Manfred Erkens

Determination of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmospheric pressure chemical ionization mass spectrometry

A selective assay of morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), morphine, codeine, codeine-6-glucuronide (C6G) and 6-monoacetylmorphine (6-MAM) based on liquid chromatography atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS) is described. The drugs were extracted from serum, autopsy blood, urine, cerebrospinal fluid or vitreous humor using C18 solid-phase extraction cartridges and subjected to LC-APCI-MS analysis. The separation was performed on an ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (5:95), using a flow-rate gradient from 0.6 to 1.1 ml/min (total analysis time was 17 min). The quantitative analysis was done using deuterated analogues of each compound. Selected-ion monitoring detection was applied: m/z 286 (for morphine, M3G-aglycone and M6G-aglycone), 289 (for morphine-d3, M3G-d3-aglycone and M6G-d3-aglycone), 300 (for codeine and C6G-aglycone), 303 (for C6G-d3-aglycone), 306 (for codeine-d6), 328 (for 6-MAM), 334 (for 6-MAM-d6), 462 (for M3G and M6G), 465 (for M3G-d3 and M6G-d3), 476 (for C6G) and 479 (for C6G-d3). The limits of quantitation were: 1 microg/l for morphine, 2 microg/l for 6-MAM, 5 microg/l for M3G, M6G and codeine and 200 microg/I for C6G. The recovery ranged from 85 to 98% for each analyte. The method appeared very selective and may be used for the routine determination of opiates in body fluids of heroin abusers and patients treated with opiates.

Reversed-phase high-performance liquid chromatographic database of retention indices and UV spectra of toxicologically relevant substances and its interlaboratory use

An HPLC identification system based on a 1-nitroalkane retention index scale and secondary retention index standards is described. The retention index values and spectral data for 383 toxicologically relevant compounds (therapeutic and illicit drugs, environmental toxins and endogenous compounds) are given. The retention data may be directly applied in any laboratory using any reversed-phase, base-deactivated column, provided that the standardized procedure is observed.

Internally Concealed Cocaine: Analytical and Diagnostic Aspects

Thirty persons arrested at Frankfurt airport for smuggling internally concealed cocaine in 1993/1994 were investigated. An X-ray examination (in all 30 cases), immunochemical examination of urine (in 27 cases) and of saliva (in 20 cases) was performed in parallel. An X-ray examination gave positive results in all examined persons. EMIT cocaine metabolite assay (cut off 300 ng benzoylecgonine (BE)/mL) was positive in eight urine samples. After reducing the cut off to 150 ng BE/mL urine, eleven samples were classified as positive. The results were confirmed by means of chromatographic determinations. These findings showed limited role of immunological examination of urine as a screening test in suspected smuggling of internally concealed drugs. All saliva samples showed negative immunochemical results. The number of concealed containers ranged from 44 to 135 per person. The amount of cocaine hydrochloride found in particular cases ranged from 242 to 1050 g net weight, divided into containers weighing from 5.7 to 13.8 g. Drug packages were obviously machine-made. The packages smuggled by a particular person were uniform. However, a distinct interpersonal variability in drug packages was observed, in regard to the number of protective layers (4-7), size, weight, and cocaine purity. This may be helpful for the identification of production site. The leaching of cocaine from selected containers was investigated in a stirring bath and was independent of the conditions applied.

An overview on the standardization of chromatographic methods for screening analysis in toxicology by means of retention indices and secondary standards

SummaryIn systematic toxicological analysis, in which a logical analytical search for a potentially harmful substance has to be carried out, chromatographic methods are of utmost importance since the retention parameter can be used for primary identification. In gas chromatography the Kováts retention index (RI) system is generally accepted for standardization of the retention. In order to increase the reproducibility of the interlaboratory RI, secondary standards for RI calculation have proven to be very useful. The best secondary standards are those that are structurally resembling the substances under investigation. For example, for general toxicological screening analysis, mixtures of drugs are recommended. Stationary phases consisting of Carbowax are most extensively used for solvents and volatile compounds; for less volatile compounds SE-30/OV-1/dimethylsilicone stationary phases have proven to be useful. For these types of stationary phases extensive data compilations comprising over 6000 substances are available for use in toxicology.

Honey with Psilocybe mushrooms: a revival of a very old preparation on the drug market?

Abstract In 1996 samples of suspicious honey preparations were confiscated at the Dutch-German border. The labels on the 50 ml jars indicated that the honey contained Stropharia cubensis (better known as Psilocybe cubensis). The jars were filled with honey with a ca. 1 cm layer of fine particles on the top. The particles were collected and subjected to microscopic and chemical analysis. By microscopy mushroom tissue (plectenchym) and spores typical for the genus Psilocybe were identified in all samples. The HPLC analysis with atmospheric pressure mass spectrometry and diode array detection revealed psilocine but psilocybine was not found. The quantitative analysis was very difficult due to the matrix problems. A search showed that the honey with Psilocybe can be purchased in Dutch coffee shops without any limitations although psilocine and psilocybine belong to listed substances according to Dutch law.

The screening for common drugs of abuse in whole blood by means of EMIT-ETS and FPIA-ADx urine immunoassays

SummaryThe purpose of the paper was to compare the performance of ETS (EMIT) and ADx (FPIA) analyzers for screening blood samples for drugs of abuse after 2 alternative pretreatment procedures (acetone precipitation and ultrafiltration). Cannabinoids, benzodiazepines and benzoylecgonine were not detectable with both assays after ultrafiltration. The detectability of morphine in blood ultrafiltrates was distinctly lower than after acetone precipitation. The comparison of results obtained with ETS and ADx after acetone precipitation showed that ETS assay is slightly more sensitive but AD is slightly more reproducible. Both assays may be used for blood examination with similar cut-off values. The ETS analyzer gave much better analytical performance (speed, flexibility) and lower reagent costs than the ADx analyzer.ZusammenfassungZum immunologischen Nachweis von Drogen in Blutproben wurde die Brauchbarkeit der ETS (EMIT)- und ADx (FPIA)-Systeme unter Einsatz zweier Probenvorbereitungsmethoden (Ultrafiltration und Acetonfällung) untersucht und die Ergebnisse miteinander verglichen. Cannabinoide (D-8-THC-COOH), Benzodiazepine (Oxazepam) und Benzoylecgonin waren nach Ultrafiltration mit beiden Systemen praktisch nicht nachweisbar. Die Nachweisbarkeit von Morphin war nach Ultrafiltration deutlich schlechter als nach vor-ausgegangener Acetonfällung. Der Vergleich der mit dem ETS- und ADx-System nach Acetonfällung erhaltenen Ergebnisse zeigt, daß der ETS-Assay etwas empfindlichere, der ADx-Assay besser reproduzierbare Meßergebnisse liefert. Beide Assays sind für Blutuntersuchungen geeignet und lassen vergleichbare cut-off-Werte zu. Das ETS-System zeigt in der Praxis eine bessere Handhabung, Geschwindigkeit und Flexibilität sowie im Vergleich zum ADx deutlich niedrigere Reagenzienkosten.

Methadon-assoziierte Todesfälle im Raum Aachen (1994–1998)

ZusammenfassungBei jedem Fünften (19/102) der zwischen Februar 1994 und Februar 1998 im Institut für Rechtsmedizin der RWTH Aachen registrierten Drogentodesfälle wurde eine Beteiligung des Opioides Methadon festgestellt. Obwohl sich die gesamte Anzahl der opiatsubstituierten Patienten in Stadt und Kreis Aachen im genannten Zeitraum etwa verzehnfachte, kam es jährlich gleichbleibend zu 4 bis 6 methadonassoziierten Todesfällen, zumeist infolge von Mischintoxikationen. Zwei Drittel aller in Methadonprogrammen substituierten Opiatabhängigen starben während der ersten drei Behandlungstage. Soweit diese Fälle polizeilich untersucht worden sind, wurde in jedem Fall von dem behandelnden Arzt eine überhöhte Methadon-Initialdosis verabreicht, darüber hinaus waren manche Patienten nicht ausreichend opiattolerant. Keiner der untersuchten Fälle war zuvor Patient in einem lege artis durchgeführten Methadonprogramm.AbstractAll drug-related deaths registered in the institute of Forensic Medicine, University of Aachen, Germany, during the time period of February 1994–February 1998 were examined and approximately 20% (19/102) were considered to be associated with methadone. Although the number of patients involved in methadone maintenance programms had increased by tenfold, the number of methadone-associated deaths remained relatively constant. Polydrug toxicity was the leading cause of death. Of the licit methadone substituted patients died two thirds during the first 3 days of treatment. According to the police investigations, all had received an induction dose that exceeded the recommended amount and some were intolerant to opiates. All fatalities showed irregularities concerning the methadone distribution practice and this highlights the need for adequate supervision of methadone treatment.

Evaluation of serum percent trisialotransferrin as potential predictive biomarker of hepatocellular dedifferentiation in chronic liver disease.

BACKGROUND AND AIM Chronic hepatitis induced liver fibrogenesis is characterized by epithelial-to-mesenchymal transition of liver parenchymal cells (hepatocytes) to fibroblast (-like cells), i.e. increasing hepatocellular dedifferentiation, ultimatively leading to the development of hepatocellular carcinoma (HCC). Up to now the spectrum of valid serum biomarkers for this process of hepatocellular dedifferentiation is very limited. We therefore investigated the dynamics of alterations in the serum transferrin isoform pattern in the pathogenetic sequence from liver fibrosis to hepatocellular carcinoma, to evaluate the suitability of one of the isoforms as potential biomarker for hepatocellular dedifferentiation in chronic liver disease. RESULTS Our data on 252 patients with hepatitis C virus (HCV) induced fibrogenic liver disease and on 43 patients with HCV induced HCC demonstrate a dynamic alteration of serum % trisialotransferrin levels in the pathogenetic sequence from early stage hepatic fibrosis to fully developed hepatocellular carcinoma, whereas serum % di- and pentasialotransferrin values seem not to be affected. We show that patients with early stage fibrosis (METAVIR stage F1) and weak fibrogenic activitiy (METAVIR grade A1) display significantly lower values of serum % trisialotransferrin compared to healthy controls, and that serum % trisialotransferrin values increased steadily parallel to an increase of fibrotic stage and grade, respectively, while finally exceeding normal values in those patients with hepatocellular carcinoma. CONCLUSION These findings propose a possible diagnostic value of serum % trisialotransferrin concentrations in the pathogenesis of hepatocellular dedifferentiation and the use of this parameter as possible predictive tumor marker in patients with chronic liver disease. Monitoring the pattern of transferrin bound sialic acid residues may thus be a helpful tool in assessing the risk of malignant degeneration in patients with chronic fibrogenic liver disease.

Stellungnahme zur Studie „Cannabis im Straßenverkehr”

AbstractIn his recent study “Cannabis im Straßenverkehr” Prof. Dr. Th. Daldrup was searching for reliable criteria assessment of drivers’ incapacity after Cannabis use. He proposed the “Cannabis influence factor” (CIF) as a measure of impairment of driving performance. The CIF is calculated from the formula: