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.

Strategies in developing high-throughput liquid chromatography protocols for method qualification of pharmacopeial monographs.

Method qualification is a key step in the development of routine analytical monitoring of pharmaceutical products. However, when relying on published monographs that describe longer method times based on older high-performance liquid chromatography column and instrument technology, this can delay the overall analysis process for generated drug products. In this study, high-throughput ultrahigh pressure liquid chromatography techniques were implemented to decrease the amount of time needed to complete a 24-run sequence to identify linearity, recovery, and repeatability for both drug assay and impurity analysis in 16 min. Multiple experimental parameters were tested to identify a range of experimental settings that could be used for the sequence while still maintaining this fast analysis time. The full sequence was replicated on a different system and with different columns, further demonstrating its robustness.

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.

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.

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.

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.

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.

Degradation of polysorbate investigated by a high-performance liquid chromatography multi-detector system with charged aerosol and mass detection.

Polysorbate 80 is widely used as a formulation component in biopharmaceutical drug products. Recent studies have shown that polysorbate 80 is readily degraded either through direct or indirect means. The degradation of polysorbate 80 causes a concern for the long-term stability of biopharmaceutical drug product, as the breakdown products of polysorbate 80 have been shown to cause adverse effects, such as formation of sub-visible and visible particles and mAb aggregation. Understanding the path and extent of degradation is of a paramount importance for the formulator during formulation development. A multi-detector HPLC system using charged aerosol and mass detection was developed and optimized for the characterization of polysorbate 80 standards. The system included a post-column make-up flow, i.e. an inverse gradient, that enabled constant eluent composition at the detectors. The inverse gradient eliminated the main source of variability for the charged aerosol detector response, thereby enabling the calculation of the mass balance between polysorbate components with different degrees of esterification. Extracted ion chromatograms of the mass detector combined with their respective retention times were used to qualitatively characterize the polysorbate samples down to the individual components. The system was applied to study the degradation of several polysorbate standards which occurred by enzymatic digestion or long-term storage. The system provided detailed information on the mechanism of degradation without the need for additional orthogonal analytical techniques.

The NORMAN Suspect List Exchange (NORMAN-SLE): facilitating European and worldwide collaboration on suspect screening in high resolution mass spectrometry.

Background: The NORMAN Association (https://www.norman-network.com/) initiated the NORMAN Suspect List Exchange (NORMAN-SLE; https://www.norman-network.com/nds/SLE/) in 2015, following the NORMAN collaborative trial on non-target screening of environmental water samples by mass spectrometry. Since then, this exchange of information on chemicals that are expected to occur in the environment, along with the accompanying expert knowledge and references, has become a valuable knowledge base for "suspect screening" lists. The NORMAN-SLE now serves as a FAIR (Findable, Accessible, Interoperable, Reusable) chemical information resource worldwide. Results: The NORMAN-SLE contains 99 separate suspect list collections (as of May 2022) from over 70 contributors around the world, totalling over 100,000 unique substances. The substance classes include per- and polyfluoroalkyl substances (PFAS), pharmaceuticals, pesticides, natural toxins, high production volume substances covered under the European REACH regulation (EC: 1272/2008), priority contaminants of emerging concern (CECs) and regulatory lists from NORMAN partners. Several lists focus on transformation products (TPs) and complex features detected in the environment with various levels of provenance and structural information. Each list is available for separate download. The merged, curated collection is also available as the NORMAN Substance Database (NORMAN SusDat). Both the NORMAN-SLE and NORMAN SusDat are integrated within the NORMAN Database System (NDS). The individual NORMAN-SLE lists receive digital object identifiers (DOIs) and traceable versioning via a Zenodo community (https://zenodo.org/communities/norman-sle), with a total of > 40,000 unique views, > 50,000 unique downloads and 40 citations (May 2022). NORMAN-SLE content is progressively integrated into large open chemical databases such as PubChem (https://pubchem.ncbi.nlm.nih.gov/) and the US EPA's CompTox Chemicals Dashboard (https://comptox.epa.gov/dashboard/), enabling further access to these lists, along with the additional functionality and calculated properties these resources offer. PubChem has also integrated significant annotation content from the NORMAN-SLE, including a classification browser (https://pubchem.ncbi.nlm.nih.gov/classification/#hid=101). Conclusions: The NORMAN-SLE offers a specialized service for hosting suspect screening lists of relevance for the environmental community in an open, FAIR manner that allows integration with other major chemical resources. These efforts foster the exchange of information between scientists and regulators, supporting the paradigm shift to the "one substance, one assessment" approach. New submissions are welcome via the contacts provided on the NORMAN-SLE website (https://www.norman-network.com/nds/SLE/). Supplementary Information: The online version contains supplementary material available at 10.1186/s12302-022-00680-6.

Localised quantitative structure-retention relationship modelling for rapid method development in reversed-phase high performance liquid chromatography.

Quantitative structure-retention relationships (QSRR) predicting the values of solute "hydrophobicity" coefficient η' in the approximate hydrophobic subtraction model (HSM) can be used to predict retention times of compounds on numerous reversed-phase (RP) columns, provided that column parameters on the corresponding stationary phases are available. In the present study, we propose a new dual clustering-based localised QSRR approach, combining P-ratio clustering (where P is the octanol-water partition coefficient) with second dominant interaction (SDI)-based clustering, to produce predictive models with an acceptable level of prediction accuracy for in silico column scoping in RP method development. QSRR models for η' values were derived for 49 compounds out of 63 in a dataset extracted from the literature, where retention data were measured under one isocratic mobile phase condition (i.e., acetonitrile-water, 50:50 [v/v]). These models gave a predictive squared correlation coefficient Qext(F2)2 of 0.83 and a root mean square error of prediction (RMSEP) of 0.14. For the modelling, a genetic algorithm-partial least square regression (GA-PLS) approach was performed using the η' values and their relevant molecular descriptors. The corresponding retention times were predicted by applying the predicted η' values of the models and the stationary phase "hydrophobicity" parameter H values for the corresponding columns to the approximate HSM, resulting in excellent accuracy and predictability (Qext(F2)2 of 0.90 and RMSEP of 0.72 min). The established QSRR approach was experimentally verified for six Thermo Scientific columns (Acclaim™ 120 C18, Acclaim Polar Advantage, Acclaim Polar Advantage II, Accucore™ aQ, Accucore Phenyl-X, and Hypersil Gold C18 columns) using two types of datasets. The first dataset consisted of eight model compounds extracted from the original dataset and retention time predictions for those compounds were then evaluated on the above columns. The result showed good agreement between predicted and observed retention times with an acceptable error in retention time predictions (slope of 0.97, Qext(F2)2 of 0.95, a mean absolute error (MAE) of 0.43 min and RMSEP of 0.61 min). The second dataset included eight test compounds not included in the original dataset, which were all classified into the η' cluster by applying a Tanimoto similarity (TS) threshold of 0.7. Similarly, predicted retention times of the test compounds were compared with their corresponding observed retention times, resulting in acceptable retention time predictions with the slope of 0.99, Qext(F2)2 of 0.93 and RMSEP of 0.52 min. Comparisons of resolution values between columns were utilised to select the most suitable columns for separations of the compounds in the respective test sets. Actual chromatograms obtained on the chosen columns showed the feasibility for effective column scoping without experimentation on numerous RP stationary phases available in the USP website, based on the predicted resolution values.

Liquid chromatography/atmospheric pressure ionization mass spectrometry with post-column liquid mixing for the efficient determination of partially oxidized polycyclic aromatic hydrocarbons.

The analytical hyphenation of micro-flow high-performance liquid chromatography (LC), with post-column liquid mixing and mass spectrometric detection (MS) was established to detect partially oxidized polycyclic aromatic hydrocarbons (oxy-PAHs) for low quantity samples. 100pmol injections of 30 reference standards could be detected in good sensitivity using either atmospheric pressure chemical ionization (APCI) and/or atmospheric pressure photoionization (APPI). The connected mass spectrometer was a single quadrupol analyzer realizing simultaneous registration of positive and negative ions in scan range width of 200 - 300Da. The ionization efficiency was compared using three ionization sources (incl. electrospray ionization (ESI)) for several oxy-PAHs. According to the mass spectra, the analytes behave differently in ionization properties. Ionization mechanism (e.g. deprotonated ions and electron captured ions) could be discussed with new inside views. Finally, the hyphenated system was applied to an exemplary aerosol extract and thus highlighting the expedient utilization of this downscaled method for real samples.

Emerging pollutants and plants--Metabolic activation of diclofenac by peroxidases.

Human pharmaceuticals and their residues are constantly detected in our waterbodies, due to poor elimination rates, even in the most advanced waste water treatment plants. Their impact on the environment and human health still remains unclear. When phytoremediation is applied to aid water treatment, plants may transform and degrade xenobiotic contaminants through phase I and phase II metabolism to more water soluble and less toxic intermediates. In this context, peroxidases play a major role in activating compounds during phase I via oxidation. In the present work, the ability of a plant peroxidase to oxidize the human painkiller diclofenac was confirmed using stopped flow spectroscopy in combination with LC-MS analysis. Analysis of an orange colored product revealed the structure of the highly reactive Diclofenac-2,5-Iminoquinone, which may be the precursor of several biological conjugates and breakdown products in planta.

LC-MS screening techniques for wastewater analysis and analytical data handling strategies: Sartans and their transformation products as an example.

A large number of anthropogenic trace contaminants such as pharmaceuticals, their human metabolites and further transformation products (TPs) enter wastewater treatment plants on a daily basis. A mixture of known, expected, and unknown molecules are discharged into the receiving aquatic environment because only partial elimination occurs for many of these chemicals during physical, biological and chemical treatment processes. In this study, an array of LC-MS methods from three collaborating laboratories was applied to detect and identify anthropogenic trace contaminants and their TPs in different waters. Starting with theoretical predictions of TPs, an efficient workflow using the combination of target, suspected-target and non-target strategies for the identification of these TPs in the environment was developed. These techniques and strategies were applied to study anti-hypertensive drugs from the sartan group (i.e., candesartan, eprosartan, irbesartan, olmesartan, and valsartan). Degradation experiments were performed in lab-scale wastewater treatment plants, and a screening workflow including an inter-laboratory approach was used for the identification of transformation products in the effluent samples. Subsequently, newly identified compounds were successfully analyzed in effluents of real wastewater treatment plants and river waters.

Study of the retention behavior in zwitterionic hydrophilic interaction chromatography of isomeric hydroxy- and aminobenzoic acids.

The retention behavior of fifteen isomeric hydroxy- and aminobenzoic acids in zwitterionic hydrophilic interaction chromatography was studied using a sulfobetaine phase (ZIC-HILIC). By an inspection of their molecular structures, the retention was related to the number, the position and hydrogen bond properties of the functional groups. The effect of the chromatographic conditions was analyzed in order to investigate the retention mechanism of the stationary phase. The increased retention observed for negative charged compounds when the mobile phase pH decreased was ascribed to a diminishing of the electrostatic repulsion with the underivatized silanol groups. Also the salt buffer concentration in the mobile was proved to have a great influence in the modulation of the electrostatic interactions. However, the retention behavior of the benzoic acids was not described by conventional ion-exchange models. Subsequently, a systematical analysis of partition, adsorption, and hydrophilic chromatographic models was presented. The results from the fittings indicated that partition processes govern mainly the ZIC-HILIC separation, but also adsorption processes via hydrogen bonds occurred for hydrogen donor analytes. Finally, the influence of the chromatographic conditions on the water enriched layer in which partition takes place has been evaluated by the elution behavior of toluene.

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.