Exploring Negative Chemical Ionization of Per- and Polyfluoroalkyl Substances via a Liquid Electron Ionization LC-MS Interface.

Per- and polyfluoroalkyl substances (PFASs) are a class of aliphatic manufactured compounds comprising fluoro-chemicals with varied functional groups and stable carbon-fluorine bonds. They are defined as "forever chemicals" due to their persistent and bioaccumulative character. These substances have been detected in various environmental samples, including water, air, soil, and human blood, posing significant health hazards. High-performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC-ESI-MS) is typically employed for the analysis of PFASs. Negative chemical ionization (NCI) is generally coupled to gas chromatography (GC) and offers high selectivity and sensitivity for compounds containing electronegative atoms, such as PFASs. The liquid electron ionization (LEI) interface is an efficient mechanism developed to robustly couple a liquid flow rate from an LC system to an EI or a CI source. This interface has been successfully utilized for pesticide determination in UHPLC-LEI-CI in negative ion mode (NCI). This work aims to evaluate different parameters involved in the ionization of PFASs analyzed in LC-LEI-NCI and subsequently develop a method for their detection in real samples. The parameters considered for this study include (i) a comparison of different CI reagent gases (methane, isobutane, and argon); (ii) the use of acetonitrile as both the chromatographic solvent and CI reagent gas; (iii) the presence of water and formic acid as chromatographic mobile phase components; and (iv) the mobile phase flow rate. The optimal combination of these parameters led to promising results. Tentative fragmentation pathways of PFASs in NCI mode are proposed based on the dissociative electron capture mechanism.

Liquid electron ionization-mass spectrometry as a novel strategy for integrating normal-phase liquid chromatography with low and high-resolution mass spectrometry.

Normal-phase liquid chromatography (NPLC) plays a pivotal role in the rapid separation of non-polar compounds, facilitating isomer separation and finding applications in various crucial areas where aprotic solvents are necessary. Similar to reversed-phase liquid chromatography (RPLC), NPLC requires a robust and sensitive detector to unequivocally identify the analytes, such as a mass spectrometer. However, coupling NPLC with mass spectrometry (MS) poses challenges due to the incompatibility between the non-polar solvents used as the mobile phase and the primary ionization techniques employed in MS. Several analytical methods have been developed to combine NPLC with electrospray ionization (ESI), but these methods are restricted to the analysis of polar compounds. In most cases, atmospheric pressure chemical ionization (APCI) becomes necessary to expand the range of analysis applications. To overcome these limitations and fully realize the potential of NPLC-MS coupling, a technique termed liquid electron ionization-mass spectrometry (LEI-MS) can be used. LEI-MS offers a straightforward solution by enabling the effective coupling of NPLC with both low and high-resolution MS. LEI allows for the comprehensive analysis of non-polar compounds and provides a powerful tool for isomer separation and precise identification of analytes. Optimal separations, mass spectral qualities, and matches with the NIST library were obtained in both configurations, demonstrating the potential of the proposed approach.

Extractive-liquid sampling electron ionization-mass spectrometry (E-LEI-MS): a new powerful combination for direct analysis.

One of modern analytical chemistry main challenges is providing as fast as possible results in different application fields. In this view, real-time analysis techniques are experiencing ever-increasing success as they can provide data quickly, almost without sample preparation steps. Most of real-time approaches are based on direct mass spectrometry (DMS), a method of analyzing samples without the need for separation or pre-treatment steps. Instead, the sample is directly introduced into the mass spectrometer for analysis. In this context, ambient ionization mass spectrometry (AIMS) techniques are widely represented and successfully used. Extractive-liquid sampling electron ionization-mass spectrometry (E-LEI-MS) represents a different analytical strategy that allows coupling ambient sampling with electron ionization (EI), avoiding any sample preparation step and providing identification based on the comparison with the National Institute of Standards and Technology (NIST) library spectra. E-LEI-MS consists of a dispositive for solvent release and sampling at ambient conditions coupled with an EI source of a single quadrupole mass spectrometer. A micromanipulator allows fine (x,y,z) positioning of a sampling tip. MS can operate in scan or SIM modes depending on the application goals and requirements. Several preliminary successful results were already obtained due to the highly informative EI mass spectra generation. The system was applied to the analysis of active ingredients in pharmaceutical tablets, pesticides on fruit peel, a drug of abuse (cocaine) determination in banknotes, and analysis of unknown components on painting surfaces. Both forensic and artwork applications allowed determining the spatial distribution of the analytes. Here we present a proof-of-concept of E-LEI-MS for targeted/non-targeted analysis and semi-quantitative detection.

Enhanced microfluidic open interface for the direct coupling of solid phase microextraction with liquid electron ionization-tandem mass spectrometry.

Solid-phase microextraction (SPME) directly coupled to MS is a widespread technique for determining small molecules in different matrices in many application fields. Here we present a modified microfluidic open interface (MOI) connected to a passive-flow-splitter device (PFS) for the direct coupling of SPME to a liquid-electron ionization (LEI) interface in a tandem mass spectrometer for the analysis of complex biological samples. No chromatographic separation is involved. The new MOI-PFS configuration was designed to speed up the sample transfer to MS, improving the signal-to-noise ratio and peak shape and leading to fast and sensitive results. MOI-PFS-LEI-MS/MS experiments were conducted using fentanyl as a model compound in water and blood serum. The method uses a C18 Bio-SPME fiber by direct immersion (3 min) in 300 µL of the sample followed by rapid desorption (1 min) in a flow isolated volume (MOI chamber, 2.5 µL) filled with 100% acetonitrile. The PFS permits the rapid transfer of a fraction of the sample into the MS via the LEI interface. The optimal conditions were obtained at a flow rate of 10 µL·min-1 and a 1:20 split ratio. Altogether, extraction, desorption, and analysis require approximately 5 min. Good interday and intraday precision, excellent linearity and LOQs in the µg·L-1 range were obtained for fentanyl in water and serum. Greenness evaluation demonstrated a limited environmental impact of this technique.

Supercritical fluid chromatography-tandem mass spectrometry of oxygen heterocyclic compounds in Citrus essential oils.

Oxygen heterocyclic compounds are secondary metabolites mainly present in the non-volatile fraction of cold-pressed Citrus essential oils. Under this denomination are included coumarins, furocoumarins, and polymethoxyflavones. These compounds possess numerous beneficial properties for human health, but the ingestion of large amounts of coumarins is often related to toxic effects, whereas the phototoxicity caused by furocoumarins and UVA exposure has been well known for a long time. This research has been aimed at the validation of an analytical approach, based on supercritical fluid chromatography coupled to tandem mass spectrometry, for the analysis of OHCs in Citrus essential oils. Among eight columns tested, packed with different stationary phases, the pentafluorophenyl allowed the best baseline separation in 8 min and by using less than 10% of methanol. Calibration curves of twenty-eight standards (coumarins, furocoumarins, polymethoxyflavones) were constructed on spiked lemon distilled essential oil and the method was validated according to the EURACHEM guidelines, by calculating linearity, limit of detection (LoD), limit of quantification (LoQ), accuracy, intra-day, and inter-day precision. Specifically, recoveries were in the 80.0-118.6% range, regression coefficients were between 0.9904 and 0.9998, the LoDs were in the 0.0004-0.0470 mg kg-1 range, the LoQs were in the 0.0014-0.1536 mg kg-1 range, and coefficients of variation were between 0.3 and 2.6% (intra-day) and 1.1 and 7.4% (inter-day). The quantitative profiles of thirteen cold-pressed Citrus essential oils were determined.

Dietary Intake of Coumarins and Furocoumarins through Citrus Beverages: A Detailed Estimation by a HPLC-MS/MS Method Combined with the Linear Retention Index System.

Official regulations concerning the maximum number of substances in food are introduced as a consequence of possible adverse effects, after oral administration. In this regard, analytical methods are necessary in order to determine specific targets. Among oxygen heterocyclic compounds (OHCs, that are furocoumarins, coumarins and polymethoxyflavones), only coumarin is subject to restriction by the Regulation (EC) No 1334/2008 of the European Parliament. Furocoumarins are known for their phototoxicity and other side effects due to their dietary intake; however, an official limit about the maximum content of these compounds in food is still missing. The lack of information about the real amount of these compounds in food is responsible for the conflicting opinions about the introduction of an official limit. The HPLC-MS/MS method here proposed, in combination with the linear retention index system, represents an innovative analytical strategy for the characterization of OHCs in citrus beverages. Several types of drinks were analysed in order to quantify 35 OHCs in total. This method is suitable for the quality control of OHCs in food and the obtained results may be considered as informative data useful for the regulatory authorities in the emission of new opinions and for a potential new regulation in this field.

Linear retention index approach applied to liquid chromatography coupled to triple quadrupole mass spectrometry to determine oxygen heterocyclic compounds at trace level in finished cosmetics.

In the European Union, cosmetic products are regulated by the Cosmetics Regulation (EC) No. 1223/2009. The Analytical Working Group of the International Fragrance Association (IFRA) suggested that a new sensitive analytical method is needed to determine psoralens in finished cosmetic products. This research provides an HPLC-MS/MS method for the quality control of 20 furocoumarins, 8 coumarins and 7 polymethoxyflavones in cosmetics. Thanks to the high sensitivity of the tandem mass spectrometry detection in Multiple Reaction Monitoring mode, psoralens contained in trace have been quantified in different products. The Limits of Quantifications were in the range 0.3-74 µg L-1. A reliable identification was achieved combining the Linear Retention Index (LRI) system with the MS and MS/MS libraries. In particular, the attribution was based on the spectra similarity filtered by the LRI parameter. The robustness and reproducibility of the LRI approach was demonstrated by the comparison of the HPLC-MS/MS results here reported with those obtained in our previous study, by using an HPLC-PDA system. This method could be taken into account for quality control of furocoumarins in cosmetics, and by the main associations, such as IFRA, for the purpose of issuing new opinions.

The retention index approach in liquid chromatography: An historical review and recent advances.

Historically, retention parameters were reliably used as identification criterion in chromatographic analytical systems. However, pure analytical standards are necessary to determine the retention behaviour of a given compound. In recent decades, mass spectrometer (MS) became the detector of choice to obtain structural information on unknown peaks, thanks to the elucidation of fragments, often arisen from the loss of specific functional groups. The cost and the level of experience of the operators is surely higher compared to the use of retention data. Therefore, the aim of the present review is to describe the efforts in the introduction of the Linear Retention Index (LRI) in routine, interlaboratory applicable identification procedures. The requirements and the main challenges will be discussed, even compared to gas chromatography methods, in which LRI is stably used for identification purposes, usually in combination with MS spectral libraries. The higher number of LC-amenable molecules and the wide range of LC mobile phase compositions make the building of universal LRI database a very challenging task. The limitations encountered in the past decades are reported, together with new proposals in order to overcome such issues.

Blood orange (Citrus sinensis) as a rich source of nutraceuticals: investigation of bioactive compounds in different parts of the fruit by HPLC-PDA/MS.

This study is part of a wider investigation aimed to sustain the economical value of the by-products generated by the Citrus industry. In particular, the protected geographic indication (PGI) red orange of sicily (known as blood orange) has been analysed by HPLC and by the enzymatic AOAC method. All the by-products contain significant amounts of biologically active compounds (limonoids and flavonoids). The decanted pulps were the most abundant, with the highest amount of flavonoids (130 g/kg) and high amount of limonoids (5.5 g/kg). Seeds were the best source of limonoids with about 10 g/kg. Low amount of anthocyanins were found only in coarse pulps and waste water. The total, the insoluble and the soluble dietary fibre (TDF, IDF and SDF respectively) were also determined. The pulps resulted to be the best source of dietary fibre, based on the amount and on the best insoluble/soluble ratio.

Combining linear retention index and electron ionization mass spectrometry for a reliable identification in nano liquid chromatography.

The present research is focused on the object to improve identification capability in liquid chromatography (LC), by creating a system as similar as possible to gas chromatography (GC), where the combination/complementarity of Linear Retention Index (LRI) and Electron Ionization Mass Spectrometry (EI-MS) data makes the identification process easy, automatic and reliable. Conversely, in LC the untargeted characterization of real-world samples is still a challenge, due to the not repeatable and poorly informative nature of typical atmospheric pressure ionization mass spectrometry, normally hyphenated to LC. In the last decades the miniaturization of LC instrumentation together with the considerable progresses in MS vacuum pump capability has made the LC-EI-MS hyphenation more feasible. In the present work, a prototypal nanoLC-EI-MS system was used for the determination of typically LC-amenable compunds, such as coumarins, furocoumarins and polymethoxyflavones in citrus essential oils. All the compounds provided high quality EI-MS spectra, evaluated by the comparison with thousands of spectra present in commercial EI-MS libraries. Spectral similarities major than 80% were achieved. Furthermore, an LRI system, based on the use of an alkyl aryl ketone homologue reference series, was proposed as additional filter to achieve a univocal identification. Then, a novel dual-filter LRI/EI-MS library was built and resulted very helpful in the case of isomeric compounds characterized by identical EI-MS spectrum, but different retention behaviour. The very low inter-day variability attained for each LRI value, together with the satisfactory chromatographic resolution of the developed method, led to a 100% reliability of the identification process based on LRI.

Comparison of different analytical techniques for the analysis of carotenoids in tamarillo (Solanum betaceum Cav.).

An on-line method based on the coupling of supercritical fluid extraction and supercritical fluid chromatography with triple quadrupole mass spectrometry detection (SFE-SFC-QqQ/MS) for the native carotenoids characterization and apocarotenoids detection in yellow tamarillo was developed for the first time; this is the first work reporting the application of this methodology for the apocarotenoids analysis in any matrix. An off-line liquid extraction (LE) and analysis by LC-PDA-MS, using both a conventional C30 (3 µm I.D. particles) and a novel C30 column with sub-2 micron particles were also performed. 31 compounds were extracted and identified by the developed SFE-SFC-QqQ/MS methodology in less than 18 min, including free carotenoids, carotenoids monoesters, carotenoids diesters and apocarotenoids in a very fast, and efficient way; moreover among those 31 compounds, 3 antheraxanthin monoesters and 9 apocarotenoids were detected in yellow tamarillo for the first time, namely apo-8'-zeaxanthinal, apo-10'-zeaxanthinal, apo-12'-zeaxanthinal, apo-14'-zeaxanthinal, apo-15'-zeaxanthinal, apo-12'-carotenal, apo-14'-carotenal and the two apocarotenoids esters apo-10'-zeaxanthinal-C4:0 and apo-8'-zeaxanthinal-C12:0. Further, the novel sub-2 micron particles C30 column, showed a better performance compared to the conventional C30 one. Finally a quantitative comparison of two selected carotenoids was performed by using SFE-SFC-QqQ/MS, SFC-QqQ/MS, and LC-PDA, which showed overall comparable results.

Development of extraction method for characterization of free and bonded polyphenols in barley (Hordeum vulgare L.) grown in Czech Republic using liquid chromatography-tandem mass spectrometry.

Complete characterizations of free and bonded phenolic compounds, presented in four cultivars of barley from two regions of Czech Republic, were achieved, using optimized solvent extraction and liquid chromatography coupled with tandem mass spectrometry. The optimization of extraction of free polyphenols was performed using Box-Behnken design and response surface methodology. The intra-day and extra-day precision of developed method were below 6% and 12%, respectively. The isolation of polyphenols bonded to the cell wall structure was carried out by a hydrolysis process. In all cultivars, p-hydroxybenzoic, p-coumaric and ferulic acids were the most abundant compounds. Their average amounts in barley samples were 17.6, 15.2 and 54.4% (m/m), respectively. The highest amount of these compounds was found in the bonded form, proving the importance of this procedure for the correct characterization of total polyphenols in food matrices.

Rapid isolation, reliable characterization, and water solubility improvement of polymethoxyflavones from cold-pressed mandarin essential oil.

Polymethoxyflavones possess many biological properties, as lipid-lowering, hypoglycaemic, anti-inflammatory, antioxidant, and anticancer activities, therefore, they may be employed as nutraceuticals or therapeutic agents. The scarcity of pure polymethoxyflavones on the market as well as their low water solubility limited in vivo studies and the use of polymethoxyflavones as food or pharmaceutical supplements. Since mandarin peels are a rich source of polymethoxyflavones, tangeretin, nobiletin, sinensetin, tetra-O-methyl scutellarein, and heptamethoxyflavone were purified from a nonvolatile residue of a cold-pressed mandarin essential oil using a multidimensional preparative liquid chromatographic system coupled with a photodiode array detector and a single quadrupole mass spectrometer. A new prototype, consisting of a nano-liquid chromatography system coupled with an electron ionization mass spectrometer, was used for the characterization of the pure isolated molecules. Finally, due to the collection of highly pure nobiletin and tangeretin, the ability of 2-hydroxypropyl-ß-cyclodextrin to enhance the water solubility of both polymethoxyflavones was evaluated by phase solubility studies and Job's plot method.