Trilinearity deviation ratio: A new metric for chemometric analysis of comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry data

Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC × GC–TOFMS) is a well-established instrumental platform for complex samples. However, chemometric data analysis is often required to fully extract useful information from the data. We demonstrate that retention time shifting from one modulation to the next, Δ²t_R, is not sufficient alone to quantitatively describe the trilinearity of a single GC × GC–TOFMS run for the purpose of predicting the performance of the chemometric method parallel factor analysis (PARAFAC). We hypothesize that analyte peak width on second dimension separations, ²W_b, also impacts trilinearity, along with Δ²t_R. The term trilinearity deviation ratio, TDR, which is Δ²t_R normalized by ²W_b, is introduced as a quantitative metric to assess accuracy for PARAFAC of a GC × GC–TOFMS data cube. We explore how modulation ratio, M_R, modulation period, P_M, temperature programming rate, T_ramp, sampling phase (in-phase and out-of-phase), and signal-to-noise ratio, S/N, all play a role in PARAFAC performance in the context of TDR. Use of a P_M in the 1–2 s range provides an optimized peak capacity for the first dimension separation (500–600) for a 30 min run, with an adequate peak capacity for the second dimension separation (12–15), concurrent with an optimized two-dimensional peak capacity (6000–7500), combined with sufficiently low TDR values (0–0.05) to facilitate low quantitative errors with PARAFAC (0–0.5%). In contrast, use of a P_M in the 5 s or greater range provides a higher peak capacity on the second dimension (30–35), concurrent with a lower peak capacity on the first dimension (100–150) for a 30 min run, and a slightly reduced two-dimensional peak capacity (3000–4500), and furthermore, the data are not sufficiently trilinear for the more retained second dimension peaks in order to directly use PARAFAC with confidence.     

Analysis of special surfactants by comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry

Multidimensional gas-chromatographic analyses of olesochemically based nonionic, anionic and several cationic surfactants in industrial cleaners are demonstrated. Comprehensive two-dimensional gas chromatography coupled with time-of-flight mass spectrometry allows the simultaneous determination of fatty alcohols, fatty alcohol sulphates and alkyl polyglucosides. In addition, the determination of fatty alcohol ethoxylates up to C₁₀EO₈ (highest degree of ethoxylation) and C₁₈EO₅ (longest C-chain at an ethoxylation degree of five) and the analysis of fatty alcohol alkoxylates that contain ethoxy (EO) and propoxy (PO) groups could be realized. Because of decomposition in the injector and a weak EI-fragmentation, cationic surfactants such as alkyl benzyl dimethyl ammonium chloride could also be identified by their characteristic fragments. Thermogravimetric analyses confirmed that the temperature in a normal GC injector is not high enough to cause thermal decomposition of esterquats. However, we could demonstrate that a modified silylation procedure forms decomposition products of esterquats in the GC injector which are detectable by GC × GC–(TOF)MS and allows the identification of such GC-atypical analytes.     

Comparison of comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry and gas chromatography-mass spectrometry for the analysis of tobacco essential oils

A sample of tobacco essential oil was analyzed using gas chromatography-mass spectrometry (GC/MS) and comprehensive two-dimensional gas chromatography coupled to a time-of-flight mass spectrometry (GC × GC/TOFMS), respectively. In the GC/MS analysis, serially coupled columns were used. By comparing the GC/MS results with GC × GC/TOFMS results, many more components in the essential oil could be found within the two-dimensional separation space of GC × GC. The quantitative determination of components in the essential oil was performed by GC × GC with flame ionization detection (FID), using a method of multiple internal standards calibration.     

Headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry for the determination of volatile compounds from marine salt

In this work, a methodology to characterise the volatile and semi-volatile compounds from marine salt by headspace solid-phase microextraction (HS-SPME) and comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry (GC × GC/TOFMS) was developed. Samples from two saltpans of Aveiro, in Portugal, with diverse locations, obtained over three years (2004, 2005, and 2007) were analysed. A 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane SPME fibre was used. The volatiles present in the headspace of the solid salt samples (crystals) were equilibrated overnight at 60 °C and extracted for 60 min prior to injection in the GC × GC/TOFMS. 157 compounds, distributed over the chemical groups of hydrocarbons, aldehydes, esters, furans, haloalkanes, ketones, ethers, alcohols, terpenoids, C₁₃ norisoprenoids, and lactones were detected across the samples. Furans, haloalkanes and ethers were identified for the first time in marine salt. The large number of co-elutions on the first column that were resolved by the GC × GC system revealed the complexity of marine salt volatile composition. The existence of a structured 2D chromatographic behaviour according to volatility, in the first dimension (¹D), and primarily polarity, in the second dimension (²D), was demonstrated, allowing more reliable identifications. The resolution and sensitivity of GC × GC/TOFMS enabled the separation and identification of a higher number of volatile compounds compared to GC–qMS, allowing a deeper characterisation of this natural product.     

Analysis of bacterial fatty acids by flow modulated comprehensive two-dimensional gas chromatography with parallel flame ionization detector/mass spectrometry

Comprehensive two-dimensional gas chromatography (GC × GC) offers an interesting tool for profiling bacterial fatty acids. Flow modulated GC × GC using a commercially available system was evaluated, different parameters such as column flows and modulation time were optimized. The method was tested on bacterial fatty acid methyl esters (BAMEs) from Stenotrophomonas maltophilia LMG 958^T by using parallel flame ionization detector (FID)/mass spectrometry (MS). The results are compared to data obtained using a thermal modulated GC × GC system. The data show that flow modulated GC × GC-FID/MS method can be applied in a routine environment and offers interesting perspectives for chemotaxonomy of bacteria.     

Quantitative assessment of moisture damage for cacao bean quality using two-dimensional gas chromatography combined with time-of-flight mass spectrometry and chemometrics

Quality control of cacao beans is a significant issue in the chocolate industry. In this report, we describe how moisture damage to cacao beans alters the volatile chemical signature of the beans in a way that can be tracked quantitatively over time. The chemical signature of the beans is monitored via sampling the headspace of the vapor above a given bean sample. Headspace vapor sampled with solid-phase micro-extraction (SPME) was detected and analyzed with comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC–TOFMS). Cacao beans from six geographical origins (Costa Rica, Ghana, Ivory Coast, Venezuela, Ecuador, and Panama) were analyzed. Twenty-nine analytes that change in concentration levels via the time-dependent moisture damage process were measured using chemometric software. Biomarker analytes that were independent of geographical origin were found. Furthermore, prediction algorithms were used to demonstrate that moisture damage could be verified before there were visible signs of mold by analyzing subsets of the 29 analytes. Thus, a quantitative approach to quality screening related to the identification of moisture damage in the absence of visible mold is presented.     

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry for metabonomics: Biomarker discovery for diabetes mellitus

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC-TOFMS) coupled with pattern recognition methods was applied to analyze plasma from diabetic patients and healthy controls. After sample preparation and GC × GC-TOFMS analysis, collected data were transformed, the peak alignment between different chromatograms was performed to generate the metabolites’ peak table, then orthogonal signal correction filtered partial least-squares discriminant analysis (OSC-PLSDA) was carried out to model the data and discover metabolites with a significant concentration change in diabetic patients. With the method above, diabetic patients and healthy controls could be correctly distinguished based on the metabolic abnormity in plasma. Five potential biomarkers including glucose, 2-hydroxyisobutyric acid, linoleic acid, palmitic acid and phosphate were identified. It was found that elevated free fatty acids were essential pathophysiological factors in diabetes mellitus which reflected either the hyperglycemia or the deregulation of fatty acids metabolism. These potential biomarkers in plasma, e.g. palmitic acid, linoleic acid and 2-hydroxybutyric acid might be helpful in the diagnosis or further study of diabetes mellitus. This study shows the practicability and advantage of GC × GC-TOFMS coupled with data analysis and mining for metabonomics in biomarker discovery.     

Amino acid analysis by using comprehensive two-dimensional gas chromatography

The separation characteristics of alkylchloroformate-derivatised amino acids (AAs) by using comprehensive two-dimensional gas chromatography (GC×GC) is reported. The use of a low-polarity/polar column set did not provide as good a separation performance as that achieved with a polar/non-polar column set, where the latter appeared to provide less correlation over the separation space. The degree of component correlation in each column set was estimated by using the correlation coefficient (r²; for ¹t_R and ²t_R data) with the low-polarity/polar and polar/low-polarity sets returning correlation coefficients of 0.86, and 0.00 respectively, under the respective conditions employed for the experiments. The 1.5-m non-polar ²D column (0.1-mm ID; 0.1-m film thickness) gave peak halfwidths of the order of 50–80 ms. Linearity of detection was good, over a three order of magnitude concentration range, with typical lower detection limit of ca. 0.01 mg L^–1, compared with 0.5 mg L^–1 for normal GC operation with splitless injection. The method was demonstrated for analysis of AAs in a range of food and beverage products, including wine, beer and honey. The major AA in these samples was proline. The Heineken beer sample had a relatively more complex and more abundant AA content compared with the other beer sample. The wine and honey samples also gave a range of AA compounds. Repetition of the sample preparation/analysis procedure for the honey sample gave acceptable reproducibility for individual AAs.     

A principal component analysis based method to discover chemical differences in comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GCxGC-TOFMS) separations of metabolites in plant samples

Comprehensive two-dimensional gas chromatography combined with time-of-flight mass spectrometry (GC × GC-TOFMS) provides high resolution separations of complex samples with a mass spectrum at every point in the separation space. The large volumes of multidimensional data obtained by GC × GC-TOFMS analysis are analyzed using a principal component analysis (PCA) method described herein to quickly and objectively discover differences between complex samples. In this work, we submitted 54 chromatograms to PCA to automatically compare the metabolite profiles of three different species of plants, namely basil (Ocimum basilicum), peppermint (Mentha piperita), and sweet herb stevia (Stevia rebaudiana), where there were 18 chromatograms for each type of plant. The 54 scores of the m/z 73 data set clustered in three groups according to the three types of plants. Principal component 1 (PC 1) separated the stevia cluster from the basil and peppermint clusters, capturing 61.84% of the total variance. Principal component 2 (PC 2) separated the basil cluster from the peppermint cluster, capturing 16.78% of the total variance. The PCA method revealed that relative abundances of amino acids, carboxylic acids, and carbohydrates were responsible for differentiating the three plants. A brief list of the 16 most significant metabolites is reported. After PCA, the 54 scores of the m/z 217 data set clustered in three groups according to the three types of plants, as well, yielding highly loaded variables corresponding with chemical differences between plants that were complementary to the m/z 73 information. The PCA data mining method is applicable to all of the monitored selective mass channels, utilizing all of the collected data, to discover unknown differences in complex sample profiles.     

A comprehensive two-dimensional gas chromatography coupled with quadrupole mass spectrometry approach for identification of C10 derivatives from decalin

A detailed mass map of C₁₀'s is required to better understand the mechanism of decalin catalytic ring opening/rearrangement. Conventional GC-FID or GC-MSD techniques could not accurately identify these isomers. Comprehensive two-dimensional gas-chromatography with MSD (GC × GC-MSD) proved to be a powerful tool for this purpose, due to its enhanced peak resolution. Analytical response quality was evaluated by the separation of two contiguous peaks and MS profile “clearness”. This allowed fragmentation study for nearly pure species. Tentative attributions, based on fragmentation-rearrangement in the MSD environment, were made after confirming that MS data bases routinely mistake olefins for cyclo-alkanes.     

Qualitative and quantitative analysis of pyrolysis oil by gas chromatography with flame ionization detection and comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry

Pyrolysis oils have attracted a lot of interest, as they are liquid energy carriers and general sources of chemicals. In this work, gas chromatography with flame ionization detector (GC-FID) and two-dimensional gas chromatography with time-of-flight mass spectrometry (GC×GC-TOFMS) techniques were used to provide both qualitative and quantitative results of the analysis of three different pyrolysis oils. The chromatographic methods and parameters were optimized and solvent choice and separation restrictions are discussed. Pyrolysis oil samples were diluted in suitable organic solvent and were analyzed by GC×GC-TOFMS. An average of 300 compounds were detected and identified in all three samples using the ChromaToF (Leco) software. The deconvoluted spectra were compared with the NIST software library for correct matching. Group type classification was performed by use of the ChromaToF software. The quantification of 11 selected compounds was performed by means of a multiple-point external calibration curve. Afterwards, the pyrolysis oils were extracted with water, and the aqueous phase was analyzed both by GC-FID and, after proper change of solvent, by GC×GC-TOFMS. As previously, the selected compounds were quantified by both techniques, by means of multiple point external calibration curves. The parameters of the calibration curves were calculated by weighted linear regression analysis. The limit of detection, limit of quantitation and linearity range for each standard compound with each method are presented. The potency of GC×GC-TOFMS for an efficient mapping of the pyrolysis oil is undisputable, and the possibility of using it for quantification as well has been demonstrated. On the other hand, the GC-FID analysis provides reliable results that allow for a rapid screening of the pyrolysis oil. To the best of our knowledge, very few papers have been reported with quantification attempts on pyrolysis oil samples using GC×GC-TOFMS most of which make use of the internal standard method. This work provides the ground for further analysis of pyrolysis oils of diverse sources for a rational design of both their production and utilization process.     

Optimization of two-dimensional gas chromatography time-of-flight mass spectrometry for separation and estimation of the residues of 160 pesticides and 25 persistent organic pollutants in grape and wine

Two-dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometric (TOFMS) method was optimized for simultaneous analysis of 160 pesticides, 12 dioxin-like polychlorinated biphenyls (PCBs), 12 polyaromatic hydrocarbons (PAHs) and bisphenol A in grape and wine. GC × GC–TOFMS could separate all the 185 analytes within 38 min with >85% NIST library-based mass spectral confirmations. The matrix effect quantified as the ratio of the slope of matrix-matched to solvent calibrations was within 0.5–1.5 for most analytes. LOQ of most of the analytes was ≤10 μg/L with nine exceptions having LOQs of 12.5–25 μg/L. Recoveries ranged between 70 and 120% with <20% expanded uncertainties for 151 and 148 compounds in grape and wine, respectively, with intra-laboratory Horwitz ratio <0.2 for all analytes. The method was evaluated in the incurred grape samples where residues of cypermethrin, permethrin, chlorpyriphos, metalaxyl and etophenprox were detected at below MRL.     

Supercritical fluid chromatography hyphenated with twin comprehensive two-dimensional gas chromatography for ultimate analysis of middle distillates

This paper reports the conditions of online hyphenation of supercritical fluid chromatography (SFC) with twin comprehensive two-dimensional gas chromatography (twin-GC × GC) for detailed characterization of middle distillates; this is essential for a better understanding of reactions involved in refining processes. In this configuration, saturated and unsaturated compounds that have been fractionated by SFC are transferred on two different GC × GC columns sets (twin-GC × GC) placed in the same GC oven. Cryogenic focusing is used for transfer of fractions into the first dimension columns before simultaneous GC × GC analysis of both saturated and unsaturated fractions. The benefits of SFC–twin-GC × GC are demonstrated for the extended alkane, iso-alkane, alkene, naphthenes and aromatics analysis (so-called PIONA analysis) of diesel samples which can be achieved in one single injection. For that purpose, saturated and unsaturated compounds have been separated by SFC using a silver loaded silica column prior to GC × GC analysis. Alkenes and naphthenes are quantitatively recovered in the unsaturated and saturated fractions, respectively, allowing their identification in various diesel samples. Thus, resolution between each class of compounds is significantly improved compared to a single GC × GC run, and for the first time, an extended PIONA analysis of diesel samples is presented.     

Factors affecting peak shape in comprehensive two-dimensional gas chromatography with non-focusing modulation

In the case of a non-focusing modulator for comprehensive two-dimensional gas chromatography (GC × GC), the systematic distortions introduced when the modulator loads the second-dimension column give rise to a characteristic peak shape. Depending on the operating conditions this systematic distortion can be the dominant component of the second-dimension elution profiles in the GC × GC peak. The present investigation involved a systematic investigation of peak shape in pulsed-flow modulation (PFM)–GC × GC. It is shown that low flow ratio can lead to significant peak skewing and increasing the flow ratio reduces the magnitude of peak skewing. Validation of the peak shape model is made by comparison with experimental data. The residuals from the fitting process (normalised to the maximum detector response) vary between –1.5% and +2.6% for an isothermal model and between –1.0% and +3.0% for a temperature-programmed model.     

Enhanced resolution comprehensive two-dimensional gas chromatography applied to the analysis of roasted coffee volatiles

The present research is based on the full exploitation of the separation power of a 0.05 mm internal diameter (ID) capillary, as a comprehensive two-dimensional (2D) GC (GC × GC) secondary column, with the objective of attaining very high-resolution second dimension separations. The aim was achieved by using a split-flow system developed in previous research [P.Q. Tranchida, A. Casilli, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 79 (2007) 2266], and a dual-oven GC × GC instrument. The column combination employed consisted of a polar 30 m × 0.25 mm ID column connected, by means of a T union, to a detector-linked high-resolution 1.1 m × 0.05 mm ID apolar analytical column and to a 0.33 m × 0.05 mm ID retention gap; the latter was connected to a manually operated split valve. As previously demonstrated, the use of a split valve enables the regulation of gas flows through both analytical columns, generating the most appropriate gas linear velocities. Comprehensive 2D GC experiments were carried out on Arabica roasted coffee volatiles (previously extracted by means of solid-phase microextraction) with the split-valve closed (equal to what can be defined as conventional GC × GC) and with the split-valve opened at various degrees. The reasons why it is absolutely not effective to use a 0.05 mm ID column as second dimension in a conventional GC × GC instrument will be discussed and demonstrated. On the contrary, the use of a 0.05 mm ID column as second dimension, under ideal conditions in a split-flow, twin-oven system, will also be illustrated and discussed.     

Measurement of retention in comprehensive two-dimensional gas chromatography using flow modulation with methane dopant

Flow modulation of methane-doped carrier gas is used to visualize the second dimension hold-up time in GC × GC continuously throughout the run. This provides an internal reference of hold-up time and presents a straightforward means of examining retention in each dimension of GC × GC. Retention factors on similar and dissimilar column pairs are examined. Stationary phase bleed is shown to be retained by the second dimension column.     

Analysis of hydroxylated polycyclic aromatic hydrocarbons in urine using comprehensive two-dimensional gas chromatography with a flame ionization detector

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in human urine is the method of choice for assessing exposure to carcinogenic compounds. The objective of this study was the development of a comprehensive two-dimensional gas chromatography (GC × GC) method using a flame ionisation detector (FID) to simultaneously determine 10 hydroxylated PAH. The method was based on enzymatic deconjugation, liquid–liquid extraction, and trimethylsilyl (TMS) derivatization of the analytes by microwave heating. Satisfactory separation was achieved. The coefficient of variance was 3.8–12.8%. LOD was 0.03–0.18 μg/L, and LOQ was 0.1–0.5 μg/L. The mean recovery was 76%. The method was applied to the analysis of urine from smokers and non-smokers.     

Comprehensive two-dimensional gas chromatography, retention indices and time-of-flight mass spectra of flavonoids and chalcones

The applicability of comprehensive two-dimensional gas chromatography (GC×GC) for flavonoids analysis was investigated by separation and identification of flavonoids in standards, and a complex matrix natural sample. The modulation temperature was optimized to achieve the best separation and signal enhancement. The separation pattern of trimethylsilyl (TMS) derivatives of flavonoids was compared on two complementary column sets. Whilst the BPX5/BPX50 (NP/P) column set offers better overall separation, BPX50/BPX5 (P/NP) provides better peak shape and sensitivity. Comparison of the identification power of GC×GC-TOFMS against both the NIST05 MS library and a laboratory (created in-house) TOFMS library was carried out on a flavonoid mixture. The basic retention index information on high-performance capillary columns with a non-polar stationary phase was established and database of mass spectra of trimethylsilyl derivatives of flavonoids was compiled. TOFMS coupled to GC×GC enabled satisfactory identification of flavonoids in complex matrix samples at their LOD over a range of 0.5-10 μg/mL. Detection of all compounds was based on full-scan mass spectra and for each compound a characteristic ion was chosen for further quantification. This study shows that GC×GC-TOFMS yields high specificity for flavonoids derived from real natural samples, dark chocolate, propolis, and chrysanthemum.     

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry detection: analysis of amino acid and organic acid trimethylsilyl derivatives, with application to the analysis of metabolites in rye grass samples

First, standard mixtures of trimethylsilyl (TMS) derivatives of amino acid and organic acid are analyzed by comprehensive two-dimensional (2D) gas chromatography (GC) coupled to time-of-flight mass spectrometry (GC × GC/TOFMS) in order to illustrate important issues regarding application of the technique. Specifically of interest is the extent to which the peak capacity of the 2D separation space has been utilized and the procedure by which the derivative standards are identified in the 2D separations using the mass spectral information. The resulting 2D separation is found to make extensive use of the GC × GC separation space provided by the complementary stationary phases employed.Second, in order to demonstrate GC × GC/TOFMS on two real sample types, trimethylsilyl metabolite derivatives were analyzed from extracts of common lawn grass samples (i.e., perennial rye grass), as a means to provide insight into both the pre and post harvest physiology. Various chemical components in the two rye grass extract samples were found to either emerge or disappear in relation to the trauma response. For example, a significant difference in the peak for the TMS derivative of malic acid was found. The successful analysis of various components was readily facilitated by the 2D separation, while a 1D separation would have produced too much peak overlap, thus impeding the analysis. The importance of using a GC × GC separation approach for the analysis of complex samples, such as metabolite extracts, is therefore demonstrated. The real-time analysis capability of GC × GC/TOFMS for multidimensional metabolite analysis makes this technique well suited to the high-throughput analysis of metabolomic samples, especially compared to slower, stopped-flow type separation approaches.     

Analysis of organic compounds of water-in-crude oil emulsions separated by microwave heating using comprehensive two-dimensional gas chromatography and time-of-flight mass spectrometry

In this work the higher peak capacity and resolution of comprehensive two-dimensional gas chromatography (GCxGC) has been successfully applied, for the first time, to tentatively identify several polar organic compounds of organic extracts of aqueous phases resulting from microwave demulsification process of water-in-crude oil emulsions. Results have shown that higher temperatures and longer exposure time to microwave irradiation produced water phases with a wider variety of polar organic compounds. The microwave process showed to be suitable for the extraction of several polar compounds classes of petroleum. The proposed microwave extraction method and GCxGC identification of polar compounds of petroleum samples are of practical interest for the petrochemical industry due to corrosion and related problems associated with these polar compounds in refinery equipments. The GCxGC/time-of-flight MS technique shows to be very important in the total separation of different classes of compounds and allows the identification of many compounds in these classes.