HPLC with laser-induced native fluorescence detection for morphine and morphine glucuronides from blood after immunoaffinity extraction.
A new immunoaffinity solid phase extraction of morphine and its phase II metabolites, morphine-3-beta-D-glucuronide and morphine-6-beta-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 (lambda=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-beta-D-glucuronide and morphine-6-beta-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.
Hupka
Y
Y
Institute of Legal Medicine, University Hospital Münster, Röntgenstrasse 23, 48149 Münster, Germany.
Beike
J
J
Roegener
J
J
Brinkmann
B
B
Blaschke
G
G
Köhler
H
H
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005453:Fluorescence; D005554:Forensic Medicine / Q000379:methods; D006801:Humans; D007834:Lasers; D009020:Morphine / Q000097:blood*; D009022:Morphine Derivatives / Q000097:blood*; D009294:Narcotics / Q000097:blood*
D009022:Morphine Derivatives; D009294:Narcotics; C035349:morphine-6-glucuronide; D009020:Morphine; C018108:morphine-3-glucuronide