大麻中未知物成分检测方案

LECO Corporation·3000 Lakeview Avenue·St. Joseph， MI 49085·Phone：800-292-6141·Fax： 269-982-8977info@leco.com·www.leco.com·ISO-9001：2008·No. FM 24045·LECO is a registered trademark of LECO Corporation.7/11-REV0Form No. 203-821-404C 2011 LECO Corporation Characterization of a Cannabinomimetic Compound inSynthetic Cannabis by Gas Chromatography-HighResolution Time of Flight Mass Spectrometry (GC-HRT) LECO Corporation； Saint Joseph， Michigan USA Keywords： Gas Chromatography； High Resolution Mass Spectrometry； Forensics； Synthetic Cannabis 1. Introduction Synthetic marijuana-type products have garnered a greatdeal of media attention over the last several months.Countless products often referred to as "incense" or "spice"are being sold at smoke shops， convenience stores， andonline sites. These products are labeled "not for humanconsumption"， but have been reported to have effectssimilar to cannabis when smoked. These reports haveprompted testing in crime laboratories across the country.Some of the products tested have been confirmed tocontain synthetic cannabinoids. The identification of thesecompounds by GC-MS can be challenging as most of themare not present in commercially available mass spectralsearch libraries. This application note shows the analysis of a commerciallyavailable "incense" product that has since been removedfrom store shelves. The product， "Mr. Smiley"， began toreceive a great deal of media attention in the MidwesternUnited States since it was reported to induce a high similarto that of cannabis when smoked. The web communityclaimed： thatittthepproduct contained： only naturalingredients， suchasisDamiana and MulleinnLeaf.Authorities were quick to disagree， stating that they hadreason to believe there were illegal compounds present. Prior to authorities banning the sale of this product， it waspurchased from a convenience store near Indianapolis， IN.The sample was taken to the LECO Life Science &Chemical Analysis Centre in St. Joseph， Ml and wassubsequently analyzed in an attempt to identify thepresence of compounds that may be contributing to the"cannabis-like" highs. The sample was analyzed by gaschromatography time-of-flight mass spectrometry (GC-TOFMS). The most abundant analyte detected in the"incense" extract by GC-TOFMS was not present in thecommercially available mass spectral search database(NIST 2008) which was used for spectral searching. Bothelectron ionization (El) and chemical ionization (Cl)experiments were conducted to verify the molecular ion.The LECO Pegasus GC-HRT (High Resolution TOFMS)utilizing Folded Flight PathTM (FFPM) technology was usedtoacquireaccurate massdataa for theeunknowncompound. The data were used to perform a formulasearch which ultimately lead to the identification of aknown cannabinoid receptor agonist. 2. Experimental Conditions Samples A 1 gram sample of the "Mr.Smiley" product was weighedinto a 20 mL scintillation vial. A10 mL aliquot of ethyl acetate was added and the sample was sonicated for 10 minutes. A portion of theethyl acetate extract was transferred to GC autosamplervials for analysis. Samples were initially analyzed byGC-TOFMS on the LECO TruTOF@ HT-TOFMS. Furthercharacterization of an unknown analyte was performedusing the LECO Pegasus GC-HRT high resolution massspectrometer. The conditions for eachsystem areincluded in the experimental section. Experimental GC-TOFMS (LECO TruTOF) GC： Agilent 7890 Injection： 1 uL， split 50：1@ 275℃ Carrier Gas： He @ 1.5 mL/min Column： Rtx-5， 10 m x0.18 mm x 0.2 um (Restek Corporation，Bellefonte， PA) GC Oven： 60℃ to 330℃ @ 50℃/min MS Transfer Line： 330℃ MS： LECO TruTOF HT Acquisition Delay： 60 seconds Spectral Acquisition Rate： 20 spectra/s Mass Range： 40-550 m/z (El)， 50-550 m/z (CI) Electron Energy： -70ev Source Temperature： 250℃(EI)，200℃(CI) CI Reagent Gas： Methane Instrument Control and Data Review： ChromaTOF Figure 1. LECO TruTOF HT (GC-TOFMS). The conditions used for the Pegasus GC-HRT analysis ofthe ethyl acetate extract of the "Mr. Smiley" product areshown below. GC-HRTOFMS (LECO Pegasus GC-HRT) GC： Agilent 7890 Injection： 1uL， split 50：1@ 275C Carrier Gas： He @ 1.5 mL/min Column： Rtx-1614，20mx0.25 mm x0.1 um (Restek Corporation， Bellefonte， PA) GC Oven： 60℃ to 330℃ @ 50C/minMS Transfer Line： 330℃ MS： LECO Pegasus GC-HRT (Electron Impact lonization) Acquisition Delay： 60 seconds Resolution Mode： High， 20m flight path Spectral Acquisition Mass Range： Rate： 20 spectra/s40-550 m/z Electron Energy： -70eV Source Temperature： 250°C Instrument Control and Data Review： ChromaTOF-HRTTM Figure 2. LECO Pegasus GC-HRT with Folded Flight Path (FFP) Technology. Figure 3. The image above provides a visual depiction of the various modesof operation made possible utilizing LECO's Folded Flight Path (FFP)technology. The Pegasus GC-HRT can operate in Ultra High (50，000 FWHM)， High(25，000 FWHM)， or Nominal (1，000 FWHM) mass resolution modes. GC-TOFMS Results Figure 4. Analytical lon Chromatogram (AIC) for ethyl acetate extract of "Mr.Smiley" product. Figure 4 above shows the analytical ion chromatogram(AIC) from the GC-TOFMS analysis of an ethyl acetateextract of the "Mr. Smiley" product on the LECO TruTOF.Several analytes were identified in the ethyl acetateextract， but the most abundant analyte detected was notpresent in the commercially available library (NIST 2008)which was used to search the spectra of the detectedcompounds. The Peak True (deconvoluted) Electron Impactionization (El) mass spectrum for the unknown analyte isshown below in Figure 5. Though it appears that themolecular ion is shown at m/z 341， additional data wasgenerated in Cl mode using methane reagent gas forverification. The methane Cl spectrum for the unknownanalyte is shown in Figure 6. The spectrum contains aprotonated molecular ion at m/z 342 as well as typicalM+29 and M+41 adducts which support the molecular ionassignment of m/z 341. Further data were generatedusing the LECO Pegasus GC-HRT to provide an accuratemass based formula assignment. Those data are shown inthe Pegasus GC-HRT results section of this applicationnote. Peak True - sample"Mr. Smiley Extract：1"， peak 51， at 1147.9 s Figure 5. El mass spectrum for unknown analyte in "Mr. Smiley" product. Figure 6. Methane Cl mass spectrum for unknown analyte in "Mr. Smiley"product. GC-HRT Results The figure below(Figure 7)shows a total ionchromatogram (TIC) and extracted ion chromatogram form/z 341.177 which is the observed accurate mass for themolecular ion of the unknown compound previouslydetected on the TruTOF unit mass resolution instrument.As stated earlier， this analyte was not present in thecommercially available mass spectral search databases. Asshown below in Figure 8， the observed accurate mass forthe molecular ion of the unknown was 341.17675. AChromaTOF-HRT formula search for the observed accuratemass suggested C24H23NO as the #1 hit with a massaccuracy of -1.95 PPM relative to the theoretical exactmass for this formula. The resulting formula (C24H23NO)was consistent with a literature search which lead to theidentification of this analyteas 1-pentyl-3-(1-naphthoyl)indole， or JWH-018， one of the most commonsynthetic cannabinoids used in "herbal incense" products.This compound was originally synthesized by Dr. John W.Huffman at Clemson University for use as a compound tostudy brain cannabinoid receptors. Figure 7. TIC "Mr. Smiley" and XIC for unidentified analyte. 5. Conclusions The Pegasus GC-HRT proved to be a valuable tool forthe detection and identification of a syntheticcannabinoid in an herbal spice sample. The accuratemass data reported to <2 PPM provided the ability tosuccessfully identify an unknown compound that wasnot present in the commercially available mass spectralsearch databases. The synthetic cannabis industry isgrowing rapidly， apparent by the sheer number ofwebsites selling herbal spice samples. Legislation isbeing passed in many states to outlaw the distributionof these products. The problem is that there are literallyhundreds of possible synthetic cannabinoid compounds，most of which are not present in the commercial massspectral libraries. The use of a high resolution massspectrometer capable of delivering the mass accuracyneeded to facilitate unknown identification will beparamount to the ability of crime labs to effectivelycharacterize these new and emerging compounds. Peak True -sample"Mr. Smiley Extract"， Peak 123， at 315.666 s Figure 8. HRT Mass spectrum and accurate mass formula search results.