Skip to main content
SpringerLink
Log in

Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood

  • Original Article
  • Published:
Forensic Science, Medicine, and Pathology Aims and scope Submit manuscript

Abstract

Purpose

Deaths among drug addicts are frequently caused by intoxication with methadone and/or morphine. These drugs are often used in combination with other drugs, such as buprenorphine. In addition, methadone is generally used as a mixture of R- and S-enantiomers. To date, a method for separation and quantitation of these specific drugs has not been developed. The aim of this study was to develop a sensitive enantioselective method for quantitation of morphine, its active metabolite morphine 6-glucuronide, buprenorphine, and R- and S-methadone, in a single analytical run.

Methods

Whole blood samples were diluted with 0.5 mol/L ammonium carbonate buffer and extracted on a Bond Elut C18 solid-phase extraction column with an automatic solid-phase extraction system. Chromatographic separation was performed on a chiral alpha-1-acid glycoprotein column with an acetonitrile/ammonium acetate buffer (10 mmol/L, pH 7.0, 22:78 v/v) mobile phase. The whole blood concentrations of the drugs were quantified by mass spectrometry using their stable isotope-labeled compounds as internal standards.

Results

The method was validated with respect to specificity, linearity, precision, limits of detection, and quantification and matrix effects. The precision (coefficient of variation) was below 15 %, and the accuracy was between 90 and 115 %.

Conclusions

This method will be useful for routine analyses in forensic laboratories where blood samples are frequently analyzed for drugs of abuse. In some cases, sudden death from methadone overdose is caused by the enantiomeric form of the methadone, which makes the enantiomer separation capability of this method important.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Simonsen KW, Normann PT, Ceder G, Vuori E, Thordardottir S, Thelander G, et al. Fatal poisoning in drug addicts in the Nordic countries in 2007. Forensic Sci Int. 2011;207(1–3):170–6.

    Article  PubMed  Google Scholar 

  2. Eap CB, Crettol S, Rougier JS, Schlapfer J, Sintra GL, Deglon JJ, et al. Stereoselective block of hERG channel by (S)-methadone and QT interval prolongation in CYP2B6 slow metabolizers. Clin Pharmacol Ther. 2007;81(5):719–28.

    Article  CAS  PubMed  Google Scholar 

  3. Strang J, Metrebian N, Lintzeris N, Potts L, Carnwath T, Mayet S, et al. Supervised injectable heroin or injectable methadone versus optimised oral methadone as treatment for chronic heroin addicts in England after persistent failure in orthodox treatment (RIOTT): a randomised trial. Lancet. 2010;375(9729):1885–95.

    Article  CAS  PubMed  Google Scholar 

  4. Rook EJ, Hillebrand MJX, Rosing H, van Ree JM, Beijnen JH. The quantitative analysis of heroin, methadone and their metabolites and the simultaneous detection of cocaine, acetylcodeine and their metabolites in human plasma by high-performance liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B. 2005;824(1–2):213–21.

    Article  CAS  Google Scholar 

  5. Taylor K, Elliott S. A validated hybrid quadrupole linear ion-trap LC-MS method for the analysis of morphine and morphine glucuronides applied to opiate deaths. Forensic Sci Int. 2009;187(1–3):34–41.

    Article  CAS  PubMed  Google Scholar 

  6. Jantos R, Skopp G. Postmortem blood and tissue concentrations of R- and S-enantiomers of methadone and its metabolite EDDP. Forensic Sci Int. 2013;226(1–3):254–60.

    Article  CAS  PubMed  Google Scholar 

  7. Al-Asmari AI, Anderson RA. Method for quantification of opioids and their metabolites in autopsy blood by liquid chromatography-tandem mass spectrometry. J Anal Toxicol. 2007;31(7):394–408.

    Article  CAS  PubMed  Google Scholar 

  8. Rodriguez-Rosas ME, Lofwall MR, Strain EC, Siluk D, Wainer IW. Simultaneous determination of buprenorphine, norbuprenorphine and the enantiomers of methadone and its metabolite (EDDP) in human plasma by liquid chromatography/mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2007;850(1–2):538–43.

    Article  CAS  PubMed  Google Scholar 

  9. Buchard A, Linnet K, Johansen SS, Munkholm J, Fregerslev M, Morling N. Postmortem blood concentrations of R- and S-enantiomers of methadone and EDDP in drug users: influence of co-medication and p-glycoprotein genotype. J Forensic Sci. 2010;55(2):457–63.

    Article  CAS  PubMed  Google Scholar 

  10. Holm KM, Linnet K. Chiral analysis of methadone and its main metabolite, EDDP, in postmortem brain and blood by automated SPE and liquid chromatography-mass spectrometry. J Anal Toxicol. 2012;36(7):487–96.

    Article  CAS  PubMed  Google Scholar 

  11. Johansen SS, Linnet K. Chiral analysis of methadone and its main metabolite EDDP in postmortem blood by liquid chromatography-mass spectrometry. J Anal Toxicol. 2008;32(7):499–504.

    Article  CAS  PubMed  Google Scholar 

  12. Rodriguez-Rosas ME, Medrano JG, Epstein DH, Moolchan ET, Preston KL, Wainer IW. Determination of total and free concentrations of the enantiomers of methadone and its metabolite (2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine) in human plasma by enantioselective liquid chromatography with mass spectrometric detection. J Chromatogr A. 2005;1073(1–2):237–48.

    Article  CAS  PubMed  Google Scholar 

  13. Wang SC, Ho IK, Wu SL, Liu SC, Kuo HW, Lin KM, et al. Development of a method to measure methadone enantiomers and its metabolites without enantiomer standard compounds for the plasma of methadone maintenance patients. Biomed Chromatogr. 2010;24(7):782–8.

    Article  CAS  PubMed  Google Scholar 

  14. Moody DE, Lin SN, Chang Y, Lamm L, Greenwald MK, Ahmed MS. An enantiomer-selective liquid chromatography-tandem mass spectrometry method for methadone and EDDP validated for use in human plasma, urine, and liver microsomes. J Anal Toxicol. 2008;32(3):208–19.

    Article  CAS  PubMed  Google Scholar 

  15. Bogusz MJ, Maier RD, Erkens M, Driessen S. Determination of morphine and its 3- and 6-glucuronides, codeine, codeine-glucuronide and 6-monoacetylmorphine in body fluids by liquid chromatography atmospheric pressure chemical ionization mass spectrometry. J Chromatogr B Biomed Sci Appl. 1997;703(1–2):115–27.

    Article  CAS  PubMed  Google Scholar 

  16. Dienes-Nagy A, Rivier L, Giroud C, Augsburger M, Mangin P. Method for quantification of morphine and its 3- and 6- glucuronides, codeine, codeine glucuronide and 6-monoacetylmorphine in human blood by liquid chromatography-electrospray mass spectrometry for routine analysis in forensic toxicology. J Chromatogr A. 1999;854(1–2):109–18.

    Article  CAS  PubMed  Google Scholar 

  17. Kolmonen M, Leinonen A, Kuuranne T, Pelander A, Ojanpera I. Hydrophilic interaction liquid chromatography and accurate mass measurement for quantification and confirmation of morphine, codeine and their glucuronide conjugates in human urine. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878(29):2959–66.

    Article  CAS  PubMed  Google Scholar 

  18. Whittington D, Kharasch ED. Determination of morphine and morphine glucuronides in human plasma by 96-well plate solid-phase extraction and liquid chromatography-electrospray ionization mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2003;796(1):95–103.

    Article  CAS  PubMed  Google Scholar 

  19. Peters FT. Method validation using LC-MS. In: Polettini A, editor. Applications of LC-MS in toxicology. London: Pharmaceutical Press; 2006. p. 43–64.

    Google Scholar 

  20. Hand CW, Moore RA, Sear JW. Comparison of whole blood and plasma morphine. J Anal Toxicol. 1988;12(4):234–5.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors thank laboratory technician Helle Terp for excellent technical assistance. The study was financially supported by The Region of Southern Denmark, Grant Number 09/13450.

Author information

Authors and Affiliations

  1. Department of Forensic Toxicology, Institute of Forensic Medicine, Winsløwparken 17B, 5000, Odense C, Denmark

    Dorte J. Christoffersen & Martin Worm-Leonhard

  2. Department of Clinical Genetics, Odense University Hospital, Sdr. Boulevard 29, 5000, Odense C, Denmark

    Charlotte Brasch-Andersen

  3. Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Sdr. Boulevard 29, 5000, Odense C, Denmark

    Per Damkier

  4. Department of Clinical Pharmacology, University of Southern Denmark, Winsløwparken 19, 5000, Odense C, Denmark

    Per Damkier & Kim Brøsen

  5. Institute of Forensic Medicine, Winsløwparken 17B, 5000, Odense C, Denmark

    Jørgen L. Thomsen

Authors
  1. Dorte J. Christoffersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Charlotte Brasch-Andersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jørgen L. Thomsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Martin Worm-Leonhard
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Per Damkier
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Kim Brøsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Dorte J. Christoffersen.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Christoffersen, D.J., Brasch-Andersen, C., Thomsen, J.L. et al. Quantification of morphine, morphine 6-glucuronide, buprenorphine, and the enantiomers of methadone by enantioselective mass spectrometric chromatography in whole blood. Forensic Sci Med Pathol 11, 193–201 (2015). https://doi.org/10.1007/s12024-015-9673-9

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12024-015-9673-9

Keywords

Search

Navigation