Detection of non-prescription heroin markers in urine with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

The planned introduction of a prescription heroin program in Germany created a need for differentiation between non-prescription and prescribed diamorphine use. The following substances were chosen as markers of non-prescription heroin: acetylcodeine (AC); its metabolites codeine (C) and codeine 6-glucuronide (C6G); papaverine (P); and noscapine (N). Typical heroin markers diamorphine (DAM) and its metabolites monoacetylmorphine (MAM) and morphine (M) were also determined. The drugs were extracted from urine samples with solid-phase extraction (C18) using standard 200-mg columns and 96-well microplates (100 mg). The extracts were examined with liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (positive ionization) in two isocratic systems. Selected ion monitoring procedures were applied for protonated molecular masses and characteristic fragments of drugs involved. The limits of detection were in the range of 0.5-1 ng/mL urine. The occurrence of selected heroin markers was investigated in 25 urine samples collected from heroin abusers (road traffic offenders and overdosed patients). C6G was found in all samples, C in 24 samples, N in 22 samples, MAM in 16 samples, P in 14 samples, DAM in 12 samples, and AC in 4 samples. The appearance of these compounds in urine reflects their pharmacokinetic properties and the composition of non-prescription heroin.

Analysis of underivatized amphetamines and related phenethylamines with high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Amphetamine, methamphetamine, illicit designer phenethylamines (MDA, MDEA, MDMA, MBDB, and BDMPEA), and other phenethylamines (benzyl-1-phenylethylamine, cathinone, ephedrine, fenfluramine, norfenfluramine, phentermine, 1-phenylethylamine, phenylpropanolamine, and propylhexedrine) were extracted from serum using a solid-phase extraction procedure. The extracts were examined with high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCI-MS). The drugs were separated on ODS column in acetonitrile/50 mM ammonium formate buffer (pH 3.0) (25:75) as a mobile phase. Full-scan mass spectra of drugs examined by means of APCI with collision-induced dissociation showed protonated molecular ions and fragments typical for particular drugs. LC-APCI-MS allowed an unequivocal differentiation of all drugs involved. The quantitation was performed using selected ion monitoring of protonated molecular ions and fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 1 to 5 microg/L serum, and the recoveries ranged from 58 to 96%. A linear response was observed for all drugs in the range from 5 to 500 microg/L. The method was applied for routine determination of amphetamine, MDMA, MDA, and MDEA in one run. Solid-phase extraction used assured simultaneous isolation of various groups of basic drugs of forensic interest (opiates, cocaines, phenethylamines, and benzodiazepines) from biofluids.

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

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 (C18 cartridges). The separation was performed on ODS column in acetonitrile-50 mM 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 microg/l, the absolute recovery was above 80% at concentration level of 10 to 100 microg/l. The method tested linear in the range from 1 to 1000 microg/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.

Determination of common drugs of abuse in body fluids using one isolation procedure and liquid chromatography--atmospheric-pressure chemical-ionization mass spectromery.

A method for determining opiate agonists (morphine, morphine-3-glucuronide, morphine-6-glucuronide, 6-monoacetylmorphine, codeine, codeine-6-glucuronide, dihydrocodeine, dihydromorphine, buprenorphine, methadone, tramadol, and ibogaine), cocaine and its metabolites (benzoylecgonine and ecgonine methyl ester) and lysergic acid diethylamide in serum, blood, urine and other biological matrices is presented. Aliquots (0.5-1.5 mL) of biological fluids were spiked with appropriate deuterated internal standards and extracted using a common solid-phase extraction method (C18 cartridges). The extracts were subjected to liquid chromatographic-atmospheric-pressure chemical-ionization mass spectrometric examination using selected ion monitoring procedures. These procedures were developed after analysis of full-scan mass spectra of examined compounds. The extraction method appeared very universal; the recoveries were high for almost all drugs and the extracts were very clean. The procedure was applied for routine forensic casework.

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.

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

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.

Morphine, morphine-3-glucuronide, morphine-6-glucuronide, and 6-monoacetylmorphine determined by means of atmospheric pressure chemical ionization-mass spectrometry-liquid chromatography in body fluids of heroin victims.

Morphine, morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and 6-monoacetylmorphine (6-MAM) were isolated from body fluids using solid-phase extraction and determined by means of atmospheric pressure chemical ionization-mass spectrometry-liquid chromatography (APCI-LC-MS) in selected ion monitoring mode. The following ions were monitored: m/z 286 for morphine; m/z 286 and 462 for M3G and M6G; m/z 211, 268, and 328 for 6-MAM; and m/z 289 for morphine-d3 (internal standard). The recoveries ranged from 82 to 89% The limits of detection were as follows: 0.1 ng/mL (morphine), 0.5 ng/mL (6-MAM), and 1 ng/mL (M3G and M6G). The analytes were determined in samples taken from 21 heroin-overdose victims. Twenty-one blood samples, 11 cerebrospinal fluid (CSF) samples, 12 vitreous humor (VH) samples, and 6 urine samples were investigated. Blood concentrations (ng/mL) of morphine ranged from 8 to 1539, of M3G from 111 to 941, of M6G from 32 to 332, and of 6-MAM from 0 to 73. The levels of morphine were correlated with glucuronide values and with 6-MAM. The concentrations of morphine, M3G, and M6G in CSF were, as a rule, lower than in blood and lower in VH than in CSF. The concentrations of morphine and molar ratios of M6G-morphine in blood and CSF were correlated. Low ratios of M3G-morphine and M6G-morphine in blood of heroin-overdose victims indicated short survival time after drug intake.

Determination of phenylisothiocyanate derivatives of amphetamine and its analogues in biological fluids by HPLC-APCI-MS or DAD.

Amphetamine (A), methamphetamine (MA), methylene dioxyamphetamine (MDA), methylenedioxyethylamphelamine (MDE), and methylenedioxymethamphetamine (MDMA), as well as eight other sympathomimetic amines (benzyl-1-phenylethylamine, ephedrine, fenfluramine, norfenfluramine, phentermine, phenylethylamine, phenylpropanolamine, and propylhexedrine), were extracted from serum or urine with ether, derivatized with phenylisothiocyanate, and subjected to high-performance liquid chromatographic (HPLC) examination in isocratic mode. Two detection arts were applied: atmospheric pressure chemical ionization (APCI) mass spectrometry (MS) and UV-spectrometry as diode array detection (DAD) or single wavelength at 250 nm. The derivatives were well-separated and showed good chromatographic behavior. Full-scan mass spectra of drugs examined by means of APCI with collision induced dissociation (APCID) contained protonated molecular ions (M+H)+ and fragments typical for particular drugs. APCID-liquid chromatography-mass spectrometry (LC-MS) appeared very selective for differentiation of all drugs involved. The quantitation with APCID was performed using selected ion monitoring (SIM) of (M+H)+ ions and selected fragments of drugs involved and their deuterated analogues. The limits of detection ranged from 0.001 mg/L (MA, MDMA, and MDE) to 0.005 mg/L (A and MDA). In HPLC-DAD, the spectra of MDMA and MDE were practically identical with maxima of 236-240 nm. Other amphetamines showed slightly different spectra with maxima of 245-250 nm. The limits of detection in UV detection amounted to 0.01-0.03 mg/L (single wavelength detector at 250 nm) or 0.05-0.1 mg/L (DAD).

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.

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.

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.

Identification power of a standardized HPLC-DAD system for systematic toxicological analysis.

High-performance liquid chromatography with photodiode-array detection (HPLC-DAD) provides two identification parameters: retention and UV spectral data. The identification power of these two parameters, expressed in standardized form (retention index and absorption maximum with the highest wavelength), is calculated using two approaches: discriminating power (DP) and mean list length (MLL). Our own HPLC database, which comprises data for more than 370 substances, is used as the basis of calculations. The identification power of both parameters applied separately is low but increases substantially when the combination of retention and spectral data is applied. Additionally, the DP and MLL values obtained for 56 acidic or neutral and 76 basic drugs examined by means of HPLC-DAD and other analytical methods (thin-layer chromatography, gas chromatography (GC), and ultraviolet (UV) detection) are compared. The on-line combination of HPLC retention index values and UV spectra, registered by means of DAD, creates an identification system in which the identification potential is slightly lower than the off-line combination of capillary GC and UV spectroscopy.

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

The 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 ADx 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.

[Digital autopsy archive using the personal computer]. / Digitales Sektions-Archiv (DSA) auf dem PC.

The development of a dissection register for Legal medicine well differentiated and considering special requirement of the subject was not achieved in history because of complexity of post mortem findings in many cases. Realizing that an all satisfying concept failed we looked for the possibility of using electronic data processing for rapid and flourish collection of autopsy results not requiring much time for the coroner. Based on personal computer software "dBase" a database program was developed easy to handle using different forms asking for characteristic circumstances of the case and essential post mortem findings. Short codes for locations and ascertainments were chosen to enable rapid selection of interesting statements as far as handling for statistical evaluations later on. Less then 10 minutes are needed for recording post mortem results in this way short time after autopsy.

[Analysis of regional trends in narcotic studies between 1980 and 1986]. / Regionale Tendenzanalyse von Betäubungsmittel-Untersuchungen zwischen 1980 und 1986.

During the period of survey, the number of narcotic drug seizures by the law, especially cannabis resin, has increased considerably. The details on this development are presented. The following main analytical results were obtained: the median concentration of THC in cannabis resin has increased up to 8.6%, in cannabis plants the THC content has fluctuated between 1% and 3%. In the heroin samples since 1982, diamorphin has predominated in the base form; the diamorphin content had dropped to 32%, which is connected with a rise simultaneous in the concentration of noscapine (up to 9%). The concentration of cocaine hydrochloride had diminished at the end of the period to 62%; on the other hand, the amphetamine sulfate content increased to 69%. LSD trips used from 10 to 120 micrograms per trip. Methadone occurred mostly in the form of tablets containing 5 mg methadone hydrochloride.

[Cyclizine as a drug of abuse?]. / Cyclizin als Rauschdroge?

Two British people going from Amsterdam to Berlin was examined at the borderline near Aix la Chapelle. Customsinspectors found besides a lot of hashish a letter hidden in a pocket lamp concerning drugs received in Amsterdam as remarks to LSD trips. Therefore a white powder found in a little paper in the purse of one person was tested for drugs. Previous examinations at the border failed and after further investigations at the Department of Forensic Medicine including mass spectrophotometry the powder was identified as Cyclizine. The analytical data like mass spectrum are described. This compound used many years for the prevention and treatment of motion sickness is off the market because of suggested teratogen effects. Previous reports about LSD-like symptoms after ingestion of large amounts of Cyclizine as the environment the powder was found let us suppose that some people will know about psychotic effects at high doses of this drug not being an inhibited narcotic.

[Toxicological aspects of fatal chloroquine poisoning over a period of several days]. / Toxikologische Aspekte beim mehrtägigen Verlauf einer tödlichen Chloroquin-Intoxikation.

The anti-inflammatory and antimalarial drug chloroquine is said to be one of the most frequently used drugs to commit suicide in the Orient and Far East. After the ingestion of large amounts of this 4-amino-chinoline derivate, signs of cardiac conduction disturbances are evidence of cardiotoxic action. For clinical treatment, hemoperfusion by activated charcoal has rather than hemodialysis proved to be partly effective for the removal of the toxic substance. Following a suicidal overdose of chloroquine tablets in a 13-year-old boy, we could confirm the efficiency of hemoperfusion by continued toxicologic examinations. The total elimination was nevertheless slow. Due to high postmortem concentrations in the blood and tissues, we believe that chloroquine is deposited and especially concentrated in the reticulohistiocytary system of the liver. Death after 5 days of clinical treatment in this case may be referred to as the first cardiac arrest with irreversible hypoxic damage of the brain.