Application of Structure-Based LC/MS Database Management ...

1 Application of Structure-Based LC/MS DatabaseManagement for forensic AnalysisCozette M. Cuppett and Michael P. BaloghWaters Corporation, Milford, MA USAA ntony Williams, Vitaly Lashin and Ilya TroiskyAdvanced Chemistry Development, Toronto, Ontario, Canada4 Chromatography Instrument: Waters 2690 Alliance System underMassLynx control Solvents: A: H2O, B: MeCN 200ml/min 30-40% B in 20 min 35 min total run time mm XTerraMS C18, 35 C column temp4 PDA Instrument: Waters Model 996 Photodiode Array Detector 210-400 nm4 Samples Standards diluted in 30/70 MeCN/H2O4 Mass Spectrometry Instrument.

2 Waters ZQ 4000under MassLynx control ESI+, centroid Scan 100-600 amu Scan time: sec Interscan delay: sec Tune page settings Capillary voltage: kV Cone voltage: 30, 50, 70 V Extractor: 6 V RF lens: V Ion energy: LM & HM resolution: Source temperature: 150 C Desolvation temperature: 300 C Cone gas: 150 l/hr Desolvation gas: 600 l/hrLC/UV/MS ConditionsOverview4 Application of LC/UV/MS to the analysis oftoxicologically relevant samples4 Demonstration of several ways in which structuralinformation may assist in sample identification Use of a Structure-Based data Management system for manualinterpretation and analysis of sample components Software tools to interpret difficult analysis cases ( , highnoise backgrounds, manual and automated fragmentassignment tools)

3 Introduction4In many cases, an automated search method can be used forsample identification Automated search results still must be manually reviewed Difficult or low concentration samples require significant manual interpretation Commercially available databases are only available with nominal massaccuracy4 Integration of structures with spectral information can provideimproved data Management and enhance library searchingcapabilities Particularly useful for manual interpretation and analysis of results Provides structure and

4 Substructure search capabilities for data mining Allows Management of multiple forms of MS data with a single chemicalstructure EI, ESI, CI, mg Waters Oasis HLB CartridgeCondition1 ml MeOH and 1 ml H2 OLoad1 ml spiked serum or Hanks solution(10:1 dilution of standards with serum or Hanks )Wash1 ml 5% MeOH in H2 OElute1 ml MeOHEvaporate and ReconstituteRoom temp in Savant Speed Vac200ml mobile phase (30% MeCN in H2O)Sample Preparation Procedurefor Spiked SamplesCopyright Waters Corporation 2001 CuppettMonoisotopic m/zNNNNClClCH3 NNOCH3 ClNNN+O-OOCH3 FNNHOOHClClHNNHOOHClNNOOHCH3 ClNN+NHO-OOClExport Data from MassLynx into ACDE xport spectrum directly fromMassLynx spectrum review windowinto ACD/ SpecManager MSmoduleThe ACD spectrum windowprovides further opportunities toprocess dataOnce the spectrum is imported intoACD/SpecManager.

5 A structure isintegrated with the spectra usingthe integrated drawing can used to removelow intensity background the spectrum window, thedata is entered into a searchabledatabase along with theappropriate user Spectrum to DatabaseCreating a UV and MS Database4 Individual benzodiazepine standards wereanalyzed under the conditions given previously All MS data (3 cone voltages used to enhancedescriptive fragmentation for each compound) wereentered into one comprehensive Database using theprocedure presented below Following a similar procedure, UV data were alsoincorporated into the database4 Chemical structures were incorporated into boththe UV and MS library entries using the integrateddrawing packageManually Searching Datain the Database4 Ways to search data By spectra By structure By substructure By individual fragmentpeaks By user-defined text By single DB or multipleDBDatabaseSearchingCapabilitiesO nce the Database is populated.

6 There are several ways in which the data may beaccessed and UV and MS LibrariesApplication of Structure-Based LC/MS DatabaseManagement for forensic AnalysisCopyright Waters Corporation 2001 CuppettManual Interpretation of LowConcentration SamplesLittle Information Available from TICsat Low ion current chromatograms (TIC) produced in the analysis of spiked serum(25 pg of each benzodiazepine on column). Little information is gained fromTICs. By visualizing data with the Map function in MassLynx, it is possible to pickpeaks out of the VTIC50 VTIC70 VTICPDATIC25 pg on columnPulling Peaks Out of the NoiseUsing the Map function in MassLynx, it is possible to manually scroll throughindividual m/z ratios collected during a scan run for each function.

7 Through thisfunctionality, peaks may be picked out that are not evident in the TICs. Forexample, with a cone voltage at 70 V, a peak at 193 m/z was found around of Hidden Peaks Ion Chromatogram193 m/zCombined Mass SpectrumOnce individual masses are distinguished from the noise, extracted ionchromatograms can be created and used to generate mass spectra. The massspectra can then be searched against MS libraries for identification. However, ifthe noise is high, extraneous peaks may diminish the utility of SearchesIn cases where manual interrogation of data is necessary, software that facilitatesthe process can translate into great time savings.

8 ACD software provides theability to easily ascertain the relative intensities of all the peaks and performsearches against MS libraries of selected peaks based on their relative SearchParametersIon Search ResultsMatch Found in DatabaseUnknown SpectrumIon Search Result in LibraryComparison of Unknown Spectrum with Match Found in LibraryThe manual selection of peaks and use of relative intensities for searches allowsotherwise unmatchable spectra to be found in the Database . Manual comparison ofthe library spectrum from the Database and the unknown spectrum is then used to assistin the determination of the identity of the Waters Corporation 2001 CuppettApplication of Structure-Based LC/MS DatabaseManagement for forensic AnalysisExport Data from MassLynx into ACD220240260280300320340360380400nm0100% Benzo_SPE_Serum_25ng_051701_2 596 ( ) Cm (593:598)4: Diode 667 ( ) Cm (665:673-(636:648+686:693))3.

9 Scan ES+ 335346394347359365377381 PDA and MS Data from MassLynxData is exported out of MassLynx intoACD/SpecManager. Thresholding isused to remove extraneous backgroundpeaks from the MS use of multi-spectral data can provide multiple reference points from which to verifycompound identity. This can lead to increased confidence in results. Here we showhow multi-spectral data can be used with the Structure-Based run: 25 ng of each benzodiazepine standard on columnSearching the Database with Multi-Spectral DataSearch Using MS DataNumber of library matchesPDA matches reflected in List AMS matches reflected in List BSimilarity betweenunknown spectrumand library spectrumSearch Using PDA DataIntersect List A and List B toconfirm matching hits fromthe PDA and MS searches1 overlapping hit from each library.

10 Flunitrazepam at 70 VDataMulti-SpectralUsingAutofragmentatio nThe identified MS spectrum may be further evaluated using ACD/SpecManager sAutofragmentation algorithm. Using a proposed structure, the software fragmentsthe molecule and compares the fragments to the fragmentPercentage of spectrumassigned fragmentsApplication of Structure-Based LC/MS DatabaseManagement for forensic AnalysisCopyright Waters Corporation 2001 CuppettSummary4 Structure-Based LC/MS Database can provide usefultools for the analysis of unknown samples Peak searching for samples containing high backgroundlevels against commercial or in-house databases Library searching of both UV and MS spectra.