21


materials (SRM) that were spiked with unknown components from the mixtures.


After participants reported their identifi cations for Phase 1, the lists of spiked components were disclosed and the participants were allowed to re-evaluate their initial fi ndings and submit those results as well. The overarching goals of this study are to answer the following questions:


What percentage of spiked standard mixtures are correctly identifi ed?


Which method and processing performs best overall? Does the complexity of the mixture/matrix impact performance?


What chemical space is being covered by each method?


What can be done to expand coverage?


What unintended components (impurities, reaction, or degradation products, etc.) are in the standard mixtures?


In environmental samples, which chemicals are detected by multiple methods and are these consistencies observed in the SRM data?


The goal of this article is to report on the fi rst phase of the trial (identifi cation of chemicals within the ten mixtures) in our laboratory.


Experimental


Our strategy for blind identifi cation (Phase 1) was to use both electron ionisation (EI) high resolution accurate mass time-of- fl ight mass spectrometry (HRAM TOF-MS) and comprehensive multidimensional gas chromatography (GCxGC). To further aid in identifi cation or confi rmation of molecular ions we performed chemical ionisation (CI) experiments with the same chromatographic parameters. The data were processed according to the following steps: (1) High Resolution Deconvolution®


(HRD®


overlap in one, two, or three dimensions simultaneously. HRD extracts these 3D features from each other and from the chemical background using a proprietary multi-dimensional deconvolution engine called Fast Accurate Robust Adaptive Deconvolution (FARAD). FARAD operates automatically without requiring preliminary knowledge of chromatographic peak shapes or an estimate of the number of coeluting components.


Experimental method parameters are described in Table 1.


Identifi cation Scoring System description


below) was applied to fi nd mathematically relevant peaks within each sample, (2) Each deconvoluted spectrum was searched against both the NIST 2017 (mainlib and replib) and a recent Cayman (v04062017) spectral library, (3) retention index (RI) values were calculated based on separate injections of a standard n-alkane mix,(4) mass accuracies of every signifi cant m/z in each deconvolved spectrum were calculated based on formulae from the top scoring library hit. The chemical ionisation data was used solely to confi rm molecular ions when needed.


The HRD algorithm treats the GCxGC-MS data as a 3-dimensional data cube populated with features or analytes which often


Hand in hand with the experimental approach, there must be a methodology to quantify confi dence in the chemical identifi cation. Since the system used (GCxGC HRAM TOF-MS) generates a signifi cant amount of characterising information as described above, we proposed a system which effectively assigns a confi dence level for each identifi ed compound. The proposal was loosely based on the systematic identifi cation methodologies proposed by others, but adapted for a GCxGC HRAM TOF-MS system. To our knowledge, this is the fi rst time this type of system has been proposed for this type of data.


Level A (Match) Every condition below must be true.


1. The forward spectral similarity score must be greater than or equal to 700 on a scale of 0-1000. Applies to EI spectra only.


2. All deconvoluted fragment m/z(s) with an abundance equal to or greater than 30% of the base m/z must have a mathematically possible formula within 5ppm based on the molecular formula of the library matched spectrum and standard valence rules.


3. A molecular ion must exist within the


deconvolved spectra. It must also be within 5ppm of the expected m/z based on the matched molecular formula. If no molecular ion is present by EI, supplemental CI data can be used to provide evidence of a molecular ion.


4. The chromatographic peak’s retention index value (Kovats n-alkane scale) must be within 50 RI units of the matched library spectrum’s median experimental Semi- Standard Non-Polar RI value. If the library match does not report this value, then the RI evaluation is ignored.


5. The reviewing analyst must be confi dent with the peak deconvolution and identifi cation.


Level B (Suspected Good Match)


1. Level B Suspected Match does not meet one (or more) of the Level A criteria.


2. The analyst feels there is not enough conclusive evidence to designate the peak as a Level A match, e.g. Similarity score is high but there are a number of similar compounds with identical or nearly identical spectra (isomers).


Note there were many other intense deconvoluted peaks in each and every sample but only those matching the above criteria were submitted to the EPA for review as part of the initial reporting (see Figure 1). Indeed, the advantages of the GCxGC and the highly mass accurate time-of-fl ight data are confi rmed in Figure 1. As observed in Figure 1, the total A+B level matches was nearly always greater than the number of components expected in each sample. This would suggest the existence of unintended reaction products as described earlier. Further, the number of A level matches was also greater than the B level in nearly every sample. However, this quality measure does not indicate the accuracy of the matches. In this study, accuracy is defi ned by the ability


Table 1. GCxGC and MS method parameters for all samples.


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