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Using ROC Curves to Optimize Discovery-Based Software with Comprehensive Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometry Brooke C. Reaser, Bob W. Wright, and Robert E. Synovec Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04991 • Publication Date (Web): 16 Feb 2017 Downloaded from http://pubs.acs.org on February 22, 2017

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Analytical Chemistry

Using ROC Curves to Optimize Discovery-Based Software with Comprehensive TwoDimensional Gas Chromatography with Time-of-Flight Mass Spectrometry

Brooke C. Reaser,1 Bob W. Wright,2 Robert E. Synovec*,1 1

Department of Chemistry, Box 351700, University of Washington, Seattle, Washington, 98198, U.S.A. 2

Pacific Northwest National Laboratory, Battelle Boulevard, P.O. Box 999, Richland, Washington, 99352, U.S.A.

Revised version of manuscript AC-2016-049919, prepared for consideration to publish in Analytical Chemistry

February 10, 2017

* CORRESPONDING AUTHOR: Tel: +1-206-685-2328; fax: +1-206-685-8665 EMAIL: [email protected]

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ABSTRACT We report a quantitative approach to optimize implementation of discovery-based

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software for comprehensive two-dimensional gas chromatography coupled with time-of-flight

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mass spectrometry (GC × GC – TOFMS). The software performs a tile-based Fisher ratio (F-

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ratio) analysis, and facilitates a supervised non-targeted analysis based upon the experimental

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design to aid in the discovery of analytes with statistically different variances between sample

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classes. The quantitative approach for software optimization uses receiver operating

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characteristic (ROC) curves. The area under the curve (AUC) for each ROC curve serves as a

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quantitative metric to optimize two key algorithm parameters: the signal-to-noise ratio (S/N)

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threshold of the data prior to calculating F-ratios at each m/z mass channel, and the number of

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these F-ratios per m/z used to calculate the average F-ratio of a tile. A total of 25 combinations

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of S/N threshold by number of m/z were studied. Fifty analytes were spiked into a diesel fuel at

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two concentration levels to produce two sample classes that should in principle produce 50

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positive instances in the ROC curves. The “sweet spot” for F-ratio analysis was determined to be

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a S/N threshold of 10 coupled with a maximum of the 10 most chemically selective m/z

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(requiring a minimum of 3 m/z), corresponding to a ~ 21% improvement in the discrimination of

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true positives relative to prior studies. This equates to an additional 9 true positives being

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discovered at a false positive probability of 0.2, and 5 additional true positives being found

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overall. Furthermore, optimization of these software parameters did not depend upon a priori

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determination of the statistically correct number of positive instances in the sample classes. The

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AUC metric appears to be suitable for the evaluation of all data analysis methods that utilize the

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proper experimental design.

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Analytical Chemistry

INTRODUCTION Comprehensive two-dimensional (2D) gas chromatography coupled with time-of-flight

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mass spectrometry (GC × GC – TOFMS) is a powerful technique for the analysis of compounds

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in complex mixtures such as those found in food quality and control,1–3 waste water,4,5

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metabolomics,6–9 and petroleum products.10–13 The inherent complexity of GC × GC – TOFMS

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data often requires advanced chemometrics for an informative analysis. Analytical approaches

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and algorithms applied to GC × GC – TOFMS data sets generally fall into two categories:

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targeted and non-targeted, with the latter further divided into supervised and unsupervised.

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Targeted analyses focus on preselected, specific analytes of interest. In contrast, discovery-based,

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non-targeted approaches aim to discover any or all analytes that may be of interest without

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requiring a priori knowledge of their character or identity. Additionally, supervised non-targeted

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approaches include classification of samples with more emphasis on experimental design.14,15

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We previously reported the development of discovery-based software for the analysis of

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GC × GC – TOFMS data. Tile-based Fisher ratio (F-ratio) analysis facilitates a supervised non-

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targeted method that uses information based upon the experimental design to aid in the discovery

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of analytes whose variances are statistically different between sample classes.15–18 Since its initial

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development,15,16 the tile-based F-ratio software has been applied to the analysis of yeast

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metabolites18 and chemically-altered diesel fuel,17 successfully elucidating statistically

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significant class-distinguishing analytes in complex sample matrices. Building upon these

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previous works, the present study serves to validate and optimize the tile-based F-ratio analysis.

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Method validation is an important part of all analytical technology developments. Several

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agencies, including the International Union of Pure and Applied Chemistry (IUPAC),19

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Eurachem,20 and the Association of Analytical Communities (AOAC)21 have published

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guidelines on the performance characteristics required to properly validate a new analytical

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method. While each agency has its own specific recommendations, they have a few key

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requirements in common: sensitivity, selectivity, accuracy, precision, working range, limit of

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detection (LOD) and limit of quantification (LOQ). While these guidelines are well established

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for new analytical methods, no such clearly published guidelines exist for the validation of new

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chemometric algorithms and software.

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Introduction of new chemometric algorithms and software should require rigorous

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validation and optimization; however, often new software algorithms are simply demonstrated

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and put into implementation, without rigorous validation. Validation may include applying the

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method on standard mixes of known composition15,16 or employing previously analyzed data

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sets18 where the “correct” answer has previously been determined. In the case of employing a

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standard mixture, the analyst will often assume that every analyte in the mixture is in fact a true

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positive when the sample matrix or analysis conditions may prevent that from actually being the

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case. The use of quantitative metrics, such as standard errors, correlation coefficients or

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statistical metrics like F-tests or Students’ t-tests, are commonly utilized for validation. The

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thorough study and evaluation of a software platform should also go beyond the validation stage.

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Key input parameters should be evaluated, to ensure algorithm optimization. Optimization of

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software algorithms and their various parameters is rarely studied in detail, likely because it can

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be a tedious and subjective process if one lacks the proper quantitative metric. To address this

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issue, herein we report the optimization of the tile-based F-ratio software15–18 using the area

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under the curve (AUC) of a receiver operating characteristic (ROC) curve as a quantitative

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metric.22,23 While the AUC for ROC curves has been applied for other purposes, implementation

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of the AUC as a quantitative metric for software optimization is presented as a new approach.

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Analytical Chemistry

ROC curves were developed during World War II in an attempt to quantify the ability of a

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radar operator to differentiate between the signals of ally and enemy aircraft.24 Since then, ROC

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curves have been used extensively in medical diagnostics,25–27 evaluating analytical method

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performance,22,28,29 and machine learning,30 but ROC curves have remained under-utilized in

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analytical and chemometric analyses.23 While machine learning and chemometric analyses are

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inherently interdisciplinary and often share similar algorithms, they have distinct differences.

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Machine learning is predominately a computer science methodology that involves computers

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learning without being explicitly programmed, while chemometrics is the application of linear

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algebra coupled with statistics to extract meaningful information from chemical systems.

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A ROC curve is a plot of the true positive probability (TPP) versus the false positive

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probability (FPP) at a particular threshold defined by the analyst, usually relating to a minimum

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signal or concentration. The terms true-positive rate (TPR) and false positive rate (FPR) are also

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used throughout the literature, interchangeably with TPP and FPP. For the purposes of this paper,

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TPP and FPP are used. The TPP, also known as the sensitivity, is equal to the sum of the true

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positives over the total number of positive instances. The FPP, also known as 1-specificity, is

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equal to the sum of the false positives over the total number of negative instances. The AUC is a

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popular metric for the quantitative, statistically-based evaluation of ROC curves. It is equivalent

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to the probability of stochastic domination in non-parametric statistics; that is, the AUC defines

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the probability that a randomly selected value will be correctly ranked as either a true positive or

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false positive. Values for the AUC range from 0.5 for a “useless” test to 1.0 for a perfect test.

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In the initial tile-based F-ratio software development, a signal-to-noise (S/N) threshold of

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3 and all of the mass channels (m/z) were utilized.15 The S/N threshold is used in the data

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reduction step prior to the calculation of F-ratios, and the number of m/z is used to calculate the

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average F-ratio value for each tile. Upon gaining more experience with the software, we have

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recognized that a rigorous evaluation of these two parameters is warranted. Accordingly, herein

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we employ the AUC metric for the ROC curve to evaluate and optimize these two key

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parameters. Each ROC curve quantitatively indicates how the S/N threshold and number of m/z

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both impact the sensitivity of the software to discover chemical features of interest. The

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hypothesis is that a higher S/N threshold (but not too high) coupled with use of less than all of

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the m/z would likely improve the capability of finding and more highly ranking true positives,

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while reducing the number of false positives.

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Fifty analytes were spiked into a diesel fuel at two similar concentration spike levels (30

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native and 20 non-native compounds), to serve as a pair of suitably complex samples to render

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challenging the software optimization using the AUC approach. Thus, the theoretical maximum

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number of positive instances is 50. The two spiked diesel samples defined two classes for F-ratio

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comparison. The S/N threshold used in the data reduction step prior to the calculation of F-ratios,

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and number of m/z used to calculate the average F-ratio values were varied, and F-ratio hit lists

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were evaluated. A total of 25 combinations of S/N threshold by number of m/z were studied.

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Varying the S/N threshold and number of m/z engenders optimization of the tile-based F-ratio

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software, to find the “sweet spot” in terms of S/N threshold, coupled with use of only the most

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chemically selective m/z (i.e., those with the highest F-ratios). Additionally, it is demonstrated

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that the number of positive instances does not need to be known prior to analysis, as the AUC

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metric still provides adequate information regarding the sensitivity of the F-ratio analysis.

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EXPERIMENTAL

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Diesel fuel (~ 1 gallon) was collected from a fueling station in the Seattle area. A nonnative internal standard, 1,2,3-trichlorobenzene (0.5170 g), was spiked into 1 L of diesel fuel to

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create a stock solution at 608 ppm trichlorobenzene. This stock solution was used for all serial

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dilutions. Two neat spike solutions were made of 30 native compounds and 20 non-native

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compounds by adding ~ 0.1 g of each component to a scintillation vial. A list of each compound

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and exact mass is provided in in Tables S1 (natives) and S2 (non-natives) in Supporting

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Information. The native and non-native compounds were then quantitatively transferred using

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stock solution to an amber bottle of known mass. Stock solution was then added to each of the

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two neat spike samples until the final mass reached ~ 100 g, creating ~ 1000 ppm native and ~

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1000 ppm non-native solutions. Approximately 80 g of the native spike solution and 8 g of the

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non-native spike solution were then transferred to a third amber bottle. Stock solution was added

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to bring the total mass of the contents to ~ 100 g, creating a spike solution containing ~ 800 ppm

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native compounds and ~ 80 ppm non-native compounds in diesel. This solution was diluted by

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half in stock solution successively until two spike solutions were obtained at the following

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nominal concentrations: 200/20 and 100/10 ppm native/non-native. The exact concentrations of

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the analytes in these spike solutions is provided in Tables S1 and S2 in Supporting Information.

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A neat standards mix with no additional solvent was created in order to find accurate retention

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times for each analyte by adding 250 µL of each compound into a scintillation vial.

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GC × GC–TOFMS data were collected using an Agilent 6890N GC (Agilent

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Technologies, Palo Alto, CA, USA) with a LECO Pegasus III TOFMS equipped with a 4D

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thermal modulator upgrade (LECO, St. Joseph, MI, USA). A reverse column configuration was

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employed with a polar 29.75 m × 250 µm × 0.25 µm Rxi-17Sil MS film primary column, 1D, and

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a nonpolar 1.5 m × 180 µm × 0.18 µm Rxi-1ms film (Restek, Bellefonte, PA, USA) secondary

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column, 2D. The GC inlet was set to 275 °C and the transfer line was set to 280 °C. Ultra-high

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purity helium (Grade 5, 99.999%, Praxair, Seattle, WA, USA) was used as the carrier gas at a

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constant flow of 2.0 mL/min. An Agilent 7683 autosampler was used to inject 1 µL for diesel

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samples or 0.1 µL of neat standards mix with a 300:1 split ratio. The primary column was

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maintained at 40 °C for 1 min, then ramped at 5 °C/min to 300 °C where it was held for 2 min.

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The secondary column and the modulator block followed the same temperature program with a

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+15 °C and +60 °C offset, respectively. The modulation period was 2 s with 0.5 s hot and cold

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pulses for each stage. The ion source was set to 225 °C, the electron impact energy was 70 eV,

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and the detector voltage was 1740 V. Mass channels, m/z 35-300, were collected at 100 spectra/s

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after a 10 s acquisition delay. Six injection replicates of each spike sample (200/20 and100/10

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ppm native/non-native with internal standard) and the stock solution (diesel with internal

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standard), and two injection replicates of the neat standards were analyzed using the GC × GC–

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TOFMS method above. These latter two injection replicates were used to determine the retention

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times of the spiked standards. A lack of variability in retention times between replicates

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indicated that two replicates were sufficient to obtain average retention times of the spikes.

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The GC × GC–TOFMS data were data processed using the instrument software

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(ChromaTOF, version 3.32, LECO, St. Joseph, MI, USA), including baseline correction, peak

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finding and peak area calculation. Peak areas were calculated at unique m/z for the 30 native

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compounds in the stock solution and in each spike sample. Standard addition method (SAM)

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plots were prepared (see Figure S1 in Supporting Information) to determine the native

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concentration of these analytes in diesel as reported in Table S1. The GC × GC–TOFMS data

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were also imported from the instrument software into Matlab R2015b (Mathworks Inc., Natick,

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MA, USA) using an in-house data converting algorithm. The data were analyzed with the in-

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house developed tile-based F-ratio software, an algorithm that elucidates chemical features that

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distinguish between two classes of samples, such as two different spike concentration levels. We

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direct the reader to previous publications and their respective supplementary information for a

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thorough description of the tile-based F-ratio software.15–18 For all F-ratio software executions, a

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1

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were employed. All chromatograms were normalized to the sum of the total ion current (TIC),

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and retention time alignment was not required. The S/N threshold and number of m/z utilized for

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the calculation of the average F-ratio were varied to find the optimum values for software

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performance. The S/N threshold values examined were 3, 6, 10, 30 and 100, while the number of

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m/z examined were 3, 6, 10, 20, and all m/z, for a total of 25 S/N by m/z combinations. At least 3

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m/z were required for a measureable F-ratio, and no F-ratio threshold minimum was employed.

D tile size of 6 s, a 2D tile size of 100 ms, a 1D cluster size of 4 s, a 2D cluster size of 60 ms

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The S/N threshold is applied by calculating the standard deviation of the noise on each

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tile region and multiplying that standard deviation by the factor given by the desired threshold

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(i.e., by a factor of 3, 6, 10, 30 or 100 per the S/N thresholds). Any m/z with a signal in the same

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tile falling below that S/N threshold is then excluded from the analysis. The number of m/z used

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for the analysis simply refers to the maximum number of m/z whose F-ratios are used to calculate

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the average F-ratio of the tile, with the minimum being 3 m/z. For example, if a tile has a feature

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containing 52 m/z occurring above the S/N threshold and the number of m/z to be examined was

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set to 10 m/z, only the 10 m/z with the highest F-ratio values would be included in calculation of

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the average F-ratio. However, if a tile has a feature having only 6 m/z, all 6 m/z would be utilized

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for the calculation of the average F-ratio even though the software was set to examine 10 m/z

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because the tile meets the criteria of having at least 3 m/z above the S/N threshold.

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RESULTS AND DISCUSSION

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The reverse column GC × GC configuration utilized a substantial portion of the 2D

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separation space, and was well suited for the separation presented in Figure 1(A) of the

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compound classes of interest in diesel fuel: alkanes, olefins, cyclic alkanes, and aromatics. The 9 ACS Paragon Plus Environment

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standards utilized for the spike study also encompass the 2D region of the chromatogram as

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shown in Figure 1(B), where each standard peak is numbered according to its respective analyte

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number in Tables S1 (native compounds) and S2 (non-native compounds).

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The F-ratio software was employed for the sample class comparison of the six injection

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replicates of each spiked diesel sample (200/20 versus 100/10 ppm native/non-native

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compounds) to generate a hit list of class distinguishing analytes. While the software was

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evaluated for 25 S/N by m/z combinations, we focus initially on the previously employed

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parameters, a S/N threshold of 3 and all m/z,15,17,18 followed by a detailed focus on the

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combination of parameters that turned out to be optimal, and finish with a summary for all 25

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combinations. The distribution of the average F-ratio values in Figure 2(A) corresponds to the

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class comparison using a S/N threshold of 3 and all m/z. The maximum of the F-ratio distribution

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is about 0.5, and the sharp peak of the distribution drops nearly to baseline at an F-ratio of about

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4. The tail of the distribution (shown in the inset figure) begins to be sparse from about 10 to

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638.9 (hit #1, cyclooctane), and contains the class distinguishing chemical features (at higher F-

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ratio) that are most likely to be true positives and thus most likely to be the spiked analytes.

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Using the known 2D retention times of the spiked standards, the hit list was evaluated. Hits that

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corresponded to spiked analytes were designated “true positives,” while the rest were designated

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“false positives.” The hit list was investigated until the 50th false positive was identified. Figure

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2(B) shows the F-ratio value versus hit number in the F-ratio hit list. The blue circles correspond

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to true positives (spiked standards), while the red circles correspond to false positives. The F-

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ratios range from 638.9 (hit #1, cyclooctane) to 3.2 (hit #83, false positive).

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Next, the results for the combination of parameters that turned out to be optimal (S/N threshold of 10 and 10 m/z) is provided in Figure 3(A). This class comparison distribution has a

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maximum F-ratio at about 1 and drops to baseline at an F-ratio of about 6.5. The area under the

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F-ratio distribution in Figure 3(A) is about 20% less than that of the curve in Figure 2(A). This

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indicates use of the stricter parameters reduced the number of total hits returned by the software

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(4,854 for S/N threshold of 10 and 10 m/z versus 5780 for S/N threshold of 3 and all m/z). The

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shape of the curve is also shifted down and to the right, indicating a shift to higher F-ratio values

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as a consequence of using the top 10 m/z for the average F-ratio calculation, decreasing the

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ability of noisy m/z to lower the average. The tail of the distribution (in the figure inset) begins to

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be sparse from about 20 to 2014 (hit #1, cyclooctane), and contains the class distinguishing

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chemical features most likely to be true positives, and thus spiked analytes. Figure 3(B) shows

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the F-ratio value versus hit number in the F-ratio hit list for this class comparison. Compared to

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the previous parameters utilized for Figure 2(B), that is S/N threshold 3; all m/z, the true positives

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(blue) are shifted up and to the left, while the false positives (red) fall down and to the right due

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to the better combination of parameters, that is S/N threshold 10; 10 m/z. The F-ratios in Figure

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3(B) range from 2014 (hit #1, cyclooctane) to 5.0 (hit #87, false positive), a much greater range

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of average F-ratio values compared to that observed in Figure 2(B). In Figure 3, the exclusion of

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non-class distinguishing m/z increases the overall average F-ratio values, prioritizes true

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positives, and therefore improves the sensitivity of the discovery-based method.

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While the results in Figures 2(B) and 3(B) were readily interpreted to gain insight into the

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F-ratio software performance, the interpretation is qualitative and not analytically rigorous.

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Therefore, using ROC curves, an AUC quantitative metric was investigated as a means to

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evaluate and optimize these two key parameters: S/N threshold and number of m/z required. The

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ROC curves for these two F-ratio analyses are presented in Figure 4(A), where the red curve

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corresponds to the parameters S/N threshold of 3 and all m/z, while the blue curve corresponds to

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the parameters S/N threshold of 10 and 10 m/z. An explanation of how the ROC curves are

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created can be found in Supporting Information. For the ROC curves shown in Figure 4(A), it

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was assumed that all 50 analytes spiked into the diesel fuel would be discovered as true positives

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such that the positive instances used for calculating the TPP was 50. For symmetry, 50 negative

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instances were evaluated for the FPP, effectively minimizing the length of the hit list evaluated

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while sufficiently investigating the ROC curves as they plateau.

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As indicated by the shift upward and to the left of the true positives in Figure 3(B), the

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blue ROC curve in Figure 4(A) shows the increase in sensitivity, or TPP, for a given FPP, or 1-

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specificity, by using S/N 10 and 10 m/z. Increasing the S/N threshold from 3 to 10 decreased the

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influence of overly noisy m/z signals. Using 10 m/z rather than all m/z ensures that the 10 largest

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F-ratios for a given tile are used to determine the average F-ratio, thus decreasing the influence

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of smaller F-ratios at m/z with spurious covariance. A quantitative metric, the AUC can aid in the

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comparison of ROC curves generated by changing the algorithm parameters. For example, the

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AUC is 0.61 for the ROC curve using a S/N threshold of 3 and all m/z, while the AUC is 0.74 for

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the ROC curve using a S/N threshold of 10 and 10 m/z. This means, for the combination of a S/N

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threshold of 10 and 10 m/z is quantitatively superior to using a S/N threshold of 3 and all m/z,

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with a ~ 21% improvement in software performance.

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One might presume that, upon software optimization, the AUC would approach the ideal

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value of 1.00. However, the experimental design provided very challenging datasets due to the

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number of native compounds naturally occurring at a high concentration in the diesel. Keeping in

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mind that native concentrations are not known a priori, some of these compounds when spiked

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in at the 200 ppm and 100 ppm level, were not apt to be discovered due to the lack of a

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statistically sufficient increase in the total concentration given by the spike. This is a major

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reason why the ROC curves level off at a sensitivity well below 1.00. For Figure 4(A), it was

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assumed that all 50 spiked analytes had a statistically sufficient concentration difference between

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the two spike levels. In a sense, the number of positive instances was not actually 50, but rather

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is likely much less. Despite this issue, the ROC curve and the AUC metric were able to

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differentiate the increase in sensitivity using a S/N threshold of 10 and 10 m/z compared to using

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a S/N threshold of 3 and all m/z.

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In order to further investigate this issue, for the purpose of determining the number of

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spiked compounds that could (or should) serve as positive instances, the native spiked analyte

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concentrations were determined using the SAM (standard addition method) with the two spike

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levels, nominally 200 ppm and 100 ppm, and the stock solution, nominally 0 ppm. The

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concentrations of the native analytes calculated from the SAM plots, as demonstrated in Figure

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S1, are provided in Table S1 in Supporting Information. Worth noting are the three analytes with

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0.0 ppm concentration in the diesel: 2,2,4-trimethylpentane, cyclohexene, and cyclooctane.

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These compounds, though having negligible concentrations in this investigation so were not

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detected, can be found regularly in other blends of diesel. For each native compound, the native

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concentration was added to the actual spike concentration (at both spike levels), and the

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concentration ratio was determined as shown in Table S1 Supporting Information. We have

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previously shown that tile-based F-ratio analysis is capable of discovering analytes at a

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concentration ratio between sample classes down to ~ 1.06.16 Accordingly, the native compounds

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that have a concentration ratio less than 1.06 are shaded in grey in Table S1. In order to

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statistically determine which of the 30 spiked native compounds were not legitimate positive

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instances, a t-test was performed as described in Harris,31 with the results provided in the last

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column of Table S1. The t-test values were calculated using the peak areas of a selective m/z for

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each native analyte in all six injection replicates of the 200 ppm, 100 ppm and the stock solution

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(that is, nominally 0 ppm). The peak areas were normalized to the internal standard. At a 95%

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confidence interval, the t-table value is 2.57 for a two-tailed test with 5 degrees of freedom, so all

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t-values less than that correspond to analytes that have no statistically significant difference in

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their concentration between the 200 ppm and 100 ppm spike levels, as designated by yellow

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highlighting in Table S1. Corresponding p-values for the t-tests were also calculated (Table S1 in

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Supporting Information with discussion). Nine analytes were determined not to be statistically

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significant positive instances. There is good agreement between the concentration ratio and the t-

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test metric for the discovery of true positives. Other than hexane, which for unknown reasons

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appears to be an outlier, the minimum concentration ratio corresponding to a true positive

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appears to be ~ 1.04, in good agreement with a previous study.16 Using the list of native

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compounds corrected using the t-test metric, ROC curves were re-made (Figure S2 in Supporting

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Information) with a TPP out of 41 total positive instances instead of the previously assumed 50

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for Figure 4(A). While the shape of each curve does not change, the AUC value obviously

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increases, from 0.61 to 0.74 for a S/N threshold of 3 and all m/z, and from 0.74 to 0.90 for a S/N

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threshold of 10 and 10 m/z. It is important to note that the analyst can draw the same conclusions

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from Figure 4(A) or Figure S2 which has important ramifications for implementing the ROC

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curve-based AUC metric. Essentially, optimization of the software parameters does not require a

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priori determination of the statistically correct number of positive instances in the sample

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classes.

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To more fully explore the effects of S/N threshold and number of m/z on F-ratio software

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performance, ROC curves were generated for the 25 combinations of S/N threshold and number

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of m/z defined in the Experimental Section. The area under the ROC curve (AUC) was calculated

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for each combination of S/N threshold and m/z, whereby the AUC provided a quantitative metric

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to assess the effect of these parameters on the discrimination of the F-ratio software. The AUC

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values for all 25 parameter combinations are shown as a heat map in where Figure 4(B)

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corresponds to AUCs determined assuming all 50 spiked analytes correspond to positive

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instances. Most notably, in Figure 4(B) the optimum parameters remain an S/N 10 coupled with

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10 m/z, producing an AUC of 0.74 assuming 50 positive instances, while the previously

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employed parameters (S/N 3 coupled with all m/z) produced an AUC of 0.61. The difference

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between these two AUC values (0.13) relative to the prior value (0.61) is 0.21, or a 21%

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improvement in the F-ratio software performance. Hence, optimization of the S/N threshold and

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number of m/z used provided a 21% increase in the discrimination of the true positives from the

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false positives. A heat map of AUCs was calculated using only the 41 statistically significant

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positive instances (Figure S3 in Supporting Information), but the conclusions remain the same,

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no a priori knowledge of the statistically different analytes is necessary.

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Beyond interpretation of the AUC, perhaps most elucidative of the increase in software

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performance is the comparison of the number of true positives discovered at different FPP

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thresholds, which can serve as alternative metrics of ROC performance (see Supporting

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Information). It may be advantageous to combine insight provided by more than one ROC curve

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metric when one set of parameters is not clearly superior, so as to result in the selection of the

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best set of parameters. For example, the previously utilized parameters (S/N 3; all m/z) found a

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total of 33 true positives, while the optimized parameters (S/N 10; 10 m/z) found a total number

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of 38 true positives of the 41 possible, statistically significant, true instances. Not only did the

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optimized parameters find 5 additional hits total (i.e., at an FPP of 1.0), but using the optimized

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parameters allowed the F-ratio algorithm to discover the vast majority of those true positives at

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much lower FPP thresholds than those required by the previously utilized parameters. For

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example, the greatest distance between the two ROC curves shown in Figure 4 occurs at an FPP

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of about 0.2. An FPP of 0.2 corresponds to when the F-ratio hit list has reached 10 false

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positives. At this FPP threshold, the optimized parameters (S/N 10; 10 m/z) have discovered 37

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of the 38 true positives that were discovered overall, which corresponds to 9 more true positives

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than the 28 true positives that the software discovered using the previous parameters (S/N 3; all

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m/z). The optimization of the parameters (using S/N 10 and 10 m/z) allowed the method to

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discover nearly all of the true positives at the cost of only 10 false positives.

333

CONCLUSION

334

The AUC is a quantitative metric to compare ROC curves to study and optimize

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chemometric software performance and to demonstrate how various input parameters employed

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by the software can be tuned for better analysis. We report the implementation of such an

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approach on the tile-based F-ratio analysis software of GC × GC – TOFMS data using diesel fuel

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spiked with 30 native and 20 non-native compounds at two nominal concentrations, 200/20 ppm

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and 100/10 ppm, native/non-native respectively. By varying the S/N threshold and the number of

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F-ratios per m/z used to calculate the average F-ratio of each tile, the AUC quantitatively

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determined the best parameters, found to be a S/N threshold of 10 and 10 m/z. Compared to the

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previously employed parameters of S/N threshold of 3 and all m/z, the use of these parameters

343

provided a ~21% improvement in the ability of the software to discriminate between true

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positives and false positives in the hit list. It also provided the method with the ability to find 9

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additional true positives at an FPP of 0.2 (10 allowed false positives) and 5 additional true

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positives overall. Furthermore, we demonstrate that the AUC metric can be used effectively

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347

without prior knowledge of whether or not the spiked analytes (i.e., the intended true positives)

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were in fact statistically significant positive instances.

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For practical application of the ROC curve-based AUC metric method to optimize other

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sample matrices and chemometric software algorithms, a few comments of merit remain. First,

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while the F-ratio software optimization used two sample classes fabricated by spiking standards

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into a diesel matrix, for other software algorithms the research goals and experimental design

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should guide the preparation of suitable samples for optimization. For example, a software

354

algorithm designed for isotopic labeling investigations of metabolomes should utilize

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isotopically labeled metabolites in a biological matrix. Second, while the software optimization

356

method was relatively time-consuming, the optimization need only be performed once prior to

357

application to numerous experiments for a given experimental design and sample type. Lastly,

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this software optimization method should be broadly applicable to many other software methods

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and investigative questions. For example, peak detection and quantification with results

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presented in peak tables, commonly applied in many software packages, could be compared15

361

and optimized using the ROC curve-based AUC metric approach.

362

ACKNOWLEDGEMENTS

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This work was supported by the Internal Revenue Service (IRS) under an Interagency Agreement

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with the U.S. Department of Energy (DOE) under Contract DE-AC-5-76RLO with the Pacific

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Northwest National Laboratory. The authors thank Dr. Brendon Parsons for his assistance

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throughout this investigation.

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SUPPORTING INFORMATION

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Table S1: Table of native spike components

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Statistical metrics for Table S1

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Table S2: Table of non-native spike components

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Figure S1: Standard addition method plot

375

Calculation of native concentration of spiked analytes

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Explanation of ROC curve creation

377

Figure S2: ROC curves with 41 positive instances

378

Figure S3: Heat map of AUC with 41 positive instances

379

Other Metrics of ROC curve performance

380

Figure S4: Heat map of TP at FPP of 0.2

381

REFERENCES

382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408

(1) Zhang, L.; Zeng, Z.; Zhao, C.; Kong, H.; Lu, X.; Xu, G. J. Chromatogr. A 2013, 1313, 245–252. (2) Cordero, C.; Liberto, E.; Bicchi, C.; Rubiolo, P.; Schieberle, P.; Reichenbach, S. E.; Tao, Q. J. Chromatogr. A 2010, 1217 (37), 5848–5858. (3) Humston, E. M.; Knowles, J. D.; McShea, A.; Synovec, R. E. J. Chromatogr. A 2010, 1217 (12), 1963–1970. (4) Prebihalo, S.; Brockman, A.; Cochran, J.; Dorman, F. L. J. Chromatogr. A 2015, 1419, 109–115. (5) Ochiai, N.; Ieda, T.; Sasamoto, K.; Takazawa, Y.; Hashimoto, S.; Fushimi, A.; Tanabe, K. J. Chromatogr. A 2011, 1218 (39), 6851–6860. (6) Castillo, S.; Mattila, I.; Miettinen, J.; Orešič, M.; Hyötyläinen, T. Anal. Chem. 2011, 83 (8), 3058–3067. (7) Welthagen, W.; Shellie, R. A.; Spranger, J.; Ristow, M.; Zimmermann, R.; Fiehn, O. Metabolomics 1 (1), 65–73. (8) Mohler, R. E.; Dombek, K. M.; Hoggard, J. C.; Young, E. T.; Synovec, R. E. Anal. Chem. 2006, 78 (8), 2700–2709. (9) Mohler, R. E.; Tu, B. P.; Dombek, K. M.; Hoggard, J. C.; Young, E. T.; Synovec, R. E. J. Chromatogr. A 2008, 1186 (1–2), 401–411. (10) Jennerwein, M. K.; Eschner, M.; Gröger, T.; Wilharm, T.; Zimmermann, R. Energy Fuels 2014, 28 (9), 5670–5681. (11) Nizio, K. D.; MGinitie, T. M.; Harynuk, J. J. J. Chromatogr. A 2012, 1255, 12–23. (12) von Mühlen, C.; Zini, C. A.; Caramão, E. B.; Marriott, P. J. J. Chromatogr. A 2006, 1105 (1–2), 39–50. (13) Kehimkar, B.; Hoggard, J. C.; Marney, L. C.; Billingsley, M. C.; Fraga, C. G.; Bruno, T. J.; Synovec, R. E. J. Chromatogr. A 2014, 1327, 132–140. (14) Pierce, K. M.; Kehimkar, B.; Marney, L. C.; Hoggard, J. C.; Synovec, R. E. J. Chromatogr. A 2012, 1255, 3–11. 18 ACS Paragon Plus Environment

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(15) Parsons, B. A.; Marney, L. C.; Siegler, W. C.; Hoggard, J. C.; Wright, B. W.; Synovec, R. E. Anal. Chem. 2015, 87 (7), 3812–3819. (16) Marney, L. C.; Christopher Siegler, W.; Parsons, B. A.; Hoggard, J. C.; Wright, B. W.; Synovec, R. E. Talanta 2013, 115, 887–895. (17) Parsons, B. A.; Pinkerton, D. K.; Wright, B. W.; Synovec, R. E. J. Chromatogr. A 2016, 1440, 179–190. (18) Watson, N. E.; Parsons, B. A.; Synovec, R. E. J. Chromatogr. A 2016, 1459, 101–111. (19) Thompson, M.; Ellison, S. L. R.; Wood, R. Pure Appl. Chem. 2002, 74 (5), 835–855. (20) Magnusson, B.; Ornemark, U. 2014. (21) Official methods of Analysis of AOAC International, 6th ed.; AOAC International, 1995. (22) Fraga, C. G.; Melville, A. M.; Wright, B. W. Analyst 2007, 132 (3), 230–236. (23) Brown, C. D.; Davis, H. T. Chemom. Intell. Lab. Syst. 2006, 80 (1), 24–38. (24) Green, D. M.; Swets, J. A. Signal Detection Theory and Psychophysics; John Wiley & Sons, Inc., 1966. (25) Metz, C. E. J. Am. Coll. Radiol. 2006, 3 (6), 413–422. (26) Baker, S. G. J. Natl. Cancer Inst. 2003, 95 (7), 511–515. (27) Carter, J. V.; Pan, J.; Rai, S. N.; Galandiuk, S. Surgery 2016, 159 (6), 1638–1645. (28) Blaise, B. J. Anal. Chem. 2013, 85 (19), 8943–8950. (29) Duraipandian, S.; Zheng, W.; Ng, J.; Low, J. J. H.; Ilancheran, A.; Huang, Z. Anal. Chem. 2012, 84 (14), 5913–5919. (30) Bradley, A. P. Pattern Recognit. 1997, 30 (7), 1145–1159. (31) Harris, D. C. Quantitative Chemical Analysis, 6th ed.; W. H. Freeman and Company: New York, NY, 2003.

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Figure 1. (A) Total ion current (TIC) chromatogram of the stock solution containing diesel fuel

435

with a ~600 ppm internal standard 1,2,3-trichlorobenzene using a reverse column GC × GC

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configuration. (B) TIC chromatogram of all 50 components in the standards mixture, including

437

30 native and 20 non-native compounds, numbered corresponding to the first column of Table S1

438

(natives) and S2 (non-natives) in Supporting Information. The “IS” indicates the internal

439

standard, 1,2,3-trichlorobenzene.

Figure Captions

440 441

Figure 2. (A) The F-ratio distribution for the preliminary hit list of the 200/20 ppm versus

442

100/10 ppm comparison using a S/N threshold of 3 and all m/z. The tail of the distribution is

443

shown in the inset figure, with the average F-ratio on a log scale so the entire tail can be viewed.

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(B) Log of average F-ratio versus hit number based upon the F-ratio distribution in (A), with true

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positives (spiked analytes) shown in blue and false positives shown in red.

446 447

Figure 3. (A) The F-ratio distribution for the preliminary hits list of the 200/20 ppm versus

448

100/10 ppm comparison at optimized conditions of the tile-based F-ratio algorithm, S/N

449

threshold of 10 and 10 m/z. The tail of the distribution is shown in the inset figure with the

450

average F-ratio on a log scale so the entire tail can be viewed. (B) Log of average F-ratio versus

451

hit number based upon the F-ratio distribution in (A), with true positives (spiked analytes) shown

452

in blue and false positives shown in red. The true positives are shifted up, to higher F-ratio

453

values, and to the left, to hit numbers toward the top of the hit list relative to Figure 2(B).

454 455

Figure 4. (A) ROC curves for the two sets of parameters studied: (red) S/N threshold of 3 and all

456

m/z (based upon the results in Figure 2), and (bue) S/N threshold of 10 and10 m/z (based upon

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the results in Figure 3). TPP is calculated assuming all 50 spiked analytes were positive

458

instances. The greatest difference between the ROC curves can be seen at an FPP of about 0.2,

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which corresponds to the allowance of 10 false positives. This difference equates to the ability of

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the software using the parameters S/N 10; 10 m/z to find 9 additional true positives than the

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previously used parameters of S/N 3; all m/z. (B) A heat map showing the area under the curve

462

(AUC) at the 25 S/N threshold and number of m/z combinations studied. The AUC was

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calculated by assuming all 50 spiked standards were positive instances. The optimum

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combination of F-ratio software parameters was determined to be a S/N threshold of 10 with 10

465

m/z for both (A) and (B).

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Figure 1(A) 190x254mm (96 x 96 DPI)

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Figure 1(B) 190x254mm (96 x 96 DPI)

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Figure 2(B) 190x254mm (96 x 96 DPI)

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Figure 4(B) 190x254mm (96 x 96 DPI)

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