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.

What ions to expect in mass-spec?

One thing that we often do in Testing Centre of University of Tartu is the analyses of the synthesis products for different labs/companies. Usually these compounds are brand new, without any standard substance available. The aim of these analyses is to confirm the identity of the product. What is often puzzling about this type of analyses is that usually it can only be assumed what kind of ions would be formed of the compounds in electrospray. For example will it be a protonated molecular ion, sodium adduct, dimer or something else? Though there are some general ideas and also intuition helping mass spectrometrist’s there is no general guidance. My student Piia focused on the ions formed in negative ESI mode in her first study. More precisely differentiating between compounds forming anions and dianions. Altogether she studied 29 compounds with at least two acidic sites, but only 9 of these gave doubly charged ions in mass spectra. Interestingly the compounds giving multiply charged ions was not related to the acidity of the compounds: there were strong acids not giving multiply charged ions and week acids giving multiply charged ions. The mechanism for this interesting phenomena was revealed by the help of linear discriminant analyses. It turned out that to produce multiply charged ions the compound has to have a suitable combination of acidity and hydrophobicity. For example the strong acids that are quite hydrophilic (and become even more hydrophilic after dissociation) do not reach the ESI droplet surface and therefore can not be ejected to the gas phase for analyses with mass spectrometer. The mistery between ions observed in positive ESI/MS mode (molecular ions, different adducts and multiply charged compounds) is yet to be solved. 

DOI: 10.1007/s13361-016-1384-2

Neonicotinoides in Estonian honey?

Our PhD student Asko Laaniste carried out an extensive study of neonicotinoides in Estonian honey samples gathered over ten years. One of the studied neonicotinoides, thiacloprid, was identified in several samples. It was observed that the fraction of samples containing thiacloprid increased from year to year, reaching up to 60% in samples form 2012. At the same time also the amount of thiaclopride imported to Estonia has increased from year to year. Our stability test showed that thiacloprid is persistent in honey and it can be assumed that these results are not influenced by the decomposition of analyte within years.

Geographical distribution of collected honey samples in Estonian counties is shown bellow. In the brackets the total number of samples acquired from corresponding region is shown (over years). The percentage shows the number of samples containing thiacloprid.

For more see DOI: 10.1080/03601234.2016.1159457

MOOC on LC-MS method validation

Already in last spring when we were writing the tutorial review on LC/MS method validation Ivo introduced us the idea of making a MOOC (massive open online course) on the same topic. By that time he had launched the first MOOC of our institute that has had hundreds of students so far, on such complex topic as measurement uncertainty.

In summer we started putting some first ideas on the “LC-MS validation course” to the paper and in autumn we started doing some first videos for clearing our ideas. By now we have been to the studio several times already and have a significant amount of course videos ready. We will cover all important aspects of method validation in the course: Trueness, Precision, Accuracy, Linearity, LoD and LoQ, Selectivity, Robustness etc. You are all welcome to post ideas or topics of specific interest! 

So we are anxiously waiting for the course to be ready by the end of spring and to launch it in September.

Optimizing sample pretreatment: temperature

In a recent semester I have been involved in several training activities. The most important of these have been the training day for water analyses laboratories organized by EKUK and the practical aspects of working in the laboratory by TÜ Katsekoda.

My lecture/seminar in the first training was about sample matrices as the source of uncertainty. In this lecture the main idea was to show how large uncertainty source may a matrix actually be. As I have been dealing with matrix effects since my masters thesis this is a very interesting point to me. On the practical aspects of working in the laboratory course my major topic was preparing samples for analyses.

Whether considering uncertainty or trueness it ends up with the understanding that a good sample pretreatment is a base for a good analytical method. It seems like an ongoing discussion on whether liquid-liquid extraction of solid phase extraction is a technique providing more possibilities for efficient sample prep. However, what I have often observed in the trainings and lectures is that one important parameter influencing sample extraction, that people do not come up with, is temperature.

Common knowledge is that higher temperatures are advantageous for extracting analytes from solid matrices and in these cases higher temperature helps to break the “bonds” between analyte and matrix. But what is not that often considered is the temperature effect on liquid-liquid extraction.

We know that water properties strongly depend on the temperature. Based on the NISTdatabase the dielectric constant of water ranges from 87.7 to 55.7. The latter value is more similar to acetonitrile then to “water” as we consider it under standard conditions. This means the enthalpy of solvatation for different species changes remarkably with temperature. For example Z. Congliang et al has observed that octanol-water distribution coefficient of an antibiotic sulfamethoxazole is reduced 5 times while working at 45 °C instead of room temperature. And for extracting the sulfamethoxazole from water with octanol the recovery would drop from 89% to 61%. Unfortunately data for lower temperatures are not available to evaluate if lower temperatures could result in higher recoveries. These effects are larger for compounds with low distribution coefficients and therefore with lower recoveries.

It is even interesting to consider how much could year-around room temperature fluctuation influence the extraction?