High-resolution Time-of-flight Mass Spectrometry Research Articles

Nanomolar activity of 4-hydrazinylphenyl benzenesulfonate against breast cancer Michigan Cancer Foundation-7 cell lines.

Hydrazine is an alkaline reduction compound which is widely used in synthesis. Based on the structure-activity analysis, to elicit antitumor activity, the presence of the N-methyl group is an absolute requirement. The aim of the research is to synthesize a new hydrazine derivate compound that has potency as a novel anti-breast cancer. 4-hydrazinylphenyl benzenesulfonate was synthesized employing reduction and diazotization methods. Structure characterization was carried out using Fourier transform infrared (FTIR), C13-nuclear magnetic resonance (NMR), H1-NMR, and High Resolution Time-of-Flight Mass Spectrometry (HR-TOF-MS). The anti-cancer activity of this compound against breast cancer Michigan Cancer Foundation-7 (MCF-7) cell line was determined using a PrestoBlue viability assay. The new of hydrazine derivative, 4-hydrazinylphenyl benzenesulfonate, has been successfully synthesized. The reduction and diazotization methods have been successfully used in the synthesis of new compound of hydrazine derivatives. Structure characterization of 4-hydrazinylphenyl benzenesulfonate was established using FTIR, C13-NMR, H1-NMR, and HR-TOF-MS. The anti-cancer activity of this compound against breast cancer MCF-7 cell line was determined using a PrestoBlue viability assay with IC50 0.00246 μg/mL or 9.32 nM. In conclusion, 4-hydrazinylphenyl benzenesulfonate was successfully synthesized as a new candidate for anti-breast cancer compound.

Tetrahydroxyphthalocyanine as a potential nonlinear optical material

We have synthesized a new zinc(II)-phthalocyanine (ZnPc) incorporating hydroxyl groups at the peripheral positions. This new zinc(II)-tetra-hydroxyphthalocyanine (1) has been characterized by high-resolution MALDI TOF MS, UV-Vis, FT-IR (ATR), and 1H- and 13C-NMR techniques, and further by the electrochemical measurements. DFT and TD-DFT computations were performed to model plausible structure and also analyze the electronic structure and optical properties of 1. The effects of the polarity and hydrogen bonding ability of the hydroxyl substituents on the peripheral positions of 1 on the optical and electrochemical properties are reported in this study. The redox behavior of 1 in dimethyl sulfoxide (DMSO) was determined by voltammetry and colorimetry supported in situ spectroelectrochemistry. The nonlinear absorption and optical limiting (OL) properties of 1 were measured in tetrahydrofuran (THF) by utilizing the open aperture Z-scan technique with nanosecond pulses at 532 nm. The sample demonstrated good reverse saturable absorption and OL behaviors. The ultrafast pump-probe experiments revealed the intersystem crossing (ISC) mechanism (triplet-triplet transition) and the nonlinear absorption mechanism of the compound. The results indicate that this new phthalocyanine (1) can be considered a potential candidate for low-power OL applications.

High performance Reversed-Phase Thin-Layer Chromatography-Desorption electrospray ionisation - time of flight high resolution mass spectrometric detection and imaging (HPTLC/DESI/ToFMS) of phytoecdysteroids

Reversed-phase high performance thin-layer chromatography (RP-HPTLC) on C18 bonded silica gel was combined with desorption electrospray ionization (DESI) and high resolution time of flight mass spectrometry (HRToFMS) to detect, characterize and image (MSI) phytoecdysteroids (plant-derived insect moulting hormones) in ethanolic extracts of members of the Silene plant family. As seen previously for silica gel, DESI provided a simple and convenient method for recovering polar polyhydoxysteroids from RP-HPTLC plates for the purposes of both the MS and MSI of extracts obtained from three species of the Silene family (Silene otites, S. nutans and S. viridiflora). Using RP-HPTLC/DESI/MSI/HRToFMS a number of ecdysteroids, including 20-hydroxyecdysone, polypodine-B, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone were identified in these extracts. Differences were noted in the mass spectra obtained depending upon both the stationary phase on which they were separated, and the temperatures used in the heated transfer line used for introduction into the ion source. Ecdysteroids detected after chromatography on C18 bonded silica showed increased fragmentation due to water loss compared to those imaged from silica. In addition, the benefits of the additional resolution provided by 2-dimensional TLC for increasing spectral quality compared to a 1-dimensional separation are demonstrated.

Improving duty cycle in the Folded Flight Path high-resolution time-of-flight mass spectrometer

The duty cycle is a critical parameter affecting the sensitivity of analyses by Time-of-Flight Mass Spectrometers with Orthogonal Accelerators (OA-TOFMS). Improving the duty cycle becomes especially critical for the analytical performance of high-resolution TOFMS (HR-TOFMS) with elongated ion paths, such as based on multi-reflecting types of mass analyzers. Currently used methods for improving the duty cycle are based on accumulating ions in various trapping devices between push pulses. These methods do not always provide the best analytical results, may create conditions for secondary reactions, and require adjusting acquisition conditions to maximize the effect. The Encoded Frequent Pushing® (EFP®) method is the spectral multiplexing technique developed for improving the duty cycle of the HR-TOFMS. We report on the implementation of the EFP in HR-TOFMS with a Folded Flight Path® mass analyzer coupled with gas chromatography (GC) and two-dimensional comprehensive GC (GCxGC) for analysis of complex mixtures. This approach is expected to benefit the results of analysis of complex samples by improving limits of detection and mass spectra quality.

Synthesis, Antioxidant Activity, and Structure–Activity Relationship of SCAP1 Analogues

Nine analogues of antioxidant peptide SCAP1 were successfully synthesised using a solid-phase method on a 2-chlorotrytil resin. The compounds were obtained in a range of yields of 7.0–57.8%. The occurrence of aggregation during the synthesis is suspected to be responsible for the poor yields. All peptides were characterized by high-resolution time-of-flight mass spectrometry (HR-TOFMS) and nuclear magnetic resonance (NMR). The antioxidant activities of the SCAP1 analogues as well as SCAP1 were analysed utilising the 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay. The results revealed that all of the analysed peptides exhibited moderate antioxidant properties. Moreover, the evaluation of the structure–activity relationship showed that the Asn residue is an important requirement for the antioxidant activity of SCAP1. The replacement of Asn with other amino acid residues (Thr, Pro, Tyr, Trp and Phe) resulted in a decrease in the IC50 values of the peptides. Notably, however, the replacement of the Lys residue with Val marginally increased the activity.

Comparison of Different Categories of Slovak Tokaj Wines in Terms of Profiles of Volatile Organic Compounds.

The present work deals with the characterization of volatile organic compounds (VOCs) in wines from the Slovak Tokaj wine region. Studied wine samples were divided into three groups—varietal wines from registered Tokaj vine varieties, film wines Tokajské samorodné dry, and naturally sweet botrytized wines Tokaj selections. The VOCs from wines were extracted using optimized solid phase microextraction (SPME) and analyzed by comprehensive two-dimensional gas chromatography (GC×GC) coupled to high-resolution time-of-flight mass spectrometry (HRTOF-MS). In total, 176 VOCs were identified in all 46 studied samples. It was found that the total number of VOCs in varietal wines was generally higher than in botrytized wines. All three studied categories showed characteristic VOC profiles with significant differences. Varietal wines were characterized by higher concentrations of esters and terpenoids originating from grapes. The presence of γ-octalactone, (E)-6-methylhept-2-en-4-one, and lack of benzaldehyde were typical for Tokajské samorodné dry. Tokaj selections expressed the highest concentration of diethyl malate, benzaldehyde, and furfurals. Several interesting trends were also observed. The concentration of fermentation products was highest in varietal wines, while long-term matured Tokaj special wines were typified by the presence of compounds related to noble-rotten raisins (2-phenylacetaldehyde, ethyl 2-phenylacetate, and 2-phenylethanol), wood (cis-whisky lactone), and aging (1,1,6-trimethyl-2H-naphthalene, furfural, and 5-methylfurfural).

Molecular Characterization of High Molecular Weight Polyesters by Matrix-Assisted Laser Desorption/Ionization High-Resolution Time-of-Flight Mass Spectrometry Combined with On-plate Alkaline Degradation and Mass Defect Analysis

Matrix-assisted laser desorption ionization high-resolution time-of-flight mass spectrometry (MALDI HR TOF MS) is a powerful tool for the molecular characterization of industrial polymers. However, accurate mass determination and resolution of isobaric ions are possible for oligomer samples only typically below m/z 3000. To cut long polymer chains into oligomers suitable for high-resolution mass spectrometry, we propose a simple “on-plate” alkaline degradation of polyesters as a sample pretreatment technique prior to the MALDI TOF MS measurement. This pretreatment can be performed on a MALDI target using a small amount of sample (μg or less) and 1 μL of alkaline reagent by simple pipetting. Informative mass spectra in the oligomeric mass range are successfully recorded but complicated by the variation of end-groups and the copolymeric composition of the degradation products. Data processing is assisted by a series of advanced Kendrick mass defect (KMD) analyses recently proposed by the authors to plot visually understandable two-dimensional maps. On-plate degradation pretreatment, high-resolution MALDI TOF MS measurements, and advanced KMD analyses are innovatively combined for the compositional characterization of bacterial poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) and industrial poly(ethylene terephthalate) samples.Graphical ᅟ

Environmental Exposure Induced Metabolic Profiling Alterations in a Migration Panel between Los Angeles and Beijing

Migration provides individuals experiencing different environmental exposure episodes naturally, which offers a chance to attribute physiological response to certain exposure factors. To reveal the mechanism of health effect under environmental exposure, we designed a migration panel study between Los Angeles and Beijing, two cities with different exposure levels. Specifically, serum samples were collected from 27 summer school exchange students three times in total, one week before their departure (LA1), and at least four weeks respectively after their arrival to Beijing (BJ) and return to Los Angeles (LA2). The metabolic profiling was obtained through untargeted metabolomics approach with a high resolution TOF-MS. Combining with a series of statistical methods like Mann-Whitney test and Orthogonal partial least squares discriminant analysis, we screened prospective markers that changed significantly between LA and BJ. The significant decrease (p<0.001, FC>1.2) of indole and its precursor tryptophan in BJ reflected the induction of AhR and CYP by higher levels of pollutants. The decreased cortisol (p<0.001, FC>1.5) was potentially due to the accelerated consumption of CYP3A, supplying further evidence of enhanced CYP activity and related xenobiotics elimination in BJ. On the other hand, the increase of IL-8 and IL-10 from cytokine analysis suggested the oxidative stress during the process. A slightly higher level of uric acid (p<0.01, FC>1.1) along with its precursors implied the upregulation of antioxidation under the environmental exposure in BJ. Generally, this study illustrated that metabolomics offered an effective tool to assess the overall health impact under subchronic exposure.

Characterizing decomposition odor from soil and adipocere samples at a death scene using HS-SPME-GC×GC-HRTOFMS

Using decomposition odor as forensic evidence remains highly controversial despite well-established gas chromatography – mass spectrometry (GC–MS) techniques. Although extensive research characterizing decomposition odor has been conducted, its use in court has been publicly debated due to its admission in the trial The State of Florida vs. Casey Marie Anthony, largely due to the perception that it was too untested. Since this case, a shift from conventional GC–MS to comprehensive two-dimensional gas chromatography – mass spectrometry (GC×GC–MS) has occurred for analyzing decomposition odor. It has become apparent that GC×GC coupled with time-of-flight mass spectrometry (TOFMS), and high-resolution TOFMS (HRTOFMS), is valuable for the analysis of volatile organic compounds (VOCs) from decomposing cadavers as a higher number of VOCs can be detected and identified confidently. This was the first application of GC×GC-HRTOFMS analyzing decomposition VOCs in a forensic case. From a decomposition site, soil from different locations around the body, and adipocere samples were collected. The main objective was to investigate the heterogeneity of the samples using GC×GC–MS. Future challenges anticipated in case scenarios were also interpreted using the generated data. VOCs identified in the soil samples reflected those identified in previous studies, while the profile of adipocere was demonstrated for the first time by GC×GC-HRTOFMS and was dominated by volatile carboxylic acids and acid esters. The power of GC×GC images in court testimony was considered to be of high potential. This case study will strengthen the reliability and evidentiary value of decomposition odor as a valid forensic field.

HPLC-HRMS Quantification of the Ichthyotoxin Karmitoxin from Karlodinium armiger.

Being able to quantify ichthyotoxic metabolites from microalgae allows for the determination of ecologically-relevant concentrations that can be simulated in laboratory experiments, as well as to investigate bioaccumulation and degradation. Here, the ichthyotoxin karmitoxin, produced by Karlodinium armiger, was quantified in laboratory-grown cultures using high-performance liquid chromatography (HPLC) coupled to electrospray ionisation high-resolution time-of-flight mass spectrometry (HRMS). Prior to the quantification of karmitoxin, a standard of karmitoxin was purified from K. armiger cultures (80 L). The standard was quantified by fluorescent derivatisation using Waters AccQ-Fluor reagent and derivatised fumonisin B1 and fumonisin B2 as standards, as each contain a primary amine. Various sample preparation methods for whole culture samples were assessed, including six different solid phase extraction substrates. During analysis of culture samples, MS source conditions were monitored with chloramphenicol and valinomycin as external standards over prolonged injection sequences (>12 h) and karmitoxin concentrations were determined using the response factor of a closely eluting iturin A2 internal standard. Using this method the limit of quantification was 0.11 μg·mL−1, and the limit of detection was found to be 0.03 μg·mL−1. Matrix effects were determined with the use of K. armiger cultures grown with 13C-labelled bicarbonate as the primary carbon source.

Characterization of Leucaena (<em>Leucaena leucephala</em>) oil by direct analysis in real time (DART) ion source and gas chromatography

For the first time, we report the characterization of triacylglycerols and fatty acids in Leucaena (Leucaena leucephala) oil [LUCO], an unexplored nontraditional non-medicinal plant belonging to the family Fabaceae. LUCO was converted to fatty acid methyl esters (FAMEs). We analyzed the triacylglycerols (TAGs) of pure LUCO and their FAMEs by time-of-flight mass spectrometry (TOF-MS) followed by multivariate analysis for discrimination among the FAMEs. Our investigations for the analysis of LUCO samples represent noble features of glycerides. A new type of ion source, coupled with high-resolution TOF-MS was applied for the comprehensive analysis of triacylglycerols. The composition of fatty acid based LUCO oil was studied using Gas Chromatography (GC-FID). The major fatty acid components of LUCO oil are linoleic acid (52.08%) oleic acid (21.26%), palmitic acid (7.91%) and stearic acid (6.01%). A metal analysis in LUCO was done by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The structural elucidation and thermal stability of LUCO were studied by FT-IR, 1H NMR, 13C NMR spectroscopic techniques and TGA-DSC, respectively. We also measured the cytotoxicity of LUCO.

Identification of related substances in tofacitinib citrate by LC-MS techniques for synthetic process optimization

A specific LC-MS method was developed for separation, identification and characterization of the process-related substances and degradation products in tofacitinib citrate. The separation was achieved on a LiChrospher C18 column (250mm×4.6mm, 5μm) by linear gradient elution of 0.1% ammonium acetate solution (pH adjusted to 4.0 by formic acid) and acetonitrile at a flow rate of 1.0mL/min. Forced degradation studies were conducted under hydrolytic (acidic, basic), oxidative, photolytic and thermal stress conditions as described in ICH. It was found that tofacitinib was stable under photolytic condition, but degraded obviously in acidic, basic, thermal and oxidative conditions. The high resolution TOF-MS and MS/MS were used for determination and structural identification of the related substances. Eleven major related substances were detected and identified as five process-related substances and six degradation products, and three of them were further synthesized and characterized by NMR spectroscopy. The most plausible mechanisms involved in the formation of the related substances were also proposed. Since related substances have a significant impact on drug safety, quality and efficacy, the data obtained are valuable for process monitoring and quality assurance of tofacitinib citrate.

Rapid quantification and isotopic analysis of dissolved sulfur species.

Dissolved sulfur species are of significant interest, both as important substrates for microbial activities and as key intermediaries in biogeochemical cycles. Species of intermediate oxidation state such as sulfite, thiosulfate, and thiols are of particular interest but are notoriously difficult to analyze, because of low concentrations and rapid oxidation during storage and analysis. Dissolved sulfur species are reacted with monobromobimane which yields a fluorescent bimane derivative that is stable to oxidation. Separation by Ultra-Performance Liquid Chromatography (UPLC) on a C18 column yields baseline resolution of analytes in under 5min. Fluorescence detection (380nm excitation, 480nm emission) provides highly selective and sensitive quantitation, and Time-of-Flight Mass Spectrometry (TOF-MS) is used to quantify isotopic abundance, providing the ability to detect stable isotope tracers (either 33 S or 34 S). Sulfite, thiosulfate, methanethiol, and bisulfide were quantified with on-column detection limits of picomoles (μM concentrations). Other sulfur species with unshared electrons are also amenable to analysis. TOF-MS detection of 34 S enrichment was accurate and precise to within 0.6% (relative) when sample and standard had similar isotope ratios, and was able to detect enrichments as small as 0.01 atom%. Accuracy was validated by comparison to isotope-ratio mass spectrometry. Four example applications are provided to demonstrate the utility of this method. Derivatization of aqueous sulfur species with bromobimane is easily accomplished in the field, and protects analytes from oxidation during storage. UPLC separation with fluorescence detection provides low-μM detection limits. Using high-resolution TOF-MS, accurate detection of as little as 0.01% 34 S label incorporation into multiple species is feasible. This provides a useful new analytical window into microbial sulfur cycling. Copyright © 2017 John Wiley & Sons, Ltd.

A cheap and simple passive sampler using silicone rubber for the analysis of surface water by gas chromatography–time of flight mass spectrometry

Water pollution events may arise rapidly, requiring a methodology that is easy to implement, fast to deploy, and sufficiently sensitive to detect the trace presence of hazardous contaminants. A cheap and easy to use silicone rubber (polydimethylsiloxane (PDMS)) miniature passive sampler is described. In order to test the methodology, pollutants were concentrated, in situ, from surface water in and around Pretoria, South Africa. The versatile sampler allowed for conventional and enhanced sensitivity, solvent-free analysis by comprehensive gas chromatography - time of flight mass spectrometry (GCxGC-TOFMS) and high resolution TOFMS (GC-HRT). Contaminants detected in surface water include caffeine, personal care products, pharmaceuticals, pesticides and polycyclic aromatic hydrocarbons.

Identification of non-regulated polycyclic aromatic compounds and other markers of urban pollution in road tunnel particulate matter

A combination of silicone rubber extraction and non-target and suspect screening by gas chromatography coupled to high-resolution time-of flight mass spectrometry was used for the identification of compounds in particulate matter (PM). Tunnel PM is a proxy for local road pollution that constitutes a hazard to the urban environment and human health. The use of silicone rubber for the extraction of PM allowed the pre-concentration of a wide range of compounds for non-target analysis while minimising the effects of the sample matrix. As expected, polycyclic aromatic compounds (PACs) constituted the major group of compounds identified, but only 5 of 50 PACs identified were amongst those regularly monitored and many of them were alkylated or contained a heteroatom. Urban markers of contamination such as organophosphate flame-retardants, phthalates, benzothiazoles, musk compounds and a plasticiser were also identified. The level of confidence for the identifications was high based on accurate mass, the pattern of fragmentation and retention. The unequivocal identification of 16 compounds, from all groups, was confirmed by co-chromatography with standards and the compounds semi-quantified. Most of the PACs identified are not regularly monitored, and the hazards they pose are therefore unknown. Some of these PACs are known to be more persistent and mobile in the environment than the EPA PAH16.

Development of a volatility and polarity separator (VAPS) for volatility- and polarity-resolved organic aerosol measurement

Discrepancies between modeled and measured atmospheric organic aerosol (OA) have highlighted the need for in situ instrumentation to better characterize the sources, formation mechanisms, and atmospheric evolution of ambient OA. We have developed the Volatility and Polarity Separator (VAPS) for hourly measurements of volatility- and polarity-resolved OA detected using high-resolution time-of-flight mass spectrometry (HR-ToF-MS). Here, atmospheric OA is inertially impacted onto a collection cell, material is transferred onto a short transfer line located inside a gas chromatography (GC) oven, the oven is heated to provide a first-dimension separation of volatility, then thermally pulsed through a short polar GC column for a second-dimension polarity separation, and finally detected by HR-ToF-MS. This novel instrument increases the mass throughput of ambient OA in comparison to traditional GC due to shorter transfer paths and passivated coatings. Molecular separation resolution is partially sacrificed for this increased mass recovery, but the high-resolution mass spectral data recovers information such as chemical classes and even some individual compounds along with elemental composition to determine aerosol oxidation states. Different techniques for interpreting and representing VAPS data are considered and its applicability to positive matrix factorization (PMF) analysis is demonstrated.Copyright © 2016 American Association for Aerosol Research

High-performance liquid chromatography–time-of-flight mass spectrometry in the identification and determination of 111 pesticides in food, feed, water, and soil

A method has been proposed for the simultaneous identification and determination of 111 pesticides from various classes in food by high-performance liquid chromatography–high-resolution time-offlight mass spectrometry combined with simple and fast sample preparation. Possibility of the identification and determination of pesticides in drinking, natural, and ground waters without sample preparation has been demonstrated. A scheme of the identification and determination of the detected analytes using the standard addition method has been suggested. The limit of detection is 0.05 (0.1) µg/(L)kg. The relative standard deviation of the results of analysis does not exceed 0.1. The time of identification is 30–40 min and time of determination is 1–2 h.

Quantitative determination of four immunosuppressants by high resolution mass spectrometry (HRMS).

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) utilizing triple-quadrupole instruments has been widely used for quantification of endogenous compounds, drugs or metabolites in clinical laboratories. In contrast, high-resolution mass spectrometry (HRMS) is typically used for compound identification due to its limited dynamic range. Recently HRMS instruments with enhanced linear dynamic range have become available. The aim of this study was to evaluate HRMS for fast quantitative applications in a clinical laboratory. A high throughput UPLC-TOF-MS assay for simultaneous quantification of cyclosporin A, tacrolimus, sirolimus and everolimus was developed. All immunosuppressants were analyzed as sodium adducts in TOF-only mode using an Agilent 6540 Q-TOF system. Extracted ion chromatograms of analytes and internal standards were created from full-scan data. The assay was evaluated and compared to an established LC-MS/MS assay according to CLSI recommendations. The novel HRMS assay has a total run time of 3 min. The assay is linear in a clinical relevant concentration range for all four immunosupressants. Method correlations vs. established LC-MS/MS assay were between R2=0.99 and R2=0.97. Total coefficients of variation (CVT) ranges were 4.5%-6.4% (tacrolimus), 7.4%-8.0% (sirolimus), 8.0%-8.8% (everolimus) and 6.1%-7.4% (cyclosporine A) for three relevant concentration levels each. High resolution TOF-MS and LC-MS/MS show equivalent quantitative performance for monitoring of cyclosporin A, tacrolimus, sirolimus and everolimus. HRMS has the potential to replace conventional LC-MS/MS in clinical laboratories because it simplifies assay development (no optimization of fragmentations and product ions necessary) and its full-scan data can provide additional information.

Qualitative analysis of a sulfur-fumigated Chinese herbal medicine by comprehensive two-dimensional gas chromatography and high-resolution time of flight mass spectrometry using colorized fuzzy difference data processing.

To investigate the chemical transformation of volatile compounds in sulfur-fumigated Radix Angelicae Sinensis. A comprehensive two-dimensional gas chromatography (GC×GC) and high-resolution time-of-flight mass spectrometry (HR-TOF/MS) with colorized fuzzy difference (CFD) method was used to investigate the effect of sulfur-fumigation on the volatile components from Radix Angelicae Sinensis. Twenty-five compounds that were found in sun-dried samples disappeared in sulfur-fumigated samples. Seventeen volatile components including two sulfur-containing compounds were newly generated for the first time in volatile oils of sulfur-fumigated Radix Angelicae Sinensis. The strategy can be successfully applied to rapidly and holistically discriminate sun-dried and sulfur-fumigated Radix Angelicae Sinensis. GC×GC-HR-TOF/MS based CFD is a powerful and feasible approach for the global quality evaluation of Radix Angelicae Sinensis as well as other herbal medicines.

Selective separation of fluorinated compounds from complex organic mixtures by pyrolysis-comprehensive two-dimensional gas chromatography coupled to high-resolution time-of-flight mass spectrometry

The usefulness of comprehensive two-dimensional gas chromatography (GC×GC) was demonstrated for the selective separation of fluorinated compounds from organic mixtures, such as kerosene/perfluorokerosene mixtures, pyrolysis products derived from polyethylene/ethylene-tetrafluoroethylene alternating copolymer mixture and poly[2-(perfluorohexyl)ethyl acrylate]. Perfluorocarbons were completely separated from hydrocarbons in the two-dimensional chromatogram. Fluorohydrocarbons in the pyrolysis products of polyethylene/ethylene-tetrafluoroethylene alternating copolymer mixture were selectively isolated from their hydrocarbon counterparts and regularly arranged according to their chain length and fluorine content in the two-dimensional chromatogram. A reliable structural analysis of the fluorohydrocarbons was achieved by combining effective GC×GC positional information with accurate mass spectral data obtained by high-resolution time-of-flight mass spectrometry (HRTOF-MS). 2-(Perfluorohexyl)ethyl acrylate monomer, dimer, and trimer as well as 2-(perfluorohexyl)ethyl alcohol in poly[2-(perfluorohexyl)ethyl acrylate] pyrolysis products were detected in the bottommost part of the two-dimensional chromatogram with separation from hydrocarbons possessing terminal structure information about the polymer, such as α-methylstyrene. Pyrolysis-GC×GC/HRTOF-MS appeared particularly suitable for the characterization of fluorinated polymer microstructures, such as monomer sequences and terminal groups.