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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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References

  1. Aderjan R, Hoffmann S, Schmitt G, Skopp G (1995) Morphine and morphine glucuronides in serum of heroin consumers and in heroin-related deaths determined by HPLC with native fluorescence detection. J Anal Toxicol 19:163–168

    CAS  PubMed  Google Scholar 

  2. Pasternak GW, Bodnar RJ, Clark JA, Inturrisi CE (1987) Morphine-6-glucuronide, a potent mu agonist. Life Sci 41:2845–2849

    Google Scholar 

  3. Gong QL, Hedner T, Hedner J, Björkmann R, Nordberg G (1991) Antinococeptive and ventilatory effects of the morphine metabolites: morphine-6-glucuronide and morphine-3-glucuronide. Eur J Pharmacol 193:47–56

    Article  CAS  PubMed  Google Scholar 

  4. Sullivan AF, McQuay HJ, Bailey D, Dickenson AH (1989) The spinal antinociceptive actions of morphine metabolites morphine-6-glucuronide and normorphine in the rat. Brain Res 482:219–224

    Article  CAS  PubMed  Google Scholar 

  5. Stain-Texier F, Boschi G, Sandouk P, Scherrmann J (1999) Elevated concentrations of morphine 6-beta-D-glucuronide in brain extracellular fluid despite low blood-brain barrier permeability. Br J Pharmacol 128:917–924

    CAS  PubMed  Google Scholar 

  6. Baker L, Dye A, Ratka A (2000) Effects of morphine glucuronides on the function of opioid receptors in human SK-N-SH cells. Neurosci Lett 281:1–4

    Google Scholar 

  7. Grung M, Skurtveit S, Ripel A, Morland J (2000) Lack of crosstolerance between morphine and morphine-6-glucuronide as revealed by locomotor activity. Pharmacol Biochem Behav 66:205–210

    Article  CAS  PubMed  Google Scholar 

  8. Brown GP, Yang K, Ouerfelli O, Standifer KM, Byrd D, Pasternak GW (1997) 3H-morphine-6beta-glucuronide binding in brain membranes and an MOR-1-transfected cell line. J Pharmacol Exp Ther 282:1291–1297

    CAS  PubMed  Google Scholar 

  9. Osborne R, Thompson P, Joel S, Trew D, Patel N, Slevin M (1992) The analgesic activity of morphine-6-glucuronide, Br J Clin Pharmacol 34:130–138

    Google Scholar 

  10. Geisslinger G, Brune K, Kobal G, Lötsch J (1997) Intravenous morphine-6-glucuronide (M6G) is devoid of analgesic activity in man. Pain 70:289–290

    Article  CAS  PubMed  Google Scholar 

  11. Lötsch J, Kobal G, Stockmann A, Brune K, Geisslinger G (1997) Lack of analgesic activity of morphine-6-glucuronide after short-term intravenous administration in healthy volunteers. Anesthesiology 87:1348–1358

    PubMed  Google Scholar 

  12. Steentoft A, Worm K, Pedersen CB, Sprehn M, Mogensen T, Sorensen MB, Nielsen E (1996) Drugs in blood samples from unconscious drug addicts after the intake of an overdose. Int J Legal Med 108:248–251

    CAS  PubMed  Google Scholar 

  13. Wright AW, Watt JA, Kennedy M, Cramond T, Smith MT (1994) Quantitation of morphine, morphine-3-glucuronide, and morphine-6-glucuronide in plasma and cerebrospinal fluid using solid-phase extraction and high-performance liquid chromatography with electrochemical detection. Ther Drug Monit 16:200–208, erratum: Ther Drug Monit (1998) 20:218

    Google Scholar 

  14. Blanchet M, Bru G, Guerret M, Bromet-Petit M, Bromet N (1999) Routine determination of morphine, morphine-3-beta-D-glucuronide and morphine-6-beta-D-glucuronide in human serum by liquid chromatography coupled to electrospray mass spectrometry. J Chromatogr A 854:93–108

    Article  CAS  PubMed  Google Scholar 

  15. Wu WS, Tsai JL (1999) Analysis of morphine and morphine-3beta-D-glucuronide in human urine by capillary zone electrophoresis with minimal sample pretreatment. Biomed Chromatogr 13:216–219

    Article  CAS  PubMed  Google Scholar 

  16. Tsai JL, Wu WS, Lee HH (2000) Qualitative determination of urinary morphine by capillary zone electrophoresis and ion trap mass spectrometry. Electrophoresis 21:1580–1586

    Article  CAS  PubMed  Google Scholar 

  17. Manetto G, Tagliaro F, Crivellente F, Pascali VL, Marigo M (2000) Field-amplified sample stacking capillary zone electrophoresis applied to the analysis of opiate drugs in hair. Electrophoresis 21:2891–2898

    Article  CAS  PubMed  Google Scholar 

  18. Vandepeer J, Trenerry VC, Keogh G (1996) Some examples of the analysis of complex samples by micellar electrokinetic capillary chromatography with photodiode array detection. Electrophoresis 17:1361–1366

    CAS  PubMed  Google Scholar 

  19. Zhang XX, Li J, Gao J, Sun L, Chang WB (2000) Determination of morphine by capillary electrophoresis immunoassay in thermally reversible hydrogel-modified buffer and laser-induced fluorescence detection. J Chromatogr A 895:1–7

    Google Scholar 

  20. Sagawa K, Kimura A, Saito Y, Inoue H, Yasuda S, Nosaka M, Tsuji T (2003) Production and characterization of a monoclonal antibody for sweat-specific protein and its application for sweat identification. Int J Legal Med 117:90–95

    PubMed  Google Scholar 

  21. De Letter E, Espeel M, Craeymeersch M et al. (2003) Immunohistochemical demonstration of the amphetamine derivatives 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in human post-mortem brain tissues and the pituitary gland. Int J Legal Med 117:2–9

    PubMed  Google Scholar 

  22. Weller MG (2000) Immunographic techniques—a critical view. Fresenius J Anal Chem 366:635–645

    Article  CAS  PubMed  Google Scholar 

  23. Van Emon JM, Gerlach CL (1998) Bioseparation and bioanalytical techniques in environmental monitoring. J Chromatogr B 715:211–228

    Google Scholar 

  24. Hage DS (1998) Survey of recent advances in analytical applications of immunoaffinity chromatography. J Chromatogr B 715:3–28

    CAS  Google Scholar 

  25. Glencross RG, Abeywardene SA, Corney SJ, Morris HS (1981) The use of oestradiol-17 beta antiserum covalently coupled to Sepharose to extract oestradiol-17 beta from biological fluids. J Chromatogr 223:193–197

    CAS  PubMed  Google Scholar 

  26. Rashid BA, Kwasowski P, Stevenson D (1999) Solid phase extraction of clenbuterol from plasma using immunoaffinity followed by HPLC. J Pharm Biomed Anal 21:635–639

    Article  CAS  PubMed  Google Scholar 

  27. Maurer HH, Schmitt CJ, Weber AA, Kraemer T (2000) Validated electrospray liquid chromatographic-mass spectrometric assay for the determination of the mushroom toxins a- and ß-amanitin in urine after immunoaffinity extraction. J Chromatogr B 748:125–135

    Article  CAS  Google Scholar 

  28. Lemm U, Tenczer J, Baudisch H, Krause W (1985) Antibody-mediated extraction of the main tetrahydrocannabinol metabolite, 11-nor-delta9-tetrahydrocannabinol-9-carboxylic acid, from human urine and its identification by gas chromatography-mass spectrometry in the sub-nanogram range. J Chromatogr 342:393–398

    Google Scholar 

  29. Feng S, El Solhly MA, Salamone S, Salem MY (2000) Simultaneous analysis of delta9-THC and its major metabolites in urine, plasma, and meconium by GC-MS using an immunoaffinity extraction procedure. J Anal Toxicol 24:395–402

    CAS  PubMed  Google Scholar 

  30. Franzelius C, Ackermann I, Deinl I, Angermaier L, Machbert G (1998) Simultaneous extraction of selected benzodiazepines and benzodiazepine-glucuronides from urine by immunoadsorption. J Anal Toxicol 22:359–362

    CAS  PubMed  Google Scholar 

  31. Deinl I, Franzelius C, Angermaier L, Mahr G, Machbert G (1999) On-line immunoaffinity extraction and HPLC analysis of flunitrazepam and its main metabolites in serum, J Anal Toxicol 23:598–602

    Google Scholar 

  32. Kerrigan S, Brooks DE (1999) Immunochemical extraction and detection of LSD in whole blood. J Immunol Methods 224:11–18

    Article  CAS  PubMed  Google Scholar 

  33. Röhrich J, Zörntlein S, Becker J (2000) Analysis of LSD in human body fluids and hair samples applying ImmunElute columns. Forensic Sci Int 107:181–190

    Article  PubMed  Google Scholar 

  34. Beike J, Köhler H, Blaschke G (1997) Antibody-mediated clean-up of blood for simultaneous HPLC determination of morphine and morphine glucuronides. Int J Legal Med 110:226–229

    Article  CAS  PubMed  Google Scholar 

  35. Beike J, Köhler H, Brinkmann B, Blaschke G (1999) Immunoaffinity extraction of morphine, morphine-3-glucuronide and morphine-6-glucuronide from blood of heroin victims for simultaneous high-performance liquid chromatographic determination. J Chromatogr B 726:111–119

    CAS  Google Scholar 

  36. Beike J, Blaschke G, Mertz A, Köhler H, Brinkmann B (1998) A specific immunoassay for the determination of morphine and its glucuronides in human blood. Int J Legal Med 112:8–14

    Article  Google Scholar 

  37. Spitzer B, Cichna M, Markl P, Sontag G, Knopp D, Niessner R (2000) Determination of 1-nitropyrene in herbs after selective enrichment by a sol-gel-generated immunoaffinity column. J Chromatogr A 880:113–120

    Article  CAS  PubMed  Google Scholar 

  38. Brown RE, Jarvis KL, Hyland KJ (1989) Protein measurement using bicinchoninic acid: elimination of interfering substances. Anal Biochem 180:136–139

    CAS  PubMed  Google Scholar 

  39. US Department of Health and Human Services, Food and Drug Administration, Center of Drug Evaluation and Research, Center of Veterinary Medicine, Guidance for Industry, Bioanalytical Validation, May 2001, BP

  40. Schmitt G, Bogusz M, Aderjan R, Meyer C (1990) Detection of morphine and codeine in blood samples with gas chromatography/mass spectrometry (NCI and PCI) for differentiating codeine use from use of heroin and morphine. Z Rechtsmed: 103:513–521

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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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