Fabritius, Marie

Giroud, Christian

RERO DOC Digital Library

LETTER TO THE EDITORLetter to the Editor regarding “Simultaneous determinationof Δ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilutionliquid chromatography tandem mass spectrometry”Marie Fabritius & Christian GiroudReceived: 7 August 2012 /Accepted: 22 October 2012 /Published online: 13 November 2012# Springer-Verlag Berlin Heidelberg 2012Dear Sir,We read with great interest the article entitled “Simultaneousdetermination of Δ9-tetrahydrocannabinol and 11-nor-9-car-boxy-Δ9-tetrahydrocannabinol in oral fluid using isotope dilu-tion liquid chromatography tandem mass spectrometry” by Leeet al. [1]. The authors describe a fast and very sensitive methodto analyse dansyl derivatives ofΔ9-tetrahydrocannabinol (THC)and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH)in oral fluid with limits of quantification of 25 and 5 pg/mL,respectively. Generally, liquid chromatography (LC)–massspectrometry (MS) is preferred over gas chromatography–MSbecause it does not require prior derivatization and extensivesample clean-up, making this approach much less tedious andtime consuming. However, this common rule has several excep-tions. In this regard, there are some substances that do not showa signal sufficiently intense with the classic ion sources com-monly used in LC-MS. Typically, the phytocannabinoids be-long to this family. To overcome this problem, derivatizationcan be used to improveMS/MS detectability [2]. In this respect,Lee et al. reported the use of a chemical derivatization approachusing isotope dilution LC-MS/MS with electrospray ionizationin positive mode to detect and quantify THC and THCCOOH.Derivatization of the phenolic group was done with dansylchloride (DC) at basic pH (10.5) at 70 °C for 5 min.This method was tested in our laboratory with oral fluidsamples spiked with a broader range of cannabinoids: THC,THCCOOH, 11-hydroxy-THC (11-OH-THC), cannabinol,cannabidiol, the two glucuronide conjugates THC–glucuro-nide (THC-gluc) and THCCOOH–glucuronide (THCCOOH-gluc) and the plant precursor of THC, Δ9-tetrahydrocannabi-nolic acid A (THC-A). All except 11-OH-THC are known tobe present in oral fluid, a few at high concentrations (THC,THC-A, cannabidiol), others in only tiny amounts (mainly theTHC metabolites and their conjugates). Since this methodinvolving derivatization followed by LC-MS/MS analysishas been advocated with other biological matrices, its evalu-ation with a more expanded range of cannabinoids is evenmore warranted.Pure and dansylated standards were analysed first to deter-mine the ion transitions and the best MS/MS parameters.Then, drug-free oral fluid samples spiked at different concen-trations (5, 25, 50, 100, 250, 500 and 1,000 pg/mL) with thewhole range of cannabinoids were pretreated, derivatized andextracted according to the protocol developed by Lee et al. [1].These samples were also extracted according to our ownprocedure, which does not include a derivatization step [3].Briefly, our method is as follows. To 250 μL of oral fluid, weadded 10μL of an internal standard solution of 5 ng/mLTHC-d3, 11-OH-THC-d3 and THCCOOH-d9. Then, the volumewas made up to 1 mL with ammonium formate buffer(10 mM, pH 6.5) and the mixture was vortexed for 5 s. OasisHLB extraction columns (Waters, Baden, Switzerland) wereconditioned with 2 mL of methanol, 2 mL of deionized waterand 2 mL of ammonium formate buffer (10 mM, pH 6.5). Thesamples were loaded by gravity. Cannabinoids were elutedwith 3 mL of methanol. The eluates were evaporated todryness and reconstituted in 150 μL of ammonium formatebuffer (5 mM, pH 6.8)–acetonitrile (70:30, v/v). For bothprocedures, the LC parameters and the MS/MS equipmentwere the same. The separation was done on a Kinetex™ C18100-Å column (150 mm×2.1 mm inner diameter, 2.6 μm),(Phenomenex, Brechbühler, Echallens, Switzerland). Gradientelution was performed using a mixture of ammonium formatebuffer at 5 mM, pH 6.8 (solvent A) and acetonitrile (solvent B)M. Fabritius :C. Giroud (*)Forensic Toxicology and Chemistry Unit,University Center of Legal Medicine,Rue du Bugnon 21,1011 Lausanne, Switzerlande-mail: christian.giroud@chuv.chAnal Bioanal Chem (2013) 405:435–437DOI 10.1007/s00216-012-6515-4at a flow rate of 400 μL/min. The initial gradient conditionswere as follows: 30% solvent B, hold for 30 s, and then linearlyincrease to reach 90% solvent B at 7 min. The conditions using90 % solvent B were maintained for 2 min. Then, the gradientwas decreased to 30% solvent B over 30 s and held for 2.5min.Then, 10 μL of the sample was injected into the LC-MS/MSsystem. Analytes were detected using an Applied BiosystemsSCIEX API 5000 triple-quadrupole mass spectrometer with aTurboIonSpray interface. As described by Lee et al., the dansy-lated derivates were registered in positive electrospray ioniza-tion mode with the same multiple reaction monitoringtransitions. In our procedure, MS transitions were recordedusing a scheduled multiple reaction monitoring mode withthree separate time windows. The first time window (from thestart to 5.1 min) was operated in negative ionization mode,whereas the second (from 5.1 to 6.4 min) and the third (from6.4 to 7.5 min) windows were operated in the positive mode.THC-A, THCCOOH and the glucuronides were analysed inthe negative mode to achieve better sensitivity.Specific analyses for THC-A and glucuronide conjugateswere also conducted. Drug-free oral fluid samples were spikedwith THC-A, THC-gluc or THCCOOH-gluc at a concentra-tion of 100 ng/mL and were analysed with the method of Leeet al. As deuterated homologues of these compounds were notcommercially available, THC-d3 was used as the internalstandard for THC-gluc and THCCOOH-d9 was selected forTHC-A and THCCOOH-gluc. Calibration curves wereobtained with six calibration levels (0.1, 0.5, 5, 10, 25 and50 ng/mL) for THC, THCCOOH, cannabidiol, cannabinoland 11-OH-THC.Therefore, two different methods in the analysis of canna-binoids in oral fluid were used and compared. What differ-entiates these two methods is the dansylation step used by Leeet al. as it combines a simple liquid–liquid extraction, whereasour method does not require any derivatization even though itinvolves a longer purification on solid-phase cartridges. Thislatter step was necessary to extract the underivatized glucuro-nide conjugates from oral fluid. Derivatization with DC wasdone at basic pH (10.5) and 70 °C for 5 min. With the methoddeveloped in our laboratory, samples were analysed straight-forwardly at almost neutral pH (6.8) and without heating. Theother parts of the methods were similar and rely upon LC-MS/MS analysis with electrospray ionization and reverse-phasechromatographic separation using a C18 column.Our results confirm and extend those of Lee et al.: thesensitivity for THC and THCCOOH detection was improved,as was that for cannabinol, cannabidiol and 11-OH-THCdetection. In this respect, the limits of detection (LOD) werelower for these five cannabinoids using the method developedby Lee et al.: the LOD of THCCOOH, 11-OH-THC andcannabinol decreased from 100 to 5 pg/mL, the LOD ofTHC decreased from 50 to 25 pg/mL and the LOD of canna-bidiol was 50 pg/mL with DC derivatization, whereas it was300 pg/mL with our method. However, we failed to detect thedansylated THC-A, THC-gluc and THCCOOH-gluc. Thereasons of their non-detection were investigated, and one ofthe most important factors could be the derivatization condi-tions. It has been proved that THC-A degrades into THC athigh temperature [4]. Incubation of DC with cannabinoids at70 °C for several minutes could lead at least to partial thermalconversion of THC-A into THC. Furthermore, the basic con-ditions (obtained with 100 mM sodium bicarbonate buffer atpH 10.5) have been proved to be sufficient to hydrolyseTHCCOOH-gluc [5] and could explain the non-detection ofthe glucuronide conjugates.To test these hypotheses, the following experiments wereperformed to assess whether THC-A is converted into THCand whether the glucuronides are hydrolysed to theircorresponding free cannabinoids during the dansylation step.Drug-free oral fluid samples were spiked with THC-A, THC-gluc or THCCOOH-gluc at a concentration of 100 ng/mL.Samples were extracted and analysed according to the proto-col developed by Lee et al. [1]. For the oral fluid specimensspiked with THCCOOH-gluc, DC-THCCOOHwas measuredat a concentration of 20 ng/mL. Taking into account theirmolar differences, we found that more than 30 % ofTHCCOOH-gluc was hydrolysed to THCCOOH during thederivatization process. For samples spiked with THC-A, DC-THC was detected at a concentration of 9 ng/mL. This levelcorresponds to 10 % of the initial concentration of THC-A.For THC-gluc samples, only 2 ng/mL DC-THC was detected(4 %). As expected, our analyses revealed that dansylationresulted in significant hydrolysis of THCCOOH-gluc toTHCCOOH. For THC-A and THC-gluc, THC was detectedin low concentrations only. These results indicate that theglucuronide conjugates of THC and THC-A are partly con-verted into THC when dansylated. Therefore, quantitativeresults of dansylated cannabinoids must be interpreted withgreat caution because they represent a partial sum of free andconjugated metabolites of THC and THC-A. However, ifqualitative results only are required, the method using dansy-lation should be preferred because it has better sensitivity. Forblood, this method is not recommended because the concen-trations of free cannabinoids could be wrongly increased.More than 80 metabolites of THC have been identified [6].In 2007, Jung et al. [7], who studied the metabolism of THC-A,reported 12 THC-A metabolites. We suggest that THC-gluc,THCCOOH-gluc, THC-A and other metabolites are also de-graded, possibly into polymers, oxidized compounds and othercannabinoids that could not be detected with our analyticalmethod. Recently, Schwilke et al. [8] suggested that the ether–glucuronide bond in THC is stabler than the ester–glucuronidebond in THCCOOH. This difference could explain the greaterhydrolysis of THCCOOH-gluc compared with THC-gluc.In the experimental design of Lee et al. [1], oral fluidsamples of suspected cannabis users were analysed. They436 M. Fabritius, C. Giroudwere not concerned with the fact that after a user has smoked ajoint, a significant amount of THC-A can be found in oralfluid [3, 9]. In this respect, THC-Awas detected at a concen-tration of 1 ng/mL byMoore et al. [9] until 8 h after a user hadsmoked a joint. In another study, we detected THC-A until theend of the experiment, this being 4 h after the first puff of thejoint had been inhaled. THCCOOH-gluc in very low concen-trations was also detected.As a conclusion, to obtain accurate measurementsand a better comparison of results between laboratories,a method able to quantify separately each cannabinoidshould be preferred. Furthermore, our results show thatsome minor cannabinoids and their metabolites whichcould be used as biomarkers to substantiate use [10]and/or to estimate the last time of use cannot be mea-sured separately after derivatization with DC. Very re-cently, another approach was suggested by Coulter et al.[11], who advocate the use of picolylamine to derivatizethe carboxylic function of THCCOOH in order toachieve the requisite sensitivity.References1. Lee PD, Chang YJ, Lin KL, Chang YZ (2012) Simultaneous determi-nation ofΔ9-tetrahydrocannabinol and 11-nor-9-carboxy-Δ9-tetrahydro-cannabinol in oral fluid using isotope dilution liquid chromatographytandem mass spectrometry. Anal Bioanal Chem 402:851–8592. Santa T (2011) Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry. Biomed Chro-matogr 25:1–103. Fabritius M, Staub C, Mangin P, Giroud C (2012) Analysis ofcannabinoids in oral fluid by liquid chromatography-tandem massspectrometry. Forensic Toxicol. doi:10.1007/s11419-012-0168-z4. Dussy FE, Hamberg C, Luginbuhl M, Schwerzmann T, BriellmannTA (2005) Isolation of Δ9-THCA-A from hemp and analyticalaspects concerning the determination of Δ9-THC in cannabis prod-ucts. Forensic Sci Int 149:3–105. Kemp PM, Abukhalaf IK, Manno JE, Manno BR, Alford DD,McWilliams ME, Nixon FE, Fitzgerald MJ, Reeves RR, WoodMJ (1995) Cannabinoids in humans. II. The influence of threemethods of hydrolysis on the concentration of THC and twometabolites in urine. J Anal Toxicol 19:292–2986. Yamamoto I, Watanabe K, Narimatsu S, Yoshimura H (1995)Recent advances in the metabolism of cannabinoids. Int J BiochemCell Biol 27:741–7467. Jung J, Kempf J, Mahler H, Weinmann W (2007) Detection of Δ9-tetrahydrocannabinolic acid A in human urine and blood serum byLC-MS/MS. J Mass Spectrom 42:354–3608. Schwilke EW, Schwope DM, Karschner EL, Lowe RH,Darwin WD, Kelly DL, Goodwin RS, Gorelick DA, Huestis MA(2009) Δ9-Tetrahydrocannabinol (THC), 11-hydroxy-THC, and11-nor-9-carboxy-THC plasma pharmacokinetics during and aftercontinuous high-dose oral THC. Clin Chem 55:2180–21899. Moore C, Rana S, Coulter C (2007) Simultaneous identification of2-carboxy-tetrahydrocannabinol, tetrahydrocannabinol, cannabi-nol and cannabidiol in oral fluid. J Chromatogr B Anal TechnolBiomed Life Sci 852:459–46410. Cone EJ, Huestis MA (2007) Interpretation of oral fluid tests fordrugs of abuse. Ann N YAcad Sci 1098:51–10311. Coulter C, Garnier M, Moore C (2012) Analysis of tetrahydrocan-nabinol and its metabolite, 11-nor-Δ9-tetrahydrocannabinol-9-car-boxylic acid, in oral fluid using liquid chromatography withtandem mass spectrometry. J Anal Toxicol 36:413–417Letter to the Editor regarding the article of Lee et al. (2012) Anal Bioanal Chem 402:851–859 437

Letter to the Editor regarding "Simultaneous determination of ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry”

http://doc.rero.ch/record/322199/files/216_2012_Article_6515.pdf

Letter to the Editor regarding "Simultaneous determination of ∆9-tetrahydrocannabinol and 11-nor-9-carboxy-∆9-tetrahydrocannabinol in oral fluid using isotope dilution liquid chromatography tandem mass spectrometry”

Abstract

Similar works

Full text

Available Versions

RERO DOC Digital Library