Should Transformation Products Change the Way We Manage Chemicals?

When chemical pollutants enter the environment, they can undergo diverse transformation processes, forming a wide range of transformation products (TPs), some of them benign and others more harmful than their precursors. To date, the majority of TPs remain largely unrecognized and unregulated, particularly as TPs are generally not part of routine chemical risk or hazard assessment. Since many TPs formed from oxidative processes are more polar than their precursors, they may be especially relevant in the context of persistent, mobile, and toxic (PMT) and very persistent and very mobile (vPvM) substances, which are two new hazard classes that have recently been established on a European level. We highlight herein that as a result, TPs deserve more attention in research, chemicals regulation, and chemicals management. This perspective summarizes the main challenges preventing a better integration of TPs in these areas: (1) the lack of reliable high-throughput TP identification methods, (2) uncertainties in TP prediction, (3) inadequately considered TP formation during (advanced) water treatment, and (4) insufficient integration and harmonization of TPs in most regulatory frameworks. A way forward to tackle these challenges and integrate TPs into chemical management is proposed.

Characterizing Powdered Activated Carbon Treatment of Surface Water Samples Using Polarity-Extended Non-Target Screening Analysis.

Advanced wastewater treatment such as powdered activated carbon (PAC) reduces the load of organic micropollutants entering the aquatic environment. Since mobile and persistent compounds accumulate in water cycles, treatment strategies need to be evaluated for the removal of (very) polar compounds. Thereby, non-targeted analysis gives a global picture of the molecular fingerprint (including these very polar molecules) of water samples. Target and non-target screening were conducted using polarity-extended chromatography hyphenated with mass spectrometry. Samples treated with different types and concentrations of PAC were compared to untreated samples. Molecular features were extracted from the analytical data to determine fold changes, perform a principal component analysis and for significance testing. The results suggest that a part of the polar target analytes was adsorbed but also some byproducts might be formed or desorbed from the PAC.

Organic Contaminants and Interactions with Micro- and Nano-Plastics in the Aqueous Environment: Review of Analytical Methods.

Micro- and nanoplastic particles are increasingly seen not only as contaminants themselves, but also as potential vectors for trace organic chemicals (TOrCs) that might sorb onto these particles. An analysis of the sorbed TOrCs can either be performed directly from the particle or TOrCs can be extracted from the particle with a solvent. Another possibility is to analyze the remaining concentration in the aqueous phase by a differential approach. In this review, the focus is on analytical methods that are suitable for identifying and quantifying sorbed TOrCs on micro- and nano-plastics. Specific gas chromatography (GC), liquid chromatography (LC) and ultraviolet-visible spectroscopy (UV-VIS) methods are considered. The respective advantages of each method are explained in detail. In addition, influencing factors for sorption in the first place are being discussed including particle size and shape (especially micro and nanoparticles) and the type of polymer, as well as methods for determining sorption kinetics. Since the particles are not present in the environment in a virgin state, the influence of aging on sorption is also considered.

Optimized hidden target screening for very polar molecules in surface waters including a compound database inquiry.

Highly polar trace organic compounds, which are persistent, mobile, and toxic (PMT) or are very persistent and very mobile (vPvM) in the aquatic environment, may pose a risk to surface water, ground water, and drinking water supplies. Despite the advances in liquid chromatography-mass spectrometry, there often exists an analytical blind spot when it comes to very polar chemicals. This study seeks to make a broad polarity range analytically accessible by means of serially coupling reversed-phase liquid chromatography (RPLC) and hydrophilic interaction liquid chromatography (HILIC) to high-resolution mass spectrometry (HRMS). Moreover, a workflow is presented using optimized data processing of nontarget screening (NTS) data and subsequently generating candidate lists for the identification of very polar molecules via an open-access NTS platform and implemented compound database. First, key input parameters and filters of the so-called feature extraction algorithms were identified, and numerical performance indicators were defined to systematically optimize the data processing method. Second, all features from the very polar HILIC elution window were uploaded to the STOFF-IDENT database as part of the FOR-IDENT open-access NTS platform, which contains additional physicochemical information, and the features matched with potential compounds by their accurate mass. The hit list was filtered for compounds with a negative log D value, indicating that they were (very) polar. For instance, 46 features were assigned to 64 candidate compounds originating from a set of 33 samples from the Isar river in Germany. Three PMT candidates (e.g., guanylurea, melamine, and 1,3-dimethylimidazolidin-2-one) were illustratively validated using the respective reference standards. In conclusion, these findings demonstrate that polarity-extended chromatography reproducibly retards and separates (very) polar compounds from surface waters. These findings further indicate that a transparent and robust data processing workflow for nontarget screening data is available for addressing new (very) polar substances in the aqueous environment.

Systematic Development of a Simultaneous Determination of Plastic Particle Identity and Adsorbed Organic Compounds by Thermodesorption-Pyrolysis GC/MS (TD-Pyr-GC/MS).

Micro-, submicro- and nanoplastic particles are increasingly regarded as vectors for trace organic chemicals. In order to determine adsorbed trace organic chemicals on polymers, it has usually been necessary to carry out complex extraction steps. With the help of a newly designed thermal desorption pyrolysis gas chromatography mass spectrometry (TD-Pyr-GC/MS) method, it is possible to identify adsorbed trace organic chemicals on micro-, submicro- and nanoparticles as well as the particle short chain polymers in one analytical setup without any transfers. This ensures a high sample throughput for the qualitative analysis of trace substances and polymer type. Since the measuring time per sample is only 2 h, a high sample throughput is possible. It is one of the few analytical methods which can be used also for the investigation of nanoplastic particles. Initially adsorbed substances are desorbed from the particle by thermal desorption (TD); subsequently, the polymer is fragmented by pyrolysis (PYR). Both particle treatment techniques are directly coupled with the same GC-MS system analyzing desorbed molecules and pyrolysis products, respectively. In this study, we developed a systematic and optimized method for this application. For method development, the trace organic chemicals phenanthrene, α-cypermethrin and triclosan were tested on reference polymers polystyrene (PS), polymethyl methacrylate (PMMA) and polyethylene (PE). Well-defined particle fractions were used, including polystyrene (sub)micro- (41 and 40 µm) and nanoparticles (78 nm) as well as 48-µm sized PE and PMMA particles, respectively. The sorption of phenanthrene (PMMA << PS 40 µm < 41 µm < PE < PS 78 nm) and α-cypermethrin (PS 41 µm < PS 40 µm < PE < PMMA < PS 78 nm) to the particles was strongly polymer-dependent. Triclosan adsorbed only on PE and on the nanoparticles of PS (PE < PS78).

Comprehensive MS-based screening and identification of pharmaceutical transformation products formed during enzymatic conversion.

In this study, transformation products (TPs) of diclofenac, mefenamic acid, and sotalol derived from peroxidase- and laccase-catalyzed transformations were studied with different mass spectrometry (MS)-based workflows. A straightforward pre-screening of enzymatic degradation rate was performed using a robotic nano-ESI source coupled to single quadrupole MS. Accurate mass data and information on molecular hydrophobicity were obtained from a serial coupling of reversed phase liquid chromatography (RPLC) with hydrophilic interaction liquid chromatography (HILIC) to a time-of-flight-mass spectrometer (ToF-MS). These parameters were combined with fragmentation information from product ion scan operated in enhanced mode (EPI) with precursor selection in Q3 and data from multiple reaction monitoring (MRM) modes using a hybrid triple quadrupole-linear ion trap-mass spectrometer (QqQ/LIT-MS). "Suspect" MRM modes did not provide a significant sensitivity improvement compared to EPI experiments. The complementarity of the data from different MS-based workflows allowed for an increase of identification confidence. Overall, this study demonstrated that dimerization, hydroxylation, and dehydration reactions were the predominant mechanisms found for diclofenac and mefenamic acid during enzyme-catalyzed transformation, whereas a degradation product was observed for the peroxidase-catalyzed conversion of sotalol. Results can contribute to understand enzymatic mechanisms and provide a basis for assessing risks and benefits of enzyme-based remediation. Graphical abstract ᅟ.

The strength in numbers: comprehensive characterization of house dust using complementary mass spectrometric techniques.

Untargeted analysis of a composite house dust sample has been performed as part of a collaborative effort to evaluate the progress in the field of suspect and nontarget screening and build an extensive database of organic indoor environment contaminants. Twenty-one participants reported results that were curated by the organizers of the collaborative trial. In total, nearly 2350 compounds were identified (18%) or tentatively identified (25% at confidence level 2 and 58% at confidence level 3), making the collaborative trial a success. However, a relatively small share (37%) of all compounds were reported by more than one participant, which shows that there is plenty of room for improvement in the field of suspect and nontarget screening. An even a smaller share (5%) of the total number of compounds were detected using both liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). Thus, the two MS techniques are highly complementary. Most of the compounds were detected using LC with electrospray ionization (ESI) MS and comprehensive 2D GC (GC×GC) with atmospheric pressure chemical ionization (APCI) and electron ionization (EI), respectively. Collectively, the three techniques accounted for more than 75% of the reported compounds. Glycols, pharmaceuticals, pesticides, and various biogenic compounds dominated among the compounds reported by LC-MS participants, while hydrocarbons, hydrocarbon derivatives, and chlorinated paraffins and chlorinated biphenyls were primarily reported by GC-MS participants. Plastics additives, flavor and fragrances, and personal care products were reported by both LC-MS and GC-MS participants. It was concluded that the use of multiple analytical techniques was required for a comprehensive characterization of house dust contaminants. Further, several recommendations are given for improved suspect and nontarget screening of house dust and other indoor environment samples, including the use of open-source data processing tools. One of the tools allowed provisional identification of almost 500 compounds that had not been reported by participants.

Monitoring enzymatic degradation of emerging contaminants using a chip-based robotic nano-ESI-MS tool.

Up to now, knowledge of enzymes capable of degrading various contaminants of emerging concern (CEC) is limited, which is especially due to the lack of rapid screening methods. Thus, a miniaturized high-throughput setup using a chip-based robotic nanoelectrospray ionization system coupled to mass spectrometry has been developed to rapidly screen enzymatic reactions with environmentally relevant CECs. Three laccases, two tyrosinases, and two peroxidases were studied for their ability to transform ten pharmaceuticals and benzotriazole. Acetaminophen was most susceptible to enzymatic conversion by horseradish peroxidase (HRP), laccase from Trametes versicolor (LccTV), and a tyrosinase from Agaricus bisporus (TyrAB). Diclofenac and mefenamic acid were converted by HRP and LccTV, whereas sotalol was solely amenable to HRP conversion. Benzotriazole, carbamazepine, gabapentin, metoprolol, primidone, sulfamethoxazole, and venlafaxine remained persistent in this study. The results obtained here emphasize that enzymes are highly selective catalysts and more effort is required in the use of fast monitoring technologies to find suitable enzyme systems. Despite the methodological limitations discussed in detail, the automated tool provides a routine on-line screening of various enzymatic reactions to identify potential enzymes that degrade CECs. Graphical abstract A chip-based robotic nano-ESI-MS tool to rapidly monitor enzymatic degradation of environmentally relevant emerging contaminants.

RPLC-HILIC and SFC with Mass Spectrometry: Polarity-Extended Organic Molecule Screening in Environmental (Water) Samples.

Trace organic compounds are important in environmental analysis because they impact water quality and introduce potential (eco)toxicological effects. Current analytical methods mostly rely on gas chromatography (GC) or reversed-phase liquid chromatography (RPLC) coupled with (tandem) mass spectrometry. However, neither method can easily separate very polar molecules. This study presents two chromatographic separation strategies, a serial RPLC-hydrophilic interaction liquid chromatography (RPLC-HILIC) coupling and an analytical scale supercritical fluid chromatography (SFC) system, and validates their separation effectiveness as polarity-extended chromatographic methods for 274 environmentally relevant compounds. Compounds tested were grouped into three polarity classes, "very polar" {log D (pH 7) below -2.5}, "polar" {log D (pH 7) -2.5 to +2}, and "non-polar" {log D (pH 7) higher than +2}). Nearly all compounds could be retained in both systems with relative standard deviations of retention times (RT; n = 6) typically between 2 and 5%. Both techniques have considerable benefits when combined with accurate mass spectrometric detection. Molecules RT and accurate mass were recorded in a database for each set up. This information was used for compound screening measurements like "hidden-target screening" in complex environmental matrices (such as wastewater treatment plant effluents). Results of both techniques are complementary and useful for all types of molecules polarity. In this study, more than 80% of the compounds found in wastewater treatment plant effluent samples possessed a negative log D (pH 7) value. This result highlights the basic necessity to include "very polar" compounds in water monitoring techniques and protocols.

Effect of Perilla frutescens Extracts on Porcine Jejunal Epithelial Cells.

Green-leaved Perilla frutescens extracts were investigated on their effect on cell proliferation of the porcine jejunal epithelial cell line, IPEC-J2, as well as on the gene expression of cell cycle or cancer-related genes. Some extracted compounds were, however, susceptible to degradation in cell culture medium, whereas others were found to be stable during the entire experimental time. Control experiments also included the assessment of H2 O2 generation in cell culture medium caused by oxidation of natural extract compounds, which was proved to be absent at low extract concentrations. A fast and significant inhibition of cell growth at low physiological extract concentrations could be observed. This finding, along with an immediate downregulation of 67 kDa laminin receptor and cyclin D1 expression, can be accounted to the presence of Perilla frutescens extract. Copyright © 2016 John Wiley & Sons, Ltd.

Comprehensive assessment of Cytochrome P450 reactions: A multiplex approach using real-time ESI-MS.

BACKGROUND: The detailed analysis of Cytochrome P450 (CYP) catalyzed reactions is of great interest, since those are of importance for biotechnical applications, drug interaction studies and environmental research. Often cocktail approaches are carried out in order to monitor several CYP activities in a single experiment. Commonly in these approaches product formation is detected and IC50 values are determined. METHODS: In the present work, the reactions of two different CYP isoforms were monitored using real-time electrospray ionization mass spectrometry. Multiplex experiments using the highly specific CYP2A6 with its corresponding substrate coumarin as well as the highly promiscuous CYP3A4 with testosterone were conducted. Product formation and substrate depletion were simultaneously monitored and compared to the single CYP experiments. The diffusion-controlled rate of reaction and conversion rates that are used as parameters to assess the enzymatic activity were calculated for all measurements conducted. RESULTS: Differences in conversion rates and the theoretical rate of reaction that were observed for single CYP and multiplex experiments, respectively, reveal the complexity of the underlying mechanisms. Findings of this study imply that there might be distinct deviations between product formation and substrate degradation when mixtures are used. CONCLUSIONS: Detailed results indicate that for a comprehensive assessment of these enzymatic reactions both product and substrate should be considered. GENERAL SIGNIFICANCE: The direct hyphenation of enzymatic reactions to mass spectrometry allows for a comprehensive assessment of enzymatic behavior. Due to the benefits of this technique, the entire system which includes substrate, product and intermediates can be investigated. Thus, besides IC50 values further information regarding the enzymatic behavior offers the opportunity for a more detailed insight.

Mass spectrometric real-time monitoring of an enzymatic phosphorylation assay using internal standards and data-handling freeware.

RATIONALE: Continuous-flow reaction detection systems (monitoring enzymatic reactions with mass spectrometry (MS)) lack quantitative values so far. Therefore, two independent internal standards (IS) are implemented in a way that the online system stability can be observed, quantitative conversion values for substrate and product can be obtained and they can be used as mass calibration standards for high MS accuracy. METHODS: An application previously developed for the MS detection of peptide phosphorylation by cAMP-dependent protein kinase A (PKA) (De Boer et al., Anal. Bioanal. Chem. 2005, 381, 647-655) was transferred to a continuous-flow reaction detection system. This enzymatic reaction, involving enzyme activation as well as the transfer of a phosphate group from ATP to a peptide substrate, was used to prove the compatibility of a quantitative enzymatic assay in a continuous-flow real-time system (connected to MS). RESULTS: Moreover (using internal standards), the critical parameter reaction temperature (including solution density variations depending on temperature) was studied in the continuous-flow mixing system. Furthermore, two substrates (malantide and kemptide), two enzyme types (catalytic subunit of PKA and complete PKA) and one inhibitor were tested to determine system robustness and long-term availability. Even spraying solutions that contained significant amount of MS contaminants (e.g. the polluted catalytic subunit) resulted in quantifiable MS signal intensities. Subsequent recalculations using the internal standards led to results representing the power of this application. CONCLUSIONS: The presented methodology and the data evaluation with available Achroma freeware enable the direct coupling of biochemical assays with quantitative MS detection. Monitoring changes such as temperature, reaction time, inhibition, or compound concentrations can be observed quantitatively and thus enzymatic activity can be calculated.

Enzymatic Assays Coupled with Mass Spectrometry with or without Embedded Liquid Chromatography.

This article reviews monitoring strategies for enzymatic assays coupled with mass spectrometric detection. This coupling has already been shown to be helpful in providing versatile and detailed knowledge about enzyme kinetics. Various available publications address two general approaches. 1) The continuous-flow setup allows real-time determination of substrate degradation. Simultaneously, resulting product or potential intermediates can be detected. 2) The online coupled continuous-flow mixing assay allows the direct coupling of an enzymatic assay to chromatographic separation of complex mixtures. The latest efforts in improving the methodology have been made with regard to miniaturization. This is especially advantageous with regard to reducing costly consumption of chemicals. Finally, these developments are applicable for diverse bioanalytical purposes in the realms of pharmaceutical, biotechnological, food, and environmental research.

Non-target screening with high-resolution mass spectrometry: critical review using a collaborative trial on water analysis.

In this article, a dataset from a collaborative non-target screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target screening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches and methods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a "fully-automated identification workflow" remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution of more spectra from standard substances into (openly accessible) databases. Graphical Abstract Matrix of identification approach versus identification confidence.

Utilization of real-time electrospray ionization mass spectrometry to gain further insight into the course of nucleotide degradation by intestinal alkaline phosphatase.

RATIONALE: Related with its ability to degrade nucleotides, intestinal alkaline phosphatase (iAP) is an important participant in intestinal pH regulation and inflammatory processes. However, its activity has been investigated mainly by using artificial non-nucleotide substrates to enable the utilization of conventional colorimetric methods. To capture the degradation of the physiological nucleotide substrate of the enzyme along with arising intermediates and the final product, the enzymatic assay was adapted to mass spectrometric detection. Therewith, the drawbacks associated with colorimetric methods could be overcome. METHODS: Enzymatic activity was comparatively investigated with a conventional colorimetric malachite green method and a single quadrupole mass spectrometer with an electrospray ionization source using the physiological nucleotide substrates ATP, ADP or AMP and three different pH-values in either methodological approach. By this means the enzymatic activity was assessed on the one hand by detecting the phosphate release spectrometrically at defined time points of enzymatic reaction or on the other by continuous monitoring with mass spectrometric detection. RESULTS: Adaption of the enzymatic assay to mass spectrometric detection disclosed the entire course of all reaction components--substrate, intermediates and product--resulting from the degradation of substrate, thereby pointing out a stepwise removal of phosphate groups. By calculating enzymatic substrate conversion rates a distinctively slower degradation of AMP compared to ADP or ATP was revealed together with the finding of a substrate competition between ATP and ADP at alkaline pH. CONCLUSIONS: The comparison of colorimetric and mass spectrometric methods to elucidate enzyme kinetics and specificity clearly underlines the advantages of mass spectrometric detection for the investigation of complex multi-component enzymatic assays. The entire course of enzymatic substrate degradation was revealed with different nucleotide substrates, thus allowing a specific monitoring of intestinal alkaline phosphatase activity.

Robustness of a method based on the serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC-MS for the analysis of phenols.

We have recently proved that the serial coupling of RP and zwitterionic hydrophilic interaction LC with mass spectrometric detection is a versatile and reliable technique to simultaneously separate polar and apolar compounds in complex samples, for example, phenols in wine. In order to evaluate the system suitability for long-term usage, the robustness of a method based on the serial coupling of RP and zwitterionic hydrophilic interaction LC was evaluated after one year of analyses comprising >1100 injections. The retention time and peak shape of phenol standards and phenols in a red wine were compared to the values previously published. Phenol retention times were shifted <30 s. However, the peak widths were increased, partially due to the deterioration of the stationary phases. Nevertheless, the method was still highly reliable for the analysis of phenols in wine.

A highly conserved arginine residue of the chitosanase from Streptomyces sp. N174 is involved both in catalysis and substrate binding.

BACKGROUND: Streptomyces sp. N174 chitosanase (CsnN174), a member of glycoside hydrolases family 46, is one of the most extensively studied chitosanases. Previous studies allowed identifying several key residues of this inverting enzyme, such as the two catalytic carboxylic amino acids as well as residues that are involved in substrate binding. In spite of the progress in understanding the catalytic mechanism of this chitosanase, the function of some residues highly conserved throughout GH46 family has not been fully elucidated. This study focuses on one of such residues, the arginine 42. RESULTS: Mutation of Arg42 into any other amino acid resulted in a drastic loss of enzyme activity. Detailed investigations of R42E and R42K chitosanases revealed that the mutant enzymes are not only impaired in their catalytic activity but also in their mode of interaction with the substrate. Mutated enzymes were more sensitive to substrate inhibition and were altered in their pattern of activity against chitosans of various degrees of deacetylation. Our data show that Arg42 plays a dual role in CsnN174 activity. CONCLUSIONS: Arginine 42 is essential to maintain the enzymatic function of chitosanase CsnN174. We suggest that this arginine is influencing the catalytic nucleophile residue and also the substrate binding mode of the enzyme by optimizing the electrostatic interaction between the negatively charged carboxylic residues of the substrate binding cleft and the amino groups of GlcN residues in chitosan.

Biochemical and molecular characterization of a thermostable chitosanase produced by the strain Paenibacillus sp. 1794 newly isolated from compost.

Chitosan raises a great interest among biotechnologists due to its potential for applications in biomedical or environmental fields. Enzymatic hydrolysis of chitosan is a recognized method allowing control of its molecular size, making possible its optimization for a given application. During the industrial hydrolysis process of chitosan, viscosity is a major problem; which can be circumvented by raising the temperature of the chitosan solution. A thermostable chitosanase is compatible with enzymatic hydrolysis at higher temperatures thus allowing chitosan to be dissolved at higher concentrations. Following an extensive micro-plate screening of microbial isolates from various batches of shrimp shells compost, the strain 1794 was characterized and shown to produce a thermostable chitosanase. The isolate was identified as a novel member of the genus Paenibacillus, based on partial 16S rDNA and rpoB gene sequences. Using the chitosanase (Csn1794) produced by this strain, a linear time course of chitosan hydrolysis has been observed for at least 6 h at 70 °C. Csn1794 was purified and its molecular weight was estimated at 40 kDa by SDS-PAGE. Optimum pH was about 4.8, the apparent Km and the catalytic constant kcat were 0.042 mg/ml and 7,588 min⁻¹, respectively. The half-life of Csn1794 at 70 °C in the presence of chitosan substrate was >20 h. The activity of chitosanase 1794 varied little with the degree of N-acetylation of chitosan. The enzyme also hydrolyzed carboxymethylcellulose but not chitin. Chitosan or cellulose-derived hexasaccharides were cleaved preferentially in a symmetrical way ("3+3") but hydrolysis rate was much faster for (GlcN)6 than (Glc)6. Gene cloning and sequencing revealed that Csn1794 belongs to family 8 of glycoside hydrolases. The enzyme should be useful in biotechnological applications of chitosan hydrolysis, dealing with concentrated chitosan solutions at high temperatures.

Serial coupling of reversed-phase and zwitterionic hydrophilic interaction LC/MS for the analysis of polar and nonpolar phenols in wine.

In the present study, an easy and efficient method based on the serial coupling of analytical reversed-phase and zwitterionic hydrophilic interaction liquid chromatography was developed for the simultaneous separation of polar and nonpolar phenols occurring in wine. The zwitterionic hydrophilic column was connected in series to the reversed-phase one via a T-piece, with which the ACN content in eluent of the second dimension was increased, in order to cope the solvent strength incompatibility between the two columns. The final mobile phase at low-flow rate (≤0.5 mL/min), high-ACN content (90%), and low-salt concentration was directed to an ESI-TOF-MS , for high accurate mass detections. The developed method was applied for the identification of target phenols in several wines. Retention time and peak width intra- and interday repeatability studies proved the reliability of the method for the simultaneous analysis of all the polar and nonpolar analytes in wine. The serial reversed-phase/zwitterionic hydrophilic interaction liquid chromatography coupling offered the possibility to enlarge the number of identified compounds and it represents a valid approach for nontarget analysis of complex samples by a single injection.