Enabling cuboid-based fisher ratio analysis using total-transfer comprehensive three-dimensional gas chromatography with time-of-flight mass spectrometry.

Comprehensive three-dimensional (3D) gas chromatography with time-of-flight mass spectrometry (GC3-TOFMS) is a promising instrumental platform for the separation of volatiles and semi-volatiles due to its increased peak capacity and selectivity relative to comprehensive two-dimensional gas chromatography with TOFMS (GC×GC-TOFMS). Given the recent advances in GC3-TOFMS instrumentation, new data analysis methods are now required to analyze its complex data structure efficiently and effectively. This report highlights the development of a cuboid-based Fisher ratio (F-ratio) analysis for supervised, non-targeted studies. This approach builds upon the previously reported tile-based F-ratio software for GC×GC-TOFMS data. Cuboid-based F-ratio analysis is enabled by constructing 3D cuboids within the GC3-TOFMS chromatogram and calculating F-ratios for every cuboid on a per-mass channel basis. This methodology is evaluated using a GC3-TOFMS data set of jet fuel spiked with both non-native and native components. The neat and spiked jet fuels were collected on a total-transfer (100 % duty cycle) GC3-TOFMS instrument, employing thermal modulation between the first (1D) and second dimension (2D) columns and dynamic pressure gradient modulation between the 2D and third dimension (3D) columns. In total, cuboid-based F-ratio analysis discovered 32 spiked analytes in the top 50 hits at concentration ratios as low as 1.1. In contrast, tile-based F-ratio analysis of the corresponding GC×GC-TOFMS data only discovered 28 of the spiked analytes total, with only 25 of them in the top 50 hits. Along with discovering more analytes, cuboid-based F-ratio analysis of GC3-TOFMS data resulted in fewer false positives. The increased discoverability is due to the added peak capacity and selectivity provided by the 3D column with GC3-TOFMS resulting in improved chromatographic resolution.

Optimization of Parameters for ROI Data Compression for Nontargeted Analyses Using LC-HRMS.

Nontargeted analyses of low-concentration analytes in the information-rich data collected by liquid chromatography with high-resolution mass spectrometry detection can be challenging to accomplish in an efficient and comprehensive manner. The aim of this study is to demonstrate a workflow involving targeted parameter optimization for entire chromatograms using region of interest (ROI) data compression uncoupled from a subsequent tile-based Fisher ratio (F-ratio) analysis, a supervised discovery-based method, for the discovery of low-concentration analytes. Soil samples spiked with 18 pesticides at nominal concentrations ranging from 0.1 to 50 ppb for a total of six sample classes served as challenging samples to demonstrate the overall workflow. Optimization of two parameters proved to be the most critical for ROI data compression: the signal threshold parameter and the admissible mass deviation parameter. The parameter optimization method workflow we introduce is based upon spiking known analytes into a representative sample and determining the number of detectable spikes and the Δppm for various combinations of the signal threshold and admissible mass deviation, where Δppm is the absolute value of the difference between the theoretical m/z and the ROI m/z. Once optimal parameters are determined providing the lowest average Δppm and the greatest number of detectable analytes, the optimized parameters can be utilized for the intended analysis. Herein, tile-based F-ratio analysis was performed on the ROI compressed data of all spiked soil samples first by applying ROI parameters recommended in the literature, referred to herein as the initial ROI parameters, and finally by the combination of the two optimized parameters. Using the initial ROI parameters, three pesticides were discovered, whereas all 18 spiked pesticides were discovered by optimizing both ROI parameters.

Minimum variance optimized Fisher ratio analysis of comprehensive two-dimensional gas chromatography / mass spectrometry data: Study of the pacu fish metabolome.

Integration of rice and fish farming, eg., pacu fish in Argentina, has raised concern that herbicides used for rice paddies may adversely affect the fish metabolome. To study this issue, tile-based Fisher ratio (F-ratio) analysis was applied to comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-TOFMS) data of pacu fish raised in an integrated rice-fish farming system (farmed class) versus fish raised in tanks (tank-raised class) to discover class-distinguishing analytes. F-ratio analysis resulted in hit lists initially dominated by artifact peaks from the sample derivatization process, as well as some redundant hits. These challenges were addressed by developing an automated artifact removal algorithm and an improvement to redundant hit removal in the tile-based F-ratio analysis using a pooled farmed fish sample, either spiked with 29 metabolites (spiked class) or unspiked (i.e., the background serving as the control class). Of the 29 spiked metabolites, 23 were discovered by standard F-ratio analysis, improving to 28 discovered using control-normalized F-ratio analysis. Standard and control-normalized F-ratio hit lists initially with 185 and 246 hits, were reduced to 56 and 49 hits, respectively, after artifact removal and removing redundant hits. Next, we returned to the F-ratio analysis of the farmed fish versus the control fish. Here, we introduce a minimum variance optimized (MVO) F-ratio calculation (MVOF-ratio) that provides a comprehensive hit list ranking. The initial MVOF-ratio analysis hit list of 537 hits was reduced to 110 hits following artifact removal. Of the 110 hits, 70 expressed a concentration ratio statistically different than 1 (p < 0.05). The MVOF-ratio hit list discovered more true positives compared to the standard, tank-normalized, and farm-normalized F-ratio hit lists, providing the combination of results from the farm-normalized and tank-normalized hit lists. A majority of analytes (54 out of 70) important for normal biological functioning of pacu fish were significantly downregulated in the farmed fish, suggesting the integrated farming system may negatively impact pacu fish quality.

Total-transfer comprehensive three-dimensional gas chromatography with time-of-flight mass spectrometry.

Although comprehensive two-dimensional (2D) gas chromatography (GC × GC) is a powerful technique for complex samples, component overlap remains likely. An intriguing route to address this challenge is to utilize the additional peak capacity and chemical selectivity provided by comprehensive three-dimensional (3D) gas chromatography (GC3), especially with time-of-flight mass spectrometry detection (GC3-TOFMS). However, the GC3-TOFMS instrumentation reported to date has employed one or both modulators with a duty cycle < 100%, making the potential gain in detection sensitivity over GC × GC modest, or perhaps even worse. Herein, we describe instrumentation for GC3-TOFMS in which both modulators provide total-transfer (100% duty cycle). Specifically, the instrument is based on the facile modification of a commercial thermally modulated comprehensive GC × GC-TOFMS platform for modulation from the 1D column to the 2D column, with recently described dynamic pressure gradient modulation (DPGM) as the second modulator from the 2D column to the 3D column, which is a total-transfer flow modulation technique. Area measurements of 1D peaks are compared to the sum of 3D peak areas to validate the assumption that total-transfer from 1D to 3D is accomplished. Additionally, peak heights were amplified by as high as a factor of 177 (x̅ = 130, s = 47) via comparison of 1D peak heights to the maximum 3D peak heights. Column selection is explored, with emphasis on the resulting peak width-at-base on each dimension and usage of 3D space as evaluation metrics. Using a nonpolar × polar × ionic liquid column combination, an effective peak capacity which considers modulation-induced broadening as high as 32,300 for select analytes was achieved (x̅ = 19,900, s = 10,700). The analytical benefits of employing three selective phases, mass spectrometry detection, and total-transfer modulation are explored with separations of a metabolomics-type sample, i.e., derivatized porcine serum, and a jet fuel spiked with various sulfur-containing compounds.

A systematic investigation of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry with dynamic pressure gradient modulation for high peak capacity separations.

Dynamic pressure gradient modulation (DPGM) in full modulation mode is optimized for comprehensive two-dimensional (2D) gas chromatography (GC × GC) with time-of-fight mass spectrometry (TOFMS) detection to obtain high peak capacity separations and demonstrate broad applicability for complex samples. A pulse valve introduces an auxiliary carrier gas flow at a T-union connecting the first dimension (1D) column to the second dimension (2D) column. At a sufficiently high auxiliary pressure (Paux) the 1D flow is temporarily stopped. Then, during each modulation period (PM) the valve is turned off briefly, a period termed the pulse width (pw), allowing the 1D effluent to essentially be reinjected onto the 2D column for the modulated separations. Modifications to the modulator assembly are provided to improve performance. Method optimization is demonstrated for a 116-component test mixture by tuning the Paux and the pw. For a PM = 2 s and 1F of 0.10 ml/min, the optimal pw and initial Paux selected were 200 ms and 330.9 kPa (33 psig), respectively. The 30 min separation of the test mixture provided a 1D peak capacity of 1nc = 330 and a 2D peak capacity of 2nc = 15, hence an ideal 2D peak capacity nc,2D = 1nc × 2nc = 4950. Likewise, the 2D peak capacity corrected for undersampling of the 1D separation was 4500 and corrected for both undersampling and sampling variation via statistical overlap theory was 4090. These results provide a 2-fold improvement in peak capacity relative to the previous DPGM study in full modulation mode for GC × GC-TOFMS. The optimized conditions were applied for a variety of applications: diesel fuel, derivatized cow serum, solid phase microextraction (SPME) of coffee headspace, and SPME of river water headspace. Additionally, the fraction of 2D separation space utilized (fcoverage), as defined by the minimum convex hull method, ranged from 0.60 to 0.85. We observed that any fcoverage correction to 2D peak capacity is highly sample dependent, since all samples, except for the diesel sample, were run with the same separation conditions, and yet the fcoverage ranged from 0.60 to 0.80.

Development of an Enhanced Total Ion Current Chromatogram Algorithm to Improve Untargeted Peak Detection.

Accurate analyte peak detection from the background noise is a fundamental step in data analysis. Often, this is initially performed on the total ion current chromatogram (TIC), which is the summed signal from all mass spectral channels. Despite the detection of many of the most abundant peaks within a chromatogram, a large fraction of peaks remains undetected in the standard TIC due to their signal being below the limit of detection. To find peaks obscured by background noise, an untargeted peak detection method termed the "enhanced TIC algorithm" was developed for comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS). The reported algorithm utilizes the entire mass spectral dimension to find regions of analytical signal above a threshold while zeroing the background noise. The resulting chromatographic data is summed together to create the enhanced TIC. The utility of the enhanced TIC algorithm is demonstrated using serial dilutions from a 10 parts-per-thousand (ppth) test mixture. For the chromatograms collected at 1 and 10 parts-per-million (ppm), the enhanced TIC algorithm recovered 62% and 93%, respectively, of the original peaks observed in the 10 ppth mixture, while the standard TIC recovered only 0% and 45%, respectively. The improvement in signal enhancement was also shown on a separation of a yeast cell metabolite extract, where the enhanced TIC found 33-64% more peaks than the standard TIC. Chromatographic simulations with increasing levels of background noise were also conducted to compare the enhanced and standard TICs in the context of statistical overlap theory (SOT). Simulated chromatograms with lower signal-to-noise were more accurately modeled by the SOT after enhanced TIC processing compared to those processed by the standard TIC. The enhanced TIC method demonstrates an immense benefit in peak discovery to improve data analysis efforts.

Dynamic pressure gradient modulation for comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detection.

Dynamic pressure gradient modulation (DPGM) is investigated for comprehensive two-dimensional gas chromatography (GC × GC) with time-of-flight mass spectrometry (TOFMS) detection. With DPGM, a commercial pneumatic "pulse" valve is opened to introduce a suitably high auxiliary gas pressure at a T-junction connecting the first dimension (1D) and second dimension (2D) columns during the modulation period (PM), temporarily stopping the 1D flow. The valve is then closed for the duration of a pulse width (pw) to "re-inject" temporally focused 1D eluate onto the 2D column for separation. This flow modulation technique is observed to be compatible with TOFMS detection using a 2D flow rate of 4 ml/min for the separation of a 90-component test mixture. A 25 min separation window using a PM = 1 s and pw = 200 ms for full modulation (and 100% duty cycle) provided an average 1Wb = 4.5 s and 2Wb = 130 ms for a 2D peak capacity of nc,2D = 2700 (100 peaks per min). The detector response enhancement factor (DREF) serves as a metric for the enhanced sensitivity of the modulated relative to the unmodulated 1D peaks, with DREFs ranging between 10 and 20 and about a 5-fold improvement in signal-to-noise ratio (S/N). The bilinear "quality" of the GC × GC data is studied using the chemometric method parallel factor analysis (PARAFAC). Since PARAFAC requires sufficiently trilinear data, the reproducibility of the 2D peak shape for a given analyte is confirmed using lack-of-fit (LOF) and percent variation (R2) metrics. The limit-of-detection (LOD) for the representative analyte hexadecane is determined using PARAFAC, providing an LOD of 0.7 ppb (±0.03 ppb) for three replicates. Seven heavily overlapped analytes are also fully resolved by PARAFAC down to the part-per-million (ppm) concentration level, producing reproducible spectra with a majority of spectral match values (MV) over 800 (RSD ≤ 7.1%). This study provides promising results for DPGM as a flow modulation technique compatible with GC × GC-TOFMS, providing high sensitivity data suitable for chemometric analysis.

Development of Ultrafast Separations Using Negative Pulse Partial Modulation To Enable New Directions in Gas Chromatography.

Partial modulation via a pulse flow valve operated in the negative pulse mode is developed for high-speed one-dimensional gas chromatography (1D-GC), comprehensive two-dimensional (2D) gas chromatography (GC × GC), and comprehensive three-dimensional gas chromatography (GC3). The pulse flow valve readily provides very short modulation periods, PM, demonstrated herein at 100, 200, and 300 ms, and holds significant promise to increase the scope and applicability of GC instrumentation. The negative pulse mode creates an extremely narrow, local analyte concentration pulse. The reproducibility of the negative pulse mode is validated in a 1D-GC mode, where a pseudosteady-state analyte stream is modulated, and 8 analytes are baseline resolved (resolution, Rs ≥ 1.5) in a 200 ms window, providing a peak capacity, nc, of 14 at unit resolution ( Rs = 1.0). Additionally, the pulse width, pw, of the pulse flow valve "injection" relationship to peak width-at-base, wb, resolution between peaks and detection sensitivity are studied. To demonstrate the applicability to GC × GC, a high-speed separation of a 20-component test mixture of similar, volatile analytes is shown. Analytes were separated on the second-dimension column, 2D, with 2 wb ranging from 7 to 12 ms, providing an exceptional 2D peak capacity, 2 nc, of ∼12 using a modulation period ( PM) of 100 ms. Next, a 12 min separation of a diesel sample using a PM of 300 ms is presented. The 1 wb is ∼4 s, resulting in a 1 nc of ∼180, and 2 wb is ∼18 ms, resulting in a 2 nc of ∼17, thus achieving a nc,2D of ∼3000 in this rapid GC × GC diesel separation. Finally, GC3 with time-of-flight mass spectrometry (TOFMS) detection using a PM of 100 ms applied between the 2D and 3D columns is reported. Narrow third dimension, 3D, peaks with 3 wb of ∼15 ms were obtained, resulting in a GC3 peak capacity, nc,3D, of ∼35 000 in a 45 min separation.