Antibody-mediated clean-up of blood for simultaneous HPLC determination of morphine and morphine glucuronides

Abstract

For the interpretation of the concentration of morphine in blood samples of heroin consumers information about the concentration of the analgesic active morphine metabolite morphine-6-glucuronide is very important. Thus a simple but specific clean-up procedure based on immuno-affinity chromatography is presented for the extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from whole blood in cases of fatal heroin overdose. The preparation of the immunoabsorber by immobilization of antibodies against morphine-3-BSA and morphine-6-KLH with carbonyldiimidazole-activated trisacrylgel is described. The separation of the extracts is achieved by HPLC using native fluorescence detection. The limits of detection for this method are 10ng for morphine and morphine glucuronides/g blood. The results for the concentration of morphine and morphine glucuronides in blood from seven cases of heroin overdose are presented. By calculating the quotients for the concentrations of morphine-6-glucuronide/morphine the time elapsed since the last intake of heroin is estimated.

Active immunisation against nicotine blocks the reward facilitating effects of nicotine and partially prevents nicotine withdrawal in the rat as measured by dopamine output in the nucleus accumbens, brain reward thresholds and somatic signs

“…KLH is a giant protein (3.5-7 MDa) composed subunits of two isoforms; it is highly immunostimulatory (Weigle 1964) and induces both a cellular (Th1) and a humoral (Th2) immune response in human (Hersh and Dyre 1974). KLH has been used as a test for human immunocompetence (Swanson and Schwartz 1967), as an immunostimulator in tumour therapy (Olsson et al 1974) and as a carrier for various haptens including drugs of abuse in preclinical research (Beike et al 1997;Ettinger et al 1997;Hieda et al 1997;de Villiers et al 2002).…”

Anti-Herpes Effect of Hemocyanin Derived from the Mollusk Rapana thomasiana

“…The hemocyanin from the mollusk keyhole limpet (Megathura crenulata), known as KLH, has been used for the above purposes. Besides these biomedical effects, KHL has been used in the diagnosis and immunotherapy of Shistosomias (Li et al, 1994), in drug addiction (Ettinger et al, 1997;Beike et al, 1997), and as a component of experimental synthetic minimal viral vaccines against AIDS (Naylor et al, 1991) and papilloma virus (Meyer et al, 1998), and exhibit also antitumour activity against different kinds of tumours (Olsson et al, 1974;Lamm et al, 2000;Jurincic-Winkler et al, 2000;Sandmaier et al, 1999;McFadden et al, 2003;Vona-Davis et al, 2004).…”

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

“…This technique has already been successfully applied for on-line or separate purification and extraction in several cases [22][23][24], e.g. for the extraction of estradiol [25], clenbuterol [26], amanitin [27], tetrahydrocannabinol [28,29], benzodiazepines and their metabolites [30,31], LSD [32,33] and morphine and its metabolites [34,35]. We developed a new immunoadsorber for the extraction of morphine and its glucuronides by the immobilisation of specific antibodies against morphine and morphine glucuronides [36] applying the solgel method.…”