in hair, the dependence of incorporation on the physico-chemical properties of the drug, the independence of drug incorporation on active melanogenesis (incorporation into non-pigmented hair) as well as the dependence of drug content on hair pigmentation are elucidated.

Influence of pigmentation on the codeine content of hair fibers in guinea pigs.

Tortoise shell guinea pigs (n = 7) were administered codeine (1 mg/mL codeine-base) in their drinking water for 3 weeks. Black, reddish-brown and white hair was collected separately from each animal before and after treatment. The hair samples were analyzed by GC/MS. The experiment showed positive results for all hair fibers with large individual variability of drug incorporation. Low drug intake resulted in small differences of the drug content in hair fibers different in color, whereas in cases of high drug intake a strong influence of hair pigmentation on the analytical results was observed. The highest drug content was always found in black hair samples, non-pigmented hair showed the lowest drug concentrations and the drug content in reddish-brown fibers was less than in black hair samples from the same animal. From the results it was concluded, that eumelanins rather than phenomelanins are the decisive factor for codeine-melanin binding in hair and the amount of drug intake was suggested to determine the relevance of hair pigmentation on the analytical results.

Buprenorphine and norbuprenorphine concentrations in human breast milk samples determined by liquid chromatography-tandem mass spectrometry.

Buprenorphine (BUP) is considered to be safe during pregnancy. However, the extent of BUP transfer into breast milk has not been investigated thoroughly. Because the drug concentration in the milk is 1 of the determinants in the assessment of the exposure risk, a rapid and sensitive LC-MS/MS method has been developed and evaluated to measure BUP and norbuprenorphine (norBUP) concentrations in milk. A solid-phase and 2 liquid-liquid extraction procedures have been compared. The lower limits of detection and quantification were 0.05 ng/mL and 0.18 ng/mL for BUP and 0.05 ng/mL and 0.20 ng/mL for norBUP, respectively, using a sample volume of 0.5 mL milk. BUP and norBUP concentrations determined from 10 random breast milk samples collected over 4 successive days from a lactating woman during buprenorphine maintenance therapy ranged from 1.0 to 14.7 and 0.6 to 6.3 ng/mL, respectively. Drug exposure of the infant may be considered to be low. Further investigations may seek to extend these preliminary findings to evaluate an infants level of BUP exposure through breast milk.

Partition coefficient, blood to plasma ratio, protein binding and short-term stability of 11-nor-Δ9-carboxy tetrahydrocannabinol glucuronide

11-Nor-Delta(9)-carboxy tetrahydrocannabinol glucuronide (THCCOOglu) is a major metabolite of tetrahydrocannabinol in blood. Despite its mass spectrometric identification already in 1980, further physicochemical data of THCCOOglu have not been established. Therefore, the octanol/buffer partition coefficient P and the blood to plasma ratio b/p for THCCOOglu concentrations of 100 and 500ng/ml were investigated. Protein binding of the glucuronide was established from spiked albumin solutions at a level of 250ng/ml as well as from authentic samples. The data were compared to those of 11-nor-Delta(9)-carboxy tetrahydrocannabinol (THCCOOH). In addition, the short-term stability of THCCOOglu in plasma at different storage temperatures was studied. Analysis was performed by LC/MS/MS. The glucuronide partition coefficient P (mean: 17.4 and 18.0 for 100 and 500ng/ml, respectively) was unexpectedly lipophilic at pH 7.4. Its blood to plasma ratios averaged 0.62 and 0.68 at 100 and 500ng/ml, respectively. THCCOOglu was highly reversibly bound to albumin (mean: 97%), and the mean fraction bound did not differ from that determined from authentic samples. THCCOOglu degraded even at a storage temperature of 4 degrees C and THCCOOH was identified as a major decomposition product.

Postmortem distribution pattern of morphine and morphine glucuronides in heroin overdose

The postmortem distribution of morphine and its metabolites was investigated in four cases of heroin overdose to evaluate some of the factors that influence intravasal blood concentrations. Variables included were the chemical stability of morphine conjugates, hemoconcentration, incomplete distribution of the drug and diffusion processes. Blood samples from different sampling sites including the aorta, the infra- and suprarenal portion of the inferior vena cava, the superior vena cava, the femoral and subclavian veins, and the right and left ventricles were examined for morphine, morphine-3-glucuronide and morphine-6-glucuronide, hematocrit and water content. Drug concentrations were determined by HPLC based on the native fluorescence of the analytes. Morphine glucuronides proved to be stable for a time period of 72 h. The water content ranged from 65 to 83% and hematocrit values from 25 to 75%, and were seen as contributory factors to the dramatic differences observed for drug concentrations from different sampling sites. The differences could neither be attributed to incomplete distribution during life-time nor to a diffusion process following the different distribution volumes of morphine and its conjugates. A definite relationship between the ratio of the molar concentrations of morphine and its glucuronides, as assessed in pharmacokinetical studies after morphine dosing, could not be established. For a better understanding more cases and changes over time and tissue concentrations should be analysed.