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Quantitative low-volume assay for simultaneous determination of fentanyl, norfentanyl, and minor metabolites in human plasma and urine by liquid chromatography—tandem mass spectrometry (LC-MS/MS)

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Abstract

A rapid and sensitive liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for simultaneous quantification of fentanyl (F), norfentanyl (NF), despropionylfentanyl (DPF), and hydroxynorfentanyl (OHNF) in human plasma and urine specimens has been developed and validated according to international guidelines. Analytes were extracted from 250-μL plasma or urine by liquid–liquid extraction. OHNF in urine affords a second extraction step and analysis with a different column. Calibration curves in plasma were linear from 0.05–10 ng/mL for F, 0.07–0.5 ng/mL for NF, 0.02–1.0 ng/ml for DPF, and 0.67–3.0 ng/mL for OHNF; in urine, from 0.09–10.0, 0.17–50, 0.08–1.0, and 1.0–5.0 ng/mL for F, NF, DPF, and OHNF, respectively. Analytical bias and intra- and inter-assay imprecision were within ±15 % of target, except for OHNF in plasma and DPF in urine at the respective lower quality control level. All analytes were stable in processed samples when stored for 24 h at room temperature. Recoveries and process efficiencies were above 82.9 and 75.1 % for all analytes in plasma and urine. The low level of DPF in plasma indicated with a matrix effect of 71.3 % moderate ion suppression, all other analytes in plasma and urine showed no matrix effects. The lower limit of quantification (LOQ) in plasma was 0.05, 0.07, 0.02 and 0.67 ng/mL for F, NF, DPF, and OHNF, respectively. In urine, the LOQ of F, NF, DPF, and OHNF were 0.09, 0.17, 0.08, and 1.28 ng/mL, respectively. This assay has been applied to human specimens collected during a clinical drug–drug interaction study.

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References

  1. Jenkins AJ (2007) In: Karch SB, Forrest R (eds) Drug abuse handbook, 2nd edn. Boca Raton, CRC Press

    Google Scholar 

  2. Clotz MA, Nahata MC (1991) Clinical uses of fentanyl, sufentanil, and alfentanil. Clin Pharm 10:581–593

    CAS  PubMed  Google Scholar 

  3. Baselt RC (2011) Disposition of toxic drugs and chemicals in man, 9th edn. Biomedical Publications, Foster City

    Google Scholar 

  4. Heishman SJ (2007) In: Karch SB, Forrest R (eds) Drug abuse handbook, 2nd edn. Boca Raton, CRC Press

    Google Scholar 

  5. Marquardt KA, Tharratt RS, Musallam NA (1995) Fentanyl remaining in a transdermal system following three days of continuous use. Ann Pharmacother 29:969–971

    CAS  PubMed  Google Scholar 

  6. Reeves MD, Ginifer CJ (2002) Fatal intravenous misuse of transdermal fentanyl. Med J Aust 177:552–553

    PubMed  Google Scholar 

  7. Dale E, Ashby F, Seelam K (2009) Report of a patient chewing fentanyl patches who was titrated onto methadone. BMJ Case Rep. doi:10.1136/bcr.01.2009.1454

    PubMed Central  PubMed  Google Scholar 

  8. Arvanitis ML, Satonik RC (2007) Transdermal fentanyl abuse and misuse. Am J Emerg Med 20:58–59

    Article  Google Scholar 

  9. Hull MJ, Juhascik M, Mazur F, Flomenbaum MA, Behonick GS (2007) Fatalities associated with fentanyl and co-administered cocaine or opiates. J Forensic Sci 52:1383–1388

    CAS  PubMed  Google Scholar 

  10. Labroo RB, Paine MF, Thummel KE, Kharasch ED (1997) Fentanyl metabolism by human hepatic and intestinal cytochrome P450 3A4: implications for interindividual variability in disposition, efficacy, and drug interactions. Drug Metab Dispos 25:1072–1080

    CAS  PubMed  Google Scholar 

  11. Schneider E, Brune K (1986) Opioid activity and distribution of fentanyl metabolites. Naunyn Schmiedebergs Arch Pharmacol 334:267–274

    Article  CAS  PubMed  Google Scholar 

  12. Peters FT, Paul LD, Mußhoff F, Aebi B, Auwärter V, Kraemer T, Skopp G (2009) Anhang B. Richtlinie der GTFCh zur Qualitätssicherung bei forensisch-toxikologischen Untersuchungen Anforderungen an die Validierung von Analysenmethoden. Toxichem Krimtech 76:185–208

    Google Scholar 

  13. Ziesenitz VC, König SK, Mahlke N, Jantos R, Skopp G, Weiss J, Haefeli WE, Mikus G (2013) Fentanyl pharmacokinetics is not dependent on hepatic uptake by organic anion-transporting polypeptide 1B1 in human beings. Basic Clin Pharmacol Toxicol 113:43–48

    Article  CAS  PubMed  Google Scholar 

  14. U.S. Department of Health and Human Services. Food and Drug Administration (2001) Guidance for industry. Bioanalytical method validation. http://www.fda.gov/cder/guidance/index.htm. Assessed 17 April 2014

  15. Matuszewski BK, Constanzer ML, Chavez-Eng CM (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 75:3019–3030

    Article  CAS  PubMed  Google Scholar 

  16. Day J, Slawson M, Lugo RA, Wilkins D (2003) Analysis of fentanyl and norfentanyl in human plasma by liquid chromatography-tandem mass spectrometry using electrospray ionization. J Anal Toxicol 27:513–516

    Article  CAS  PubMed  Google Scholar 

  17. Huynh NH, Tyrefors N, Ekman L, Johansson M (2005) Determination of fentanyl in human plasma and fentanyl and norfentanyl in human urine using LC-MS/MS. J Pharm Biomed Anal 37:1095–1100

    Article  CAS  PubMed  Google Scholar 

  18. Cooreman S, Deprez C, Martens F, Van Bocxlaer J, Croes K (2010) A comprehensive LC-MS-based quantitative analysis of fentanyl-like drugs in plasma and urine. J Sep Sci 33:2654–2662

    Article  CAS  PubMed  Google Scholar 

  19. Verplaetse R, Tytgat J (2010) Development and validation of a sensitive ultra performance liquid chromatography tandem mass spectrometry method for the analysis of fentanyl and its major metabolite norfentanyl in urine and whole blood in forensic context. J Chromatogr B 878:1987–1996

    Article  CAS  Google Scholar 

  20. Andresen H, Gullans A, Veselinovic M, Anders S, Schmoldt A, Iwersen-Bergmann S, Mueller A (2012) Fentanyl: toxic or therapeutic? Postmortem and ante mortem blood concentrations after transdermal fentanyl application. J Anal Toxicol 36:182–194

    Article  CAS  PubMed  Google Scholar 

  21. Scarth JP, Spencer HA, Timbers SE, Hudson SC, Hillyer LL (2010) The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance. Drug Test Anal 2:1–10

    CAS  PubMed  Google Scholar 

  22. Lu C, Jia JY, Gui YZ, Liu GY, Shi XJ, Li SJ, Yu C (2010) Development of a liquid chromatography-isotope dilution mass spectrometry method for quantification of fentanyl in human plasma. Biomed Chromatogr 24:711–716

    Article  CAS  PubMed  Google Scholar 

  23. Maruyama Y, Hosoya E (1969) Studies on the fate of fentanyl. Keio J Med 18:59–70

    Article  CAS  PubMed  Google Scholar 

  24. Feierman DE, Lasker JM (1996) Metabolism of fentanyl, a synthetic opioid analgesic, by human liver microsomes. Role of CYP3A4. Drug Metab Dispos 24:932–939

    CAS  PubMed  Google Scholar 

  25. Guitton J, Désage M, Alamercery S, Dutruch L, Dautraix S, Perdrix JP, Brazier JL (1997) Gas chromatographic-mass spectrometry and gas chromatographic-Fourier transform infrared spectroscopy assay for the simultaneous identification of fentanyl metabolites. J Chromatogr B Biomed Sci Appl 693:59–70

    Article  CAS  PubMed  Google Scholar 

  26. Tharp AM, Winecker RE, Winston DC (2004) Fatal intravenous fentanyl abuse: four cases involving extraction of fentanyl from transdermal patches. Am J Forensic Med Pathol 25:178–181

    Article  PubMed  Google Scholar 

  27. Poklis A, Backer R (2004) Urine concentrations of fentanyl and norfentanyl during application of Duragesic transdermal patches. J Anal Toxicol 28:422–425

    Article  CAS  PubMed  Google Scholar 

  28. Teske J, Weller JP, Larsch K, Tröger HD, Karst M (2007) Fatal outcome in a child after ingestion of a transdermal fentanyl patch. Int J Legal Med 121:147–151

    Article  PubMed  Google Scholar 

  29. Coopman V, Cordonnier J, Pien K, Van Varenbergh D (2007) LC-MS/MS analysis of fentanyl and norfentanyl in a fatality due to application of multiple Durogesic transdermal therapeutic systems. Forensic Sci Int 169:223–227

    Article  CAS  PubMed  Google Scholar 

  30. Musshoff F, Trafkowski J, Kuepper U, Madea B (2006) An automated and fully validated LC-MS/MS procedure for the simultaneous determination of 11 opioids used in palliative care, with 5 of their metabolites. J Mass Spectrom 41:633–640

    Article  CAS  PubMed  Google Scholar 

  31. Marier JF, Lor M, Potvin D, Dimarco M, Morelli G, Saedder EA (2006) Pharmacokinetics, tolerability, and performance of a novel matrix transdermal delivery system of fentanyl relative to the commercially available reservoir formulation in healthy subjects. J Clin Pharmacol 46:642–653

    Article  CAS  PubMed  Google Scholar 

  32. Tateishi T, Wood AJ, Guengerich FP, Wood M (1995) Biotransformation of tritiated fentanyl in human liver microsomes. Monitoring metabolism using phenylacetic acid and 2-phenylethanol. Biochem Pharmacol 50:1921–1924

    Article  CAS  PubMed  Google Scholar 

  33. Portier EJ, de Blok K, Butter JJ, van Boxtel CJ (1999) Simultaneous determination of fentanyl and midazolam using high-performance liquid chromatography with ultraviolet detection. J Chromatogr B Biomed Sci App 723:313–318

    Article  CAS  Google Scholar 

  34. Silverstein JH, Rieders MF, McMullin M, Schulman S, Zahl K (1993) An analysis of the duration of fentanyl and its metabolites in urine and saliva. Anesth Analg 76:618–621

    Article  CAS  PubMed  Google Scholar 

  35. Koch DE, Isaza R, Carpenter JW, Hunter RP (2004) Simultaneous extraction and quantitation of fentanyl and norfentanyl from primate plasma with LC/MS detection. J Pharm Biomed Anal 34:577–584

    Article  CAS  PubMed  Google Scholar 

  36. Peer CJ, Shakleya DM, Younis IR, Kraner JC, Callery PS (2007) Direct-injection mass spectrometric method for the rapid identification of fentanyl and norfentanyl in postmortem urine of six drug-overdose cases. J Anal Toxicol 31:515–521

    Article  CAS  PubMed  Google Scholar 

  37. Pihlainen K, Grigoras K, Franssila S, Ketola R, Kotiaho T, Kostiainen R (2005) Analysis of amphetamines and fentanyls by atmospheric pressure desorption/ionization on silicon mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry and its application to forensic analysis of drug seizures. J Mass Spectrom 40:539–545

    Article  CAS  PubMed  Google Scholar 

  38. Clavijo CF, Thomas JJ, Cromie M, Schniedewind B, Hoffman KL, Christians U, Galinkin JL (2011) A low blood volume LC-MS/MS assay for the quantification of fentanyl and its major metabolites norfentanyl and despropionyl fentanyl in children. J Sep Sci 34:3568–3577

    Article  CAS  PubMed  Google Scholar 

  39. Anderson DT, Muto JJ (2000) Duragesic transdermal patch: postmortem tissue distribution of fentanyl in 25 cases. J Anal Toxicol 24:627–634

    Article  CAS  PubMed  Google Scholar 

  40. Van Rooy HH, Vermeulen MP, Bovill JG (1981) The assay of fentanyl and its metabolites in plasma of patients using gas chromatography with alkali flame ionisation detection and gas chromatography–mass spectrometry. J Chromatogr 223:85–93

    Article  PubMed  Google Scholar 

  41. Strano-Rossi S, Alvarez I, Tabernero MJ, Cabarcos P, Fernández P, Bermejo AM (2011) Determination of fentanyl, metabolite and analogs in urine by GC/MS. J Appl Toxicol 31:649–654

    Article  CAS  PubMed  Google Scholar 

  42. Bansal R, Aranda JV (1996) High performance liquid chromatography microassay for the simultaneous determination of fentanyl and its major metabolites in biological samples. J Liquid Chromatogr Relat Technol 19:353–364

    Article  CAS  Google Scholar 

  43. Goromaru T, Matsuura H, Yoshimura N, Miyawaki T, Sameshima T, Miyao J, Furuta T, Baba S (1984) Identification and quantitative determination of fentanyl metabolites in patients by gas chromatography–mass spectrometry. Anesthesiol 61:73–77

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Legal Medicine and Traffic Medicine, University Hospital, Voss-Str. 2, 69115, Heidelberg, Germany

    Nina Sophia Mahlke & Gisela Skopp

  2. Department of Clinical Pharmacology and Pharmacoepidemiology, University Hospital, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

    Victoria Ziesenitz & Gerd Mikus

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  1. Nina Sophia Mahlke
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  2. Victoria Ziesenitz
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  3. Gerd Mikus
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  4. Gisela Skopp
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Correspondence to Gisela Skopp.

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Mahlke, N.S., Ziesenitz, V., Mikus, G. et al. Quantitative low-volume assay for simultaneous determination of fentanyl, norfentanyl, and minor metabolites in human plasma and urine by liquid chromatography—tandem mass spectrometry (LC-MS/MS). Int J Legal Med 128, 771–778 (2014). https://doi.org/10.1007/s00414-014-1040-y

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