What can we learn from mass spectrometry about charged droplets?

Charged droplets occur everywhere in the world. They are created by the oceans (known as see spray aerosols), near waterfalls and in thunderstorm clouds. Such droplets are expected to play significant role in environmental processes. Similar droplets are also created in electrospray ionization (ESI) source.

Mari Ojakivi joined our group three years ago to conduct her bachelor thesis with us. We were then very strongly interested in the way different additives influence ionization efficiencies in ESI. So she started studying how different acids, salts and bases influence the ionization of some amines in charged water droplets. Soon, some extremely interesting results were revealed that allowed us to make much wider conclusions about charged droplets.

We were able to pinpoint, that protonation of the amines is strongly dependent on the type of additives present in the droplets and is virtually independent of the pH of the solution used for “preparing” the droplets. In “normal” solutions the protonation determined solely by the pH of the solution. This led us to conclude that some of the additives change something about the droplets that other additives do not affect. It turned out, that the factor determining the protonation is the cation present near the surface of the charged droplets, as the protonation mostly takes place on the surface. Cations, such as hydronium ion (droplet A in the picture), that are strong acids protonate the compounds, while weak acids, such as ammonium cation (droplet B in the picture), do not. If both types of cations are present in the solution, the protonation is determined by the ion that has higher affinity for the droplets surface. We were also able to find support for our model from the molecular dynamics simulations carried out in Prof. Konermann’s group.

Why is the protonation in charged droplets at all important? Protonation is one of the fundamental properties of compounds; it may catalyze reactions, break up or induce complexation, change conformation of the macromolecules, etc. Therefore, it can be assumed, that the reactions and processes taking place in charged droplets also depend on the protonation. 

Modifying the Acidity of Charged Droplets. Mari Ojakivi, Jaanus Liigand, Dr. Anneli Kruve. Chemistry Select doi.org/10.1002/slct.201702269

Illustration of droplets surface for droplets that do not contain ammonium anion (A) and that do contain ammonium ions (B). The latter significantly decreases the protonation of the compounds in the droplet. Picture by M. and K. Ojakivi.

IsrAnalytica2017

Last week there were two awesome analytical chemistry events in Tel Aviv. On Monday 23th of January there was an international workshop on validation of test methods, human errors and measurement uncertainty of results organized by Dr. Ilya Kuselman. In this event I had an amazing chance to present my experiences in the validation in this workshop. My lecture was about handling different matrices during validation of LC/MS method. It was such a pleasure to share my academic knowledge with practitioners and to receive their feedback. 

From 24^th to 25^th the IsrAnalytica conference was held. The conference hosted four parallel sessions, around thousand visitors from universities, industries, regulatory bodies, etc. The topics of the presentations ranged from forensic, to food, to innovative apparatus. The conference also hosted a massive exhibition area with nearly hundred companies. For example check out this massive rotary evaporator instrument for the factories. 

Choosing analyses conditions to get matrix effect free?

Recently my first single-authored paper became public. It concerns the solvent effect on electrospray ionization efficiency and matrix effect. As we have talked about ionization efficiency and its solvent effects also previously let's focus on matrix effects this time. 

As LC/ESI/MS is the tool for various fields in routine analyses (quality control over pesticides but also mycotoxins in the food we eat, drug monitoring in patients body, etc) to solving highly demanding scientific questions (finding biomarkers, decoding nervous signalling, etc). However, one thing haunting quantitation in these analyses is the matrix effect. The influence of sample compounds (called matrix compounds; for example the flavor compounds in tomato) on the ionization of our target analyte (e.g. the monitored mycotoxin in tomato). This effect may both give results that are either higher or lower than the actual content. Though underestimation is more common. Meaning that the mycotoxin may actually exceed the allowed limits, but matrix effect may hinder our ability to detect this. This makes matrix effect very important and the need to remove its origin becomes obvious.  

This is how matrix effect looks on a chromatogram. 

Though, since I started interacting with this field in the beginning of my Master's thesis, one thing has definitely improved: the awareness of the presence of matrix effect. Still much unclarity exists in the fundamental level. There have been numerous mechanisms proposed to the origin of matrix effect, however some of them are contradictory or have not been reproduced latter. However, there is one general mathematical model proposed by Christie Enke in 1997, which states that matrix effect is caused by the fact that more two compounds compete for the surface charge in the ESI droplets and that the extent of matrix effect is related to the magnitude of these affinities. However, it is unknown of what these affinities depend on. In one of my previous works I discovered that this affinity is well correlated with ionization efficiencies for ionisable compounds1.  

During investigating ionization efficiencies dependents on solvent composition3 naturally the question arouse: if compounds' ionization efficiencies depend on the solvent composition why shouldn’t the matrix effect do2? And it does! It was observed that solvent compositions that provide higher ionization efficiencies produce more matrix effect. Higher ionization efficiencies are usually considered beneficial because they allow analysing smaller analyte quantities (limit of quantitation, LoQ) and increase sensitivity. So if you tune your method for lower LOD-s it will automatically be less robust for matrix effects. It turns out that in ESI/MS you have to balance between low LoD-s and little matrix effect, while choosing your solvent.  

With this study I was able to uncover the true dual nature of the processes occurring in the charged nanodroplets in ESI. Though the practical conclusions are not according to every analyst dreams4 it effectively demonstrates the dual side of the processes occurring in micro compartments such as nanodroplets. Therefore, I find that these nanodroplets are capable of much more and we shall hear about these again.  

1 It is known that also compounds that do not give ESI/MS signal also produce matrix effect; however, this process is expected to follow a different mechanism.  

2 Previously different solvent conditions have been also used to reduce or eliminate matrix effect, but with the aim of providing better separation of the analyte and matrix components.  

3 Solvent with higher organic content tends to yield higher ionization efficiencies.  

4 It would be best to have lowest LoD-s, no matrix effect, high sensitivity, etc all at once.  

Our publications

Electrospray ionization studies

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, Anneli Kruve
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.

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
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.


Predicting ESI/MS Signal Change for Anions in Different Solvents
Anneli Kruve, Karl Kaupmees
Doi: 10.1021/acs.analchem.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 physicochemical 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.

Adduct Formation in ESI/MS by Mobile Phase Additives
Anneli Kruve, Karl Kaupmees
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. Although 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.

pH Effects on Electrospray Ionization Efficiency
Jaanus Liigand, Asko Laaniste, Anneli Kruve
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.

Influence of mobile phase, source parametersand source type on electrospray ionization efficiency in negative ion mode

Anneli Kruve

Doi: 10.1002/jms.3790

The effect of organic solvent content on ionization efficiency (sensitivity) is compared for different ESI sources in negative mode. It was observed that ionization efficiency in ESI with thermal focusing (such as Jet Stream source) is little affected by the organic solvent content while in conventional ESI ionization efficiency can be significantly (by an order of magnitude) increased with increasing organic solvent content (both acetonitrile and methanol). However pure acetonitrile is not recommended for such measurements as it yields poor repeatability. Additionally though organic solvent content increase results in higher ionization efficiency it unfortunately also increases ionization suppression.

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

Piia Liigand, Karl Kaupmees, Anneli Kruve

Doi:10.1007/s13361-016-1384-2

The factors influencing observation of doubly charged ions in mass spectra was studied on the example of small acidic (carboxylic acids, phenols, sulphonic acids) compounds with at least two ionisable sites in ESI negative mode. It was observed that being only a strong acid (meaning that the compound is present in solution as two valent anion) is not sufficient for observing doubly charged ion in mass spectra. Additionally two compound needs to be sufficiently hydrophobic. Also, if sufficiently hydrophobic compounds which are not expected to be present as divalent anions in solution, may give doubly charged ions in mass spectra.

Establishing Atmospheric Pressure ChemicalIonization Efficiency Scale

Riin Rebane, Anneli Kruve, Piia Liigand, Jaanus Liigand, Koit Herodes, Ivo Lieto

Doi: 10.1021/acs.analchem.5b04852

Transferability of the Electrospray IonizationEfficiency Scale between Different Instruments

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

Doi: 10.1007/s13361-015-1219-6

Influence of source and mass analyser type on ionization efficiency scales was studied on the example of ESI positive mode. It is demonstrated that ionization efficiency scales can be successfully transferred between different instruments.

Effect of Mobile Phase on ElectrosprayIonization Efficiency

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

Doi:10.1007/s13361-014-0969-x

The mobile phase pH and organic solvent content effect on ionization efficiency (sensitivity) of basic or neutral compounds in ESI positive mode was studied. It was observed that ionization efficiency changes with pH for compounds which have pK_a in the range of varied pH. Compounds that are permanently charged in solution or are not charged at all in solution will not change their ionization efficiency in ESI. Also organic solvent content influences ionization efficiency.

Negative Electrospray Ionization viaDeprotonation: Predicting the Ionization Efficiency

Anneli Kruve, Karl Kaupmees, Jaanus Liigand, Ivo Leito

Doi: 10.1021/ac404066v

Introducing an ionization efficiency scale for electrospray negative ionization. Altogether ionization efficiencies (related to sensitivity) of 64 acidic compounds (carboxylic acids, phenols, imides, sulphonic acids, sulphonamides, etc) was measured and modelled. It was observed that ionization efficiency can be explained by the ionization degree in solution and charge delocalization of the formed anion. The missprediction of the proposed model was 3 times while the measured ionization efficiencies varied over six orders of magnitude.

Sodium adduct formationefficiency in ESI source

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

Doi: 10.1002/jms.3218

The sodium adduct formation efficiency (SAFE) scale was introduced and showed that the order compounds is unaffected by the sodium content in the mobile phase. Also it was observed that SAFE can not be explained by the solvent/vacuum distribution coefficient of formed species nor with strength of the formed adduct species (described by partial charged on the heteroatom associated with sodium and bond length between the hetero atom and sodium).

Electrospray IonizationEfficiency Scale of Organic Compounds

Merit Oss, Anneli Kruve, Koit Herodes, Ivo Leito

Doi: 10.1021/ac902856t

Towards the electrosprayionization mass spectrometry ionization efficiency scale of organic compounds

Ivo Leito, Koit Herodes, Merito Huopolainen, Kristina Viroo, Allan Künnapas, Anneli Kruve, Risto Tanner

Doi: 10.1002/rcm.3371

Defences in our group: Asko Laaniste and Hanno Evard

We are happily back from IMSC in Toronto and a very important celebration in our group has occurred! On the last day of August two PhD theses were defended in our group by Asko Laaniste and Hanno Evard.
Asko wrote his thesis on ionization sources. Specifically focusing on the comparison of different sources for pesticides. The main conclusion of his work is, that conventional electrospray ionization (ESI) is better than generally expected. Additionally Asko studied the possibilities of 3R nebulizer developed in our lab for ESI source. As an opponent Prof. Risto Kostiainen from University of Helsinki served.
Hanno on the other hand solved a very crucial question of LoD determination. It has been often bothering our lab that the methodology used for LoD determination in various papers can not be clearly understood. So Hanno compared all common methods used to determine LoD and observed that the approach using calibration function residuals to estimate the noise of the method (suggested by International Conference of Harmonization, ICH) gives results closest to the VIM (Vocabulário Internacional de Metrologia) definition. The opponent in the defense was Dr Emilia Vasileva-Veleva from Marine Environmental Studies Laboratory, International Atomic Energy Agency, Monaco.
 Both theses can be accessed form here:
Asko Laaniste, Comparison and optimisation of novel mass spectrometry ionisation sources
Hanno Evard, Estimating limit of detection for mass spectrometric analysis methods

Asko and Hanno celebrating the defense.

Happy supervisor with a defense gift.

We will be in IMSC

A small part of our group is going to International Mass Spectrometry Conference from 20th to 26th of August in Toront and present the latest results of our studies. The results are so hot that these are not yet available in the journals:

M-T-053 pH effects on electrospray ionization efficiency Jaanus Liigand
W-T-019 Ionization efficiency of oligopeptides and small hydrophilic molecules in ESI/MS Piia Liigand
W-T-137 Predicting concentrations of small molecules without standard substances in LC/ESI/MS via ionization efficiency scales Anneli Kruve

See you in Toronto .... or after that! We will definitely report the coolest talks and posters and how it went for us!

NonTarget2016

Today the importance of environmental analyses has been generally accepted. However conventional environmental monitoring programs are somewhat limited in scope as these programs only search for compounds that have been previously found to exist in contaminated samples. These compounds make up only a fraction of compounds that may be present in target samples overall. In addition to previously found compounds there is a large number of contaminants we are not aware of and for sure some of these compounds are of importance. These compounds have not yet made their way to the emerging contaminants lists.

The lack of these monitoring programs comes from the fact that with the conventional analytical techniques it is even impossible to monitor “everything” as most of the techniques require standard substances for analyses. However if a compound has not yet been identified it is also unlikely (if not impossible) to have a standard for this substance. Currently the importance of analyses of unknown compounds been increasingly noticed. The detection of such unknown compounds is called non-target analyses referring that there aren’t any target compounds, which are specifically looked for. Such non-target analyses can today almost only be done with high-resolution mass spectrometry (HRMS). The beauty of HRMS data compared to conventional target analyses is that you can go back to your data and re-examine if one or another compound was in the sample at any time. This could be done even years later when we have a lot more information on the contaminants..

NonTarget2016, picture from www.eawag.ch

From 29th of May until 3th of June a NonTarge 2016 conference was held in Ascona, Switzerland, specifically dedicated to such analyse. Our Master’s student Gunnar Printsmann who has started to study the organic pollutants in Estonian groundwater received a stipendium to this Europe’s largest conference specifically dedicated to non-target analyses.

In the conference there were present most analytical laboratories dealing with non-target analyses today. In general, it was proposed that approximately 5-10% of the HRMS peaks observed in spectrum can actually be identified via non-target analyses. However, several institutions are working to improve this situation. One thing of urgent need is the list of suspect substances. During last year we have tried to conduct our own suspects list of pesticides, detergents, pharmaceuticals, industrial chemicals etc. Similar lists are also available or being developed under names Tracefinder, NORMAN, etc. Another software that is being developed in several places and which is of great interest also for other MS users not only in non-target screening is the MS/MS transitions predictor. 

We hope to report on Estonian ground water non-target analyses in a near future.