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  37th TIAFT Triennial Meeting Cracow

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Opening Lectures

Interpretation of Analytical Results

Drugs of Abuse and Doping

Validation of Analytical Procedures

Analysis of Alternative Materials

Driving under the Influence

Free Topics


  Validation of Analytical Results

Evaluation of Abselut® SPE Columns for Broad Spectrum Drug Screening in Plasma and Urine Using GC-FID and HPLC-DAD

dezeuw.jpg Jan Bosman, Jaap Wijsbeek, Jan Piet Franke and Rokus A. de Zeeuw*
Department of Analytical Chemistry and Toxicology, University Centre for Pharmacy, NL-9713 AV Groningen, The Netherlands

Abselut is a new SPE-sorbent, designed to extract a wide range of toxicologically relevant substances from biofluids. It is a highly crosslinked, special polymeric sorbent with hydrophilic and lipophilic properties. This polymeric nature is claimed to preclude channelling, pH instability and nonspecific binding. In this study, we have evaluated this new sorbent for broad spectrum drug screening.

The Abselut cartridges tested had a bed mass of 50 mg and a 3 ml or 10 ml specimen reservoir and were capable of extracting an extensive range of analytes with high (>75%) and reproducible (CV <5%) recoveries. Yet, because retention is based on a single mechanism, the pH of the extraction procedure is important: acidic analytes are best retained at acidic pH, whereas neutrals and basic substances are best retained at pH values of 7-9. Urine matrix interferences could be removed by adding a small percentage of methanol to the wash fluid. For plasma, removal of matrix interferences required the addition of salt (1M) to the phosphate buffer used to dilute plasma 1:1. Cleanliness of the chromatograms and recoveries were evaluated by GC-FID and HPLC-DAD.

The major advantages of the Abselut material were its rapid mass transfer, excellent flow properties and easy and reproducible handling.

Keywords: SPE, Systematic Toxicological Analysis, Abselut.



Selectivity of Photodiode Array UV-Spectra for Substance Identification in Systematic Toxicological Analysis

Matthias Herzler*, Fritz Pragst, Sieglinde Herre and Michael Rothe
Institute of Legal Medicine, Humboldt-University, Hannoversche Strasse 6, D-10115 Berlin, Germany

There are several limitations for the use of UV/VIS-spectra for substance identification, since they have only a small number of broad maxima and minima, and compounds with the same chromophoric electron system may have very similar or even identical spectra. On the other hand the new generation of photodiode array detectors (DAD) enables the operator to measure well resolved and reproducible spectra of amounts as low as 0.1 ng, and advanced library search software can distinguish between spectra with very small differences.

In this investigation based on a library of more than 2.000 UV-spectra of toxic compounds, the selectivity of HPLC/DAD for substance identification in Systematic Toxicological Analysis (STA) was examined. All compounds were classified with respect to their chromophore structure using a chemical database software (ISIS Base), showing that the variety of the absorbing p-electron system is much higher than primarily assumed. Even within larger groups of substances with the same chromophor like phenyl-substituted aliphatic compounds, mono- and dihydroxy substituted benzene derivatives, phenothiazines, xanthines, barbiturates, benzodiazepines, opiates and steroids, differences between spectra are found resulting from electronic and steric effects of nonconjugated groups. This was also shown by cross-searching of the entire library.

Furthermore parameters interfering with a spectral library search such as electronic noise, sample matrix background, chromatographic peak overlap, and the kind and concentration of organic modifier (acetonitrile, methanol, ethanol, isopropanol) in the mobile phase were evaluated. The quality and consistency of spectra measured with detectors of different manufacturers (Hewlett Packard, Kontron and Shimadzu) were compared. As a result HPLC/DAD including the additional use of a retention parameter was shown to be a sensitive and selective method for STA. It was applicable to more than 80% of the toxicologically relevant compounds, and enabled substance identification down to 10 ng/ml with a high day to day reproducibility and with different instruments. There are advantages and disadvantages compared to GC/MS, and both methods should be regarded as complementary rather than competing with each other.

Keywords: Photodiode Array Detector, Selectivity of UV-spectra, Substance Identification, Systematic Toxicological Analysis, UV-spectra Library of Toxic Compounds.



Screening for Drugs in Serum by ESI/CID and Library Searching

Wolfgang Weinmann*1, Michaela Renz1, Michal Svoboda2
1 Institute of Legal Medicine, University of Freiburg, Albertstrasse 9, D-79104 Freiburg, Germany
2 Perkin-Elmer Biosystems, Paul-Ehrlich Str. 17, D-63225 Langen, Germany

In-source collisionally induced dissociation (ESI/CID) of drugs was investigated using single-quadrupole mode with a Perkin Elmer/SCIEX API 365 IonSpray™ mass spectrometer. A mass spectra library of drugs (more than 600 compounds) was set up with three different orifice voltages. This library was used successfully for the identification of drugs and metabolites in serum in therapeutic concentrations (e.g. antidepressants, tranquilizers, neuroleptics).

For standardisation of fragment ion formation the influence of variation of several parameters such as spray-voltage, acetonitrile-content and pH of buffer was investigated. Changes in orifice-voltage had the greatest impact on in-source CID, while other parameters only had minor effects. Using haloperidol as the calibration substance, one molecule ion (MH+ 376) and two fragment ions (F1+ 165, F2+ 123) were formed. Characteristic breakdown curves were obtained with characteristic ion ratios and intercepts. These breakdown curves could be used for tuning of TRCID with different PE/SCIEX API instruments. Thus, reproducible TRCID spectra of drugs were achieved with different Ionspray sources and PE/SCIEX API mass spectrometers at three orifice-voltages (20, 50 and 80 V). With these preconditions a mass spectral library of approx. 600 drugs was set-up by LC/ESI/CID-MS analysis of 200 ng of each substance.

For general-unknown drug screening in serum samples liquid-liquid as well as solid-phase extraction procedures were worked out, and drugs and metabolites could be identified in therapeutic and toxic concentrations by LC-ESI/CID-MS with library searching.
A full list of library entries and more information about the Ionspray/CID-mass spectra library is available via internet (http://www.chemicalsoft.de).

Keywords: ESI/CID, LC/MS, Mass Spectra Library.



The Combination of Two-Dimensional Thin Layer Chromatography and Remission Spectrometry - a Chromatographic Technique with High Identification Power for Systematic Toxicological Analysis

demme.jpg Ulrich Demme*, Bjorn Ahrens, Annelies Klein and Rolf Werner
Institute of Forensic Medicine, University of Jena, Germany

After a short review of new tendencies in thin layer chromatography, the combination of TLC and remission spectra for the identification of unknown substances will be described in detail.

It is necessary to consider the extent to which identification of sunbstances is dependant on the spectral shape of the substance mass in the TLC-spot. The increase of separation power while using two dimensional thin layer chromatography will be demonstrated.

Discrimination and identification power of this chromatographic procedure are calculated (using a database of more than 100 substances) and compared with other chromatographic techniques (e.g. GC-MS). These calculations were performed not only - as described in literature - with respect to retention parameters, but also taking into account response and spectra shape, drug concentrations in blood and recovery.

These calculations show that the combination of two dimensional thin layer chromatography and remission spectra provides very high values for discrimination and identification power, comparable with or higher than other chromatographic techniques.

Furthermore, from a more practical point of view, the advantages and disadvantages of this combination are presented - again in comparison to other techniques.

Finally, some examples of application in forensic toxicology are given, esp. the general unknown analysis of blood (in autopsy and other cases). The identification and quantification of benzodiazepines and other psychotropic drugs, the quantification of antiarrhythmic drugs and b-adrenergic antagonists agents as well as of fluorescent drugs will also be mentioned.

To sum up, it can be said that modern thin layer chromatography (e.g. as described above) is not the chromatography of the underdog but a sophisticated chromatographic method with high identification power and some advantages in comparison to other chromatographic procedures.

Keywords: Two-Dimensional Thin Layer Chromatography, Remission Spectrometry, Identification Power.



Simultaneous Analysis of Sildenafil and Metabolites in Serum and Urine by High-Performance Liquid Chromatography with Diode-Array Detection

Marc Deveaux*1, Bertille Willaume1, Francis Collier2, Valery Hedouin1, Gilles Tournel1, Didier Gosset1
1Institut de Medecine Legale, place Theo Varlet, 59000 Lille, France
2Hospital Jeanne de Flandre, C.H.R.U., 59037 Lille cedex, France

Sildenafil (Viagra®, Laboratoire Pfizer, Orsay, France) is a selective inhibitor of type V phosphodiesterase and has the potential to improve penile erectile function in cases of simultaneous sexual stimulation. The main metabolite (UK-103,320) has a†similar potency. Forensic toxicologists have to be able to analyse this drug in body fluids in cases of misuse or recreational use. The aim of this work was to develop a†simple assay for extraction and analysis of both compounds in serum and urine, instead of the dialyse*** and the HPLC method published by Cooper [J.A.†Chromatogr. B, 701 (1997), 87-95].

After a single dose of 25 or 50 mg, blood and urine of two patients (68 and 75 year old, with normal liver function) were collected over a 5 hour period. Optimisation of the method was obtained with the following conditions. Extraction was performed on 2 ml samples of serum and urine by 5 ml of chlorform/2-propanol/n-heptane (60/14/26) with trazodone as internal standard. The HPLC system consisted of a Waters 600 E pump, a 996 photo diode-array detector and a†Millenium V3.0 software, operating with a C8 Waters Symmetry® column. The mobile phase was a phosphate buffer pH 4.5/acetonitrile gradient phase with a†variable flow rate. The column was heated to 30°C.

This procedure is shown to be selective for sildenafil, metabolite UK-103,320, two others metabolites and the internal standard. Absorbance was detected at 211†nm wavelength for all eluted compounds. Recovery is 96% in serum and urine. The procedure is linear in the range 20-2000 ng/ml. The LOD is 10 ng/ml and the LOQ is 20 ng/ml. At serum concentrations of 500 ng/ml, the CV of the method is less than 1%. Blood sildenafil concentrations are within the therapeutic range (70-315 ng/ml) and urine sildenafil concentrations range from 180 to 1600 ng/ml.

This simple assay shows similar performance for the estimation of sidenafil and main metabolites. We also describe separation and identification of a new sidenafil matabolite. This assay may be used in forensic cases on gastric content and cardiac blood.

Keywords: Sildenafil, Metabolites, Blood, HPLC-DAD.



Extraction and Analysis of Flunitrazepam and Metabolites in Urine using SPE-HPLC-DAD

Anya L. Hunt* and D. Zeitlin
1Forensic Science Unit, University of Strathclyde, 204 George St., C11XW Glasgow, UK

Flunitrazepam (Rohypnol) rapidly dissolves in water and is almost tasteless. During the last decade there have been a number of reports of the drug being added to the drinks of the unsuspecting inducing an inhibited but conscience state. During this period many young woman have reported very similar happenings while potentially under the influence of the drug. They have all approached the police 2 or 3 days later confused and alleging possible rape. Their memory is hazy and many report flashbacks. As a result of these reports the drug has become widely known as "The Date Rape Drug".

Rohypnol is the N-methyl-2'fluoro analogue of Nitrazepam. It is available in a†number of Western European countries for use as a hypnotic. It is routinely administered orally or by intravenous injection in doses of 2 mg. The drug undergoes biotransformation via N-demethylation, 3-hydroxlation and glucuronidation and reduction of the nitro group to an amine with subsequent acetylation. The drug half life is 10-70 hours (mean 25), the bioavailability 80-90% and volume of distribution approximately 4 l/kg.

Since the drug half life is relatively short and due to the time lapse between ingestion and report of the incident a very sensitive method of detection is required for the longer acting metabolites. This project has looked at optimisation of the extraction process used for the extraction and preconcentration of a number of the major urinary metabolites. Solid phase extraction utilising Bond Elut Certify speciality phases have achieved good results. The extracts were analysis using a†Varian HPLC equipped with a Hypersil High purity Elite analytical column in Reverse phase mode and Diode array detection (fixed wavelength 254 nm). The method was found to both repeatable and reproducible with a limit of detection of 1-8 µg/l. This presentation will discuss the optimisation processes used in this study and present statistical data on the method performance.



Sensitive Determination of Mianserin and Setiptiline in Body Fluids by Gas Chromatography with Surface Ionization Detector (GC-SID)

ishii.jpg Akira Ishii*1, Hiroshi Seno2, Kanako Watanabe-Suzuki2, Takeshi Kumazawa3, Osamu Suzuki2 and Yoshinao Katsumata
1Department of Legal Medicine and Bioethics, Nagoya University School of Medicine, 65 Tsuruna-cho, Showa-ku, Nagoya 466-8550, Japan
2Department of Legal Medicine, Hamamatsu University School of Medicine, 3600 Handa-cho, Hamamatsu 431-3192, Japan 3Department of Legal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Tokyo 142-8555, Japan

Mianserin and setiptiline, tetracyclic antidepressants, have been found measurable by gas chromatography with surface ionization detector (GC-SID). GC-SID is highly selective and sensitive for compounds containing tertiary amino groups; thus it is suitable for the determination of these antidepressants.

A GC-14A gas chromatograph with an SID system (Shimadzu) was used. We extracted mianserin and setiptiline (25 or 5 ng) spiked to 1 ml body fluids by solid-phase extraction using Bond Elut C18 cartridges before analyses.

By GC-SID, the peaks of mianserin and setiptiline appeared at 14.3 and 15.7 min, respectively. Recoveries of the compounds by solid-phase extraction were above 85%. Mianserin could be quantitated using setiptiline as internal standard and vice versa. Linearities were quite good in the range of 1.25 - 50 ng/ml for mianserin, and 2.5 - 100 ng/ml for setiptiline. The detection limits of mianserin and setiptiline were about 1.0 and 2.0 ng/ml, respectively. The sensitivities by GC-SID for these compounds were about 10-20 times higher than those by GC-nitrogen phosphorus detector (NPD). Our method seems useful in forensic toxicology, because of its sensitivity and specificity for these compounds.

Keywords: Mianserin, Setiptiline, Gas Chromatography with Surface Ionization Detector.