PUBLICATIONS

Our poster presentations from conferences can be found here.

Standard substances free quantification makes LC/ESI/MS non-targeted screening of pesticides in cereals comparable between labs

TingtingWang, JaanusLiigand, Henrik LauritzFrandsen, JørnSmedsgaard, AnneliKruve

Food Chemistry 2020, 318

DOI: 10.1016/j.foodchem.2020.126460

LC/ESI/MS is the technique of choice for qualitative and quantitative food monitoring; however, analysis of a large number of compounds is challenged by the availability of standard substances. The impediment of detection of food contaminants has been overcome by the suspect and non-targeted screening. Still, the results from one laboratory cannot be compared with the results of another laboratory as quantitative results are required for this purpose. Here we show that the results of the suspect and non-targeted screening for pesticides can be made quantitative with the aid of in silico predicted electrospray ionization efficiencies and this allows direct comparison of the results obtained in two different laboratories. For this purpose, six cereal matrices were spiked with 134 pesticides and analysed in two independent labs; a high correlation for the results with the R2 of 0.85.

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

Artur Gornischeff, AnneliKruve, Riin Rebane

Journal of Chromatography A 2020

DOI: 10.1016/j.chroma.2020.461012

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.

Strategies for Drawing Quantitative Conclusions from Nontargeted Liquid Chromatography−High-Resolution Mass Spectrometry Analysis

Anneli Kruve

Analytical Chemistry 2020

DOI: 10.1021/acs.analchem.9b03481

This Feature aims at giving an overview of different possibilities for quantitatively comparing the results obtained from LC−HRMS-based nontargeted analysis. More specifically, quantification via structurally similar internal standards, different isotope labeling strategies, radiolabeling, and predicted ionization efficiencies are reviewed.

Quantification for non-targeted LC/MS screening without standard substances

Jaanus Liigand, Tingting Wang, Joshua Kellogg, Jørn Smedsgaard, Nadja Cech, Anneli Kruve

Scientific Reports 2020

DOI: 10.1038/s41598-020-62573-z

Non-targeted and suspect analyses with liquid chromatography/electrospray/high-resolution mass spectrometry (LC/ESI/HRMS) are gaining importance as they enable identification of hundreds or even thousands of compounds in a single sample. Here, we present an approach to address the challenge to quantify compounds identified from LC/HRMS data without authentic standards. The approach uses random forest (RF) regression to predict the response of the compounds in ESI/HRMS with a mean error of 2.2 and 2.0 times for ESI positive and negative mode, respectively. We observe that the predicted responses can be transferred between different instruments via a regression approach.
Furthermore, we applied the predicted responses to estimate the concentration of the compounds without the standard substances. The approach was validated by quantifying pesticides and mycotoxins in six different cereal samples. For applicability, the accuracy of the concentration prediction needs to be compatible with the effect (e.g. toxicology) predictions. We achieved the average quantification error of 5.9 times, which is well compatible with the accuracy of the toxicology predictions.

Instrumental techniques in the analysis of natural red textile dyes

Pilleriin Peets, Signe vahur, Anneli Kruve, Tõiv Haljasorg, Koit Herodes, Todd Pagano, Ivo Leito

Journal of Cultural Heritage 2020, 42, 19-27

DOI: 10.1016/j.culher.2019.09.002

Various dyes present in historical objects can be indicative of different usage, age and origin of the artifacts. Knowledge about the composition and origin of textile dyes is essential for the preservation and conservation of the items. In this paper, we compare different instrumental techniques to detect and identify natural red dyes from historical textiles without the need for standard substances of the dye components. Several instrumental techniques were used, including liquid chromatography with photodiode array, fluorescence and mass -spectrometric detectors, as well as Fourier transform ion cyclotron resonance mass spectrometry with electrospray ionization and matrix-assisted laser desorption/ionization sources. Seven natural red dye sources were investigated, including dyer’s madder, redwood, logwood, sandalwood, kermes, American cochineal and bloodred webcap (Cortinarius sanguineus). The dye components of the latter source have not been characterized before with all of these analytical techniques. A substantial library of chromatograms and mass spectra, along with absorption and fluorescence spectra (altogether 113 chromatograms/spectra), of dyes and/or dye components were recorded and a comparison of the utility of the different analytical techniques in the analysis of the dye sources is provided. The usefulness of the assembled library of chromatograms/spectra is demonstrated on the analysis of several historical textile samples from museum artifacts..

The NORMAN Association and the European Partnership for Chemicals Risk Assessment (PARC): let’s cooperate!

Valeria Dulio, et al.

Environmental Sciences Europe 2020, 32, 100

DOI:  10.1186/s12302-020-00375-w

The Partnership for Chemicals Risk Assessment (PARC) is currently under development as a joint research and innovation programme to strengthen the scientific basis for chemical risk assessment in the EU. The plan is to bring chemical risk assessors and managers together with scientists to accelerate method development and the production of necessary data and knowledge, and to facilitate the transition to next-generation evidence-based risk assessment, a non-toxic environment and the European Green Deal. The NORMAN Network is an independent, well-established and competent network of more than 80 organisations in the field of emerging substances and has enormous potential to contribute to the implementation of the PARC partnership. NORMAN stands ready to provide expert advice to PARC, drawing on its long experience in the development, harmonisation and testing of advanced tools in relation to chemicals of emerging concern and in support of a European Early Warning System to unravel the risks of contaminants of emerging concern (CECs) and close the gap between research and innovation and regulatory processes. In this commentary we highlight the tools developed by NORMAN that we consider most relevant to supporting the PARC initiative: (i) joint data space and cutting-edge research tools for risk assessment of contaminants of emerging concern; (ii) collaborative European framework to improve data quality and comparability; (iii) advanced data analysis tools for a European early warning system and (iv) support to national and European chemical risk assessment thanks to harnessing, combining and sharing evidence and expertise on CECs. By combining the extensive knowledge and experience of the NORMAN network with the financial and policy-related strengths of the PARC initiative, a large step towards the goal of a non-toxic environment can be taken.

Our poster presentations from conferences can be found here.

Ion-Mobility Mass Spectrometry for the Rapid Determination of the Topology of Interlocked and Knotted Molecules

Anneli Kruve, Kenji Caprice, Roy lavendomme, Jan M. Wollschlaeger, Stefan Schoder, Henrik V. Schröder, Jonathan R. Nitschke, Fabien B.L. Cougnon, Christoph A. Schalley

Angewandte Chemie 2019, 58(33), 11324-11328

DOI: 10.1002/anie.201904541

A rapid screening method based on traveling‐wave ion‐mobility spectrometry (TWIMS) combined with tandem mass spectrometry provides insight into the topology of interlocked and knotted molecules, even when they exist in complex mixtures, such as interconverting dynamic combinatorial libraries. A TWIMS characterization of structure‐indicative fragments generated by collision‐induced dissociation (CID) together with a floppiness parameter defined based on parent‐ and fragment‐ion arrival times provide a straightforward topology identification. To demonstrate its broad applicability, this approach is applied here to six Hopf and two Solomon links, a trefoil knot, and a [3]catenate.

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

Riin Rebane, Anneli Kruve, Jaanus Liigand, Piia Liigand, Artur Gornischeff, Ivo Leito

Rapid Communications in Mass Spectrometry 2019, 33(23), 1834-1843

DOI: 10.1002/rcm.8545

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.

Our poster presentations from conferences can be found here.

Influence of the amino acid composition on the ionization efficiencies of small peptides

Piia Liigand, Karl Kaupmees and Anneli Kruve

Journal of Mass Spectrometry 2019, 54(6), 481-487

DOI: 10.1002/jms.4348

Electrospray ionization is widely used to generate gas phase ions from a variety of molecules ranging from small ions to large proteins, while the ionization mechanism is claimed to depend on the size of the molecule. For small molecules, the ionization efficiency, the amount of gas phase ions produced in the electrospray process, depends on the properties of the compound. Here we show that the amino acid composition also influences the ionization efficiency of the oligopeptide. Additionally, we show that the ionization efficiencies of oligopeptides consisting of more than 5 amino acid residues are very similar to one another and assuming equal ionization efficiencies is feasible. Therefore, accurate ionization efficiency predictions are needed mainly for small oligopeptides. For these oligopeptides, the amino acid composition can be used to estimate the ionization efficiency in an easy to use manner.

ESI outcompetes other ion sources in LC/MS trace analysis

Asko Laaniste, Ivo Leito, Anneli Kruve

Analytical and Bioanalytical Chemistry 2019, 411(16), 3533-3542

DOI: 10.1007/s00216-019-01832-z

Electrospray ionization is widely used to generate gas phase ions from a variety of molecules ranging from small ions to large proteins, while the ionization mechanism is claimed to depend on the size of the molecule. For small molecules, the ionization efficiency, the amount of gas phase ions produced in the electrospray process, depends on the properties of the compound. Here, we show that the amino acid composition also influences the ionization efficiency of the oligopeptide. Additionally, we show that the ionization efficiencies of oligopeptides consisting of more than five amino acid residues are very similar to one another, and assuming equal ionization efficiencies is feasible. Therefore, accurate ionization efficiency predictions are needed mainly for small oligopeptides. For these oligopeptides, the amino acid composition can be used to estimate the ionization efficiency in an easy to use manner.

Optimization of flow splitting and make‐up flow conditions in liquid chromatography/electrospray ionization mass spectrometry

Jaanus Liigand, Ronald de Vries, Filip Cuyckens

Rapid Communications in Mass Spectrometry 2019, 33(3), 314-322

DOI: 10.1002/rcm.8352

Rationale
In liquid chromatography/mass spectrometry (LC/MS) the LC flow is often split prior to the mass spectrometer, for instance, when collecting fractions of the separated sample for other purposes or when less sensitive parallel detection is applied. The aim of this study is to optimize the actual split ratio and make‐up flow composition.
Methods
Different types of splitters were evaluated in combination with a make‐up flow. A home‐made 1/10 T‐piece splitter and commercial 1/10, 1/100 and 1/250 splitters were evaluated by continuous and accurate measurements of the actual split ratio throughout the LC gradient. The make‐up flow composition was optimized for maximum electrospray ionization (ESI)‐MS sensitivity in the positive mode using ESI efficiency measurements.
Results
Altogether 22 different solvent conditions were tested on 20 pharmaceutical compounds with a wide variety of functional groups and physicochemical properties (molecular weight, logP, and pKa). Methanol/10 mM formic acid in water (90/10) provided on average the best results.
Conclusions
Methanol/10 mM formic acid in water (90/10) proved to be the best make‐up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.

Anion-driven encapsulation of cationic guests inside pyridine[4]arene dimers

Anniina Kiesila, Jani O. Moilanen, AnneliKruve, Christoph A. Schalley, Perdita Barran, Elina Kalenius

Bielstein Journal of Organic Chemistry 2019, 15, 2486-2492

DOI:  10.3762/bjoc.15.241

Pyridine[4]arenes have previously been considered as anion binding hosts due to the electron-poor nature of the pyridine ring. Herein, we demonstrate the encapsulation of Me4N+ cations inside a dimeric hydrogen-bonded pyridine[4]arene capsule, which contradicts with earlier assumptions. The complexation of a cationic guest inside the pyridine[4]arene dimer has been detected and studied by multiple gas-phase techniques, ESI-QTOF-MS, IRMPD, and DT-IMMS experiments, as well as DFT calculations. The comparison of classical resorcinarenes with pyridinearenes by MS and NMR experiments reveals clear differences in their host-guest chemistry and implies that cation encapsulation in pyridine[4] arene is an anion-driven process.

Our poster presentations from conferences can be found here.

Semi‐quantitative non‐target analysis of water with LC/HRMS: how far are we?

Anneli Kruve

Rapid Communications in Mass Spectrometry 2018, in press

DOI: 10.1002/rcm.8208

Combining high‐resolution mass spectrometry (HRMS) with liquid chromatography (LC) has considerably increased the capability of analytical chemistry. Among others, it has stimulated the growth of the non‐target analysis, which aims at identifying compounds without their preceding selection. This approach is already widely applied in various fields, such as metabolomics, proteomics, etc. The applicability of LC/HRMS‐based non‐target analysis in environmental analyses, such as water studies, would be beneficial for understanding the environmental fate of polar pollutants and evaluating the health risks exposed by the new emerging contaminants. During the last five to seven years the use of LC/HRMS‐based non‐target analysis has grown rapidly. However, routine non‐target analysis is still uncommon for most environmental monitoring agencies and environmental scientists. The main reasons are the complicated data processing and the inability to provide quantitative information about identified compounds. The latter shortcoming follows from the lack of standard substances, considered so far as the soul of each quantitative analysis for the newly discovered pollutants. To overcome this, non‐target analyses could be combined with semi‐quantitation. This Perspective aims at describing the current methods for non‐target analysis, the possibilities and challenges of standard substance‐free semi‐quantitative analysis, and proposes tools to join these two fields together.

Ionisation efficiencies can be predicted in complicated biological matrices: A proof of concept

Piia Liigand, Jaanus Liigand, Filip Cuyckens, Rob J. Vreeken and Anneli Kruve

Analytica Chimica Acta 2018, 1032, 68-74

DOI: 10.1016/j.aca.2018.05.072

The importance of metabolites is assessed based on their abundance. Most of the metabolites are at present identified based on ESI/MS measurements and the relative abundance is assessed from the relative peak areas of these metabolites. Unfortunately, relative intensities can be highly misleading as different compounds ionise with vastly different efficiency in the ESI source and matrix components may cause severe ionisation suppression. In order to reduce this inaccuracy, we propose predicting the ionisation efficiencies of the analytes in seven biological matrices (neat solvent, blood, plasma, urine, cerebrospinal fluid, brain and liver tissue homogenates). We demonstrate, that this approach may lead to an order of magnitude increase in accuracy even in complicated matrices. For the analyses of 10 compounds, mostly drugs, in negative electrospray ionisation mode we reduce the predicted abundance mismatch compared to the actual abundance on average from 660 to 8 times. The ionisation efficiencies were predicted based on i) the charge delocalisation parameter WAPS and ii) the degree of ionisation α, and the prediction model was subsequently validated based on the cross-validation method ‘leave-one-out’.

Modifying the Acidity of Charged Droplets

Mari Ojakivi, Jaanus Liigand and Anneli Kruve

Chemistry Select 2018, 3, 335 – 338

DOI: 10.1002/slct.201702269

The concept of acidity in confined spaces is up to date poorly understood; especially, in case of media violating electroneutrality. Here, we describe the acidity of charged droplets via their ability to protonate simple nitrogen bases and we propose ways to modify the protonation efficiency with the help of additives. We observed that the protonation of compounds in charged water droplets is independent of solution-phase acidity; instead, it can be adjusted with the help of additive type. On the other hand, the extent of protonation in charged methanol droplets can be adjusted with the
conventional approach of changing the pH.

Quantitative and sensitive mapping of imidacloprid on plants using Multiphoton Electron Extraction Spectroscopy

Anneli Kruve, Valery Bulatov and Israel Schechter

Chemical Physics, in press

DOI: 10.1016/j.chemphys.2018.03.031

Neonicotinoids, including imidacloprid, are extensively used for plant protection against insects. Unfortunately, these effective pesticides are one of the reasons for the decline of the bee population in recent decades. Ensuring application of minimal pesticide quantity and preventing excess requires a fast method for monitoring the coverage on plants. We present a new, method based on multiphoton electron extraction spectroscopy (MEES), for detecting, quantifying and mapping of imidacloprid coverage on plants. Imaging and quantitative analyses were demonstrated on several plant surfaces including olive and mint leaves and orange peel. Method provides both low detection limits (down to nanogram level) and good trueness. This method is fast and can be directly performed with no sample pre-treatment, thus, it is a good candidate for field analyses.

pH Effects on Electrospray Ionization Efficiency

Jaanus Liigand, Asko Laaniste and Anneli Kruve

J. Am. Soc. Mass Spectrom. 2017,28( 3), 461 – 469

DOI: 10.1007/s13361-016-1563-1

Electrospray ionization efficiency is known to be affected by mobile phase composition. In this paper, a detailed study of analyte ionization efficiency dependence on mobile phase pH is presented. The pH effect was studied on 28 compounds with different chemical properties. Neither pKa nor solution phase ionization degree by itself was observed to be sufficient at describing how aqueous phase pH affects the ionization efficiency of the analyte. Therefore, the analyte behavior was related to various physicochemical properties via linear discriminant analyses. Distinction between pH-dependent and pH-independent compounds was achieved using two parameters: number of potential charge centers and hydrogen bonding acceptor capacity (in the case of 80% acetonitrile) or polarity of neutral form of analyte and pKa (in the case of 20% acetonitrile). It was also observed that decreasing pH may increase ionization efficiency of a compound by more than two orders of magnitude.

Think Negative: Finding the Best Electrospray Ionization/MS Mode for Your Analyte

Piia Liigand, Karl Kaupmees, Kristjan Haav, Jaanus Liigand,Ivo Leito, Marion Girod, Rodolphe Antoine and Anneli Kruve

Anal. Chem. 2017, 89( 11), 5665 – 5668

DOI: 10.1021/acs.analchem.7b00096

For the first time, the electrospray ionization efficiency (IE) scales in positive and negative mode are united into a single system enabling direct comparison of IE values across ionization modes. This is made possible by the use of a reference compound that ionizes to a similar extent in both positive and negative modes. Thus, choosing the optimal (i.e., most sensitive) ionization conditions for a given set of analytes is enabled. Ionization efficiencies of 33 compounds ionizing in both modes demonstrate that, contrary to general practice, negative mode allows better sensitivity for 46% of such compounds whereas the positive mode is preferred for only 18%, and for 36%, the results for both modes are comparable.

The Evolution of Electrospray Generated Droplets is Not Affected by Ionization Mode

Piia Liigand, Agnes Heering (Suu), Karl Kaupmees, Ivo Leito, Marion Girod, Rodolphe Antoine and Anneli Kruve

J. Am. Soc. Mass Spectrom. 2017, 28 (10), 2124-2131

DOI: 10.1007/s13361-017-1737-5

Ionization efficiency and mechanism in ESI is strongly affected by the properties of mobile phase. The use of mobile-phase properties to accurately describe droplets in ESI source is convenient but may be inadequate as the composition of the droplets is changing in the plume due to electrochemical reactions occurring in the needle tip as well as continuous drying and fission of droplets. Presently, there is paucity of research on the effect of the polarity of the ESI mode on mobile phase composition in the droplets. In this paper, the change in the organic solvent content, pH, and droplet size are studied in the ESI plume in both ESI+ and ESI– ionization mode. We introduce a rigorous way – the absolute pH (pHabsH2O) – to describe pH change in the plume that takes into account organic solvent content in the mobile phase. pHabsH2O enables comparing acidities of ESI droplets with different organic solvent contents. The results are surprisingly similar for both ionization modes, indicating that the dynamics of the change of mobile-phase properties is independent from the ESI mode used. This allows us to conclude that the evolution of ESI droplets first of all proceeds via the evaporation of the organic modifier and to a lesser extent via fission of smaller droplets from parent droplets. Secondly, our study shows that qualitative findings related to the ESI process obtained on the ESI+ mode can almost directly be applied also in the ESI– mode.

Adduct Formation in ESI/MS by Mobile Phase Additives

Anneli Kruve and Karl Kaupmees

J. Am. Soc. Mass Spectrom. 2017, 28 (5), 887-894

DOI: 10.1007/s13361-017-1626-y

Adduct formation is a common ionization method in electrospray ionization mass spectrometry (ESI/MS). However, this process is poorly understood and complicated to control. We demonstrate possibilities to control adduct formation via mobile phase additives in ESI positive mode for 17 oxygen and nitrogen bases. Mobile phase additives were found to be a very effective measure for manipulating the formation efficiencies of adducts. An appropriate choice of additive may increase sensitivity by up to three orders of magnitude. In general, sodium adduct [M+Na]+ and protonated molecule [M+H]+ formation efficiencies were found to be in good correlation, however, the former were significantly more influenced by mobile phase properties. Though the highest formation efficiencies for both species were observed in water/acetonitrile mixtures not containing additives, the repeatability of the formation efficiencies was found to be improved by additives. It is concluded that mobile phase additives are powerful, yet not limiting factors, for altering adduct formation.

Imine-based [2]catenanes in water

Kenji Caprice, Marion Pupier, Anneli Kruve, Christoph Schalley and Fabien B. L. Cougnon

Chemical Science 2017, 28 (5), 887-894

DOI: 10.1039/C7SC04901C

We report the efficient condensation of imine-based macrocycles from dialdehyde A and aliphatic diamines Bn in pure water. Within the libraries, we identified a family of homologous amphiphilic [2]catenanes, whose self-assembly is primarily driven by the hydrophobic effect. The length and odd-even character of the diamine alkyl linker dictate both the yield and the conformation of the [2]catenanes, whose particular thermodynamic stability further shifts the overall equilibrium in favour of imine condensation. These findings highlight the role played by solvophobic effects in the self-assembly of complex architectures.

Laser Applications in Chromatography

Anneli Kruve and Israeli Schechter

Advances in Chromatography 2017, 55, 23-49

WEB: 10.1039/C7SC04901C

In this chapter we give overview of the state of the art methods using lasers in chromatographic detection systems. More precisely, we will focus on Surface Enhances Raman Spectroscopy (SERS), Laser Induced Fluorescence (LIF), Multiangle Light Scattering (MALS) and different methods allowing connecting chromatography and mass spectrometry (MS), such as Single Photon Ionization (SPI), Multiphoton Ionization (MPI), Matrix Assisted Laser Desorption/Ionization (MALDI) together with novel ambient laser induced ionization methods. We introduce the principle of these methods, describe the advantages, possible interferences and technical solutions used. For each method we also describe up to date applications in several fields together with the obtained advantages over conventional detectors.

Determination of glyphosate in surface water with high organic matter content

Vahur Toss, Ivo Leito, Sergei Yurchenko, Rene Freiberg and Anneli Kruve

Environ Sci Pollut Res 2018, 66 (1), 7880-7888

DOI: 10.1007/s11356-017-8522-7

In this paper, we investigate the sample preparation and analysis process in order to achieve adequate results for surface water collected from rivers that flow through swamps and are consequently rich in organic matter. We show that matrix effects in glyphosate determination can be reduced by optimizing sample volume, liquid chromatography (LC) mobile phase buffer concentration and pH as well as gradient speed. Also, aspects of derivatization procedure (borate buffer concentration, fluorenylmethyloxycarbonyl chloride concentration) and their influence on accuracy are considered in detail. We encountered a cross-talk effect in the mass spectra, interfering with quantization during analysis, which was removed by optimizing MS parameters. As a result it was demonstrated that isotope-labelled internal standard with just one 13C atom is sufficient for the analysis. All these aspects were found to strongly impact the accuracy of the glyphosate determination but have received little or no attention in earlier works. We propose a reliable solid phase extraction and LC/ESI/MS/MS method for determination of glyphosate in organic-rich waters and demonstrate that LoD can be decreased by about two times using an ESI nebulizer with a modified design.

Predicting ESI/MS Signal Change for Anions in Different Solvents

Anneli Kruve and Karl Kaupmees

Anal Chem 2017, 89 (9), 5079-5086.

DOI: 10.1021/acsanalchem.7b00595

LC/ESI/MS is a technique widely used for qualitative and quantitative analysis in various fields. However, quantification is currently possible only for compounds for which the standard substances are available, as the ionization efficiency of different compounds in ESI source differs by orders of magnitude. In this paper we present an approach for quantitative LC/ESI/MS analysis without standard substances. This approach relies on accurately predicting the ionization efficiencies in ESI source based on a model, which uses physico-chemical parameters of analytes. Furthermore, the model has been made transferable between different mobile phases and instrument setups by using a suitable set of calibration compounds. This approach has been validated both in flow injection and chromatographic mode with gradient elution.

Ionization Efficiency of Doubly Charged Ions Formed from Polyprotic Acids in Electrospray Negative Mode

Piia Liigand, Karl Kaupmees and Anneli Kruve

J. Am. Soc. Mass Spectrom. 2016, 27 (7), 1211-1218

DOI: 10.1007/s13361-016-1384-2

The ability of polyprotic acids to give doubly charged ions in negative mode electrospray was studied and related to physicochemical properties of the acids via linear discriminant analysis (LDA). It was discovered that the compound has to be strongly acidic (low pKa1 and pKa2) and to have high hydrophobicity (logPow) to become multiply charged. Ability to give multiply charged ions in ESI/MS cannot be directly predicted from the solution phase acidities. Therefore, for the first time, a quantitative model to predict the charge state of the analyte in ESI/MS is proposed and validated for small anions. Also, a model to predict ionization efficiencies of these analytes was developed. Results indicate that acidity of the analyte, its octanol-water partition coefficient, and charge delocalization are important factors that influence ionization efficiencies as well as charge states of the analytes. The pH of the solvent was also found to be an important factor influencing the ionization efficiency of doubly charged ions.

Establishing Atmospheric Pressure Chemical Ionization Efficiency Scale

Riin Rebane, Anneli Kruve, Piia Liigand, Jaanus Liigand, Koit Herodes and Ivo Leito

Anal. Chem. 2016, 88( 7), 3435 – 3439

DOI: 10.1021/acs.analchem.5b04852

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.

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

Asko Laaniste, Ivo Leito, Riin Rebane, Rünno Lõhmus, Ants Lõhmus, Fredrik Punga and Anneli Kruve

J. Environ. Sci. Health. Part B 2016, 51, 455-464.

DOI: 10.1080/03601234.2016.1159457

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 (MRL) set by the European Union (EU). The other neonicotinoids were not detected. The proportion of thiacloprid-positive samples for the different years correlates well with the data on thiacloprid imports into Estonia, indicating that honey contamination with neonicotinoids can be estimated based on import data.

Influence of mobile phase, source parameters and source type on electrospray ionisation efficiency in negative ion mode

Anneli Kruve

J. Am. Soc. Mass Spectrom. 2016, 51 (8), 596-602.

DOI: 10.1002/jms.3790

Electrospray ionisation (ESI) efficiency is known to be affected by the properties of the analytes, source design and source parameters. In this study, the ionisation efficiency of 17 acidic compounds at various conditions in ESI negative ion mode was evaluated. Namely, the influence of organic solvent content in the mobile phase, ionisation source parameters, ionisation source geometry and functionality (conventional ESI, ESI with thermal focusing and with additional internal nebuliser gas) was studied. It was observed that the ionisation efficiency in thermal focusing ESI is only marginally affected by the organic solvent composition, while for conventional ESI and ESI with internal nebuliser gas, the ionisation efficiency increases significantly with increasing organic modifier content. For all ionisation sources and mobile phase compositions, the ionisation efficiency values between different setups showed good correlation.

Transferability of the Electrospray Ionization Efficiency Scale between Different Instruments

Jaanus Liigand,Anneli Kruve, Piia Liigand, Asko Laaniste, Marion Girod, Rodolphe Antoine and Ivo Leito

J. Am. Soc. Mass Spectrom. 2015, 26( 11), 1923 – 1930

DOI: 10.1007/s13361-015-1219-6

For the first time, quantitative electrospray (ESI) ionization efficiencies (IE), expressed as logIE values, obtained on different mass-spectrometric setups (four mass analyzers and four ESI sources) are compared for 15 compounds of diverse properties. The general trends of change of IE with molecular structure are the same with all experimental setups. The obtained IE scales could be applied on different setups: there were no statistically significant changes in the order of ionization efficiency and the root mean of squared differences of the logIE values of compounds between the scales compiled on different instruments were found to be between 0.21 and 0.55 log units. The results show that orthogonal ESI source geometry gives better differentiating power and additional pneumatic assistance improves it even more. It is also shown that the ionization efficiency values are transferable between different mass-spectrometric setups by three anchoring points and a linear model. The root mean square error of logIE prediction ranged from 0.24 to 0.72 depending on the instrument. This work demonstrates for the first time the inter-instrument transferability of quantitative electrospray ionization efficiency data.

Unified pH Values of Liquid Chromatography Mobile Phases

Agnes Suu, Lauri Jalukse, Jaanus Liigand, Anneli Kruve, Daniel Himmel, Ingo Krossing, Marti Rosés and Ivo Leito

Anal. Chem. 2015, 87( 5), 2623–2630

DOI: 10.1021/ac504692m

This work introduces a conceptually new approach of measuring pH of mixed-solvent liquid chromatography (LC) mobile phases. Mobile phase pH is very important in LC, but its correct measurement is not straightforward, and all commonly used approaches have deficiencies. The new approach is based on the recently introduced unified pH (pHabs) scale, which enables direct comparison of acidities of solutions made in different solvents based on chemical potential of the proton in the solutions. This work represents the first experimental realization of the pHabs concept using differential potentiometric measurement for comparison of the chemical potentials of the proton in different solutions (connected by a salt bridge), together with earlier published reference points for obtaining the pHabs values (referenced to the gas phase) or pHabsH2O values (referenced to the aqueous solution). The liquid junction potentials were estimated in the framework of Izutsu’s three-component method. pHabs values for a number of common LC and LC–MS mobile phases have been determined. The pHabs scale enables for the first time direct comparison of acidities of any LC mobile phases, with different organic additives, different buffer components, etc. A possible experimental protocol of putting this new approach into chromatographic practice has been envisaged and its applicability tested. It has been demonstrated that the ionization behavior of bases (cationic acids) in the mobile phases can be better predicted by using the pHabsH2O values and aqueous pKa values than by using the alternative means of expressing mobile phase acidity. Description of the ionization behavior of acids on the basis of pHabsH2O values is possible if the change of their pKa values with solvent composition change is taken into account.

Paper spray ionization mass spectrometry: Study of a method for fast-screening analysis of pesticides in fruits and vegetables

Hanno Evard, Anneli Kruve, Rünno Lõhmus and Ivo Leito

J. Food Compos. Anal. 2015, 41, 221-225.

DOI: 10.1016/j.jfca.2015.01.010

New faster and simpler methods for determination of pesticides in agricultural products are necessary as requirements for food safety are becoming increasingly stringent. One possibility is to introduce a fast, easy and low-cost screening method before liquid chromatography mass spectrometry analyses. We hereby present a systematic proof of concept study of paper spray mass spectrometry method for pesticide detection as a screening method. Two sampling approaches – wiping the surface with paper and applying the sample homogenate directly on the paper – were used. The wiping method was more extensively studied for imazalil and thiabendazole originally present in oranges.. For homogenized samples three matrices – oranges, tomatoes and grapes – and five pesticides of different chemical nature and polarity – thiabendazole, aldicarb, imazalil, methomyl and methiocarb – were chosen. It has been shown that limits of detection below maximum residue levels can be achieved for both methods. The methods are therefore suitable for fast screening of samples. Moreover, the wiping method was also applied for 11 samples – oranges, grapefruits, lemons, limes, mandarins, tomatoes, apples, pears, strawberries, grapes and sweet peppers – from the local supermarket to screen for different pesticides. Three positive samples for thiabendazole and imazalil and one positive sample for only imazalil were found.

Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part I

Anneli Kruve, Riin Rebane, Karin Kipper, Maarja-Liisa Oldekop, Hanno Evard, Koit Herodes, Pekka Ravio and Ivo Leito

Analytica Chimica Acta 2015, 870, 29-44

DOI: 10.1016/j.aca.2015.02.017

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.

Tutorial review on validation of liquid chromatography–mass spectrometry methods: Part II

Anneli Kruve, Riin Rebane, Karin Kipper, Maarja-Liisa Oldekop, Hanno Evard, Koit Herodes, Pekka Ravio and Ivo Leito

Analytica Chimica Acta 2015, 870, 8-28.

DOI: 10.1016/j.aca.2015.02.016

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 MSMS) 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.

Negative Electrospray Ionization via Deprotonation: Predicting the Ionization Efficiency

Anneli Kruve, Karl Kaupmees, Jaanus Liigand and Ivo Leito

Anal. Chem. 2014, 86( 10), 4822–4830

DOI: 10.1021/ac404066v

Electrospray ionization (ESI) in the negative ion mode has received less attention in fundamental studies than the positive ion electrospray ionization. In this paper, we study the efficiency of negative ion formation in the ESI source via deprotonation of substituted phenols and benzoic acids and explore correlations of the obtained ionization efficiency values (logIE) with different molecular properties. It is observed that stronger acids (i.e., fully deprotonated in the droplets) yielding anions with highly delocalized charge [quantified by the weighted average positive sigma (WAPS) parameter rooted in the COSMO theory] have higher ionization efficiency and give higher signals in the negative-ion ESI/MS. A linear model was obtained, which equally well describes the logIE of both phenols and benzoic acids (R2 = 0.83, S = 0.40 log units) and contains only an ionization degree in solution (α) and WAPS as molecular parameters. Both parameters can easily be calculated with the COSMO-RS method. The model was successfully validated using a test set of acids belonging neither to phenols nor to benzoic acids, thereby demonstrating its broad applicability and the universality of the above-described relationships between IE and molecular properties.

Effect of Mobile Phase on Electrospray Ionization Efficiency

Jaanus Liigand,Anneli Kruve,Ivo Leito, Marion Girod and Rodolphe Antoine

J. Am. Soc. Mass Spectrom. 2014, 25( 11), 1853 – 1861

DOI: 10.1007/s13361-014-0969-x

Electrospray (ESI) ionization efficiencies (IE) of a set of 10 compounds differing by chemical nature, extent of ionization in solution (basicity), and by hydrophobicity (tetrapropylammonium and tetraethylammonium ion, triethylamine, 1-naphthylamine, N,N-dimethylaniline, diphenylphthalate, dimethylphtahalate, piperidine, pyrrolidine, pyridine) have been measured in seven mobile phases (three acetonitrile percentages 20%, 50%, and 80%, and three different pH-adjusting additives, 0.1% formic acid, 1 mM ammonia, pH 5.0 buffer combination) using the relative measurement method. MS parameters were optimized separately for each ion. The resulting relative IE data were converted into comparable logIE values by anchoring them to the logIE of tetrapropylammonium ion taking into account the differences of ionization in different solvents and thereby making the logIE values of the compounds comparable across solvents. The following conclusions were made from analysis of the data. The compounds with pKa values in the range of the solution pH values displayed higher IE at lower pH. The sensitivity of IE towards pH depends on hydrophobicity being very strong with pyridine, weaker with N,N-dimethylaniline, and weakest with 1-naphthylamine. IEs of tetraalkylammonium ions and triethylamine were expectedly insensitive towards solution pH. Surprisingly high IEs of phthalate esters were observed. The differences in solutions with different acetonitrile content and similar pH were smaller compared with the pH effects. These results highlight the importance of hydrophobicity in electrospray and demonstrate that high hydrophobicity can sometimes successfully compensate for low basicity.

Comparison of different methods aiming to account for/overcome matrix effects in LC/ESI/MS on the example of pesticide analyses

Anneli Kruve and Ivo Leito

Analytical methods 2013, 5(12), 3035-3044.

DOI: 10.1039/C3AY26551J

In this paper some of the most common methods for overcoming matrix effects in LC/ESI/MS (matrix-matched calibration, standard addition, post-column standard infusion, extrapolative dilution, and post-column flow splitting) are compared according to their ability to give both true and accurate results for pesticide determination in complicated matrices such as onion and garlic. In order to provide a quantitative comparison we use a measure of accuracy that would account for both average trueness and scatter of the results. Extrapolative dilution and standard addition were found to give results statistically insignificantly different from the correct values. In addition extrapolative dilution – a hybrid approach for both reducing and correcting for matrix effects – was found to result in the highest accuracy of the measurements.

Ensuring repeatability and robustness of poly(glycidyl methacrylate-co-ethylene dimethacrylate) HPLC monolithic columns of 3 mm id through covalent bonding to the column wall

Asko Laaniste, Anneli Kruve and Ivo Leito

J Sep Sci 2013, 15(36), 2458-2463

DOI: 10.1002/jssc.201300133

Two different methods to reinforce the poly(glycidyl methacrylate-co-ethylene dimethacrylate) HPLC monolithic columns of 3 mm id in a glass column reservoir were studied: composite columns with polymeric particles in the monolith and surface treatment of the reservoir wall. Of the two methods used to counter the mechanical instability and formation of flow channels (composite columns and column wall surface treatment), we demonstrated that proper column wall surface treatment was sufficient to solve both problems. Our study also indicated that no surface treatment is efficient, and of the methods studied silanization in acidified ethanol solution and constant renewal of the reaction mixture (dynamic mode) proved to be the most effective. As a result of this study, we have been able to prepare repeatable and durable methacrylate HPLC columns with good efficiencies.

Enhanced Nebulization Efficiency of Electrospray Mass Spectrometry: Improved Sensitivity and Detection Limit

Anneli Kruve, Ivo Leito, Koit Herodes, Asko Laaniste and Rünno Lõhmus

J Am. Soc. Mass Specrom. 2012, 12(23), 2051-2054.

DOI: 10.1007/s13361-012-0475-y

A novel electrospray nebulizer has been designed, which includes an additional nebulization gas capillary inside the liquid capillary. This design offers significantly enhanced ionization efficiency compared with the classic nebulizer design and leads to improved sensitivity (by three to 10 times) and decreases the detection limit, on an average 10 times. We see these results as the first step in the design of ESI nebulizers offering improved sensitivity and higher robustness. Possible future developments would include optimization of the dimensions of the capillaries as well as testing the nebulizer for other matrices and analytes.