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Forensic Toxicology

, Volume 31, Issue 1, pp 151–163 | Cite as

Analysis of cannabinoids in oral fluid by liquid chromatography–tandem mass spectrometry

  • Marie Fabritius
  • Christian Staub
  • Patrice Mangin
  • Christian Giroud
Short Communication

Abstract

A sensitive method was developed for quantifying a wide range of cannabinoids in oral fluid (OF) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). These cannabinoids include ∆9-tetrahydrocannabinol (THC), 11-hydroxy-∆9-tetrahydrocannabinol (11-OH-THC), 11-nor-9-carboxy-∆9-tetrahydrocannabinol (THCCOOH), cannabinol (CBN), cannabidiol (CBD), ∆9-tetrahydrocannabinolic acid A (THC-A), 11-nor-9-carboxy-∆9-tetrahydrocannabinol glucuronide (THCCOOH-gluc), and ∆9-tetrahydrocannabinol glucuronide (THC-gluc). Samples were collected using a Quantisal™ device. The advantages of performing a liquid–liquid extraction (LLE) of KCl-saturated OF using heptane/ethyl acetate versus a solid-phase extraction (SPE) using HLB copolymer columns were determined. Chromatographic separation was achieved in 11.5 min on a Kinetex™ column packed with 2.6-μm core–shell particles. Both positive (THC, 11-OH-THC, CBN, and CBD) and negative (THCCOOH, THC-gluc, THCCOOH-gluc, and THC-A) electrospray ionization modes were employed with multiple reaction monitoring using a high-end AB Sciex API 5000™ triple quadrupole LC–MS/MS system. Unlike SPE, LLE failed to extract THC-gluc and THCCOOH-gluc. However, the LLE method was more sensitive for the detection of THCCOOH than the SPE method, wherein the limit of detection (LOD) and limit of quantification (LOQ) decreased from 100 to 50 pg/ml and from 500 to 80 pg/ml, respectively. The two extraction methods were successfully applied to OF samples collected from volunteers before and after they smoked a homemade cannabis joint. High levels of THC were measured soon after smoking, in addition to significant amounts of THC-A. Other cannabinoids were found in low concentrations. Glucuronide conjugate levels were lower than the method’s LOD for most samples. Incubation studies suggest that glucuronides could be enzymatically degraded by glucuronidase prior to OF collection.

Keywords

Tetrahydrocannabinol Cannabinoids Oral fluid Saliva Tandem mass spectrometry Beta-glucuronidase 

Notes

Acknowledgments

The authors thank the Swiss National Science Foundation (FNS_320030_127507/1) and the Faculty of Biology and Medicine (interdisciplinary grant) at the University of Lausanne for their financial support.

References

  1. 1.
    Kelly E, Darke S, Ross J (2004) A review of drug use and driving: epidemiology, impairment, risk factors and risk perceptions. Drug Alcohol Rev 23(3):319–344. doi: 10.1080/09595230412331289482 PubMedCrossRefGoogle Scholar
  2. 2.
    Choo RE, Huestis MA (2004) Oral fluid as a diagnostic tool. Clin Chem Lab Med 42(11):1273–1287. doi: 10.1515/CCLM.2004.248 PubMedCrossRefGoogle Scholar
  3. 3.
    Molnar A, Lewis J, Doble P, Hansen G, Prolov T, Fu S (2011) A rapid and sensitive method for the identification of delta-9-tetrahydrocannabinol in oral fluid by liquid chromatography–tandem mass spectrometry. Forensic Sci Int. doi: 10.1016/j.forsciint.2011.01.045 PubMedGoogle Scholar
  4. 4.
    Moore C, Rana S, Coulter C, Day D, Vincent M, Soares J (2007) Detection of conjugated 11-nor-Delta9-tetrahydrocannabinol-9-carboxylic acid in oral fluid. J Anal Toxicol 31(4):187–194PubMedGoogle Scholar
  5. 5.
    Lee D, Milman G, Barnes AJ, Goodwin RS, Hirvonen J, Huestis MA (2011) Oral fluid cannabinoids in chronic, daily Cannabis smokers during sustained, monitored abstinence. Clin Chem 57(8):1127–1136. doi: 10.1373/clinchem.2011.164822 PubMedCrossRefGoogle Scholar
  6. 6.
    Milman G, Barnes AJ, Schwope DM, Schwilke EW, Darwin WD, Goodwin RS, Kelly DL, Gorelick DA, Huestis MA (2010) Disposition of cannabinoids in oral fluid after controlled around-the-clock oral THC administration. Clin Chem 56(8):1261–1269. doi: 10.1373/clinchem.2009.141853 PubMedCrossRefGoogle Scholar
  7. 7.
    Milman G, Schwope DM, Schwilke EW, Darwin WD, Kelly DL, Goodwin RS, Gorelick DA, Huestis MA (2011) Oral fluid and plasma cannabinoid ratios after around-the-clock controlled oral Delta(9)-tetrahydrocannabinol administration. Clin Chem 57(11):1597–1606. doi: 10.1373/clinchem.2011.169490 PubMedCrossRefGoogle Scholar
  8. 8.
    Moore C, Rana S, Coulter C (2007) Simultaneous identification of 2-carboxy-tetrahydrocannabinol, tetrahydrocannabinol, cannabinol and cannabidiol in oral fluid. J Chromatogr B Analyt Technol Biomed Life Sci 852(1–2):459–464. doi: 10.1016/j.jchromb.2007.02.016 PubMedGoogle Scholar
  9. 9.
    Coles R, Clements TT, Nelson GJ, McMillin GA, Urry FM (2005) Simultaneous analysis of the Delta9-THC metabolites 11-nor-9-carboxy-Delta9-THC and 11-hydroxy-Delta9-THC in meconium by GC–MS. J Anal Toxicol 29(6):522–527PubMedGoogle Scholar
  10. 10.
    ElSohly MA, Feng S (1998) Delta 9-THC metabolites in meconium: identification of 11-OH-delta 9-THC, 8 beta, 11-diOH-delta 9-THC, and 11-nor-delta 9-THC-9-COOH as major metabolites of delta 9-THC. J Anal Toxicol 22(4):329–335PubMedGoogle Scholar
  11. 11.
    Fabritius M, Staub C, Mangin P, Giroud C (2012) Distribution of free and conjugated cannabinoids in human bile samples. Forensic Sci Int. http://dx.doi.org/10.1016/j.forsciint.2012.08.013
  12. 12.
    Feng S, ElSohly 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(6):395–402PubMedGoogle Scholar
  13. 13.
    Gronewold A, Skopp G (2011) A preliminary investigation on the distribution of cannabinoids in man. Forensic Sci Int 210(1–3):e7–e11. doi: 10.1016/j.forsciint.2011.04.010 PubMedCrossRefGoogle Scholar
  14. 14.
    Lin DL, Lin RL (2005) Distribution of 11-nor-9-carboxy-Delta9-tetrahydrocannabinol in traffic fatality cases. J Anal Toxicol 29(1):58–61PubMedGoogle Scholar
  15. 15.
    Marchei E, Pellegrini M, Pacifici R, Palmi I, Lozano J, Garcia-Algar O, Pichini S (2006) Quantification of Delta9-tetrahydrocannabinol and its major metabolites in meconium by gas chromatographic-mass spectrometric assay: assay validation and preliminary results of the “meconium project”. Ther Drug Monit 28(5):700–706. doi: 10.1097/01.ftd.0000245380.95186.13
  16. 16.
    Moore C, Lewis D, Becker J, Leikin J (1996) The determination of 11-nor-delta 9-tetrahydrocannabinol-9-carboxylic acid (THCCOOH) in meconium. J Anal Toxicol 20(1):50–51PubMedGoogle Scholar
  17. 17.
    Papoutsis I, Nikolaou P, Dona A, Pistos C, Stefanidou M, Spiliopoulou C, Athanaselis S (2012) A validated GC–MS method for the determination of ∆9-tetrahydrocannabinol and 11-nor-∆9-tetrahydrocannabinol-9-carboxylic acid in bile samples. Forensic Toxicol 30(1):51–58. doi: 10.1007/s11419-011-0126-1 Google Scholar
  18. 18.
    Badawi N, Simonsen KW, Steentoft A, Bernhoft IM, Linnet K (2009) Simultaneous screening and quantification of 29 drugs of abuse in oral fluid by solid-phase extraction and ultraperformance LC–MS/MS. Clin Chem 55 (11):2004–2018. doi: 10.1373/clinchem.2008.122341 Google Scholar
  19. 19.
    Chi E, Cole J (2010) Detecting Marijuana in Saliva. Forensic Mag 7(5):17–20Google Scholar
  20. 20.
    Concheiro M, de Castro A, Quintela O, Cruz A, Lopez-Rivadulla M (2008) Determination of illicit and medicinal drugs and their metabolites in oral fluid and preserved oral fluid by liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 391(6):2329–2338. doi: 10.1007/s00216-008-2135-4 PubMedCrossRefGoogle Scholar
  21. 21.
    Day D, Kuntz DJ, Feldman M, Presley L (2006) Detection of THCA in oral fluid by GC–MS–MS. J Anal Toxicol 30(9):645–650PubMedGoogle Scholar
  22. 22.
    Laloup M, Ramirez Fernandez Mdel M, Wood M, De Boeck G, Henquet C, Maes V, Samyn N (2005) Quantitative analysis of delta9-tetrahydrocannabinol in preserved oral fluid by liquid chromatography–tandem mass spectrometry. J Chromatogr A 1082(1):15–24Google Scholar
  23. 23.
    Niedbala RS, Kardos KW, Fritch DF, Kardos S, Fries T, Waga J, Robb J, Cone EJ (2001) Detection of marijuana use by oral fluid and urine analysis following single-dose administration of smoked and oral marijuana. J Anal Toxicol 25(5):289–303PubMedGoogle Scholar
  24. 24.
    Oiestad EL, Johansen U, Christophersen AS (2007) Drug screening of preserved oral fluid by liquid chromatography–tandem mass spectrometry. Clin Chem 53(2):300–309. doi: 10.1373/clinchem.2006.074237 Google Scholar
  25. 25.
    Quintela O, Andrenyak DM, Hoggan AM, Crouch DJ (2007) A validated method for the detection of Delta 9-tetrahydrocannabinol and 11-nor-9-carboxy- Delta 9-tetrahydrocannabinol in oral fluid samples by liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. J Anal Toxicol 31(3):157–164PubMedGoogle Scholar
  26. 26.
    Sergi M, Bafile E, Compagnone D, Curini R, D’Ascenzo G, Romolo FS (2009) Multiclass analysis of illicit drugs in plasma and oral fluids by LC–MS/MS. Anal Bioanal Chem 393(2):709–718. doi: 10.1007/s00216-008-2456-3 PubMedCrossRefGoogle Scholar
  27. 27.
    Simoes SS, Ajenjo AC, Franco JM, Vieira DN, Dias MJ (2009) Liquid chromatography/tandem mass spectrometry for the qualitative and quantitative analysis of illicit drugs and medicines in preserved oral fluid. Rapid Commun Mass Spectrom 23(10):1451–1460. doi: 10.1002/rcm.4020 PubMedCrossRefGoogle Scholar
  28. 28.
    Simonin J, Salquebre G, Cirimele V, Kintz P (2007) Screening for illicit drugs in oral fluid by LC–MS/MS. Ann Toxicol Anal 19(2):141–150CrossRefGoogle Scholar
  29. 29.
    Gritti F, Guiochon G (2010) Performance of columns packed with the new shell Kinetex-C18 particles in gradient elution chromatography. J Chromatogr A 1217(10):1604–1615. doi: 10.1016/j.chroma.2010.01.008 PubMedCrossRefGoogle Scholar
  30. 30.
    Gritti F, Leonardis I, Shock D, Stevenson P, Shalliker A, Guiochon G (2010) Performance of columns packed with the new shell particles, Kinetex-C18. J Chromatogr A 1217(10):1589–1603. doi: 10.1016/j.chroma.2009.12.079 PubMedCrossRefGoogle Scholar
  31. 31.
    Moore C, Vincent M, Rana S, Coulter C, Agrawal A, Soares J (2006) Stability of Delta(9)-tetrahydrocannabinol (THC) in oral fluid using the Quantisal collection device. Forensic Sci Int 164(2–3):126–130. doi: 10.1016/j.forsciint.2005.12.011 PubMedCrossRefGoogle Scholar
  32. 32.
    Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Chiap P, Cohen N, Compagnon PA, Dewe W, Feinberg M, Lallier M, Laurentie M, Mercier N, Muzard G, Nivet C, Valat L, Rozet E (2007) Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal—part II. J Pharm Biomed Anal 45(1):70–81. doi: 10.1016/j.jpba.2007.06.013 PubMedCrossRefGoogle Scholar
  33. 33.
    Hubert P, Nguyen-Huu JJ, Boulanger B, Chapuzet E, Cohen N, Compagnon PA, Dewe W, Feinberg M, Laurentie M, Mercier N, Muzard G, Valat L, Rozet E (2007) Harmonization of strategies for the validation of quantitative analytical procedures. A SFSTP proposal—part III. J Pharm Biomed Anal 45(1):82–96. doi: 10.1016/j.jpba.2007.06.032 PubMedCrossRefGoogle Scholar
  34. 34.
    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(13):3019–3030PubMedCrossRefGoogle Scholar
  35. 35.
    Chauncey HH, Lionetti F, Winer RA, Lisanti VF (1954) Enzymes of human saliva. I. The determination, distribution, and origin of whole saliva enzymes. J Dent Res 33(3):321–334PubMedCrossRefGoogle Scholar
  36. 36.
    Ghadge MS, Raste AS (2006) Place of salivary beta glucuronidase activity in head and neck cancers. Indian J Clin Biochem 21(1):196–198PubMedCrossRefGoogle Scholar
  37. 37.
    Lamster IB, Kaufman E, Grbic JT, Winston LJ, Singer RE (2003) Beta-glucuronidase activity in saliva: relationship to clinical periodontal parameters. J Periodontol 74(3):353–359. doi: 10.1902/jop.2003.74.3.353 Google Scholar
  38. 38.
    Pil K, Verstraete A (2008) Current developments in drug testing in oral fluid. Ther Drug Monit 30(2):196–202. doi: 10.1097/FTD.0b013e318167d563 PubMedCrossRefGoogle Scholar
  39. 39.
    Hazekamp A, Peltenburg A, Verpoorte R, Giroud C (2005) Chromatographic and spectroscopic data of cannabinoids from Cannabis sativa L. J Liq Chromatogr Relat Technol 28(15):2361–2382CrossRefGoogle Scholar
  40. 40.
    Poklis JL, Thompson CC, Long KA, Lichtman AH, Poklis A (2010) Disposition of cannabichromene, cannabidiol, and Delta(9)-tetrahydrocannabinol and its metabolites in mouse brain following marijuana inhalation determined by high-performance liquid chromatography–tandem mass spectrometry. J Anal Toxicol 34(8):516–520PubMedGoogle Scholar
  41. 41.
    Barni-Comparini I, Ferri S, Centini F (1984) Cannabinoid level in the leaves as a tool for the early discrimination of cannabis chemiovariants. Forensic Sci Int 24(1):37–42PubMedCrossRefGoogle Scholar
  42. 42.
    Skopp G, Potsch L (2002) Stability of 11-nor-delta(9)-carboxy-tetrahydrocannabinol glucuronide in plasma and urine assessed by liquid chromatography–tandem mass spectrometry. Clin Chem 48(2):301–306PubMedGoogle Scholar
  43. 43.
    Humphrey SP, Williamson RT (2001) A review of saliva: normal composition, flow, and function. J Prosthet Dent 85(2):162–169. doi: 10.1067/mpr.2001.113778 PubMedCrossRefGoogle Scholar
  44. 44.
    Skopp G, Potsch L, Mauden M, Richter B (2002) Partition coefficient, blood to plasma ratio, protein binding and short-term stability of 11-nor-Delta(9)-carboxy tetrahydrocannabinol glucuronide. Forensic Sci Int 126(1):17–23PubMedCrossRefGoogle Scholar
  45. 45.
    Cone EJ, Huestis MA (2007) Interpretation of oral fluid tests for drugs of abuse. Ann N Y Acad Sci 1098:51–103. doi: 10.1196/annals.1384.037 PubMedCrossRefGoogle Scholar
  46. 46.
    Smink BE, Hofman BJ, Dijkhuizen A, Lusthof KJ, de Gier JJ, Egberts AC, Uges DR (2008) The concentration of oxazepam and oxazepam glucuronide in oral fluid, blood and serum after controlled administration of 15 and 30 mg oxazepam. Br J Clin Pharmacol 66(4):556–560. doi: 10.1111/j.1365-2125.2008.03252.x PubMedCrossRefGoogle Scholar
  47. 47.
    Watanabe K, Oguri K, Yoshimura H (1979) Synthesis of delta 8-tetrahydrocannabinol glucuronide and sulfate, and their metabolic disposition in rats. Chem Pharm Bull 27(12):3009–3014PubMedCrossRefGoogle Scholar
  48. 48.
    Scheidweiler KB, Desrosiers NA, Huestis MA (2012) Simultaneous quantification of free and glucuronidated cannabinoids in human urine by liquid chromatography tandem mass spectrometry. Clin Chim Acta Int J Clin Chem 413(23–24):1839–1847. doi: 10.1016/j.cca.2012.06.034 CrossRefGoogle Scholar
  49. 49.
    Wang RE, Tian L, Chang YH (2012) A homogeneous fluorescent sensor for human serum albumin. J Pharm Biomed Anal 63:165–169. doi: 10.1016/j.jpba.2011.12.035 PubMedCrossRefGoogle Scholar
  50. 50.
    Fanali G, Cao Y, Ascenzi P, Trezza V, Rubino T, Parolaro D, Fasano M (2011) Binding of delta9-tetrahydrocannabinol and diazepam to human serum albumin. IUBMB Life 63(6):446–451. doi: 10.1002/iub.466 PubMedCrossRefGoogle Scholar

Copyright information

© Japanese Association of Forensic Toxicology and Springer Japan 2012

Authors and Affiliations

  • Marie Fabritius
    • 1
  • Christian Staub
    • 1
  • Patrice Mangin
    • 2
  • Christian Giroud
    • 1
  1. 1.Forensic Toxicology and Chemistry UnitUniversity Center of Legal MedicineLausanneSwitzerland
  2. 2.Unit of Forensic MedicineUniversity Center of Legal MedicineLausanneSwitzerland

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