Second-dimension Retention Times Research Articles

Lipid Wizard: Analysis Software for Comprehensive Two-Dimensional Liquid Chromatography-Mass Spectrometry-Based Lipid Profiling.

Lipids play a significant role in life activities and participate in the biological system through different pathways. Although comprehensive two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) has been developed to profile lipid abundance changes, lipid identification and quantification from 2DLC-MS data remain a challenge. We created Lipid Wizard, open-source software for lipid assignment and isotopic peak stripping of the 2DLC-MS data. Lipid Wizard takes the peak list deconvoluted from the 2DLC-MS data as input and assigns each isotopic peak to the lipids recorded in the LIPID MAPS database by precursor ion m/z matching. The matched lipids are then filtered by the first-dimension retention time (1D RT), followed by the second-dimension retention time (2D RT), where the 2D RT of each lipid is predicted using an equivalent carbon number (ECN) model. The remaining assigned lipids are used for isotopic peak stripping via an iterative linear regression. The performance of Lipid Wizard was tested using a set of lipid standards and then applied to study the lipid changes in the livers of mice (fat-1) fed with alcohol.

A deep learning-based simulator for comprehensive two-dimensional GC applications.

Among the main approaches for predicting the spatial positions of eluates in comprehensive two-dimensional gas chromatography, the still under-explored computational models based on deep learning algorithms emerge as robust and reliable options due to their high adaptability to the structure and complexity of the data. In this work, an open-source program based on deep neural networks was developed to optimize chromatographic methods and simulate operating conditions outside the laboratory. The deep neural networks models were fit to convenient experimental predictors, resulting in scaled losses (mean squared error) equivalent to 0.006 (relative average deviation=8.56%, R2 =0.9202) and 0.014 (relative average deviation=1.67%, R2 =0.8009) in the prediction of the first- and second-dimension retention times, respectively. Good compliance was observed for the main chemical classes, such as environmental contaminants: volatile, semivolatile organic compounds, and pesticides; biochemistry molecules: amino acids and lipids; pharmaceutical industry and personal care products and residues: drugs and metabolites; among others. On the other hand, there is a need for continuous database updates to predict retention times of less common compounds accurately. Thus, forming a collaborative database is proposed, gathering voluntary findings from other users.

Top-Down Approach to Retention Time Prediction in Comprehensive Two-Dimensional Gas Chromatography-Mass Spectrometry.

In this contribution, we describe a novel modeling approach to predicting retention times (tr) in comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC-ToF-MS) with a particular emphasis on the second-dimension (2D) retention time predictions (2tr). This approach is referred to as a "top-down" approach in that it breaks down the complete GC × GC separation into two independent one-dimensional gas chromatography separations (1D-GC). In this regard, both dimensions, that is, first dimension (1D) and second dimension (2D) are treated separately, and the cryogenic modulator is simply considered as a second consecutive injection device. Separate 1D-GC tr predictions are performed on both dimensions using the same flow rate as the one deployed in the conventional GC × GC system. The separate tr predictions are then combined to account for the two-dimensional separation. This model was applied to 24 analytes from 2 standard mixtures (Grob Test Mix and Fragrance Materials Test Mix) and assessed across 9 GC × GC chromatographic conditions. The experimental and predicted chromatographic retention space occupations were assessed by using the convex hull approach defined by the Delaunay triangulation. The predicted percentage of space occupation corresponded favorably with the experimental values. Furthermore, the top-down approach enabled an accurate prediction of the 2tr of all investigated analytes, providing an average 2tr modeling error of 0.26 ± 0.01 s.

Biomarkers of photosynthetic sulfur bacteria in Lower Cretaceous crude oils, East Brazilian marginal basin

Diagenetic aromatic carotenoids and degradation products, which are derived from the carotenoids of photosynthetic sulfur bacteria, were characterized in the aromatic fraction of Brazilian Cretaceous crude oils (NE Brazil, Sergipe-Alagoas Basin) by high-temperature comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (HT-GC × GC–TOFMS). Unresolved complex mixtures (UCM), coeluting compounds, and limited mass spectrometric identification were noted by traditional one-dimensional (1D) GC–MS screening of aromatic compounds. Group-type separation on a two-dimensional chromatographic plane, first- and second-dimension retention times (1tR and 2tR, respectively), and mass spectra enabled the assignment of alkylated mono-, di-, tri-, and tetra-aryl hydrocarbons with confidence. A series of diagenetic C40 carotenoids and degraded products, which are likely indicators for green-brown sulfur bacteria, were detected. This finding corroborates previous results, and isorenieratane was relatively more abundant than 2,3,6-trimethylaryl isoprenoids. Moreover, atropisomeric biphenyls (m/z 237), which coeluted using 1D GC–MS analyses, were resolved by GC × GC and identified by mass spectrometry. The identification of minor abundances of chlorobactane, diagenetic biphenyl chlorobactane, and related compounds, for the first time in these Brazilian Cretaceous oils, may indicate a contribution of green sulfur bacteria (Chlorobiaceae) and photic zone euxinia in shallow waters.

Occurrence of extended tetracyclic polyprenoid series in crude oils

A homologous series of tetracyclic polyprenanes from C24 to C41 was identified in crude oils from Espírito Santo Basin (Brazil) by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry (GC×GC–TOFMS) and gas chromatography–tandem mass spectrometry (GC–MS-MS) analyses. These products were separately characterized by elution order in first- and second-dimension retention times (1tR and 2tR, respectively), time-of-flight mass spectra (TOFMS), MRM chromatograms, product ion spectra including diagnostic ions at m/z 191, m/z 259 and m/z 149 + 14n (n = 0–17), the latter a key mass fragment for the evaluation of side chain extension at the C-18 position in tetracyclic polyprenoids. The tetracyclic compounds were thus identified as scalarane-like structures related to abundant C30 tetracyclic polyprenoids, an important indicator of input from fresh to brackish water paleoenvironments.

Thermodynamic-based retention time predictions of endogenous steroids in comprehensive two-dimensional gas chromatography.

This work evaluates the application of a thermodynamic model to comprehensive two-dimensional gas chromatography (GC × GC) coupled with time-of-flight mass spectrometry for anabolic agent investigation. Doping control deals with hundreds of drugs that are prohibited in sports. Drug discovery in biological matrices is a challenging task that requires powerful tools when one is faced with the rapidly changing designer drug landscape. In this work, a thermodynamic model developed for the prediction of both primary and secondary retention times in GC × GC has been applied to trimethylsilylated hydroxyl (O-TMS)- and methoxime-trimethylsilylated carbonyl (MO-TMS)-derivatized endogenous steroids. This model was previously demonstrated on a pneumatically modulated GC × GC system, and is applied for the first time to a thermally modulated GC × GC system. Preliminary one-dimensional experiments allowed the calculation of thermodynamic parameters (ΔH, ΔS, and ΔC p ) which were successfully applied for the prediction of the analytes' interactions with the stationary phases of both the first-dimension column and the second-dimension column. The model was able to predict both first-dimension and second-dimension retention times with high accuracy compared with the GC × GC experimental measurements. Maximum differences of -8.22 s in the first dimension and 0.4 s in the second dimension were encountered for the O-TMS derivatives of 11β-hydroxyandrosterone and 11-ketoetiocholanolone, respectively. For the MO-TMS derivatives, the largest discrepancies were from testosterone (9.65 ) for the first-dimension retention times and 11-keto-etiocholanolone (0.4 s) for the second-dimension retention times.

Application of thermodynamic-based retention time prediction to ionic liquid stationary phases

First- and second-dimension retention times for a series of alkyl phosphates were predicted for multiple column combinations in GC×GC. This was accomplished through the use of a three-parameter thermodynamic model where the analytes' interactions with the stationary phases in both dimensions are known. Ionic liquid columns were employed to impart unique selectivity for alkyl phosphates, and it was determined that for alkyl phosphate compounds, ionic liquid columns are best used in the primary dimension. Retention coordinates for unknown phosphates are predicted from the thermodynamic parameters of a set standard alkyl phosphates. Additionally, we present changing retention properties of alkyl phosphates on some ionic liquid columns, due to suspected reaction between the analyte and column. This makes it difficult to accurately predict their retention properties, and in general poses a problem for ionic liquid columns with these types of analytes.

Bayesian Approach for Peak Detection in Two-Dimensional Chromatography

A new method for peak detection in two-dimensional chromatography is presented. In a first step, the method starts with a conventional one-dimensional peak detection algorithm to detect modulated peaks. In a second step, a sophisticated algorithm is constructed to decide which of the individual one-dimensional peaks have been originated from the same compound and should then be arranged in a two-dimensional peak. The merging algorithm is based on Bayesian inference. The user sets prior information about certain parameters (e.g., second-dimension retention time variability, first-dimension band broadening, chromatographic noise). On the basis of these priors, the algorithm calculates the probability of myriads of peak arrangements (i.e., ways of merging one-dimensional peaks), finding which of them holds the highest value. Uncertainty in each parameter can be accounted by adapting conveniently its probability distribution function, which in turn may change the final decision of the most probable peak arrangement. It has been demonstrated that the Bayesian approach presented in this paper follows the chromatographers' intuition. The algorithm has been applied and tested with LC × LC and GC × GC data and takes around 1 min to process chromatograms with several thousands of peaks.

Probability of failure of the watershed algorithm for peak detection in comprehensive two-dimensional chromatography

The watershed algorithm is the most common method used for peak detection and integration in two-dimensional chromatography. However, the retention time variability in the second dimension may render the algorithm to fail. A study calculating the probabilities of failure of the watershed algorithm was performed. The main objective was to calculate the maximum second-dimension retention time variability, Delta(2)t(R,crit), above which the algorithm fails. Several models to calculate Delta(2)t(R,crit) were developed and evaluated: (a) exact model; (b) simplified model and (c) simple-modified model. Model (c) gave the best performance and allowed to deduce an analytical expression for the probability of failure of the watershed algorithm as a function of experimental Delta(2)t(R), modulation time and peak width in the first and second dimensions. It could be demonstrated that the probability of failure of the watershed algorithm under normal conditions in GCxGC is around 15-20%. Small changes of Delta(2)t(R), modulation time and/or peak width in the first and second dimension could induce subtle changes in the probability of failure of the watershed algorithm. Theoretical equations were verified with experimental results from a diesel sample injected in GC x GC and were found to be in good agreement with the experiments.

Dependence of Effective Peak Capacity in Comprehensive Two-Dimensional Separations on the Distribution of Peak Capacity between the Two Dimensions

One of the basic tenets of comprehensive two-dimensional chromatography is that the total peak capacity is simply the product of the first- and second-dimension peak capacities. As formulated, the total peak capacity does not depend on the relative values of the individual dimensions but only on the product of the two. This concept is tested here for the experimentally realistic situation wherein the first-dimension separation is undersampled. We first propose that a relationship exists between the number of observed peaks in a two-dimensional separation and the effective peak capacity. We then show here for a range of reasonable total peak capacities (500-4000) and various contributions of peak capacity in each dimension (10-150) that the number of observed peaks is only slightly dependent on the relative contributions over a reasonable and realistic range in sampling times (equal to the first-dimension peak standard deviation, multiplied by 0.2-16). Most of this work was carried out under the assumption of totally uncorrelated retention times. For uncorrelated separations, the small deviations from the product rule are due to the "edge effect" of statistical overlap theory and a recently introduced factor that corrects for the broadening of first-dimension peaks by undersampling them. They predict that relatively more peaks will be observed when the ratio of the first- to the second-dimension peak capacity is much less than unity. Additional complications are observed when first- and second-dimension retention times show some correlation, but again the effects are small. In both cases, deviations from the product rule are measured by the relative standard deviations of the number of observed peaks, which are typically 10 or less. Thus, although the basic tenet of two-dimensional chromatography is not exact when the first dimension is undersampled, the deviations from the product rule are sufficiently small as to be unimportant in practical work. Our results show that practitioners have a high degree of flexibility in designing and optimizing experimental comprehensive two-dimensional separations.

Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry peak sorting algorithm

We report a novel peak sorting method for the two-dimensional gas chromatography/time-of-flight mass spectrometry (GC × GC/TOF-MS) system. The objective of peak sorting is to recognize peaks from the same metabolite occurring in different samples from thousands of peaks detected in the analytical procedure. The developed algorithm is based on the fact that the chromatographic peaks for a given analyte have similar retention times in all of the chromatograms. Raw instrument data are first processed by ChromaTOF (Leco) software to provide the peak tables. Our algorithm achieves peak sorting by utilizing the first- and second-dimension retention times in the peak tables and the mass spectra generated during the process of electron impact ionization. The algorithm searches the peak tables for the peaks generated by the same type of metabolite using several search criteria. Our software also includes options to eliminate non-target peaks from the sorting results, e.g., peaks of contaminants. The developed software package has been tested using a mixture of standard metabolites and another mixture of standard metabolites spiked into human serum. Manual validation demonstrates high accuracy of peak sorting with this algorithm.

Comprehensive two-dimensional liquid chromatography with ultraviolet, evaporative light scattering and mass spectrometric detection of triacylglycerols in corn oil

An improved comprehensive two-dimensional (LC × LC) HPLC system for the analysis of triacylglycerols was developed. In the first-dimension, a Ag(I)-coated cation exchanger (250 mm × 2.1 mm, 5 μm) was employed with a gradient from 100% MeOH to 6% MeCN in MeOH at 20 μL/min. Using a 10-way valve with two switching loops, 1 min sections of the first-dimension were introduced in the second-dimension consisting of a 30 mm × 4.6 mm C18 (1.8 μm) column with an isocratic mobile phase of methanol–methyl tert-butyl ether (70:30) at 3.0 mL/min. As the second-dimension solvent was stronger than the first-dimension solvent, focusing in the second-dimension took place, leading to better separations than in previously reported analyses in which hexane was the main constituent of the first-dimension eluent. Compounds differing by 2 in their partition number were baseline separated in the second-dimension. Detection took place by UV at 210 nm, evaporative light scattering and (+)-atmospheric pressure chemical ionisation-MS with the latter giving the best results. Corn oil was investigated and 44 compounds could be detected: 34 triacylglycerols (TAGs), 8 oxygenated TAGs, and 2 TAGs containing a trans double bond. Data manipulation allowed the construction of contour plots and the automated calculation of the first- and second-dimension retention times and peak areas. Quantitative results are compared with a fatty acid methyl ester analysis, and with literature data.

Development of an algorithm for peak detection in comprehensive two-dimensional chromatography

A method for peak detection in two-dimensional chromatography is presented. The algorithm applies first the methods developed for peak detection in one-dimensional chromatography to detect peaks in one dimension. In a second step, a decision tree is applied to decide which one-dimensional peaks are originated from the same compound and have to be ‘merged’ into one two-dimensional peak. To this end, different features of the peaks (second-dimension peak regions and second-dimension retention times) are compared and different criteria (common peak regions, retention time differences, unimodality in the first dimension) are applied. Different options can be used, depending on the nature of the data. The user controls this decision tree by establishing several options and “switches”. The algorithm was tested with GC×GC chromatograms obtained for a commercial air-freshener sample, detecting and merging the modulated peaks belonging to the same compound. Recommendations for the set of options and switches are given. A utility that calculates and sums peak areas from merged peaks is added to facilitate automated quantification. Although the algorithm was developed for GC×GC, its application to comprehensive two-dimensional liquid chromatography (LC×LC) data should at most require minor modifications.

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection for the trace analysis of flavour compounds in food

The practicability and potential of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–TOF-MS) for the analysis of complex flavour mixtures in food were studied. With the determination of key flavour targets in dairy samples as an example, it was demonstrated that GC×GC dramatically improves the separation. As a consequence, identification and, more importantly, quantification down to the ng/g level can be performed more reliably: background interferences largely disappear. Next to the peak table generated from the GC–TOF-MS software after data processing, the additional use of well-ordered patterns in the 2D-plane and information from second-dimension retention times can substantially help the identification of unknowns. The technique was successfully used for an evaluation of extraction techniques and the characterisation of different types of samples.

Unravelling the composition of very complex samples by comprehensive gas chromatography coupled to time-of-flight mass spectrometry: Cigarette smoke

The potential and current limitations of comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC×GC–TOF-MS) for the analysis of very complex samples were studied with the separation of cigarette smoke as an example. Because of the large number of peaks in such a GC×GC chromatogram it was not possible to perform manual data processing. Instead, the GC–TOF-MS software was used to perform peak finding, deconvolution and library search in an automated fashion; this resulted in a peak table containing some 30 000 peaks. Mass spectral match factors were used to evaluate the library search results. The additional use of retention indices and information from second-dimension retention times can substantially improve the identification. The combined separation power of the GC×GC–TOF-MS system and the deconvolution algorithm provide a system with a most impressive separation power.

Comprehensive Two-Dimensional Gas Chromatography with Fast Enantioseparation

The development of fast chiral analysis for use in comprehensive two-dimensional gas chromatography in which a short second dimension enantioselective capillary column provides a route to precise measurement of chiral ratios of enantiomers is described. Retention times as short as 8 s are reported for (+/-)-limonene, with adequate enantioseparation maintained (Rs approximately 1.0) on a 1-m cyclodextrin derivative-coated capillary column. Sufficiently fast elution on the second column was achieved by using GC/ MS in which the subambient pressure (vacuum outlet) conditions promote increased diffusion coefficients and higher component volatility; a 4-fold reduction of second-dimension retention time was observed, as compared with ambient pressure outlet conditions. The enantiomeric distribution of several monoterpene compounds in bergamot essential oil is reported as a demonstration of the method. Total analysis time of the target components was approximately 8.5 min.

Comprehensive Two-Dimensional Gas Chromatography with a Rotating Thermal Desorption Modulator and Independently Temperature-Programmable Columns

In comprehensive two-dimensional gas chromatography, two individual separations are coupled by means of a rotating thermal desorption modulator interface. The injection pulse introduced via the interface onto the second column should be as short as possible. Parameters affecting the modulator operation are studied. In the set-up used in this study, the temperature of the second column can be programmed independently from that of the first column. Optimization of the second-dimension separation to minimize peak broadening and maximize resolution is discussed and an elegant approach to determine second-dimension retention times using a non-constant modulation frequency is demonstrated. The high separation power of the comprehensive system is demonstrated by the analysis of technical and biota samples containing chlorinated biphenyls and toxaphene.

Comprehensive Two-Dimensional Gas Chromatography with a Rotating Thermal Desorption Modulator and Independently Temperature-Programmable Columns

In comprehensive two-dimensional gas chromatography, two individual separations are coupled by means of a rotating thermal desorption modulator interface. The injection pulse introduced via the interface onto the second column should be as short as possible. Parameters affecting the modulator operation are studied. In the set-up used in this study, the temperature of the second column can be programmed independently from that of the first column. Optimization of the second-dimension separation to minimize peak broadening and maximize resolution is discussed and an elegant approach to determine second-dimension retention times using a non-constant modulation frequency is demonstrated. The high separation power of the comprehensive system is demonstrated by the analysis of technical and biota samples containing chlorinated biphenyls and toxaphene.