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HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction

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Abstract

A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-ß-D-glucuronide and morphine-6-ß-D-glucuronide is described. An immunoadsorber was applied which was created for the first time by the immobilisation of specific antibodies (polyclonal, host: rabbit) by the sol-gel method. The extraction method in combination with high performance liquid chromatography-fluorescence determination has been validated and shown to be applicable to blood samples of heroin victims in a low concentration range. Blood extracts were essentially free of interfering matrix components when compared to C8-extracts. Additionally, a novel, sensitive and selective detection system for wavelength-resolved analysis of laser-induced fluorescence coupled to HPLC was developed. The analytes were excited with a frequency tripled Ti:Sa laser (λ=244 nm quasi cw). The total emission spectrum was recorded with a detection system consisting of an imaging spectrograph and a back-illuminated CCD camera. This technique of detection, combined with an extended optical path (at least 6 mm could be illuminated by the laser), resulted in an optimal fluorescence intensity of the analytes. The method permitted the analysis of morphine, morphine-3-ß-D-glucuronide and morphine-6-ß-D-glucuronide in a low concentration range and could be applied to a complex matrix such as postmortem blood samples because analyte peaks could be discriminated from matrix peaks by their characteristic emission spectra.

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Authors and Affiliations

  1. Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse 23, 48149 , Münster, Germany

    Y. Hupka, J. Beike, B. Brinkmann & H. Köhler

  2. Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Hittorfstrasse 58–62, 48149 , Münster, Germany

    G. Blaschke

  3. Faculty of Physics, University of Bielefeld, Universitätsstrasse 25, 33615 , Bielefeld, Germany

    J. Roegener

Authors
  1. Y. Hupka
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  2. J. Beike
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  3. J. Roegener
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  4. B. Brinkmann
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  5. G. Blaschke
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  6. H. Köhler
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Correspondence to H. Köhler.

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Hupka, Y., Beike, J., Roegener, J. et al. HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction. Int J Legal Med 119, 121–128 (2005). https://doi.org/10.1007/s00414-004-0501-0

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  • DOI: https://doi.org/10.1007/s00414-004-0501-0

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