Summary recommendations on "Analytical methods for substances in the Watch List under the Water Framework Directive".

The Watch List (WL) is a monitoring program under the European Water Framework Directive (WFD) to obtain high-quality Union-wide monitoring data on potential water pollutants for which scarce monitoring data or data of insufficient quality are available. The main purpose of the WL data collection is to determine if the substances pose a risk to the aquatic environment at EU level and subsequently to decide whether a threshold, the Environmental Quality Standards (EQS) should be set for them and, potentially to be listed as priority substance in the WFD. The first WL was established in 2015 and contained 10 individual or groups of substances while the 4th WL was launched in 2022. The results of monitoring the substances of the first WL showed that some countries had difficulties to reach an analytical Limit of Quantification (LOQ) below or equal to the Predicted No-Effect Concentrations (PNEC) or EQS. The Joint Research Centre (JRC) of the European Commission (EC) organised a series of workshops to support the EU Member States (MS) and their activities under the WFD. Sharing the knowledge among the Member States on the analytical methods is important to deliver good data quality. The outcome and the discussion engaged with the experts are described in this paper, and in addition a literature review of the most important publications on the analysis of 17-alpha-ethinylestradiol (EE2), amoxicillin, ciprofloxacin, metaflumizone, fipronil, metformin, and guanylurea from the last years is presented.

Quantitative electrospray ionization efficiency scale: 10 years after.

RATIONALE: The first comprehensive quantitative scale of the efficiency of electrospray ionization (ESI) in the positive mode by monoprotonation, containing 62 compounds, was published in 2010. Several trends were found between the compound structure and ionization efficiency (IE) but, possibly because of the limited diversity of the compounds, some questions remained. This work undertakes to align the new data with the originally published IE scale and carry out statistical analysis of the resulting more extensive and diverse data set to derive more grounded relationships and offer a possibility of predicting logIE values. METHODS: Recently, several new IE studies with numerous compounds have been conducted. In several of them, more detailed investigations of the influence of compound structure, solvent properties, or instrument settings have been conducted. IE data from these studies and results from this work were combined, and the multilinear regression method was applied to relate IE to various compound parameters. RESULTS: The most comprehensive IE scale available, containing 334 compounds of highly diverse chemical nature and spanning 6 orders of magnitude of IE, has been compiled. Several useful trends were revealed. CONCLUSIONS: The ESI ionization efficiency of a compound by protonation is mainly affected by three factors: basicity (expressed by pKaH in water), molecular size (expressed by molar volume or surface area), and hydrophobicity of the ion (expressed by charge delocalization in the ion or its partition coefficient between a water-acetonitrile mixture and hexane). The presented models can be used for tentative prediction of logIE of new compounds (under the used conditions) from parameters that can be computed using commercially available software. The root mean square error of prediction is in the range of 0.7-0.8 log units.

Removal of hazardous substances in municipal wastewater treatment plants.

Chemical pollution poses a threat to the aquatic environment and to human health. Wastewater treatment plants are the last defensive line between the aquatic environment and emissions of pollutants. This study focuses on identification of most relevant hazardous substances in Estonian municipal wastewater and their fate in the treatment process. During this study, seasonal wastewater and sewage sludge samples were collected from nine municipal wastewater treatment plants and analyzed for 282 hazardous substances, including EU (n = 45) and Estonian (n = 31) priority substances. Results of this study show that several substances that are subject to international restrictions (e.g. Stockholm Convention) are still present in untreated sewage. Wastewater treatment systems that had a greater level of complexity (TEC >5) were more successful in removing hazardous substances. Statistical analyses showed that removal efficiency of organic hazardous substances had significant (p-value <0.05) linear correlation with removal efficiencies of chemical oxygen demand (COD) and total suspended solids (TSS), but a monotonic relationship with operators' competency. This study showed that operators' competency had a strong influence on the stability of the wastewater treatment efficiency and removal of organic hazardous substances.

Characterization of wines with liquid chromatography electrospray ionization mass spectrometry: Quantification of amino acids via ionization efficiency values.

Quantification of analysis results for the suspect and non-targeted screening is essential for obtaining meaningful insight from the measurements. Ionization efficiency predictions is a possible approach to enable quantitation without standard substances. This is, however, especially challenging for the analysis carried out by combining the full scan mode either with fragmentation experiments in data-dependent or data-independent acquisition mode. Here we investigate the correlation of ionization efficiency values measured in full scan mode with the response factors measured in multiple reaction monitoring (MRM) mode for derivatized amino acids. We observe good correlation (R2 of 0.80) for 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatized amino acids. This encourages the use of the measured ionization efficiency values to estimate amino acid concentrations in different beverages. We apply the measured ionization efficiency values for estimating the concentration of amino acids for measurements done both in full scan as well as in MRM mode in wines and beers. We show that the calculated concentrations are in very good correlation with measured values (R2 of 0.71 to 1.00). The method possesses average trueness of 70.5% and shows an insignificant matrix effect.

Matrix interference in LC-ESI-MS/MS analysis of metanephrines in protein precipitated plasma samples.

Metanephrine and normetanephrine are measured in blood plasma to diagnose different diseases. Simpler sample preparation procedures are preferred but tend to yield less purified extracts. Therefore, thorough investigation of matrix effects is required. In this work, several sample preparation methods and chromatographic modes were compared for liquid chromatography tandem mass spectrometric (with electrospray ionization; LC-ESI-MS/MS) analysis of metanephrine and normetanephrine in blood plasma. Protein precipitation with methanol was found to be sufficient for sample preparation and pentafluorophenyl column provided adequate chromatographic separation. A new cheaper and less labor-intensive approach is proposed where necessary quantitation limits are achieved through a sample preparation containing only protein precipitation and dilution of the sample extract. Matrix effects for different sample preparation methods and the use of isotope-labeled internal standards were evaluated. Unusual interference to D3-labeled internal standard of normetanephrine was discovered - signal of interfering compound increased while the matrix effects were reduced by dilution, e.g. dilution eliminates matrix suppression on interfering compound. The results stress the need to monitor interfering compounds and evaluate matrix effects at every step of method development. Matrix effects and interferences can be different for analytes and their corresponding isotopically labeled internal standards. This means that the use of isotopically labeled internal standards cannot guarantee accuracy of obtained results. New method allows quantification of the low nanomolar concentrations of metanephrine and normetanephrine in plasma samples.

Ionization efficiency ladders as tools for choosing ionization mode and solvent in liquid chromatography/mass spectrometry.

RATIONALE: The choice of mobile phase components and optimal ion source, mainly electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI), is a crucial part in liquid chromatography/mass spectrometry (LC/MS) method development to achieve higher sensitivity and lower detection limits. In this study we demonstrate how to rigorously solve these questions by using ionization efficiency scales. METHODS: Four ionization efficiency scales are used: recorded with both APCI and ESI sources and using both methanol- and acetonitrile-containing mobile phases. Each scale contains altogether more than 50 compounds. In addition, measurements with a chromatographic column were also performed. RESULTS: We observed a correlation between calibration graph slopes under LC conditions and logIE values in ESI (but not APCI) thereby validating the use of logIE values for choosing the ion source. Most of the studied compounds preferred ESI as an ion source and methanol as mobile organic phase. APCI remains the ion source of choice for polycyclic aromatic hydrocarbons. For APCI, both acetonitrile and methanol provide similar ionization efficiencies with few exceptions. CONCLUSIONS: Overall the results of this work give a concise guideline for practitioners in choosing an ion source for LC/MS analysis on the basis of the chemical nature of the analytes.

A systematic approach toward comparing electrospray ionization efficiencies of derivatized and non-derivatized amino acids and biogenic amines.

Ionization efficiency (IE) in mass spectrometry (MS) has been studied for many different compounds, and different IE scales have been constructed in order to quantitatively characterize IE. In the case of MS, derivatization has been used to increase the sensitivity of the method and to lower the limits of detection. However, the influence of derivatization on IE across different compounds and different derivatization reagents has not been thoroughly researched, so that practitioners do not have information on the IE-enhancing abilities of different derivatization reagents. Moreover, measuring IE via direct infusion of compounds cannot be considered fully adequate. Since derivatized compounds are in complex mixtures, a chromatographic method is needed to separate these compounds to minimize potential matrix effects. In this work, an IE measurement system with a chromatographic column was developed for mainly amino acids and some biogenic amines. IE measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) were carried out, and IE scales were constructed with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate. Additionally, eluent composition effects on ionization were investigated. Results showed that derivatization increases IE for most of the compounds (by average 0.9 and up to 2-2.5 logIE units) and derivatized compounds have more similar logIE values than without derivatization. Mobile phase composition effects on ionization efficiencies were negligible. It was also noted that the use of chromatographic separation instead of flow injection mode slightly increases IE. In this work, for the first time, IE enhancement of derivatization reagents was quantified under real LC/ESI/MS conditions and obtained logIE values of derivatized compounds were linked with the existing scale.

Dependence of matrix effect on ionization polarity during LC-ESI-MS analysis of derivatized amino acids in some natural samples.

Matrix effect, the influence of co-eluting components on the ionization efficiency of the analyte, affects the trueness and precision of the LC-ESI-MS analysis. Derivatization can reduce or eliminate matrix effect, for example, diethyl ethoxymethylenemalonate (DEEMM) derivatives have shown less matrix effect compared to other derivatives. Moreover, the use of negative ion mode can further reduce matrix effect. In order to investigate the combination of derivatization and different ionization modes, an LC-ESI-MS/MS method using alternating positive/negative ion mode was developed and validated. The analyses in positive and negative ion modes had comparable limit of quantitation values. The influence of ESI polarity on matrix effect was investigated during the analysis of 22 DEEMM-derivatized amino acids in herbal extracts and honeys. Sample dilution approach was used for the evaluation of the presence of matrix effect. Altogether, 4 honeys and 11 herbal extracts were analyzed, and the concentrations of 22 amino acids in the samples are presented. In the positive ion mode, matrix effect was observed for several amino acid derivatives and the matrix effect was stronger in honey samples compared to the herbal extracts. The negative ion mode was free from matrix effect, with only few exceptions in honeys (average relative standard deviation over all analytes and matrices was 8%; SD = 7%). The matrix effect was eliminated in the positive ion mode by sample dilution and agreement between concentrations from the two ion modes was achieved for most amino acids. In conclusion, it was shown that the combination of derivatization and negative ion mode can be a powerful tool for minimizing matrix effect in more complicated applications.

Determination of neonicotinoids in Estonian honey by liquid chromatography-electrospray mass spectrometry.

The aim of the study was to provide a comprehensive overview of neonicotinoid pesticide residues in honey samples for a single country and compare the results with the import data for neonicotinoid pesticides. The levels of four neonicotinoid pesticides, namely thiamethoxam, imidacloprid, acetamiprid, and thiacloprid, were determined in 294 honey samples harvested from 2005 to 2013 from more than 200 locations in Estonia. For the analyzed honey samples, 27% contained thiacloprid, and its levels in all cases were below the maximum residue level set by the European Union. The other neonicotinoids were not detected. The proportion of thiacloprid-positive samples for different years correlates well with the data on thiacloprid imports into Estonia, indicating that honey contamination with neonicotinoids can be estimated based on the import data.

Establishing Atmospheric Pressure Chemical Ionization Efficiency Scale.

Recent evidence has shown that the atmospheric pressure chemical ionization (APCI) mechanism can be more complex than generally assumed. In order to better understand the processes in the APCI source, for the first time, an ionization efficiency scale for an APCI source has been created. The scale spans over 5 logIE (were IE is ionization efficiency) units and includes 40 compounds with a wide range of chemical and physical properties. The results of the experiments show that for most of the compounds the ionization efficiency order in the APCI source is surprisingly similar to that in the ESI source. Most of the compounds that are best ionized in the APCI source are not small volatile molecules. Large tetraalkylammonium cations are a prominent example. At the same time, low-polarity hydrocarbons pyrene and anthracene are ionized in the APCI source but not in the ESI source. These results strongly imply that in APCI several ionization mechanisms operate in parallel and a mechanism not relying on evaporation of neutral molecules from droplets has significantly higher influence than commonly assumed.

Tutorial review on validation of liquid chromatography-mass spectrometry methods: part II.

This is the part II of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC-MS) and discuss specific issues that arise with MS (and MS-MS) detection in LC (as opposed to the "conventional" detectors). The Part II starts with briefly introducing the main quantitation methods and then addresses the performance related to quantification: linearity of signal, sensitivity, precision, trueness, accuracy, stability and measurement uncertainty. The last section is devoted to practical considerations in validation. With every performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to handle it, specifically in the case of LC-MS methods.

Tutorial review on validation of liquid chromatography-mass spectrometry methods: part I.

This is the part I of a tutorial review intending to give an overview of the state of the art of method validation in liquid chromatography mass spectrometry (LC-MS) and discuss specific issues that arise with MS (and MS/MS) detection in LC (as opposed to the "conventional" detectors). The Part I briefly introduces the principles of operation of LC-MS (emphasizing the aspects important from the validation point of view, in particular the ionization process and ionization suppression/enhancement); reviews the main validation guideline documents and discusses in detail the following performance parameters: selectivity/specificity/identity, ruggedness/robustness, limit of detection, limit of quantification, decision limit and detection capability. With every method performance characteristic its essence and terminology are addressed, the current status of treating it is reviewed and recommendations are given, how to determine it, specifically in the case of LC-MS methods.

Development of amino acid derivatization reagents for liquid chromatography electrospray ionization mass spectrometric analysis and ionization efficiency measurements.

Derivatization is one of the most common ways for improving chromatographic separation and sensitivity for LC-ESI-MS analysis. The aim of this work was to design new derivatization reagents for LC-ESI-MS analysis of amino acids which would (1) provide good reversed phase chromatographic separation, (2) most importantly, provide low detection limits, (3) be easily synthesized, (4) produce derivatives which are less susceptible to matrix influences and (5) have convenient derivatization procedure with stable derivatives suitable for automatization. In the current work two new LC-ESI-MS compatible derivatization reagents have been designed and synthesized, dibenzyl ethoxymethylene malonate (DBEMM) and benzyl ethyl ethoxymethylene malonate (EBEMM). The DBEMM meets all the goals set with instrumental detection limits as low as 1 femtomole for amino acids and 40 attomole for selenoamino acids.

Study of the matrix effects and sample dilution influence on the LC-ESI-MS/MS analysis using four derivatization reagents.

For liquid chromatographic analysis of amino acids involving derivatization and mass-spectrometric detection, it becomes more important to evaluate the presence of matrix effects in complex samples. This is somewhat complicated for amino acid analysis where analyte free sample matrix is often unavailable. In this work, matrix effects were investigated using post-column infusion method for 9-fluorenylmethyl chloroformate (FMOC-Cl) derivatives of ß-Ala, Gly and Phe and diethyl ethoxymethylenemalonate (DEEMM) derivative of ß-Ala. While for DEEMM derivatives, the main signal suppression was due to the borate buffer, in case of FMOC-Cl, other FMOC-derivatives caused signal suppression. Analysis of amino acids in tea and honey with DEEMM, FMOC-Cl, p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) and dansyl chloride (DNS) showed that amino acid concentrations found with different reagents do not agree well. Sample dilution experiments indicated that the sample matrix affected the analysis results obtained with DEEMM the least, but with FMOC-Cl, TAHS and DNS, sample dilution had an influence on the results. When sample dilution and extrapolative dilution approach were applied on the latter results, an agreement of amino acid concentrations measured with different reagents was achieved within relative standard deviation (RSD) of 22% for most cases.

Matrix influence on derivatization and ionization processes during selenoamino acid liquid chromatography electrospray ionization mass spectrometric analysis.

Considering the importance of derivatization in LC/ESI/MS analysis, the objective of this work was to develop a method for evaluation of matrix effect that would discriminate between matrix effect due to the derivatization reaction yield and from the ESI. Four derivatization reagents (TAHS, DEEMM, DNS, FMOC-Cl) were studied with respect to matrix effects using two selenoamino acids and onion matrix as model system. A novel method for assessing matrix effects of LC/ESI/MS analyses involving derivatization is proposed, named herein post-derivatization spiking, that allows evaluating effect of matrix on ESI ionization without derivatization reaction yield contribution. The proposed post-derivatization spiking method allowed to demonstrate that the reason of reduced analytical signal can be signal suppression in ESI (as in case of DNS derivatives with matrix effects 38-99%), alteration of derivatization reaction yield (TAHS, matrix effects 92-113%, but reaction yields 20-50%) or both (FMOC-Cl, matrix effects 28-88% and reaction yields 50-70%). In case of DEEMM derivatives, matrix reduces reaction yield but enhances ESI/MS signal. A method for matrix effect evaluation was developed. It was also confirmed that matrix effects can be reduced by dilution.

Comparison of amino acid derivatization reagents for LC-ESI-MS analysis. Introducing a novel phosphazene-based derivatization reagent.

Amino acid analysis with high performance liquid chromatography with electrospray ionization mass spectrometry (LC-ESI-MS) is an emerging method. For more sensitive analysis, derivatization is used and next to commercially available derivatization reagents such as dansyl chloride (DNS), 9-fluorenylmethyl chloroformate (FMOC-Cl) and diethyl ethoxymethylenemalonate (DEEMM), new derivatization reagents are designed specially for LC-ESI-MS, like p-N,N,N-trimethylammonioanilyl N'-hydroxysuccinimidyl carbamate iodide (TAHS) which provides very low limits of detection. In this work, a novel phosphazene based derivatization reagent (FOSF) that provides comparable limits of quantitation (LoQ) to TAHS is introduced. Moreover, a thorough comparison between FOSF, TAHS, DNS, FMOC-Cl and DEEMM is carried out for 7 different amino acids - Arg, Asp, Gly, ß-Ala, Pro, Trp and Phe. This is a first time that thorough comparison is carried out on the same instrument for amino acid derivatization reagents. Results on the same instrument for five amino acid derivatization reagents show that novel reagents are sensitive with LoQ values around 80 fmol but have disadvantages such as problematic chromatographic separation. Next to novel reagents, DEEMM offers very good LoQ-s (average of 150 fmol) and wide dynamic linear range.

Influence of boric acid on electrospray ionization efficiency.

Liquid chromatography electrospray ionization mass-spectrometry (LC-ESI-MS) was used to analyze 9-fluorenylmethylmethoxycarbonyl chloride (Fmoc-Cl) and diethyl ethoxymethylenemalonate (Deemm) derivatives of three amino acids and five other compounds. Influence of boric acid on their ionization was investigated and dramatic impact on the signal was observed. The strongest signal suppression (6% of signal remains) was observed for the Deemm derivative of beta-Alanine (with ammonium acetate in eluent). With only formic acid as the eluent pH modifier, signal enhancement was observed, being largest for Fmoc-Cl derivative of Phenylalanine, 267%. Investigation of the influence of boric acid shows that it is a possible signal enhancer for LC-ESI-MS analysis.

Comparison of three buffer solutions for amino acid derivatization and following analysis by liquid chromatography electrospray mass spectrometry.

For reversed phase separation amino acids are usually derivatized. Several derivatization reactions are carried out at basic pH. In the present work, influence of three basic buffer solutions on liquid chromatography electrospray ionization mass-spectrometric (LC-ESI-MS) analysis of amino acid derivatives was studied. Borate buffer--the most common derivatization buffer--was found to influence ESI ionization up to 23 min retention time. For 9-fluorenylmethylmethoxycarbonyl chloride (Fmoc-Cl derivatization) carbonate buffer should be preferred as it provides higher responses. Hexafluoroisopropanol (HFIP) buffer improves chromatographic peak shapes and responses for diethyl ethoxymethylenemalonate (DEEMM) derivatives.

Analysis of selenomethylselenocysteine and selenomethionine by LC-ESI-MS/MS with diethyl ethoxymethylenemalonate derivatization.

A simple method to identify and determine Selenomethionine (SeMet) and Selenomethylselenocysteine (Se-MeSeCys) with diethyl ethoxymethylenemalonate (DEEMM) derivatization and LC-ESI-MS/MS determination was developed. Separation of SeMet and Se-MeSeCys was achieved in 15.3 minutes. The calibration graph was linear in the range of 0.32 pmol to 49 pmol for SeMet and 0.34 pmol to 40 pmol for Se-MeSeCys. To prevent oxidation of SeMet, 2-mercaptoethanol was introduced to the calibration solutions. Detection limits were 0.1 pmol, which are comparable to LC-ICP-MS analysis. The developed method therefore offers an alternative to LC-ICP-MS offering similar sensitivity and additionally allows identification. The method was used to determine Se-MeSeCys and SeMet in onion samples.

A sensitive method for free amino acids analysis by liquid chromatography with ultraviolet and mass spectrometric detection using precolumn derivatization with diethyl ethoxymethylenemalonate: application to the honey analysis.

In case of some foods and drinks, their amino acid content is demonstrated to correlate with the botanical and/or geographical origin of the plant. In present work a method for amino acid analysis in honey was developed and validated. The method consists of sample preparation (including solid phase extraction), derivatization with diethyl ethoxymethylenemalonate and liquid chromatographic analysis. Full separation of 23 amino acids was achieved. All steps were extensively studied and optimized for analytical performance. Although ultraviolet (UV) detection provides sufficient sensitivity for honey analysis, all the steps of the method were designed to be compatible with mass spectrometric (MS) detection. The developed method has been applied to analysis of nearly 200 Estonian honey samples.