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, in press

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.

Tutorial on Estimating Limit of Detection on the example of LC-MS analysis: Part I

Hanno Evard, Anneli Kruve and Ivo Leito

Analytical Chimica Acta 2016, 942, 23-39.

DOI: 10.1016/j.aca.2016.08.043

A large body of literature exists on limit of detection (LOD), but there is still a lot of confusion about this important validation parameter. This confusion mainly stem from its statistically complex background. The goal of this two part tutorial is to discuss and clarify the topic of LOD for the practitioners. Part I of the tutorial contains theoretical discussion (without excessively sophisticated statistics) and part II contains examples on the basis of experimental data. LOD and other definitions related to it are reviewed, and their estimation and use are discussed. The assumptions, practicality and results of different approaches to estimate LOD are compared. Different aspects of the analytical method that strongly influence LOD estimates (e.g. linearity, scedasticity and day-to-day variability of LOD) together with experimental design considerations when estimating LOD are discussed.
The two main conclusions of this tutorial are: (1) the choice of how to estimate LOD should be based on the purpose of the analytical method that is being validated (e.g. large effort should not be made to estimate LOD for a method that is not used for detecting traces in the vicinity of LOD), and (2) LOD estimates are strongly dependant on different assumptions and the used approach, and therefore caution must be exercised when using the estimate or when comparing different estimates.
A decision tree is proposed for estimating and monitoring LOD. A detailed working procedure for estimating LOD is presented. This tutorial focuses on LC-MS/MS and specific problems associated with this technique. Several topics are pointed out on which further research and discussion is needed.

Tutorial on Estimating Limit of Detection on the example of LC-MS analysis: Part II

Hanno Evard, Anneli Kruveand Ivo Leito

Analytical Chimica Acta 2016, 942, 40-49.

DOI: 10.1016/j.aca.2016.08.042

In part II of this tutorial investigated approaches of estimating the limit of detection (LOD) are applied to experimental data from LC-MS measurements. Important practical aspects specific for LC-MS and related to LOD are reviewed. Results of different tests of estimating linearity and scedasticity are compared. LOD estimates obtained with different approaches (for both simple characterization of the analysis method and accurate interpretation of the results) are applied to the data and the obtained values are compared. As a conclusion a decision tree is proposed for estimating LOD for analytical methods using the LC-MS technique.

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.

Sodium adduct formation efficiency in ESI source

Anneli Kruve, Karl Kaupmees, Jaanus Liigand, Merit Oss and Ivo Leito

J. Mass Spectrom. 2013, 48( 6), 695-702

DOI: 10.1002/jms.3218

Formation of sodium adducts in electrospray (ESI) has been known for long time, but has not been used extensively in practice, and several important aspects of Na+ adduct formation in ESI source have been almost unexplored: the ionization efficiency of different molecules via Na+ adduct formation, its dependence on molecular structure and Na+ ion concentration in solution, fragmentation behaviour of the adducts as well as the ruggedness (a prerequisite for wider practical use) of ionization via Na+ adduct formation. In this work, we have developed a parameter describing sodium adducts formation efficiency (SAFE) of neutral molecules and have built a SAFE scale that ranges for over four orders of magnitude and contains 19 compounds. In general, oxygen bases have higher efficiency of Na+ adducts formation than nitrogen bases because of the higher partial negative charge on oxygen atoms and competition from protonation in the case of nitrogen bases. Chelating ability strongly increases the Na+ adduct formation efficiency. We show that not only protonation but also Na+ adduct formation is a quantitative and reproducible process if relative measurements are performed.

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.

Accounting for matrix effects of pesticide residue liquid chromatography/electrospray ionisation mass spectrometric determination by treatment of background mass spectra with chemometric tools

Anneli Kruve, Koit Herodes and Ivo Leito

Rapid Commun. Mass Spectrom. 2011, 25(9), 1159–1168.

DOI: 10.1002/rcm.4971

Matrix effect (ME) – ionisation suppression or enhancement – in liquid chromatography/electrospray ionisation mass spectrometry (LC/ESI-MS) is caused by matrix components co-eluting with the analytes. ME has a complex and not fully understood nature. ME is also highly variable from sample to sample making it difficult to compensate for. In this work it was studied whether the background ion signals in scanned mass spectra of the LC effluent at the retention time of the analyte offer some insight into the presence and extent of matrix effect. Matrix effects for six pesticides – thiabendazole, carbendazime, methomyl, aldicarb, imazalil and methiocarb – in garlic and onion samples used in the study varied from 1% (suppression 99%) to 127% (enhancement 27%) depending on the pesticide and sample. Also, standards in solvent and solvent blanks were included in the study. The ions most strongly varying from sample to sample – and therefore best describing the changes in sample composition and ME – were selected for quantification according to principal component analysis (PCA) for all six pesticides under study. These ions were used to account for ME via partial least-squares (PLS) regression. The calibration set was constructed from 19 samples and standards and the obtained calibration function was validated with seven samples and standards. The average errors from the test set were from 0.05 to 0.27 mg/kg for carbendazim and imazalil, respectively (the respective average pesticide concentrations were 0.22 and 0.88 mg/kg). The PLS results were significantly more accurate compared to the conventional solvent calibration resulting in average errors from 0.07 to 0.69 mg/kg for carbendazime and methiocarb, respectively.

Feasibility of capillary liquid chromatography-microchip-atmospheric pressure photoionization-mass spectrometry for pesticide analysis in tomato

Anneli Kruve, Koit Herodes and Ivo Leito

Analytica Chimica Acta 2011, 696(1-2), 77–83

DOI: 10.1016/j.aca.2011.04.006

A new and sensitive analytical method, using capillary liquid chromatography (capLC) with a microfabricated heated nebulizer chip for atmospheric pressure photoionization and tandem mass spectrometry (µAPPI-MS/MS), was developed for the analysis of selected carbamate pesticides in a vegetable matrix. The performance of the method was evaluated, using six pesticides, namely oxamyl, methomyl, aldicarb, carbofuran, pirimicarb, and methiocarb, and ditalimfos as an internal standard in a tomato matrix. The limits of detection achieved with capLC-μAPPI-MS/MS method in the positive ion mode were low, ranging from 0.25 ng/ml for pirimicarb to 5 ng/ml for oxamyl and methomyl, corresponding to 5 and 0.25 µg/kg for tomato samples, respectively, which are clearly below the maximum residue limits for them in fruits and vegetables. The repeatability of the method ranged from 2.9-13.9% (RSD) at a low (0.05 μg/ml) concentration level. An adequate linearity (r2 = 0.984-0.999) at a concentration range from 0.005 to 5.0 μg/ml was observed for all pesticides. The results obtained show that the capLC-μAPPI-MS/MS method developed could be used for the analysis of selected pesticides from tomato.

Study of liquid chromatography/electrospray ionization mass spectrometry matrix effect on the example of glyphosate analysis from cereals

Anneli Kruve, Ragne Auling, Koit Herodes and Ivo Leito

Rapid Commun. Mass Spectrom. 2011, 25(21), 3252–3258.

DOI: 10.1002/rcm.5222

Glyphosate is one of the most common pesticides used in the pre‐harvest treatment of cereals. This paper examines the matrix effect of glyphosate liquid chromatography/electrospray ionization mass spectrometric (LC/ESI‐MS) analysis in wheat and rye. The matrix effect (ionization suppression) was found to be dependent on sample particle size taken for the extraction. If samples are ground to very small particles severe ionization suppression occurs. For lower glyphosate contents (<1 mg/kg) the signal may even be suppressed by more than 90%. The matrix effect was found to be dependent on the matrix – rye showed significantly stronger ionization suppression than wheat, although these matrices are not very different. The matrix effect also depends on the concentration of glyphosate in the post‐extraction spiked samples. It is demonstrated that the isotope‐labelled standard 13C2‐glyphosate undergoes different ionization suppression than glyphosate and is therefore not efficient in compensating for matrix effect. At the same time the extrapolative dilution approach allows to efficiently compensate for matrix effect.

Optimization of electrospray interface and quadrupole ion trap mass spectrometer parameters in pesticide liquid chromatography/electrospray ionization mass spectrometry analysis

Anneli Kruve, Koit Herodes and Ivo Leito

Rapid Commun. Mass Spectrom. 2010, 24(7), 919–926

DOI: 10.1002/rcm.4470

Optimization of both the ionization process and ion transportation in the mass spectrometer is of crucial importance in order to achieve high sensitivity and low detection limits and acceptable accuracy in liquid chromatography/electrospray ionization mass spectrometry (LC/ESI‐MS) analysis. In this paper four optimization procedures of electrospray interface and quadrupole ion‐trap mass spectrometer parameters (ESI‐MS) (nebulizer gas and drying gas flow rate, end plate voltage, capillary voltage, skimmer voltage, octopoles direct current and radio frequency, trap drive and lens voltages) were studied on three pesticides – thiabendazole, aldicarb and imazalil. The results demonstrate that the methodology of optimization strongly influences the effectiveness of finding true optima of the operating parameters. Both eluent flow rate and composition during optimization have to mimic the situation during real analysis as closely as possible in order to achieve parameters giving the highest sensitivity. Therefore, post‐column addition of analyte to the mobile phase identical in composition to the one in which analyte elutes during real analysis combined with software‐based optimization was found to be the most effective and fastest method for achieving intensity maxima. The parameters most strongly affecting ion formation and transportation, hence sensitivity, were capillary voltage, direct current of the first octopole, trap drive and the second lens for all pesticides under study. In addition to sensitivity and detection limit matrix effect was considered in the optimization process. It was found that the matrix effect can be reduced but not eliminated by adjusting the ESI and MS parameters. The optimal parameters from the point of view of the matrix effect can only be found with factorial design. Parameters giving higher sensitivity tended to be more affected by matrix effect causing higher ionization suppression by co‐eluting compounds.

Electrospray ionization matrix effect as an uncertainty source in HPLC/ESI-MS pesticide residue analysis

Anneli Kruve, Koit Herodes and Ivo Leito

Journal of AOAC International 2010, 93, 306–314.

DOI:  10.1016/j.aca.2009.07.060

The matrix effects in HPLC/electrospray ionization (ESI)-MS analysis are difficult to compensate for because of their large variability. It is, therefore, often more practical to include uncertainty due to the matrix effect into the uncertainty budget rather than try to compensate. This work presents an empirical approach–the matrix effect graph approach–for estimating the uncertainty due to the matrix effect in HPLC/ESI-MS analysis of pesticide residues in fruits and vegetables. At certain time intervals (1 month), a calibration graph using extracts of different fruits/vegetables as calibration solutions is prepared, and a regression line is fitted through these data. These fruits/vegetables may be either from the commodity group of the samples or from different commodity groups. The relative residuals of the calibration point peak areas are calculated and plotted against the measurement time. We term the resulting graph the matrix effect graph. The root mean square of the relative residuals is calculated and used as the estimate of relative uncertainty of the sample peak areas caused by the matrix effect. The matrix effect graph obtained over fruits/vegetables from different commodity groups can also be used to identify fruits/vegetables with extreme matrix effects. The matrix effect graph approach was used for determination of thiabendazole, aldicarb, imazalil, and methiocarb and was validated with tomato, cucumber, and sweet corn matrixes at the 0.5 mg/kg concentration level. When different commodity groups were used to compile the matrix effect graph, results of analysis of all samples agreed with the spiked concentrations within the expanded uncertainties (at k=2 level). When the matrix effect graph was compiled using fruits from the same commodity group as the analyzed samples (fruiting vegetables in this case), agreement was found in 98% of the cases.

Electrospray ionization efficiency scale of organic compounds

Merit Oss, Anneli Kruve, Koit Herodes and Ivo Leito

Anal. Chem. 2010, 82(7), 2865–2872

DOI: 10.1021/ac902856t

Ionization efficiency (IE) of different compounds in electrospray ionization (ESI) source differs widely, leading to widely differing sensitivities of ESI-MS to different analytes. An approach for quantifying ESI efficiencies (as logIE values) and setting up a self-consistent quantitative experimental ESI efficiency scale of organic compounds under predefined ionization conditions (ionization by monoprotonation) has been developed recently. Using this approach a logIE scale containing 62 compounds of different chemical nature and ranging for 6 orders of magnitude has been established. The scale is based on
over 400 relative IE (ΔlogIE) measurements between more than 250 different pairs of compounds. To evaluate which molecular parameters contribute the most to the IE of a compound linear regression analysis logIE values and different molecular parameters were carried out. The two most influential parameters in predicting the IE in ESI source are the pKa and the molecular volume of the compound. This scale and the whole approach can be a tool for practicing liquid chromatographists and mass spectrometrists. It can be used in any mass-spectrometry laboratory and we encourage practitioners to characterize their analytes with the logIE values so that a broad knowledge base on electrospray ionization efficiencies of compounds would eventually develop.

Combating matrix effects in LC/ESI/MS: The extrapolative dilution approach

Anneli Kruve, Koit Herodes and Ivo Leito

Analytica Chimica Acta 2009, 651(1), 75–80

DOI: 10.1016/j.aca.2009.07.060

Liquid chromatography electrospray mass spectrometry – LC/ESI/MS—a primary tool for analysis of low volatility compounds in difficult matrices – suffers from the matrix effects in the ESI ionization. It is well known that matrix effects can be reduced by sample dilution. However, the efficiency of simple sample dilution is often limited, in particular by the limit of detection of the method, and can strongly vary from sample to sample.
In this study matrix effect is investigated as the function of dilution. It is demonstrated that in some cases dilution can eliminate matrix effect, but often it is just reduced. Based on these findings we propose a new quantitation method based on consecutive dilutions of the sample and extrapolation of the analyte content to the infinite dilution, i.e. to matrix-free solution.
The method was validated for LC/ESI/MS analysis of five pesticides (methomyl, thiabendazole, aldicarb, imazalil, methiocarb) in five matrices (tomato, cucumber, apple, rye and garlic) at two concentration levels (0.5 and 5.0 mg/kg). Agreement between the analyzed and spiked concentrations was found for all samples. It was demonstrated that in terms of accuracy of the obtained results the proposed extrapolative dilution approach works distinctly better than simple sample dilution.
The main use of this approach is envisaged for (a) method development/validation to determine the extent of matrix effects and the ways of overcoming them and (b) as a second step of analysis in the case of samples having analyte contents near the maximum residue limits (MRL).

Towards the electrospray ionization mass spectrometry ionization efficiency scale of organic compounds

Ivo Leito, Koit Herodes, Merit Huopolainen, Kristina Virro, Allan Künnapas, Anneli Kruve and Risto Tanner

Rapid Commun. Mass Spectrom. 2008, 22(3), 379–384

DOI: 10.1002/rcm.3371

An approach that allows setting up under predefined ionization conditions a rugged self‐consistent quantitative experimental scale of electrospray ionization (ESI) efficiencies of organic compounds is presented. By ESI ionization efficiency (IE) we mean the efficiency of generating gas‐phase ions from analyte molecules or ions in the ESI source. The approach is based on measurement of relative ionization efficiency (RIE) of two compounds (B1 and B2) by infusing a solution containing both compounds at known concentrations (C1 and C2) and measuring the mass‐spectrometric responses of the protonated forms of the compounds (R1 and R2). The RIE of B1 and B2 is expressed as logRIE(B1, B2) = log[(R1 · C2)/(C1 · R2)]. The relative way of measurement leads to cancellation of many of the factors affecting IE (ESI source design, voltages in the source and ion transport system, solvent composition, flow rates and temperatures of the nebulizing and drying gases). Using this approach an ESI IE scale containing ten compounds (esters and aromatic amines) and spanning over 4 logRIE units has been compiled. The consistency of the scale (the consistency standard deviation of the scale is s = 0.16 logRIE units) was assured by making measurements using different concentration ratios (at least 6‐fold concentration ratio range) of the compounds and by making circular validation measurements (the logRIE of any two compounds was checked by measuring both against a third compound).

Matrix effects in pesticide multi-residue analysis by liquid chromatography-mass spectrometry

Anneli Kruve, Allan Künnapas, Koit Herodesand Ivo Leito

J. Chromatogr. A 2008, 1187, 58–66.

DOI: 10.1016/j.chroma.2008.01.077

Three sample preparation methods: Luke method (AOAC 985.22), QuEChERS (quick, easy, cheap, effective, rugged and safe) and matrix solid-phase dispersion (MSPD) were applied to different fruits and vegetables for analysis of 14 pesticide residues by high-performance liquid chromatography with electrospray ionization–mass spectrometry (HPLC/ESI/MS). Matrix effect, recovery and process efficiency of the sample preparation methods applied to different fruits and vegetables were compared. The Luke method was found to produce least matrix effect. On an average the best recoveries were obtained with the QuEChERS method. MSPD gave unsatisfactory recoveries for some basic pesticide residues. Comparison of matrix effects for different apple varieties showed high variability for some residues. It was demonstrated that the amount of co-extracting compounds that cause ionization suppression of aldicarb depends on the apple variety as well as on the sample preparation method employed.