Prediction of ionization efficiencies in biological matrices

In the recent years, we have made significant progress in ionization efficiency studies. In our previous publications, we have shown that our approach can be applied in both electrospray positive and negative mode (we have recently also enabled comparing the logIE values measured in both ESI modes numerically), that our approach is transferable between different instruments with various ESI source geometries from all major mass spectrometric systems vendors, and that it is also transferable between different eluent compositions.

The logIE approach is already applicable in nice clean matrices such as neat solvents. We wanted to see if our approach also works with more difficult matrices, such as bodily fluids and tissues. The results were recently published in Analytica Chimica Acta.

In this study, we took a representative set of 10 compounds, including drugs, e.g., naproxen and lincomycin. As matrices, we used blood, plasma, urine, cerebrospinal fluid, brain and liver tissue homogenates and neat solvent to compare with. We carried out a simple and robust sample pretreatment of protein precipitation. We measured the IEs in a worst-case scenario in flow injection mode without any chromatographic separation.

As with different instrumental setups and solvents, the IEs vary between biological matrices, but the order of the compounds remains roughly the same. These variations between different matrices and variations between a certain biological matrix and neat solvent demonstrate that matrix affects ionization efficiencies and also the prediction models of ionization efficiencies. This, in turn, shows that matrix affects the importance of properties of compounds in the prediction model.

Even though the effects are big and matrix effect is strong we were happy to see that the correlations between IEs measured in the neat solvent and IEs measured in a biological matrix are in good correlation (R2 from 0.7 to 0.9). These good correlations were a promising start to predict IEs in biological matrices similarly to previous predictions in the neat solvent. The most accurate model was obtained for the solvent with a mismatch of 2 times which was also expected since it is the cleanest matrix. But also for liver and brain tissues the mismatch of the model is only 3-fold.

The correlation between predicted and calculated IEs is good with the average mismatch over all biological matrices of 8 times. This means that the accuracy of standard substance free quantitation has been improved by more than an order of magnitude for the set of compounds used in this study.

More details can be found in the paper published in Analytica Chimica Acta. Piia Liigand also gave a talk on the topic in ASMS which can be found here. More papers by our group on the topic of ionization efficiencies can be found here.

Is it possible to use data below the limit of quantitation in pharmacokinetic studies?

Results below the limit of quantification (BLQ) are generally not reported or reported without explicit numbers, leaving pharmacokinetic (PK) and pharmacodynamic (PD) studies wishing for more information to improve their model parameters. But the laboratory cannot release actual values BLQ since the quality of that data has never been assessed.

Possible solutions to this unfortunate situation were investigated in a recent perspective article led by our group and published in Bioanalysis. The article titled: “Utilization of data below the analytical limit of quantitation in pharmacokinetic analysis and modelling: promoting interdisciplinary debate”, expanse the so far used statistical salvage of information, by an experimental addition to investigate the quality of data BLQ.

By directing this article towards the pharmacometrician, the analytical scientist and the regulatory personnel, we hope to encourage an inter-disciplinary discussion to improve the situation by finding ways to use BLQ data in PK/PD studies, in order to enhance the quality of the obtained pharmacokinetic models. Several ways were proposed for moving forward, in particular improving/modifying method validation guidelines for enabling to use BLQ data and leaving the decision regarding whether and how to incorporate the BLQ data into a PK/PD model to the data analyst and not the analytical chemist.

ASMS 2018 in San Diego

Piia Liigand giving her talk

On 3rd-7th June three members of our group focusing on ionization efficiency studies Dr. Anneli Kruve-Viil, PhD students Piia Liigand and Jaanus Liigand, participated in the 66th Annual Conference of American Society for Mass Spectrometry (ASMS) in San Diego, CA. The conference was a huge success. There were altogether about 7500 scientists participating, ca 200 oral presentations and 3300 of posters. We had two oral presentations and one poster presentation.

The conference was kicked off by a presentation by Lisa Shipley from Merck who gave a very informative talk about smart trials and moving to patient-centric clinical trials – most probably the future of clinical trials. It was very inspiring to see how a field that is considered to be relatively conservative is picking up the most modern technical solutions such as clever packages and home-based sample collection.

The first full day of the conference started with a presentation by our PhD student Piia, who gave a talk about achieving more accurate semi-quantitative analysis by predicting electrospray ionization efficiencies. She first gave an overview of the research carried out so far and then, introduced her latest results. She showed that with the help of using ionization efficiencies, concentration estimation in various biological matrices was improved by more than an order of magnitude.

Jaanus Liigand presenting his poster

Jaanus presented a poster on semiquantitative LC/ESI/MS via ionization efficiency prediction. He presented the simple and user-friendly approach of predicting ionization efficiencies using only 2D structures. We are now able to predict ionization efficiencies in both ESI positive and negative mode and in different solvent compositions without time-consuming DFT-COSMO calculations. Additionally, we have shown that using ionization efficiency predictions the validation with LC gradient elution resulted in 2.7-fold mispredicted concentrations compared to 44-fold mispredictions using directly peak areas. We were happy to see that numerous groups were interested in our studies and fruitful discussions about the results did not stop until the end of the poster session.

Dr. Anneli Kruve-Viil giving her talk

Dr. Anneli Kruve-Viil presented some of the latest results obtained in FU Berlin. She talked about using mass spectrometry and ion mobility spectrometry for investigating interlocked nature of catenanes and knots. These results will soon be shared with everyone.

In conclusion, this year’s ASMS was very successful for our group. We saw a lot of interest in our work, met our collaborators and got some new friends and possible future collaborators. We are already looking forward to the next ASMS in Atlanta. We are also grateful for our financial supporters, who made this conference possible to us: instrumentation provided by Estonian Center of Analytical Chemistry (www.akki.ee), Ministry of Education and Research of Estonia by smart specialization doctoral stipend, Graduate School of Functional Materials and Technologies, and Alexander von Humboldt Foundation.

More information about the group focusing on ionization efficiency studies can be found on the webpage kruvelab.com

Recent study emphasizes that pesticide traces in Estonian groundwater under farmlands exceed the allowed limit

Sum of pesticide traces in samples from 2016-2017. Source: Estonian Environmental Research Centre

Our joint research fellow with Estonian Environmental Research Centre Dr. Riin Rebane and joint PhD candidate Siiri Saaver participated in the study “Analysis of pesticide traces and dynamics in surface and groundwater” . In this study, 137 surface and groundwater samples from all over Estonia were analysed with a targeted screening of 135 pesticides. From 137 analysed samples 49 pesticide traces were detected in more than half of the samples. In 34 samples 9 pesticides and their metabolites exceeded the allowed concentration level of 0.1 μg/L. In 9 samples pesticides and their transformation products exceeded 0.5 μg/L. Interestingly, the main compound found (in 26 samples) was chloridazon-desphenyl, which is a metabolite of pesticide chloridazon that is not registered on Estonian market and therefore not sold, which makes the determination of the origin of this compound complicated. It needs further research whether it is a remnant of Soviet times and slowly moving in the soil towards groundwater or if it is in use now.
This study was also reported in news portal Novaator and in environmental TV show Osoon

Moreover, there are hundreds of different pesticides and targeted screening methods usually look for compounds that are banned or known to be problematic. This study used the broader list of pesticides than regular yearly monitoring of pesticides indicating that broader coverage of potential pollutants is needed. Therefore, there is a need for the suspect and non-target screening methods for detecting more compounds and their metabolites.

In collaboration with Estonian Environmental Research Centre, our group has also contributed to studying and developing the non-target approach. Namely, Gunnar Printsmann developed in his master thesis suspect and non-target screening method for groundwater using high-resolution mass spectrometry which also included pollutants database. He found and confirmed a new industrial pollutant dibutyl phthalate in one sample from North-Eastern Estonia.

Belgian Society for Mass Spectrometry 2018 Annual Meeting

Our PhD student Jaanus is currently doing research in the group of Prof. Edwin De Pauw at the University of Liege in Belgium and he had a nice opportunity to take part in a 21st annual meeting of Belgian Society for Mass Spectrometry that was held on 29th of March in Liege. The meeting was organized in honour of Prof. Edwin De Pauw to summarize his work as he intended to retire after this year. Luckily for the mass spectrometric community, he will be actively involved in the research at least for four coming years.

Prof. Scott McLuckey opened the day with a presentation about strategies for achieving enzyme-like specificity in the gas-phase fragmentation of peptide and proteins. He showed different strategies how to cleave for example specifically at asparagine or lysine.

Prof. Andrea Sinz presented the results of the development of cleavable cross-linkers. Additionally, she pointed out that although cleavable crosslinkers are available on the market most of the groups determining the protein structures still use the noncleavable crosslinkers and do not take advantage of additional information provided by cleavable crosslinkers.

Dr. Valérie Gabelica presented the results of the studies of nucleic acid noncovalent complexes in native MS. She said that by knowing and studying the fundamentals of ESI processes one gets also a real insight into phenomena seen in different applications (in her case, nucleic acid noncovalent complexes). She also pointed out that unfortunately, scientific community publishes solely positive results which result in biased and non-universal theories.

Jaanus Liigand also had the possibility to present the results of our studies of standard substance free quantitation in LC/ESI/MS analysis and industry showed a great interest in our approach which could be beneficial for their needs (see the poster here).

It was a really nice day full of interesting presentations and discussions.

Analytical chemists fighting food fraud

Every year Estonian University of Life Sciences organises a conference called “Healthy animal and healthy food” where Dr. Riin Rebane made a presentation “Fight against food fraud” which explained the ever-expanding role of analytical chemists in food science. Reasons for food fraud vary, but are almost always for monetary gain and therefore food fraud is in constant progress. One good example is honey analysis, where for decades there has been a change in methods in order to identify whether honey is real or whether it is identified with correct botanical or geographical origin. As a natural product, no two honeys are identical and this makes identification further more challenging for the chemists. One of the possible methods is amino acid analysis since the amino acid content can be like a fingerprint for honeys and in University of Tartu we have analysed few hundreds of Estonian honeys and have seen that that foreign honeys do differ in most cases and also that there is a correlation between the amino acid content and botanical origin. But nevertheless, even this method might not work every time and chemists are looking towards methods such as nuclear magnetic resonance spectroscopy and even DNA-analysis to get better certainty for determining the origin of honey.

The summary based on the presentation was also reported in the newspaper Maaleht.

European Mass Spectrometry Conference 2018

Dr. Anneli Kruve giving her talk at EMSC 2018
Dr. Anneli Kruve giving her talk at EMSC 2018

Dr. Anneli Kruve, a leader of research team focussing on ionization efficiency studies in electrospray, is currently a Humboldt fellow in Freie Universität Berlin and visited the 1st European Mass Spectrometry Conference in Saarbrücken (Germany) this month. She describes some of the highlights of this conference in her blog, you can read the post below.

 

Last week I had a chance to take part in the European Mass Spectrometry Conference that was hosted by DGMS (German Society for Mass Spectrometry) and SFSM (French Society for Mass Spectrometry). Below I share a few key ideas from this nice conference that took place in Saarbrücken over 5 days.

The conference was opened with a plenary lecture by Prof. Alain van Dorsselaer who summarized the main work he and his group has done on mass spec during the last 30 years. One of the key ideas, that came up several times in his talk referred to the fact that endless possibilities are accompanied by extreme data load. The amount of data in LC/MS/MS is huge and it is very complicated to analyse these massive data sets. Several other scientists, including Prof. Andreas Roempp and his group, also stressed the importance of transparent and open source data analyses and storage that could eventually simplify the data treatment. These ideas strongly resonate with my own ideas of applying more data science tools in primary data treatment in mass spectrometry, as today the data processing is by far limiting the progress in several fields of analytical mass spectrometry. Mostly this is the case for fields, where the science is still in the “discovery” stage; meaning that the scientists aim at finding the important compounds and yet do not know which these compounds could be. Such fields include metabolomics, proteomics, environmental science, etc.

Prof. Philippe Schmitt Kopplin stressed the importance of high throughput in metabolic sample analyses and explained why dissolve-and-shoot approach (flow-injection or infusion combined with MS) is often most practical. Also, he showed several case studies where marker compounds could be reliably identified with this simple approach if accompanied with efficient and accurate data processing. A particularly interesting example was a case study of 170-year-old wine from the bottom of the Baltic Sea.

Prof. Carsten Engelhard showed an extremely clever, almost brilliant, method to analyse nanoparticles with simple dilution & infusion experiment. The infusion of homogeneous solution to ICP-MS instrument causes an almost constant signal with small random variations. However, if the solution of nanoparticles is infused to ICP-MS, most of the time there is no signal (only noise). When one of the nanoparticles enters the plasma a signal suddenly occurs causing a peak in the chronogram. The height of the signal reflects the size of the nanoparticle and the number of peaks per volume indicates the concentration of the nanoparticles.

Prof. Thomas Kraemer introduced us to the world of forensic analyses. Particularly, he focused on MALDI imaging techniques, that allow revealing drug intake or exposure to toxic compounds. For this purpose his lab is using two types of samples, the traditional hair and lately also toenails, to overcome the problem arising for hairless people. Interestingly, the single hair analysis also reveals time-resolved information with high precision; therefore, allowing to distinguish between one-time and long time exposures.

You can check out more posts from our team studying ionization efficiencies kruvelab.com

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 sea 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 Mass Spectrometry lab three years ago to conduct her bachelor thesis with us. Mari 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 to make much wider conclusions about charged droplets.

It became possible 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 is 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. Cations, such as hydronium ion, which are strong acids protonate the compounds, while weak acids, such as ammonium cation, 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. The support for this model was found 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.

The results were published in ChemistrySelect . 

Sponge Spray – New Approach for Direct Sampling and Analysis by MS

DSC_0294Mass spectrometry is currently probably the No 1 technique for determining trace-level components in complex (especially biomed-related) mixtures. The key issues in such applications are sample preparation, sample introduction to MS and ionization of components of interest (analytes). Big efforts are continually made to improve any of them.

In his recent development – Sponge spray – Max Hecht, an AMS graduate, now PhD student at UT attempts improvements in all of the above issues. The elegant approach proposed by Max utilizes a volumetric sampling device – a hydrophilic sponge, which after absorbing a predetermined amount of sample (e.g. blood or urine), can be directly used for sample introduction to MS and ionizing the analytes.

The seriousness of the work has been demonstrated by the fact that it was accepted for publication by Analytical Chemistry, the top journal in the field. The published article Sponge Spray – Reaching New Dimensions of Direct Sampling and Analysis by MS is now available from the journal website.

Further developments of this approach may lead e.g. to fast medical diagnosis MS methods that, contrary to the current situation with MS in medicine, could be applied as “bed-side” diagnosis tools in hospitals.

(Photo: Max Hecht working with the sponge spray ion source)

 

LC-MS Validation tutorial review and online course at Euro Mass Spectrometry 2017

Leito_LCMS_Validation_EuroMS_2017On 21.06.17 Ivo Leito gave a talk titled Review on validation of liquid chromatography–mass spectrometry methods at the Euro Mass Spectrometry 2017 meeting (London, UK). The talk focused on the two-part Tutorial review on validation of liquid chromatography–mass spectrometry (LC-MS) methods

as well as on the related on-line course (MOOC) LC-MS Method Validation and the ValChrom validation software.

The reception of this talk was one of the warmest during the meeting! Several participants came later to say words of thank for offering such a valuable resource to the LC-MS community. There were also some interesting ideas proposed regarding topics that could be covered in the online course. LC-MS and MiC issues, such as validation, are among core competences of the UT Analytical Chemistry research group. The tutorial review, the on-line course, as well as the ValChrom software together form a nice outcome of joining these competences together.

Concerning the topics of the meeting in general, they were remarkably diverse and not so heavily dominated by biomedical MS as is often the case at mass spectrometry meetings. Interesting presentations were given on gas-phase ion processes, different laser techniques in MS (e.g. for analysis of solids without sample preparation), advanced catalysis studies by MS, LC-ICP-DRC-MS for trace element speciation, the possibility of making a high-end LC-HRMS system an “open access” system within an organization, etc.