Built on the foundation of several years of research on ionization efficiency by Kruve et al. there is now an easy-to-use tool to quantify analytes in LC/ESI/MS analysis without the use of standard substances.
The availability of standard substances is one of the main limitations in LC/ESI/MS analysis, especially in the fields of metabolomics, environmental analysis as well as illegal substance monitoring. If no commercial standards are available, the only choices so far have been to either (1) synthesise these in-house which is very expensive and time-consuming or (2) use other compounds for quantification and ignore the possibility of vastly different response factors. The latter choice could lead to errors up to 10 million times. Now, a third, overwhelmingly faster, cost-effective and accurate option has been developed.
A team consisting of scientist associated with Chair of Analytical Chemistry has launched their company Quantem Analytics aimed at providing standard-substance-free quantification solutions for LC/ESI/MS analysis. They combine the fundamental research[1][2][3] in the field of mass spectrometry with data science to provide the first solution to situations where there simply are no standard substances available for quantification. Quantem uses machine learning to predict response factors of analytes taking into account the eluent composition at the retention time. Their novel approach is applicable to:
Numerous types of analytes with logP from -10 to +10 and molar mass below 1500 Daltons;
Different matrices, e.g. urine, plasma, liver, and cereal;
All common eluent compositions, both in terms of organic modifiers and additives;
Both positive and negative mode ESI;
Gradient elution, including different flow rates;
This, in turn, opens various new possibilities:
Switching to an approach where your quantification is not limited by the availability of standard substances but rather your ability to identify the peaks;
Quantification of more than 1000 peaks within 24 h;
Retrospective analysis. Quantification of analysis data acquired even years ago;
Direct comparison between standard-substance-free analysis results obtained on different instruments and even in different labs opening the door for large scale collaboration in the field of quantitative non-target analysis;
The accuracy of the Quantem predictions is high, the average error is below 5 times, i.e. if the method predicts a concentration of 1 ppm the true values is probably in the range of 0.2 – 5 ppm. In the vast majority of cases, this is sufficient input for making data-driven decisions.
If you have any further questions you can contact Quantem through https://quantem.co
It is a pleasure to see the real-life application of one’s research. Even more so if discoveries are made during “routine” work. We aimed to develop and validate a UHPLC-MS/MS assay suitable for the quantifying simultaneously two important cardiovascular drugs – milrinone and dobutamine. Dobutamine has a half-life of 2 minutes and unfortunately also undergoes rapid degradation in plasma samples, which we managed to get under control by the addition of ascorbic acid (vitamin C). The study aims to determine the pharmacokinetics of these drugs in neonates and paediatric patients, which restricted the sample amount for the assay. Nonetheless, we achieved the full validation using only 20 µL of human plasma for the analysis, while still reaching the required lowest limit of quantification of 1 ng/mL. This was facilitated by the use of ammonium fluoride as an eluent additive, which provided a significant signal enhancement in positive electrospray ionisation (an effect not entirely understood yet). This collaborative study was recently published in Clinical Mass Spectrometry.
The Workshop will take place in Tartu (Dorpat conference centre) on May 20th and 21st. The programme of the workshop, as well as registration and abstract submission are available at the event website: https://eurachem2019.akki.ut.ee/
The workshop addresses the current status of analytical method validation in general and specifically validation of the non-targeted methods (i.e. ones where the analyte is not defined beforehand). Non-targeted methods are an especially noteworthy part of the workshop programme, because their validation involves specific issues (since analyte is not known it is not possible to make validation experiments with it) and is significantly less developed than validation of targeted methods (i.e. the “normal” analytical methods, where the analyte is known beforehand). At the same time non-targeted methods are becoming increasingly important in environmental protection, food safety, different omics areas, etc.
Some example topics of the workshop are: Validation of targeted methods: where are we? Validation of non-targeted methods – differences from targeted methods. Detection of a multitude of (unknown) components in complex samples: criteria for identification. Managing the huge amounts of complex data from non-targeted methods. Software solutions for validation.
Compact overview of the workshop can be found in the 2nd circular.
These events mark the 30th anniversary of Eurachem and are jointly organized by Eurachem and ECAC (University of Tartu, Tallinn University of Technology and the Estonian Environmental Research Centre).
The measured ionization efficiencies in different solvent systems. The colour of the box plot background shows the organic modifier (90%): Green – Methanol, blue – Acetonitrile, red – Acetone, orange – Isopropanol. The letter shows the water phase additive (10 mM): F – Formic acid, O – Oxalic acid, P – Propionic acid
Recently a study in Rapid Communications in Mass Spectrometry was published. In this study, we optimized the actual split ratio and make-up flow composition in LC/ESI/MS analysis to increase the signal in radio detector and enhance the sensitivity of electrospray ionization.
High methanol content increases electrospray ionization efficiency
We studied make-up flow composition with a set of 20 pharmaceuticals covering 21 different make-up flow compositions. We studied methanol, acetonitrile, isopropanol and acetone as organic modifiers. As we studied positive mode the acidic additives under investigation were formic acid, oxalic acid and propionic acid. DMSO and sulfolane as additives were studied as well. The Methanol/10 mM formic acid in water (90/10) proved to be the best make‐up flow composition in relation to the average sensitivity obtained. Stronger acidic conditions using oxalic acid or higher formic acid concentrations had a clear positive effect on the sensitivity of compounds with low ionization efficiency.
Split-ratios remain stable over main part of the gradient
The second part of this study was testing and monitoring different split ratios (1:10, 1:100 and 1:250) with different splitters (Alliance flow‐splitter kit and homemade T-piece splitter). To monitor the actual split ratio haloperidol solution was added with infusion pump post-column while a haloperidol-d4 solution was added as a make‐up flow by the ISM pump after the splitter. The tested split ratios were relatively stable over the main part of the gradient but showed some variation at very low and very high organic conditions. Differences were larger with methanol compared with acetonitrile containing solvent compositions and when applied without a column or with very long connecting tubing.
I thank Janssen Pharmaceutica and especially Dr Filip Cuyckens for the internship position for letting to gain experience in an industry setting and study these effects. Additionally, I thank Erasmus+ mobility and Smart specialization doctoral stipend for funding my stay.
Main concern while measuring ionization efficiency (IE) of derivatized compounds is that derivatized compounds are in complex mixtures. To overcome this problem, we developed a chromatographic method to separate these compounds and measure their ionization efficiencies.
If ionization efficiencies of derivatized compounds are measured without chromatographic separation in a complex mixture, which includes analytes, derivatization reagents and different by-products, then matrix effects could occur. This problem can be solved if we first separate these compounds from each other and then measure ionization efficiencies.
Chromatographic method
To separate different compounds, we used a simple chromatographic gradient elution method. One might think, that if compounds have different retention time, then they also elute at different organic phase percentages which obviously affects measured ionization efficiency values. The effect is not so significant, but we could manage to model it and take it into account to level all newly measured ionization efficiency values to a previously measured scale.
logIE measurements
We carried out ionization efficiency measurements with liquid chromatography electrospray ionization mass spectrometry (LC/ESI/MS) and constructed IE scales with a calibration curve for compounds with and without derivatization reagent diethyl ethoxymethylenemalonate (DEEMM). Additionally, we investigated eluent composition effects on ionization. Measured compounds were mainly amino acids but also included some biogenic amines.
Results
We saw, that
Derivatization increases IE for most of the compounds (by an average of 0.9 and up to 2 – 2.5 logIE units) and derivatized compounds have more similar logIE values than their underivatized variants.
Measurement of derivatized compounds is possible from the mixture when using chromatographic separation technique.
It was also noted, that using chromatographic separation instead of flow injection mode tends to slightly increase ionization efficiency.
We managed to link all our results with existing ionization efficiency scale and therefore widen the scale and get comparable logIE values for measured derivatized compounds.
The last part of the talk is devoted to the Eurachem 2018 General Assembly and Workshop that will take place in Tartu on May 20-21, 2018. The topic of the workshop is “Validation of targeted and non-targeted methods of analysis”.
Dr. Anneli Kruve defended her PhD thesis – “Matrix effects in liquid-chromatography electrospray mass-spectrometry” in University of Tartu in 2011 under the supervision of prof. Ivo Leito and dr. Koit Herodes. Right after she got her first personal funding and established the electrospray ionization efficiency studies group. In addition to understanding the ionization mechanism for small molecules in electrospray, she is interested in using charged nanodroplets generated by electrospray as an organic synthesis medium. In 2016 Dr. Anneli Kruve moved to Haifa, Israel to do her first post-doc at the Technion’s Schulich Faculty of Chemistry in Prof. Israel Schechter’s group. Right now, she is a Humboldt Fellow in the Institute of Chemistry and Biochemistry at Freie Universität Berlin in Germany in Prof. Christoph Schalley’s group.
In addition to excellent science, she is spreading the knowledge as she has designed and is actively lecturing different courses (instrumental analysis lab, statistics in analytical chemistry to name some) in analytical chemistry and statistics in analytical chemistry. She has also supervised several PhD and numerous MSc and BSc students.
Early inspiration
“I got started in organic synthesis but quickly joined a biomedical lab that needed students to operate a HPLC. From there, moving to LC/MS was a natural step. The ability to incorporate various skills from IT to synthesis with mass spectrometry-based research has kept me motivated to stay on this path.”
Research
“My field is structural and quantitative characterization of compounds with LC/ESI/IMS/MS. A key focus of mine is developing the possibility of giving a quantitative context to non-target LC/HRMS data without the need for standard substances.”
Greatest achievement
“I have already seen many students moving from our lab to the workforce and receiving positive feedback from their employers – that definitely makes a former supervisor happy!”
Prediction
“Mass spectrometry has already changed almost everything; its impact will increase further, as we make non-target screening more efficient with effective algorithms, and make it quantitative. I also have a dream technique: I sometimes wonder what the world would look like with MS-(gas phase)NMR . . .” (The Analytical Scientist)
We are very happy and proud that Dr. Anneli Kruve received this well-deservedly recognition of being nominated to the Top 40 Under 40 Power List by the Analytical Scientist!
From the 9th to the 13th of September MSACL EU 2018 Annual Congress took place in the cosy city of Salzburg, Austria. This conference was designed to mass spectrometry audience mainly focusing on clinical applications. I had the opportunity to participate in this inspiring conference. Moreover, I had the possibility to give a talk. I introduced our studies on standard substance free quantitation in LC/ESI/MS analysis.
Lightning talks to encourage young scientists
Prof. Jerzy Adamski kicked off the conference with the plenary lecture on the topic “Metabolomics Messages on Human Health and Diseases”. As one of the main goals of this conference is to inspire and encourage the young scientists the next session was lightning talks. Poster presenters had 90 seconds and one slide to present their research and cultivate interest in their topic. Additional possibility to get to know mature scientist was the program “meet the experts”. There were short meetings as well as booth and poster tours with experts. I had the possibility to analyze some posters together with Dr. Oleg Mayboroda from Leiden University Medical Centre. It was nice to see how the experts look at the posters and how and which questions they ask. Dr. Mayboroda gave a checklist of how to assess the goodness of the PCA plot presented on posters.
Scientific highlights of the conference
There were 5 parallel sessions every day. For me, the three highlights were as follows. First of all, Dr. Mario Thevis gave a talk about mass spectrometry in sports drug testing. Also, Prof. David Millington gave an interesting talk about the role of tandem mass spectrometry in newborn screening. It was encouraging to see how the methods developed by his team save around 2500 newborns on yearly basis. Furthermore, Dr. Lyudmilla Yanshole gave a talk about using postmortem tissues for biomarker identification. It is an interesting study as for lots of diseases it is very difficult to get tissue of healthy species.
Standard substance free quantification LC/ESI/MS
I had the possibility to present for the first time a talk at an international conference. For that, I am most grateful for the MSACL organization and the scientific committee. I introduced the recent results on the feasibility of standard substance free (semi)quantitation in LC/ESI/MS using ionization efficiency prediction models. We have measured more than 2000 ionization efficiency values. We have incorporated 21 solvent compositions. The models are developed based on descriptors calculated from the 2D structure of compounds and the predictions perform well even in complex matrices, namely, bodily fluids. Our average misprediction of ionization efficiencies is less than 3.5 times mismatch. I thank the audience for fruitful discussions on the topic.
Salzburg is a lovely city with stunning views of the Alps. Furthermore, the organizer made our stay at the conference very enjoyable with the nice coffee breaks and warm buffet for lunch and dinner which were offered every day.
From 26 to 31 of August Florence hosted a vast majority of mass spectrometry scientist in the course of 22nd International Mass Spectrometry Conference. Among these were also our group members researchers Anneli Kruve and Riin Rebane, PhD students Piia Liigand and Jaanus Liigand, MSc student Mari Ojakivi and a visiting PhD student Tingting Wang from the Technical University of Denmark. We had a chance to present altogether 5 posters at the conference. Our posters can be found here. As is the case for IMSC, the topics ranged as wide as it can be for mass spec and always there was something to see and hear. We were happy to see that there was a lot of interest in our work. Congratulations to Anneli, whose poster received Chemistry – A European journal poster award! The conference was accompanied by delicious Italian food and nice warm weather. We enjoyed every second of this highly intensive week and are looking forward to IMSC 2020 in Rio.
Anneli Kruve: It is no secret that I am very keen on non-target screening methods, so I really enjoyed all the talks and posters regarding non-target screening and compound identification. My favorite talk was actually also in the field of compound identification given by Prof. Rober Mistrik in the Thermo lunch seminar. He very nicely explained the Precursor Ion Fingerprinting strategy and explained some of the new tips and tricks which are now combined in the Mass Frontier 8.0. It also very strongly resonated with the perspective I recently wrote about semi-quantitative non-target screening. I also have to mention the plenary lecture by Prof. Marco Leona from Metropolitan Museum of Art, who did not focus specifically on mass spec but gave us a really nice overview of scientific research of art. I constantly kept thinking how beautiful it is to have a background in chemistry: one can, virtually, combine all interest and make a great new whole. Prof. Leona, for example, was combining beautiful artworks, history of humankind, the biology of colorful bugs and plants, and on top of that mass spectrometry. And I definitely also felt honored to receive my best poster prize awarded by Chemistry – A European Journal!
Riin Rebane: It was my first experience of Florence as well as IMSC and when the first one greeted with wonderful food, the second one gave food for thought. It was a thorough overview of the latest advances in mass spectrometry and an insight into different works of scientists from academia as well as industry, therefore, it was certainly a challenge to grasp it all. But despite that, it was also nice to see that sessions towards practical applications of mass spectrometry in for example food or environmental analysis, were so popular that arriving late meant that all seats were already taken by other enthusiasts.
Mari Ojakivi: IMSC 2018 was the first scientific conference that I attended and as a master’s student it was an eye-opening experience. It was very nice to see so many scientists introducing their brilliant work with a mass spec. Seeing all the different applications in forensics, food and environmental science, as well as fundamental research in all the -omics fields made me understand how powerful tool mass spectrometry is. What is more, presenting my own work and seeing other scientists being so interested in it, verified that I am doing good and important science.
Piia Liigand: I thoroughly enjoyed the IMSC 2018 in Florence. It was a nice opportunity to meet and talk to scientists from all over the world. I was also very positively surprised to see so much interest in my poster and I truly enjoyed the discussions I had while presenting my poster. Thanks to these discussions I now have even more understanding of the importance of the science we do and what to do next. I also enjoyed meeting friends from past conferences and catching up on their research in more detail. Of course, the food, wine and general ambiance of Florence was an excellent environment to do all that.
Jaanus Liigand: I enjoyed the IMSC 2018 in Florence as it offered a very wide set of different interesting topics of mass spectrometry field. The inspiring presentations, fruitful discussions, interdisciplinary ideas, and excellent community made it a very enjoyable conference. My favorite session was environmental MS where Prof. Susan Richardson gave in her keynote a very nice overview of the recent developments in this field followed by interesting presentations. Additionally, I enjoyed meeting friends from previous conferences and discussing their and our research. Of course, the historical city – Florence – with the food and wine scenery made the week even more enjoyable.
Tingting Wang: It was a really memorable experience to participate IMSC in Florence, and I enjoyed the atmosphere and all the academic events there. It’s so impressive how wide are the application fields that mass spectrometry can do in the world by looking through a thousand posters. It’s hard to say which presentation or poster was my favorite, but I do pay more attention to the non-target screening strategy applied in food control, environmental, metabolomics, and proteomics study, especially the workshop on the second day about non-target analysis. It was interesting and very informative because unknown screening by high-resolution mass spectrometry is the topic of my PhD project, how to combine target and non-target screening, how to develop a more precise identification and quantification strategy for the non-target even unknowns, and how to prioritize unknowns are the problems I am always facing. It is nice that I could see these similar studies with various workflows from other researchers, and have the chance to listen to their presentations and even talk with those people to get inspiration for my research.
Anneli, Riin, Mari, Jaanus and Piia presenting their posters.
This week two PhD students of our group, Rūta Veigure and Max Hecht had a chance to present their research in the 7th European Association for Chemical and Molecular Sciences (EuCheMS) Congress in Liverpool, organized by the European chemistry society. The conference offered a 5 day packed programme and celebrated the organization of different chemists in societies all over the world. For example, the Estonian chemical society is turning 100 years this year.
Rūta Veigure presented a poster on “Widening the range of eluent additives for LC-MS analysis to improve the retention of drug-like molecules”, while Max Hecht delivered a talk on the “The Evolution of Sponge spray for direct Sampling and Analysis by MS”.
Liverpool itself offered a variety of sights and enjoyable experiences, and as being the home-town of Beatles, there was even a chance to enjoy the tribute band “The Bootleg Beatles” during one of the Congress’ evenings.
