Rolf-Dieter Maier

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.

Monitoring of olanzapine in serum by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry

Abstract A selective assay of olanzapine with liquid chromatography atmospheric pressure chemical ionization (LC–APCI–MS, positive ions) is described. The drug and internal standard (ethyl derivative of olanzapine) were isolated from serum using a solid-phase extraction procedure (C 18 cartridges). The separation was performed on ODS column in acetonitrile–50 m M ammonium formate buffer, pH 3.0 (25:75). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection (SIM) was applied with the following ions: m/z 313 and 256 for olanzapine and m/z 327 and 270 for the internal standard for quantitation. The limit of quantitation was 1 μg/l, the absolute recovery was above 80% at concentration level of 10 to 100 μg/l. The method tested linear in the range from 1 to 1000 μg/l and was applied for therapeutic monitoring of olanzapine in the serum of patients receiving (Zyprexa™) and in one case of olanzapine overdose. Olanzapine in frozen serum samples and in frozen extracts was stable over at least four weeks. The examinations of urine extracts from patients receiving olanzapine revealed peaks of postulated metabolites (glucuronide and N -desmethylolanzapine).

Poor reproducibility of in-source collisional atmospheric pressure ionization mass spectra of toxicologically relevant drugs.

The purpose of the study was to examine the intra- and interlaboratory reproducibility of mass spectra obtained with liquid chromatography-atmospheric pressure ionization mass spectrometry (LC--API-MS) both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) modes. Toxicologically relevant drugs of different polarity were selected as test substances: morphine-6-glucuronide, 6-monoacetylmorphine, codeine, lysergic acid diethylamide, methylenedioxymethamphetamine. The study was performed in two laboratories using identical instruments and in one using a slightly different instrument. Basic instrument settings and mobile phase were identical in all laboratories. Mass spectra of drugs were taken at four collision energy voltages and using mobile phase of different composition (four concentration levels of acetonitrile and of ammonium formate buffer). The experiments demonstrated that mass spectra of given drugs, obtained in identical conditions with identical instruments, may show very different degrees of fragmentation. Mass spectra obtained with different instruments differed profoundly not only in the degree of fragmentation, but also different fragments and adducts were observed. Short-term intralaboratory reproducibility of mass spectra was satisfactory. On the other hand, the long-term experiments showed different degrees of fragmentation of APCI-generated mass spectra at nominally identical fragmentation energy. The changes in the composition of the mobile phase (concentration of organic modifier or buffer molarity) did not affect the reproducibility of fragmentation to any relevant degree. The study showed that the interlaboratory exchange and use of mass spectrum library, generated by single-quadrupole (LC--API-MS instruments, is hardly feasible at the moment, even under very carefully standardized conditions.

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.

Determination of flunitrazepam and its metabolites in blood by high-performance liquid chromatography–atmospheric pressure chemical ionization mass spectrometry

A selective assay of flunitrazepam (F) and its metabolites 7-aminoflunitrazepam (7-AF), N-desmethylflunitrazepam (N-DF) and 3-hydroxyflunitrazepam (3-OHF) with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS, positive ions) is described. The drugs were isolated from serum, blood or urine using a solid-phase extraction procedure previously applied to various drugs of abuse. F-d3 and 7-AF-d3 were used as internal standards. The drugs were separated on ODS column in acetonitrile-50 mM ammonium formate buffer, pH 3.0 (45:55, v/v). After analysis of mass spectra taken in full scan mode, a selected-ion monitoring detection was applied with following ions: m/z 284 (7-AF and F), 287 (7-AF-d3 and F-d3), 314 (F), 300 (N-DF and 3-OHF), 317 (F-d3), 330 (3-OHF). The limits of detection were: 0.2 microg/l for F and 7-AF, 1 microg/l for N-DF and 3-OHF. The method was linear in the range 1-500 microg/l, the recoveries ranged from 92 to 99%. The method was applied for determination of F and metabolites in clinical and forensic samples. LC-APCI-MS seems to be a method of choice for these compounds.

Applicability of various brands of mixed-phase extraction columns for opiate extraction from blood and serum.

Four commercially available types of mixed-phase solid-phase extraction (SPE) columns (Bond Elut Certify, Isolute Confirm HCX, Chromabond Drug and Bakerbond Narc-2) were examined in order to compare the extraction efficiencies and chromatographic purity of extracts. The absolute recovery of morphine, 6-monoacetylmorphine and codeine was examined in blood and serum (ten samples each at two concentration levels), using SPE columns of the same batch. GC-MS (ion trap) and HPLC with amperometric detection were used for quantitation. A distinct variability in extraction recovery was observed among the same batches of all brands of SPE columns. All extracts were chromatographically pure and no interfering peaks were observed, neither in GC-MS nor in HPLC examinations, but in some extracts large peaks of plasticizers were identified. The measurements of flow velocities of the same samples of blood or serum through the SPE columns of the same batch showed very large variability of random character. The morphometric analysis of particles was performed for two batches of each sort of SPE columns by means of an image analysing system. Symmetrical distribution of particle size was observed only in Chromabond MN Drug packing, while in other cartridges large fractions of fine particles and nonhomogenous distribution were found. Only in one case the morphometric findings were pretty concordant with the data available from the manufacturer; in two cases, observed data varied considerably from that expected, and in one case no information was available at all. The study showed generally that there was room for improvement in the quality of mixed-phase SPE columns.

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.

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: