Reference measurement procedure for Delta9-tetrahydrocannabinol in serum.

An isotope dilution gas chromatography/mass spectrometry (ID-GC/MS) reference measurement procedure for Delta9-tetrahydrocannabinol (THC) in serum was developed and validated. The method complies with the concept of a ratio primary reference measurement procedure. The uncertainty was determined for two concentrations of THC in serum (1 ng/mL and 2.4 ng/mL). The calculation procedure is based on the Guide to the Expression of Uncertainty in Measurement (GUM). The relative expanded uncertainty was found to be less than 2% for both concentration levels, corresponding to a 95% confidence interval. For the reference method, it was shown that the measurement of THC within the concentration range covered by the current threshold values is very accurate. The method has the potential to provide traceability for the methods used in practical forensics.

The role of active metabolites in dihydrocodeine effects.

OBJECTIVE: The metabolism of dihydrocodeine to dihydromorphine, a high affinity mu-opioid receptor ligand in membrane homogenates, is catalyzed by CYP2D6. However, it is not clear whether an active CYP2D6 enzyme is required for opioid receptor-mediated effects in man after standard dihydrocodeine doses. METHODS: Whole cell opioid-receptor affinity and effects on cAMP accumulation of dihydrocodeine and its metabolites were determined in differentiated SH-SY5Y neuroblastoma cells. In a double-blind, 2-period, placebo-controlled randomized crossover pilot study the pharmacokinetics of dihydrocodeine (60 mg single dose) and its metabolites were examined in 5 phenotyped extensive (EMs) and 4 poor metabolizers (PMs) for CYP2D6, and pharmacodynamics were evaluated using a pain threshold model and dynamic pupillometry. RESULTS: Displacement binding and cAMP accumulation experiments showed clearly higher affinities (100- and 50-fold) and activities (180- and 250-fold) of dihydromorphine and dihydromorphine-6-glucuronide, respectively, whereas the other metabolites had similar or lower affinities and activities as compared to dihydrocodeine. The clinical study revealed no significant difference in plasma or urine pharmacokinetics between EMs and PMs for dihydrocodeine and its glucuronide. Dihydromorphine and its glucuronides were detectable in EMs only. A clear reduction of initial pupil diameters was observed up to 6 hours postdose in both PMs and EMs, with no obvious differences between CYP2D6 phenotypes. In the pain threshold model no effects were observed in either group. CONCLUSION: CYP2D6 phenotype has no major impact on opioid receptor-mediated effects of a single 60 mg dihydrocodeine dose, despite the essential role of CYP2D6 in formation of highly active metabolites.

Ritonavir increases loperamide plasma concentrations without evidence for P-glycoprotein involvement.

BACKGROUND: The antidiarrheal drug loperamide is frequently used to treat ritonavir-associated diarrhea in patients with human immunodeficiency virus. The absence of marked central opioid effects has been attributed to its low bioavailability and its poor penetration of the blood-brain barrier, both of which might be altered by ritonavir, a potent P-glycoprotein and cytochrome P4503A inhibitor. METHODS: A 16-mg dose of loperamide was administered to 12 healthy male and female volunteers together with either 600 mg of ritonavir or placebo. Detailed pharmacokinetics of loperamide and its metabolites were determined over 72 hours. Central opioid effects were measured by evaluation of pupil diameter, cold pressor test, and transcutaneous PCO2 and PO2. RESULTS: Ritonavir caused a major pharmacokinetic interaction, increasing the area under the concentration-time curve of loperamide from 104 +/- 60 h x pmol/ml after placebo to 276 +/- 68 h. pmol/ml and delayed formation of the major metabolite desmethylloperamide (time to reach maximum concentration after drug administration [t(max)], 7.1 +/- 2.6 hours versus 19.6 +/- 9.1 hours). The urinary metabolic ratio of loperamide increased 3 times whereas the total molar amount of loperamide and metabolites excreted in urine remained unchanged. No central pharmacodynamic effects were observed after coadministration of loperamide with either ritonavir or placebo. CONCLUSION: This study demonstrates a major metabolic interaction probably by cytochrome P4503A4 with no evidence of P-glycoprotein involvement. This might explain the lack of central effects after ritonavir.

Analysis of cocaine, benzoylecgonine, ecogonine methyl ester, and ecgonine by high-pressure liquid chromatography-API mass spectrometry and application to a short-term degradation study of cocaine in plasma.

A method for the determination of cocaine (COC), benzoylecgonine (BE), ecgonine methyl ester (EME), and ecgonine (ECG) in plasma by liquid chromatography-mass spectrometry (LC-MS-MS) was developed. The analytes were isolated from human plasma by subsequent solid-phase extraction and were separated on a Zorbax Eclipse XDB-C8 narrow-bore column using an ammonium acetate buffer/acetonitrile/methanol gradient. A Turbolonspray source was used for ionization. The analytes were characterized by their particular molecular ion and several fragments. Multiple reaction monitoring (MRM) was used for isolation and quantitation. The assay was rapid, highly sensitive, and reliable. The method was applied to monitor the in vitro degradation of cocaine in plasma. Fresh unpreserved and preserved (0.25% KF) plasma samples were spiked with 1,000 ng cocaine/mL. Aliquots of both series were stored at 4 degrees C and 20 degrees C and were analyzed at selected storage times of up to 15 days. In all samples, degradation of cocaine that was dependent on storage time and temperature and on sample preservation could be observed. The formation of BE did not occur to a significant extent (< 12%, referred to the initial concentration of COC), and its concentration was slightly higher in preserved compared with unpreserved plasma at both storage temperatures chosen. EME was formed in considerably higher amounts compared to BE. As already observed for COC, its formation and degradation were dependent on storage time, temperature, and preservation. EME is suggested to be the major source of ECG, which was detectable in all samples after 1-2 days of storage. Although the degradation of COC was shown to be highly dynamic in nature, the sum of all hydrolysis products of COC accounted for the initial COC concentration at any particular time of storage. Therefore, production of hitherto unknown degradation products of COC seems unlikely. Moreover, the common transformation product of BE and EME appeared to be stable, and ECG is suggested as a promising postcollection artifact.

Saliva testing after single and chronic administration of dihydrocodeine.

In the present study, concentrations of dihydrocodeine and its metabolites in saliva and serum were compared after single low-dose and chronic high-dosage administration of the drug. In the first investigation, blood and saliva were collected periodically from six subjects after oral administration of 60 mg dihydrocodeine. In the second study, 20 subjects on oral dihydrocodeine maintenance provided single samples of blood and saliva simultaneously. Serum protein binding of salivary analytes and their recovery from the adsorbing material of the collection device as well as pH values of saliva samples were determined. The fluids were analyzed for dihydrocodeine and the major metabolites by high-performance liquid chromatography. In the single dose study dihydrocodeine was the only analyte found in saliva for up to 12-24 h post-dose. The half-life of dihydrocodeine in saliva was about twice that found in blood. The ratios of saliva/serum concentrations ranged from 1.2 to 17.0. After chronic high-dosage use, dihydrocodeine was the main salivary analyte and N-nordihydrocodeine was present in a few samples. Saliva/serum concentration ratios of dihydrocodeine were strongly dependent on the pH value of saliva and, to a lesser extent, on serum-protein binding. The saliva/serum ratios were more similar after chronic administration. The data suggest a passive diffusion process as the underlying mechanism for the transport of dihydrocodeine into saliva. After both single and chronic use, the presence of the drug in saliva can be used as evidence of recent substance administration.

[Distribution of morphine and morphine glucuronides in body tissue and fluids--postmortem findings in brief survival]. / Verteilung von Morphin und Morphinglucuroniden in Körpergeweben und -flüssigkeiten--Postmortale Befunde bei kurzer Uberlebenszeit.

An intoxication following administration of morphine, tramadol and atracurium in a suicide case is reported. The route of administration and the amount of the particular drug were known from the investigation of the death scene and the findings of the postmortem examination. Tramadol was present in the gastric contents as well as in blood, liver, kidney and brain samples, whereas the drug could not be detected in muscle. All body fluids and tissues investigated contained morphine as well as its 3- and 6-glucuronides with the exception of muscle tissue. The concentrations of morphine and its glucuronide metabolites were determined by LC/MS following solid phase extraction. Interestingly, the concentration of M6G in brain, liver and kidney were close to the concentration of M3G in the particular tissue. This phenomenon might be explained by a preferential hydrolysis of M3G or by a preferential formation of M6G postmortem. Measurement of morphine and M6G in femoral blood and cerebrospinal fluid may be a useful indicator in rapid deaths.

Ethyl glucuronide in human hair.

Ethyl glucuronide (EtG) is considered to be a promising candidate marker of alcohol consumption, but exhibits a short window of detection in blood or urine. Keratinized tissues are known to retain foreign substances and to provide a greater retrospective window of detection than body fluids. Therefore, post-mortem hair, skin swabs, and stratum corneum samples were collected from four subjects with a reported history of alcohol misuse and from seven subjects with a report of regular, socially accepted drinking behaviour, and were investigated for EtG. Additionally, certain specimens were collected from three children, who had not yet consumed any alcoholic beverages. EtG was detectable in most of the hair and stratum corneum samples as well as in perspiration stains from alcohol-consuming subjects. The results indicated that EtG might be formed locally in very small and highly variable amounts. The most important finding was that EtG cannot be expected to be generally detectable in keratinized tissues or perspiration stains from alcohol-drinking subjects, whereas a positive result is always associated with recent alcohol consumption.

The detection of dihydrocodeine and its main metabolites in cases of fatal overdose.

The levels of dihydrocodeine found in impaired individuals and in fatalities show a wide overlap in the ranges. Among other factors, the genetically controlled metabolism of dihydrocodeine should play an important role in dihydrocodeine toxicity. For the first time, the most important metabolites of dihydrocodeine were investigated in femoral blood from three fatal cases by simultaneous determination using HPLC and native fluorescence for detection. The amount of parent drug always exceeded dihydrocodeine-glucuronide formation and dihydromorphine concentrations ranged from 0.16-0.21 mg/L. The similar binding affinities of dihydromorphine and morphine to mu-opioid receptors suggest similar pharmacological effects and adverse reactions. The determination of the pharmacologically active metabolites should help to clarify the cause of death in fatal cases especially if a relatively low concentration of the parent drug is found.

Determination of ethyl glucuronide, a minor metabolite of ethanol, in human serum by liquid chromatography-electrospray ionization mass spectrometry.

A rapid and sensitive determination procedure using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) has been developed for the determination of ethyl glucuronide (EtG) in human serum. Samples were precipitated with methanol, centrifuged and the supernatant was evaporated to dryness followed by reconstitution with distilled water. As mobile phase 30 mM ammonium acetate-acetonitrile (30:70, v/v) was utilized. The base peak observed at m/z 221 was the [M-H]- ion of EtG, which was detectable in satisfactory sense. The detection limit was 0.03 microg/ml in the selected ion monitoring mode. A calibration graph constructed for EtG in serum gave good linearity over the range from 0.1 to 25 microg/ml. This paper also presents the application of this LC-ESI-MS procedure to the analysis of authentic serum samples.

The influence of the route of administration: a comparative study at steady state of oral sustained release morphine and morphine sulfate suppositories.

Steady state pharmacokinetics of morphine (M), morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G) were investigated in 6 patients with intractable cancer pain administered orally with MST (Mundipharma, Limburg, Germany) and, subsequently, rectally with MSR to make a judgment whether orally administered morphine can be replaced by rectally administered morphine. The parent drug and glucuronide metabolites were measured simultaneously using high-performance liquid chromatography (HPLC) and native fluorescence detection. The mean morphine area under the curve (AUC) value (0-8 h) was smaller (434.3 +/- 170.2 nmolL(-1)h) in the oral administration than in the rectal administration (574.8 +/- 285.0 nmolL(-1)h) (p < 0.05). The rectal administration resulted in less production of M3G and M6G. There were no significant differences in the mean steady state concentrations (C(ss)) of morphine, M3G, and M6G between the oral and rectal administrations (p > 0.05). The median AUC ratio--M3G/M and M6G/M, 12.58 and 1.85--following MSR rectal administration was smaller than following MST oral administration in 6 patients (19.97 and 2.59; p < 0.05), whereas the median AUC ratio M3G/M6G in the rectal dosing was 6.24 (range 5.2-7.6) was almost the same as the median ratio M3G/M6G in the oral dosing was 6.49 (range 5.8-8.5; p > 0.1). Four of the 6 patients had a greater Cmax of M3G and M6G after oral administration than after rectal administration. The same 4 had lower fluctuation rates for morphine, M3G (p < 0.05), and M6G (p < 0.05) after rectal administration. Therefore, during chronic morphine treatment, it still seems difficult to decide whether oral administration can be replaced by rectal administration.

Fatal chloroquine intoxication.

A suicidal ingestion of an unknown quantity of Resochin (chloroquine) tablets is described. Although chloroquine is known since 1934, intoxications due to chloroquine overdose are rather rare in European countries. The authors report on a new and fast method of analysing and determining the chloroquine concentration in body fluids and postmortem specimens. The analytes were extracted from alkalinized samples into ethyl acetate before GC/MS analysis. The analyses of chloroquine were performed without any complex sample clean-up steps and, in addition, with little sample material. The proposed method resulted in a rapid procedure most useful in cases of deliberate poisoning with the anti-inflammatory and antimalarial drug chloroquine.

Formation and clearance of active and inactive metabolites of opiates in humans.

The results of recent investigations of the analgesic and the nonanalgesic effects of opioid glucuronides are relevant to the research on drug abuse in forensic toxicology. As has been shown for heroin, knowledge of the state of distribution and elimination of active and inactive metabolites and glucuronides offers new possibilities in forensic interpretation of analytic results. Because of similar metabolic degradation, calculation of the time-dependent ratio of the concentration of morphine and its glucuronide metabolites in blood or serum allows a rough estimation of increased dosage and of time elapsed since the last application. Drug effects can be examined with respect to individual case histories, including overdose and survival time if the patient died. However, different methods of administration and the strong influence of different volumes or compartments of distribution of parent compounds and metabolites on concentrations in human body tissues require careful use of glucuronide concentration data. In Germany, dihydrocodeine (DHC) is prescribed as a heroin substitute, and relative overdoses are needed to be effective. DHC metabolism was studied in three patients who died from overdoses. All metabolites (dihydrocodeine-6-glucuronide [DHC6], nor-DHC [NDHC], dihydromorphine [DHM], nor-DHM [NDHM], and DHM-3- and 6-glucuronide [DHM3G, DHM6G]) were determined using HPLC and fluorescence detection. Concentrations of DHM (0.16 mg/L to 0.22 mg/L serum) were found. The DHM glucuronide ratios were similar to those of morphine. Receptor binding studies showed that the binding affinity of DHM to porcine mu-receptor was higher than that of morphine, and DHM6G's binding affinity was as high as that of morphine-6-glucuronide (M6G). Metabolites may play an important role in the effectiveness of DHC in substitution and toxicity. Because of enzyme polymorphism, the formation of DHC poses a risk for proper dosage in patients who are either poor or extensive metabolizers. The distribution of opioid glucuronides in cerebral spinal fluid in relation to transcellular transport in central nervous tissue is discussed with respect to the receptor binding of opiates and drug effect.

Postmortem distribution of dihydrocodeine and metabolites in a fatal case of dihydrocodeine intoxication.

A report of a fatal dihydrocodeine ingestion under substitution therapy is given. Quantitation of dihydrocodeine, dihydromorphine, N-nordihydrocodeine, dihydrocodeine-6-, dihydromorphine-6- and dihydromorphine-3-glucuronide was performed simultaneously after solid-phase extraction prior to HPLC analysis, and the analytes were detected using their native fluorescence. Postmortem concentrations of blood samples from different sampling sites as well as from liver, kidney and cerebrum are reported. A hair sample was investigated to prove long-term use of the substitute drug. Site-to-site differences of the analytes from blood samples were very small. The partition behavior of the opioid glucuronides depended on the hematocrit value of the particular blood sample. Most important findings seemed that dihydromorphine and dihydromorphine-6-glucuronide concentrations decisively contributed to the toxicity of dihydrocodeine. This case report outlines that in dihydrocodeine related deaths the concentrations of the pharmacologically active metabolites should additionally be determined for reliable interpretation.

Helicobacter pylori infection decreases gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol in man.

BACKGROUND/AIMS: Ethanol is metabolized by alcohol dehydrogenase in the human stomach. This metabolism contributes to the so-called first-pass metabolism of ethanol which is affected by gender, medication, and morphological alterations of the gastric mucosa. Recently, it has been shown that Helicobacter pylori is capable to oxidize ethanol to acetaldehyde in vitro. Since H. pylori also injures gastric mucosa, the present study examines the effect of this bacterium on gastric alcohol dehydrogenase activity and systemic availability of ethanol in vivo. METHODS: Thirteen volunteers (7 men and 6 women, aged 18-52 years) with gastric H. pylori infection diagnosed by a positive CLO test and positive gastric histology received ethanol (0.225 g/kg) either orally or intravenously before and after H. pylori elimination to determine systemic availability of ethanol. In addition, gastric biopsy specimens were taken from all subjects before and after H. pylori elimination for histological assessment of mucosal alterations and determinations of gastric alcohol dehydrogenase activity and phenotype of the enzyme. RESULTS: In the presence of H. pylori the first-pass metabolism of ethanol was found to be significantly reduced (625 +/- 234 vs. 1,155 +/- 114 mg/dl/min, p = 0.046). This reduction of first-pass metabolism of ethanol was associated with a significant decrease in alcohol dehydrogenase activity (4.8 +/- 1.5 vs. 12.1 +/- 2.3 nmol/mg protein x min, p < 0.05) and an increase in the severity of mucosal damage as determined by a histological score (p < 0.05). CONCLUSIONS: H. pylori infection leads to gastric mucosal injury which is associated with a decrease in gastric alcohol dehydrogenase activity and first-pass metabolism of ethanol. Ethanol metabolism by H. pylori does not play an important role in vivo. However, gastric morphology is one important factor determining systemic availability of ethanol in man.

A preliminary study on the distribution of morphine and its glucuronides in the subcompartments of blood.

The distribution of morphine, morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) in whole blood, plasma, and packed erythrocytes was studied. Parameters investigated were the hematocrit values (10, 42, 44, and 71%) and the water content of the samples. The blood-to-plasma ratio of morphine concentrations was unaffected by variations in hematocrit and water content, whereas the corresponding ratios for M3G and M6G were strongly influenced. Ratios were 0.53 to 0.65 and 0.52 to 0.62 in specimens with average hematocrit values (42 and 44%, respectively), and the ratios were 0.81 or 0.89 (hematocrit 10%) and 0.27 or 0.28 (hematocrit 71%) in blood samples with different hematocrit values. In contrast to the morphine conjugates, morphine was highly bound to or partitioned into red blood cells (beta e = 55.9). Although the present data are limited, they already demonstrate that conclusions drawn from pharmacokinetic studies and transferred to parent drug to metabolite ratios resulting from forensic blood samples may be biased by the particular biological matrix under investigation.

[Fatal poisoning with clozapine and perazine. A case report]. / Tödliche Vergiftung mit Clozapin und Perazin. Eine Falldarstellung.

An intoxication following an apparent overdose of clozapine (Leponex) and perazine (Taxilan) is reported. There was a wide range of variation in postmortem blood and tissue concentrations of clozapine, desmethyclozapine and perazine. Clozapine/norclozapine blood and tissue ratios and perazine-pill-fragments in the gastric content could be used as a sign of suspected acute clozapine and perazine overdose.

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.

An in vitro experiment for postmortem vascular permeation. The passage of morphine and morphine glucuronides across a vascular wall.

A venous blood sample taken at autopsy cannot be considered to represent the antemortem blood concentration of a particular substance. Autolytic processes cause disintegration and increasing permeability of the physiological and anatomical barriers such as vascular walls and lead to changes in substance concentrations. In the present study, the experimental design represents an in vitro postmortem simulation of a drug substance crossing a venous wall. The postmortem behavior of morphine, morphine-3- and morphine-6-glucuronide was investigated. A Chien-Valia-diffusion chamber with a patch of inferior vena cava as diffusion barrier was used. For optimal simulation of postmortem events, vein sampling was restricted to selected autopsy cases. Parameters for the analysis of diffusion across the vascular tissue were dependence on time, temperature, and initial substance concentrations. The penetration behavior simulating venous efflux and influx of the substances was studied by different orientation of the venous wall in the experiments. Rhodamine B was used as a model substance to visualize the binding to the tissue and the passage across the venous wall. The permeation of morphine, morphine-3- and morphine-6-glucuronide across a vein tissue was found to be mainly dependent on the disintegration of the vascular wall and on the postmortem time period as well as on concentration gradients. From the data of this preliminary in vitro study, it can be concluded that a lag time for transvascular diffusion exists postmortem. However, it could be demonstrated, that adsorption to and penetration into the vascular tissue may alter intraluminal blood concentrations even at an early stage of the postmortem time period.

Plasma concentrations of heroin and morphine-related metabolites after intranasal and intramuscular administration.

The disposition of heroin and its metabolites was investigated in four healthy male volunteers following intranasal administration of 6 and 12 mg heroin hydrochloride. In addition, two doses of 6 mg heroin hydrochloride were injected intramuscularly for comparison of pharmacokinetic parameters. Serum samples were analyzed for heroin, 6-acetylmorphine, and morphine by solid-phase extraction-gas chromatography-mass spectrometry. The concentration of morphine glucuronides was determined by high-performance liquid chromatography based on the native fluorescence of the conjugates. Major findings were rapidly rising and declining terminal phases for heroin and 6-acetylmorphine and slowly declining phases of morphine and metabolites after both routes of administration. The area under the curve values of morphine-3-glucuronide depended on dose but not on route of administration. The apparent terminal half-lives of morphine-3-glucuronide ranged from 2.2 to 5.2 h for intranasally administered heroin and were 3.0 and 1.7 h for the intramuscularly applied drug. A mean morphine-3-glucuronide-heroin area-under-curve ratio of 93 for the intranasal route as compared with 38 for the intramuscular route demonstrated that circulating amounts of heroin were about half the size after intranasal administration of the same dose.

Fatal poisoning with the antidepressive agent opipramol.

An ingestion of an unknown quantity ( < or = 3000 mg) of opipramol dihydrochloride in a suicide case is described. Quantitation of opipramol and its deshydroxyethyl metabolite was performed simultaneously after liquid-liquid extraction from alkalinized samples prior to HPLC analysis. Postmortem concentrations of opipramol and of the metabolite in body fluids and organs are given. The distribution pattern of opipramol in tissue was comparable to findings from animal studies. The results showed the high uptake of opipramol in parenchymal tissues resulting in a low blood concentration. A possibly low contribution of deshydroxyethyl opipramol to the antidepressive effect may be concluded as the concentration was considerably lower in brain compared to other organs.