Unraveling the Double Bond Position of Fatty Acids by GC-MS Using Electron Capture APCI and In-Source Fragmentation Patterns.

The position of double bonds in unsaturated fatty acids is strongly connected to their biological effects, but their analytical characterization is still challenging. However, the ionization of unsaturated fatty acids by a GC-APCI leads to regiospecific in-source fragment ions, which can be used to identify the double bond position. The fragment ions are oxidized species that occur mostly at the double bond closest to the carboxylic acid group. This effect can be further promoted by using benzaldehyde as a gas-phase reactant. This allows the identification of the Δ-notation of the fatty acid, and based on additional information such as m/z and retention time, it is possible to annotate the corresponding fatty acid. The developed method also enables the quantification of fatty acids in one step with high selectivity and sensitivity. Moreover, rare fatty acids can be identified in suspected target approaches that are often not available as standards. This was demonstrated by analyzing fish oil samples that provide a complex mixture of highly unsaturated fatty acids and by identifying rare fatty acids such as hexadecatetraenoic acid (FA 16:4 Δ6).

Comprehensive two-dimensional gas chromatography with flow modulator coupled via tube plasma ionization to an atmospheric pressure high-resolution mass spectrometer for the analysis of vermouth volatile profile.

The analysis of complex samples is a big analytical challenge due to the vast number of compounds present in these samples as well as the influence matrix components could cause in the methodology. In this way, comprehensive two-dimensional gas chromatography coupled to mass spectrometry (GC × GC-MS) is a very powerful tool to achieve the characterization of complex samples. Nevertheless, due to possible coelutions occurring in these matrices, mixed spectra are generally obtained with electron ionization (EI) which could extremely complicate the identification of the analytes. Thereby, new methodology setups are required to improve the confidence on the identification in non-targeted determinations. Here, we present a high-throughput methodology consisting of GC × GC with flow modulation coupled to high-resolution atmospheric pressure mass spectrometry (HRMS) via a novel tube plasma ion source (TPI). The flow modulator allows to easily automate the GC × GC method compared to traditional cryo-modulators, while the soft ionization provided by TPI helps to preserve the [M]+• or [M+H]+ ions, thus increasing the confidence in the identification. Additionally, the combination of a flow modulation with an atmospheric pressure mass spectrometer significantly improves the sensitivity over flow modulated GC × GC-EI-MS methods because no split is required. This methodology was applied to the analysis of a complex sample such as vermouth where the volatile profile is usually considered by consumers as a product quality indicator since it raises the first sensations produced during its consumption. Using this approach, different classes of compounds were tentatively identified in the sample, including monoterpenes, terpenoids, sesquiterpenoids and carboxylic acid, and carboxylate esters among others, showing the great potential of a GC × GC-TPI-qTOF-MS platform for improving the confidence of the identifications in non-targeted applications.

Multi-2D LC × LC as a Novel and Powerful Implement for the Maximum Separation of Complex Samples.

Achieving complete information about the chemical composition of complex samples requires the use of multianalytical platforms able to maximize the acquisition of high-quality data for unequivocal identification. However, this process requires long analysis times and several instruments. Food analysis is one of the analytical fields where the analysis of very complex samples has a huge impact. One of these complex samples is vermouth, a fortified wine based on the maceration of a large number of herbs, fruits, barks, seeds, and leaves. The application of conventional or even advanced analytical techniques like comprehensive two-dimensional (2D) liquid chromatography (LC × LC) does not provide enough separation power to resolve the complete profile of this sample. In this work, a novel 2DLC strategy called multi-2D LC × LC is developed. This new setup consists of the use of two different columns with different separation properties in the second dimension (2D) that can be selected during the LC × LC analysis accordingly to the chemical nature of the compounds eluted from the first dimension (1D). The vermouth sample was analyzed using a 1D-PFP and a combination of HILIC (from 0 to 30 min) and C18 (from 30 to the end) columns in the 2D. This setup increased both the peak capacity and the orthogonality of the analysis in comparison to the use of only one of the columns in the 2D. Multi-2D LC × LC is presented as an integrated 2DLC tool that maximizes the separation capacity for very complex samples.

Review on atmospheric pressure ionization sources for gas chromatography-mass spectrometry. Part I: Current ion source developments and improvements in ionization strategies.

The use of atmospheric pressure ionization (API) sources has become very popular for gas chromatography-mass spectrometry (GC-MS) determinations. GC-API-MS shows important advantages over traditional vacuum ionization sources such as a lower fragmentation preserving the molecular or quasi-molecular ion, the combination of GC separations with advanced mass spectrometers typically developed for liquid chromatography (LC) systems, a significantly higher sensitivity, or the reduction of costs due to the capability to use the same MS for both LC- and GC- couplings. For these reasons, the development of new API sources and GC-API-MS platforms has exponentially increased during the last years. Thus, this review is mainly focused on the last advances in GC-API-MS instrumentation. New setups and couplings on extensively reported API techniques as well as the development of new API sources for GC-MS coupling are thoroughly discussed. Moreover, novel ionization strategies have been reviewed to overcome some of the drawbacks of GC-API-MS methodologies.

Review on atmospheric pressure ionization sources for gas chromatography-mass spectrometry. Part II: Current applications.

The application of atmospheric pressure ionization (API) sources in gas chromatography-mass spectrometry (GC-MS) determinations is becoming more popular since they have shown great capabilities to sort out the main drawbacks of vacuum ionization techniques like electron ionization (EI) and chemical ionization (CI). The development of new API techniques and set-ups have grown in the last decades, opening the field of GC-MS to new applications and facing some of the major issues in current analytical methodologies such as the requirement of a compromise between sensitivity and selectivity. Thus, this review is mainly focused on the use of GC-API-MS in different application fields such as food analysis (food safety and food metabolomics), environmental analysis, clinical analysis, drug and pharmaceutical analysis, and petroleomics, among others. The methodologies have been critically reviewed to compare the performance of different API sources and approaches, highlighting the main contributions to overcoming some of the major limitations of the current methodologies as well as the new perspectives that GC-API-MS might open in the different fields.

Quantification and identification of bile acids in saliva by liquid chromatography-mass spectrometry: Possible non-invasive diagnostics of Barrett's esophagus?

Bile acids are a group of steroid compounds essential for lipid digestion. However, when bile acids are refluxed into the stomach and the esophagus, during the so called duodenogastroesophageal reflux, they can have a detrimental effect on the esophageal epithelium and cause pathological changes of esophageal tissue, e.g., Barrett's esophagus (BE). The levels of bile acids in saliva could therefore serve as possible biomarkers for the diagnostics of BE. In this work, we focused on optimization of sample collection and preparation by solid-phase extraction and subsequent quantification of 11 bile acids (unconjugated, glycine-conjugated) in saliva from healthy volunteers and BE patients by ultra-high-performance liquid chromatography coupled to triple-quadrupole tandem mass spectrometry. Moreover, high resolution MS (Orbitrap-MS) was utilized for identification of new bile acids in saliva. Methods for saliva collection including simple spitting and the Salivette® saliva collection system were compared; the latter was found to be unsuitable due to excessive retention of bile acids in the cotton swab. Methanol with 0.1% formic acid were selected for protein precipitation and bile acid extraction prior to SPE. Separation was performed in gradient elution of methanol and 0.1% formic acid in less than 10 min. Saliva from BE patients contained higher levels of almost all bile acids, and the tested groups could be distinguished by principal component analysis. In untargeted analysis by high resolution MS, taurine-conjugated bile acids and glycine-conjugated dihydroxy-bile acid sulfate were identified in saliva from healthy volunteers. We propose that analysis of salivary bile acids including taurine conjugates could be applicable in diagnostics of BE, following a larger clinical study.

Potential of atmospheric pressure ionization sources for the analysis of free fatty acids in clinical and biological samples by gas chromatography-mass spectrometry.

Because of the central role of fatty acids in biological systems, their accurate quantification is still important. However, the impact of the complex matrix of biologically and clinically relevant samples such as plasma, serum, or cells makes the analysis still challenging, especially, when free non-esterified fatty acids have to be quantified. Here we developed and characterized a novel GC-MS method using pentafluorobenzyl bromide as a derivatization agent and compared different ionization techniques such as atmospheric pressure chemical ionization (APCI), atmospheric pressure chemical photoionization (APPI), and negative ion chemical ionization (NICI). The GC-APCI-MS showed the lowest limits of detection from 30 to 300 nM for a broad range of fatty acids and a similar response for various fatty acids from a chain length of 10 to 20 carbon atoms. This allows the number of internal standards necessary for accurate quantification to be reduced. Moreover, the use of pentafluorobenzyl bromide allows the direct derivatization of free fatty acids making them accessible for GC-MS analysis without labor-intense sample pretreatment.

Development of a Tube Plasma Ion Source for Gas Chromatography-Mass Spectrometry Analysis and Comparison with Other Atmospheric Pressure Ionization Techniques.

A tube plasma ionization (TPI) open-air source for gas chromatography-mass spectrometry (GC-MS) was developed. This source is based on an inverse low temperature plasma configuration where the pin inner electrode is applying the high voltage and the grounded electrode is the housing itself. The ionization possibilities were tested by using an EPA mix of priority contaminants, showing that 68% of the analytes could undergo both proton-transfer and charge-exchange reactions. The potential of using different discharge gases (He and Ar) to ionize the analytes and auxiliary gases (He, N2, O2, and synthetic air) to transport the ions toward the MS was carefully investigated. Additionally, the addition of water was also tested to show the different ionization trends in the TPI source. Finally, the ionization by TPI under both dry and wet conditions was compared with other gas-phase atmospheric pressure ionization sources showing TPI could ionize a wider range of compounds (97%) than atmospheric pressure chemical ionization (APCI, 95%) and atmospheric pressure photoionization (APPI, 87%). Besides, the detection capability of TPI was better than APCI and APPI, achieving instrumental limits of detection down to 3 fg on column, which demonstrates the great potential of this ionization source for GC-MS determinations.

Differentiation of industrial hemp strains by their cannabinoid and phenolic compounds using LC × LC-HRMS.

Cannabis is an ancient plant that has been used for therapeutic and recreational purposes. Nowadays, industrial hemp, a variety with low concentration of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) and high concentration of non-psychoactive cannabinoids, is getting more and more interest in the food, pharmaceutical, and cosmetic industry. However, cannabis not only contains cannabinoids as bioactive components but also other metabolites like terpenes and phenolic compounds, and the content of these interesting secondary metabolites greatly differs with the genetic variety of the plant. Due to the huge complexity of composition of the cannabis matrix, in this work, a comprehensive two-dimensional liquid chromatography (LC × LC) method has been developed as a very power separation technique coupling a pentafluorophenyl (PFP) and a C18 in the first and second dimensions. Two industrial hemp strains (cookie and gelato) were analyzed to determine the difference in their content of cannabinoids and phenolic compounds. To do this, a new demodulation process was applied for the first time to transform 2D raw data into 1D data which allowed carrying out the chemometric analysis needed to determine the statistical differences between the hemp strains. The cookie strain presented a total of 41 cannabinoid markers, while the gelato strain presented more representative phenolic compounds, in total 24 phenolic compounds were detected as potential markers of this sample. These differences in the chemical composition could determine the industrial destiny of the different hemp strains.

Analysis of Dechlorane Plus and related compounds in gull eggs by GC-HRMS using a novel atmospheric pressure photoionization source.

Here, a new gas chromatography-atmospheric pressure photoionization-high-resolution mass spectrometry (GC-APPI-HRMS) method combined with selective pressurized liquid extraction (sPLE) has been developed for the selective determination of Dechlorane Plus (DP) and its related compounds in gull egg samples used as a bioindicator of contamination. To the best of our knowledge, this is the first time these compounds have been analyzed by GC-MS using atmospheric pressure photoionization (APPI). Negative ion dopant-assisted APPI using vapors of diethyl ether and a source temperature of 250 °C provided high ionization efficiencies and mass spectra characterized by intense in-source fragment ions as well as the presence of molecular ion and characteristic cluster ions containing oxygen atoms in their chemical structure. This made it possible to improve the selectivity in the determination of these compounds compared to that obtained with traditional GC-MS ion sources. Under optimized conditions, the sPLE GC-APPI-HRMS (Orbitrap) method provided high recoveries (> 91%), good precisions (RSD% < 12%), and low method limits of detection (0.1-3.5 pg g-1 wet weight). The developed methodology has been applied to the determination of DP and related compounds in eggs of two gull species (L. michahellis and L. audouinii) from several Spanish protected areas. The results obtained showed significant differences in the DP concentration profiles in eggs from different gull breeding locations and between gull species of the same protected area. These results demonstrated the good performance of the GC-APPI-HRMS system to achieve a selective and sensitive determination of DP and related compounds in complex environmental samples.

Analysis of hydroxylated phenylalkylamine stimulants in urine by GC-APPI-HRMS.

A gas chromatography-atmospheric pressure photoionization-high-resolution mass spectrometry (GC-APPI-HRMS) method was developed for the determination of eight phenylalkylamine stimulants in urine samples. Spiked urine samples were hydrolyzed, processed by solid-phase extraction, and derivatized before analysis. Two derivatization reactions were studied: the formation of trimethylsilyl (TMS) derivatives with N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and trimethylsilyl/trifluoroacetyl (TMS/TFA) derivatives with MSTFA and N-methyl-bis (trifluoroacetamide) (MBTFA) as derivatization reagents. Gas chromatography of both derivatives was performed with a 100% dimethylsiloxane column and a good separation of all isomeric compounds was achieved. To maximize the signal of the protonated molecule [M+H]+, the APPI most critical parameters were optimized. Three solvents were tested as dopant agents, with acetone yielding the lower in-source collision-induced dissociation (CID) fragmentation. The acquisition was performed in full scan and product ion scan (parallel reaction monitoring, PRM) using a quadrupole-Orbitrap mass analyzer (35,000 FWHM at m/z 200) in positive ion detection mode. At the optimal working conditions, the full scan method was evaluated for the fulfillment of identification requirements in doping analysis. Selectivity, limits of detection, matrix effect, and precision were estimated to validate the method for confirmation purposes and its applicability was tested by the analysis of spiked samples as well as by the analysis of samples obtained after the administration of some of the compounds to healthy volunteers. Results were compared with those obtained by GC-electron ionization-MS, demonstrating that the GC-APPI-HRMS method improved selectivity and sensibility, achieving lower limits of detection and satisfactory reproducibility.

Fragmentation studies of neutral per- and polyfluoroalkyl substances by atmospheric pressure ionization-multiple-stage mass spectrometry.

The establishment of fragmentation pathways has a great interest in the identification of new or unknown related compounds present in complex samples. On that way, tentative fragmentation pathways for the ions generated by atmospheric pressure ionization of neutral per- and polyfluorinated alkyl substances (PFASs) have been proposed in this work. Electrospray (ESI), atmospheric pressure chemical ionization (APCI) and photoionization (APPI) were evaluated using mobile phases and source conditions that enhance the ionization efficiency of ions generated. A hybrid mass spectrometer consisting of a linear ion trap and an Orbitrap was used to combine the information of both multiple-stage mass spectrometry (MSn) and mass accuracy measurements to characterize and establish the genealogical relationship between the product ions observed. The ionization mechanisms to generate ions such as [M-H]-, [M]-•, and [M+O2]-• or the in-source collision-induced dissociation (CID) fragment ions in each API source are discussed in this study. In general, fluorotelomer olefins (FTOs) ionized in negative-ion APCI and APPI generated the molecular ion, while fluorotelomer alcohols (FTOHs) also provided the deprotonated molecule. Besides, fluorooctane sulfonamides (FOSAs) and sulfonamido-ethanols (FOSEs) led to the deprotonated molecule and in-source CID fragment ions, respectively. The fragmentation pathways from these precursor ions mainly involved initial α,ß-eliminations of HF units and successive losses of CF2 units coming from the perfluorinated alkyl chain. Moreover, FTOHs and FOSEs showed a high tendency to generate adduct ions under negative-ion ESI and APPI conditions. The fragmentation study of these adduct ions has demonstrated a strong interaction with the attached moiety. Graphical abstract.

Negative-ion atmospheric pressure ionisation of semi-volatile fluorinated compounds for ultra-high-performance liquid chromatography tandem mass spectrometry analysis.

In this work, the feasibility of negative-ion atmospheric pressure chemical ionisation (APCI) and atmospheric pressure photoionisation (APPI) for ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) determination of fluorotelomer alcohols (FTOHs), fluorinated octanesulfonamides (FOSAs) and fluorinated octanesulfonamido-ethanols (FOSEs) was evaluated. The study of the effect of mobile phase composition on the atmospheric pressure ionisation of these compounds indicated that methanol/water mixtures provided the best responses in APCI, while acetonitrile/water with a post-column addition of toluene as dopant was the most appropriated mixture in APPI. Under the optimal working conditions, most of the target compounds produced the ion [M-H]- as base peak, although in-source collision-induced dissociation fragment ions in APCI and APPI and superoxide adduct ions [M+O2]-• in APPI were also present. These ions proved to be more useful as precursor ions for MS/MS determination than the adduct ions generated in electrospray. Although the UHPLC-APCI-MS/MS method allowed the determination of these semi-volatile compounds at low concentration levels, the analysis by UHPLC-APPI-MS/MS provided the lowest limits of detection and it was applied to the analysis of water samples in combination with solid-phase extraction. Quality parameters demonstrated the good performance of the proposed method, providing low method limits of detection (0.3-6 ng L-1), good precision (RSD % < 5%) and an accurate quantification (relative error % < 14%). Among the river water samples analysed by the developed method, 4:2 FTOH and N-EtFOSA were determined at 30 and 780 ng L-1, respectively.