Control-Normalized Fisher Ratio Analysis of Comprehensive Two-Dimensional Gas Chromatography Time-of-Flight Mass Spectrometry Data for Enhanced Biomarker Discovery in a Metabolomic Study of Orthopedic Knee-Ligament Injury.

An innovative form of Fisher ratio (F-ratio) analysis (FRA) is developed for use with comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) data and applied to the investigation of the changes in the metabolome in human plasma for patients with injury to their anterior cruciate ligament (ACL). Specifically, FRA provides a supervised discovery of metabolites that express a statistically significant variance in a two-sample class comparison: patients and healthy controls. The standard F-ratio utilizes the between-class variance relative to the pooled within-class variance. Because standard FRA is adversely impacted by metabolites expressed with a large within-class variance in the patient class, "control-normalized FRA" has been developed to provide complementary information, by normalizing the between-class variance to the variance of the control class only. Thirty plasma samples from patients who recently suffered from an ACL injury, along with matched controls, were subjected to GC × GC-TOFMS analysis. Following both standard and control-normalized FRA, the concentration ratio for the top 30 "hits" in each comparison was obtained and then t-tested for statistical significance. Twenty four out of 30 metabolites plus the therapeutic agent, naproxen (24/30), passed the t-test for the control-normalized FRA, which included 8/24 unique to control-normalized FRA and 16/24 in common with the standard FRA. Likewise, standard FRA provided 21/30 metabolites passing the t-test, with 5/21 undiscovered by control-normalized FRA. The complementary information obtained by both F-ratio analyses demonstrates the general utility of the new approach for a variety of applications.

Enhancing gas chromatography-time of flight mass spectrometry data analysis using two-dimensional mass channel cluster plots.

A novel data reduction and representation method for gas chromatography time-of-flight mass spectrometry (GC-TOFMS) is presented that significantly facilitates separation visualization and analyte peak deconvolution. The method utilizes the rapid mass spectral data collection rate (100 scans/s or greater) of current generation TOFMS detectors. Chromatographic peak maxima (serving as the retention time, tR) above a user specified signal threshold are located, and the chromatographic peak width, W, are determined on a per mass channel (m/z) basis for each analyte peak. The peak W (per m/z) is then plotted against its respective tR (with 10 ms precision) in a two-dimensional (2D) format, producing a cluster of points (i.e., one point per peak W versus tR in the 2D plot). Analysis of GC-TOFMS data by this method produces what is referred to as a two-dimensional mass channel cluster plot (2D m/z cluster plot). We observed that adjacent eluting (even coeluting) peaks in a temperature programmed separation can have their peak W vary as much as ∼10-15%. Hence, the peak W provides useful chemical selectivity when viewed in the 2D m/z cluster plot format. Pairs of overlapped analyte peaks with one-dimensional GC resolution as low as Rs ≈ 0.03 can be visually identified as fully resolved in a 2D m/z cluster plot and readily deconvoluted using chemometrics (i.e., demonstrated using classical least-squares analysis). Using the 2D m/z cluster plot method, the effective peak capacity of one-dimensional GC separations is magnified nearly 40-fold in one-dimensional GC, and potentially ∼100-fold in the context of comparing it to a two-dimensional separation. The method was studied using a 73 component test mixture separated on a 30 m × 250 µm i.d. RTX-5 column with a LECO Pegasus III TOFMS.

Analytical Determination of the Severity of Potato Taste Defect in Roasted East African Arabica Coffee.

The quality of East African coffee beans has been significantly reduced by a flavor defect known as potato taste defect (PTD) due to the presence of 2-isopropyl-3-methoxypyrazine (IPMP) and 2-isobutyl-3-methoxypyrazine (IBMP). Therefore, the aims of this study were to determine the correlation between these methoxypyrazines and the severity of odor attributed to PTD and discover additional analytes that may be correlated with PTD using Fisher ratio analysis, a supervised discovery-based data analysis method. Specialty ground roasted coffees from East Africa were classified as clean (i.e., no off-odor), mild, medium, or strong PTD. For the samples examined, IPMP was found to discriminate between non-defective and defective samples, while IBMP did not do so. Samples affected by PTD exhibited a wide range of IPMP concentration (1.6-529.9 ng/g). Except for one sample, the IPMP concentration in defective samples was greater than the average IPMP concentration in the non-defective samples (2.0 ng/g). Also, an analysis of variance found that IPMP concentrations were significantly different based on the severity of odor attributed to PTD (p < 0.05). Fisher ratio analysis discovered 21 additional analytes whose concentrations were statistically different based on the severity of PTD odor (p < 0.05). Generally, analytes that were positively correlated with odor severity generally had unpleasant sensory descriptions, while analytes typically associated with desirable aromas were found to be negatively correlated with odor severity. These findings not only show that IPMP concentration can differentiate the severity of PTD but also that changes in the volatile analyte profile of coffee beans induced by PTD can contribute to odor severity.

Development of gas chromatographic pattern recognition and classification tools for compliance and forensic analyses of fuels: A review.

Gas chromatography (GC) is undoubtedly the analytical technique of choice for compositional analysis of petroleum-based fuels. Over the past twenty years, as comprehensive two-dimensional gas chromatography (GC × GC) has evolved, fuel analysis has often been highlighted in scientific reports, since the complexity of fuel analysis allows for illustration of the impressive peak capacity gains afforded by GC × GC. Indeed, several research groups in recent years have applied GC × GC and chemometric data analysis to demonstrate the potential of these analytical tools to address important compliance (tax evasion, tax credits, physical quality standards) and forensic (arson investigations, oil spills) applications involving fuels. None the less, routine use of GC × GC in forensic laboratories has been limited largely by (1) legal and regulatory guidelines, (2) lack of chemometrics training, and (3) concerns about the reproducibility of GC × GC. The goal of this review is to highlight recent advances in one-dimensional GC (1D-GC) and GC × GC analyses of fuels for compliance and forensic applications, to assist scientists in overcoming the aforementioned hindrances. An introduction to 1D-GC principles, GC × GC technology (column stationary phases and modulators) and several chemometric methods is provided. More specifically, chemometric methods will be broken down into (1) signal preprocessing, (2) peak decomposition, identification and quantification, and (3) classification and pattern recognition. Examples of compliance and forensic applications will be discussed with particular emphasis on the demonstrated success of the employed chemometric methods. This review will hopefully make 1D-GC and GC × GC coupled with chemometric data analysis tools more accessible to the larger scientific community, and aid in eventual widespread standardization.

Methods of discovery-based and targeted metabolite analysis by comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detection.

The investigation of naturally volatile and derivatized metabolites in biological tissues by comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC-TOFMS) can provide highly complex and information-rich data for comprehensive metabolomics analysis. The addition of the second separation dimension with GC × GC provides additional chemical selectivity, and the fast scanning time of TOFMS offers benefits in chemical selectivity and overall peak capacity compared to traditional one-dimensional (1D) GC. Furthermore, methods of derivatization to facilitate volatility and thermal stability, the most prominent being the silylation of organic compounds, have extended the use of GC as an important metabolomics tool. The highly information-rich data from GC × GC-TOFMS benefits from sophisticated comprehensive targeted and nontargeted algorithmic software methods. Herein, we detail a robust derivatization and instrumental method for metabolomics analysis and provide a brief overview of possible methods for data analysis.

Analysis of commercial beverage products by size exclusion chromatography coupled with UV-vis absorbance detection and dynamic surface tension detection.

Multidimensional analysis of instant coffee and barley beverage samples using size exclusion chromatography (SEC) combined with a dynamic surface tension detector (DSTD) and a UV-vis absorbance detector (UV) is reported. A unique finding of this study was the action of the tetrabutylammonium (TBA) cation as a modifying agent (with bromide as the counter anion) that substantially increased the surface pressure signal and sensitivity of many of the proteins in the chromatographically separated samples. The tetrabutylammonium bromide (TBAB) enhancement of the surface pressure signal was further investigated by studying the response of 12 commercial standard proteins (alpha-lactalbumin, beta-lactoglobulin, human serum albumin (HSA), albumin from chicken egg white (OVA), bovine serum albumin (BSA), hemoglobin, alpha-chymotrypsinogen A, cytochrome C, myoglobin, RNase A, carbonic anhydrase, and lysozyme) in buffer performed using flow injection analysis (FIA) coupled with the DSTD with and without various concentrations of TBAB. The FIA-DSTD data show that 1mM TBAB enhances sensitivity of HSA detection, by lowering the limit of detection (LOD) from 2mg/mL to 0.1mg/mL. Similarly, the LOD for BSA was reduced from 1mg/mL to 0.2mg/mL. These FIA-DSTD experiments allowed the detection conditions to be optimized for further SEC-UV/DSTD experiments. Thus, the SEC-UV/DSTD system has been optimized and successfully applied to the selective analysis of surface-active protein fractions in a commercial instant coffee sample and in a soluble barley sample. The complementary selectivity of using the DSTD relative to an absorbance detector is also demonstrated.

Flow injection analysis with diode array absorbance detection and dynamic surface tension detection for studying denaturation and surface activity of globular proteins.

In this article, a multidimensional dynamic surface tension detector (DSTD), in a parallel configuration with a UV-visible diode array absorbance detector, is presented in a novel flow injection analysis (FIA) application to study the effects of chemical denaturants urea, guanidinium hydrochloride (GdmHCl), and guanidinium thyocyanate (GdmSCN) on the surface activity of globular proteins at the liquid-air interface. The DSTD signal is obtained by measuring the changing pressure across the liquid-air interface of 4-mul drops repeatedly forming at the end of a capillary using FIA. The sensitivity and selectivity of the DSTD signal is related to the surface-active protein concentration in aqueous solution combined with the thermodynamics and kinetics of protein interaction at a liquid-air drop interface. Rapid on-line calibration and measurement of dynamic surface tension is applied, with the surface tension converted into surface pressure results. Continuous surface tension measurement throughout the entire drop growth is achieved, providing insight into kinetic behavior of protein interactive processes at the liquid-air drop interface. Specifically, chemical denaturation of 12 commercial globular proteins-chicken egg albumin, bovine serum albumin, human serum albumin, alpha-lactalbumin (alpha-Lac), myoglobin, cytochrome c, hemoglobin, carbonic anhydrase, alpha-chymotrypsinogen A, beta-lactoglobulin (beta-LG), lysozyme, and glyceraldehyde-3-phosphate-dehydrogenase-is studied in terms of surface pressure (i.e., surface activity) after treatment with increasing concentrations of urea, GdmHCl, and GdmSCN in the 0-8, 0-6, and 0-5 M ranges, respectively. For several of these proteins, the spectroscopic absorbance changes are monitored simultaneously to provide additional information prior to drop formation. Results show that surface pressure of proteins generally increases as the denaturant concentration increases and that effectiveness is GdmSCN > GdmHCl > urea. Protein unfolding curves obtained by plotting surface pressure as a function of denaturant concentration are presented and compared with respect to unfolding curves obtained by using UV absorbance and literature data. Kinetic information relative to the protein adsorption to the air-liquid interface of two proteins, alpha-Lac and beta-LG (chosen as representative proteins for comparison), denatured by the three denaturants is also studied and discussed.

High-throughput screening of protein surface activity via flow injection analysis-pH gradient-dynamic surface tension detection.

Using flow injection analysis (FIA), a pH gradient is blended in real time with a protein sample as the pH-dependent protein surface activity is measured by a dynamic surface tension detector (FIA-pH-DSTD). This instrumental system was developed as a high-throughput method for the screening of protein surface activity at the air/liquid interface as a function of pH. This method utilizes the continuous flow, drop-based dynamic surface tension detector in combination with flow injection sample introduction and blending of a steady-state concentration of protein sample with a pH gradient ranging from pH 2.0 to pH 11.5. Dynamic surface tension is measured through the differential pressure across the air/liquid interface of repeatedly growing and detaching drops. Continuous surface tension measurement is achieved for each eluting drop of 2-s length (2 muL), providing insight into both the kinetic and thermodynamic behaviors of molecular orientation processes at the liquid/air interface. Three-dimensional data are obtained, with surface tension first converted to surface pressure, which is collected as a function of elution time versus drop time. In FIA-pH-DSTD, a commercial pH probe is used to measure pH during elution time, enabling surface pressure throughout drop time to be subsequently plotted as a function of eluting pH. An automated DSTD calibration procedure and data analysis method is applied, which allows simultaneous use of two different solvents, permitting real-time dynamic surface tension data to be obtained. The method was applied to the analysis of 14 commercial purified proteins, yielding characteristic features of surface activity as a function of pH. The reproducibility of the measurement and selectivity advantage of the DSTD was shown for the analysis of serum albumins from various mammalian sources. Several applications were also suggested and discussed in order to show the potential of the method for protein and food chemistry studies and in the study of protein-polymer interactions.

Determination, by dynamic surface-tension analysis, of the molar mass of proteins denatured in guanidine thiocyanate.

A drop-based dynamic surface-tension detector (DSTD) has been used to study the dynamic surface tension behavior of proteins denatured in guanidine thiocyanate (GndSCN). The dynamic surface tension at the air-liquid interface is obtained by measuring the internal pressure of drops that grow and detach at a specified rate. In the method the sample of interest is injected and subsequently flows to the DSTD-sensing capillary tip. For this work, a novel DSTD calibration procedure utilizing two distinct mobile phases is applied. Here, the mobile phases are aqueous with different constituents, for example GndSCN and phosphate buffer, either added or omitted. The dual-mobile phase calibration procedure gives the analyst the capability of making protein measurements in a GndSCN-phosphate buffer mobile phase, while measuring a calibration standard in another mobile phase, such as water, in which the surface tension of the calibration standard is readily available. Results are presented with drop volumes of either 2 microL (i.e. 2-s drops) or 7 microL (i.e. 7-s drops) for proteins varying in molar mass from 12,000 to 330,000 g mol(-1). We demonstrate that the DSTD can be used to determine the molar mass of proteins denatured in GndSCN. The method applies a regime where the denatured protein is detected by surface-active properties, and selectivity with regard to molar mass is contained in the dynamic component of the DSTD signal. The dynamic surface pressure signals of the denatured proteins suggest that diffusion plays a large role in the kinetics of the surface activity. The limit of detection for the denatured proteins studied ranged from 3 mg L(-1) to 14 mg L(-1). The DSTD, coupled with the novel dual-mobile phase calibration procedure, can be used to investigate the fundamental properties of proteins. Insight into the behavior at the air-liquid interface for native and denatured proteins is achieved; this is a novel tool for studying protein denaturation, complementary to other common approaches such as spectroscopy and calorimetry. Furthermore, the reported method could be widely applied to the study of effects on the interfacial properties of proteins after a variety of chemical and physical modifications that are possible with the dual-mobile phase calibration procedure.