INTRODUCTION LC/MS LIBRARY CREATION - Waters

1 [ white paper ]C r e at i n g a n d U s i n g L C / Ms a n d L C / Ms / Ms L i b r a r i e sI N T R O D U C T I O NMass spectral libraries are a valuable tool for the rapid identifi-cation of unknown compounds in a sample. Mass spectra provide both compound molecular weight and fragmentation information. GC/MS spectral libraries containing EI (electron impact) spectra have been in widespread use for the last few decades and there are many comprehensive commercially available libraries such as the National Institute of Standards and Technology (NIST ) and Wiley1. T he use of LC/MS has become more widespread in the last 15 years due to the development of the electrospray ionization technique. Until recently, there were no commercially available spectral librar-ies for compounds not amenable to GC/MS.

2 In 2005, NIST added a LIBRARY of MS/MS spectra to the NIST 05 Mass Spectral LIBRARY (NIST/EPA/NIH) product2. T his LIBRARY was created on a variety of tandem quadrupole and ion trap mass spectrometers and contains 5,191 spectra of 1,943 compounds. However, spectra produced on different instrumentation types from a variety of instrument vendors can vary significantly in terms of ion abundance and spec-tral content3,4. T his paper describes the process of creating both LC/MS and LC/MS /MS libraries for use on Waters single and tandem quadru-pole instruments. Details on the use of LC/MS (/MS) libraries for sample screening for unknown compounds and the corroboration or exclusion of compounds tentatively identified from a previous analysis are also included.

3 T he use of Waters ChromaLynx soft-ware for automated LIBRARY searching is also C / M S L I B R A R Y C R E AT I O NT he use of in-source Collision Induced Dissociation (CID) allows the use of single quadrupole mass spectrometers to produce spectra that provide both precursor and product ion information. CID is carried out by increasing the cone voltage causing the analyte to fragment into its product ions. T here are two distinct approaches which can be taken when creat-ing LC/MS libraries. T he first is to create one spectral entry per compound. In this case, the cone voltage is optimized to create spectra which contain characteristic fragmentation, while maintain-ing the integrity of the molecular ion at 10-25% to insure accurate, although tentative identification.

4 Spectra containing two or more distinct product ions are preferable. Figure 1 shows a spectrum for the compound nicotine created using this approach. Note the molecular ion, 163 m/z, is approximately 15% of the intensity of the most intense fragment, 130 m/z and there are six distinct product ions in the 1. LC/MS spectrum of nicotine.[ white paper ][ white paper ]T he second approach is to create multiple spectra per compound by collecting spectra at various cone voltages. For example, in the Waters Toxicology LIBRARY , there are spectra for the compound nicotine at six cone voltages: 15, 30, 45, 60, 75 and 90 V. T he benefit of this approach is that it results in multiple matches for the same compound at different degrees of fragmentation, providing additional confidence in the LIBRARY match.

5 Figure 2 shows spectra of the compound nicotine from 15 to 90 V. T he spectrum at 15 V contains only the molecular ion, the spectra at 30 and 45 V also contain the molecular ion and some product ions. T he molecular ion is not visible in the spectra collected at 60 V or above, and the product ion distribution has changed such that the product ion at 132 m/z is not present in the spectra collected at 75 or 90 he recommended procedure for the addition of spectra to a LIBRARY is to inject a high concentration of the compound (10 25 ng) on column using a generic gradient and the mobile phase additives which will be used for future sample screening. For small mol-ecule applications, full scan spectra should be collected from 70 800 m/z at the optimum cone voltage or at various cone voltages.

6 W hen the acquisition is complete, the spectrum can be extracted from the chromatogram by combining the spectra under the chromatographic peak and doing a background subtract. Retention time and cone voltage information should be added to the LIBRARY entry at this time for future use with automated LIBRARY C / M S / M S L I B R A R Y C R E AT I O NLC/MS/MS libraries are created in a similar manner to LC/MS librar-ies, but fragmentation is carried out in the collision cell rather than in the source. T here are also two approaches which can be taken when creating LC/MS /MS libraries. In both cases, the spectra should be collected at the cone voltage which gives the greatest response for the molecular ion. T he first method is to create spectra with at least two product ions while maintaining the integrity of the molecular ion at 10 25% by optimizing the collision energy.

7 T he spectrum for the compound nicotine obtained using the approach is shown in Figure he second technique is to add multiple spectra for the same com-pound at various collision energies, for example, 10, 25 and 40 V. T he benefit of this approach is the provision of LIBRARY matches at various degrees of fragmentation, providing additional confidence in the LIBRARY match.[ white paper ]Figure 2. LC/MS spectra of nicotine at various cone 3. LC/MS /MS product ion spectrum of nicotine.[ white paper ][ white paper ]S A M P L E S C R E E N I N G U S I N G L C / M S L I B R A R I E SSample screening by LC/MS is typically carried out using MS scan functions at various cone voltages. An appropriate MS tune file, with the resolution and ion energy settings adjusted to achieve unit mass resolution, should be used.

8 Ideally, the mobile phase constituents should be the same as those used in the CREATION of the LIBRARY so that the proper screening data will be searched against. T he LC column for the sample screening should also be the same chemistry, particle size and dimensions as the column used to create the LIBRARY if retention time information is to be hen data collection for the sample screening has been completed, LIBRARY searching can be carried out either manually or automati-cally using ChromaLynx software. If manual LIBRARY searching is carried out, the chromatogram for each of the scan functions should be examined individually and the spectra for every peak on every chromatogram should be searched against the LIBRARY . Figure 4 shows the seven scan chromatograms obtained from the sample screening of a water sample spiked with five compounds.

9 For this example, it would take approximately 30 minutes to manually search these T O M AT E D L I B R A RY S E A R C H I N G : CH RO M ALY NxManual searching of spectral libraries is a time consuming task and requires a knowledgeable user. Waters ChromaLynx software automates the processing of LC/MS and LC/MS /MS data. T he ChromaLynx chromatographic deconvolution algorithm allows effi-cient peak location, peak detection, and generation of clean mass spectra. T he deconvoluted spectra can be automatically searched against libraries and the results displayed in a customizable browser. ChromaLynx uses the NIST based search algorithm for LIBRARY searching. Spectral match factors are calculated and can be used to determine how good the match is between the LIBRARY spectrum and compound spectrum from a particular sample.

10 Match factors range from 1000 for a perfect match, to zero for spectra having no common peaks. If multiple spectra for the same compound exist in the LIBRARY and several matches are found, the average fit factor is also calculated. In general, a compound with a match factor >700 is considered a possible candidate. Results can be filtered against specified cone voltages or cone voltage ranges, ionization polarity, and retention time limits. T he candidate list of identified compounds, chromatograms, component spectra, LIBRARY spectra and LIBRARY match factors are all viewable in the ChromaLynx Identify browser. Figure 5 shows a ChromaLynx dataset for LC/MS screening analysis of a water sample spiked with nicotine, hexazinone, prometryn, methiocarb and diazinon. T he match factors for all five compounds identified are >700, indicat-ing close matches with the LIBRARY 4.