New publication – retention studies in C18 column using novel fluorinated eluent additives

It is essential to understand the interaction between the analyte, mobile phase and the column chemistry for the best possible separation of molecules. Of all three, the mobile phase in HPLC is the easiest to modify and thus, usually contains different eluent additives or buffers. While using mass spectrometry undoubtedly increases the variety of analytes possible to detect, it also introduces limiting factors such as the requirement of the volatility of the eluent. Most common additives in LC-MS systems are formic and acetic acid, their ammonium salts as well as ammonium bicarbonate. In our recent paper, we have proposed novel – fluorinated, eluent additives (hexafluoroisopropanol, hexafluoro-tert-butyl alcohol, trifluoroethanol as well as nonafluoro-tert-butyl alcohol and perfluoropinacol).  Their influence was shown on rather simple exemplary molecules, which are widely spread over different logP values, containing protonated and deprotonated acids and bases. All novel fluorinated eluent additives demonstrated a strong influence on basic polar analytes in basic medium – they drastically increased retention. A decrease in retention was observed for acidic analytes when these novel eluent additives were used.

Moreover, current research displays a comprehensive overview of retention mechanisms for nonafluoro-tert-butyl alcohol and is the first time ever when perfluoropinacol has been introduced as eluent additive for reversed phase chromatography. Additionally, the influence on MS signal was studied when fluoroalcohols were used as eluent additives. This is also the first time when the absolute pH (pHabs) scale was used for expressing the mobile phase pH.

Current fundamental research forms a basis for a better understanding of the influence of fluoroalcohols as eluent additives and will help in the assay development in a wide range of applications.

This work is part of a larger endeavor – to promote a wider usage the unified pH scale (pHabs) by the research and technology communities, which is currently in progress via the UnipHied.

The UnipHied project is funded from the EMPIR programme (project 17FUN09) co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation programme.

 

 

17th Nordic Mass Spectrometry Conference in Espoo

In the last week of August, the 17th Nordic Mass Spectrometry conference was held in Espoo, Finland. With around 150 participants from Nordic countries, the conference covered various different topics within the field of mass spectrometry.

Anneli making her presentation at the 17th NordicMS

From our group, Anneli Kruve presented some of the most recent results in the field of quantitative suspect screening for pesticides and mycotoxins. Riin Rebane presented a poster about analyzing metanephrine (MN) and normetanephrine (NMN) and also about unusual matrix interferences related to the analysis (results have been published here). Artur Gornischeff presented some very exciting results on the ionization studies of derivatized amino acids which have also been published here.

  • Riin with her poster
  • Artur with his poster

NordicMS is a mass spectrometry conference taking place every three years and hosted in turns by nordic countries. This time it was Finland’s turn. The three-day conference had roughly 150 attendees; as expected, primarily from Finland and other Nordic countries. The conference covered all major areas of application of mass spectrometry: food characterization, metabolomics, drug discovery, proteomics, doping discovery and forensics. Topics covered analysis from the sea bottom (Hanna Niemikoski’s presentation about the identification of novel chemical warfare agents) up to the sky (Kari Hartonen’s presentation about sampling from air with drones).

The conference showed that even in the small circle of Nordic countries the research in the field of mass spectrometry is diverse and every mass spectrometry enthusiast will find his or her spot in the field.

We are happy to introduce: Dr. Piia Liigand and Dr. Jaanus Liigand

In August, additionally two of our PhD students, Piia Liigand and Jaanus Liigand, successfully defended their PhD theses. Both Piia and Jaanus worked intensively and successfully on understanding the ionization process in electrospray ionization source (ESI) and applying this knowledge for quantifying compounds with LC/ESI/MS without standard substances. Congratulations to the fresh doctors! Piia is continuing as a lab manager in TBD-Biodiscovery and Jaanus continues his scientific career at the University of Alberta in Prof. Wishart group.

On 15th of August, Piia Liigand defended her PhD thesis on “Expanding and improving methodology and applications of ionization efficiency measurements”. The opponent of Piia was Prof. Dr. Susan D. Richardson from the University of South Carolina. Over the last four years, Piia has contributed significantly into the development of ionization efficiency measurement methodology. She has been able to develop a method for measuring the ionization efficiencies for both small molecules as well as oligopeptides. Most of all, she has shown that the predicted ionization efficiencies can be applied for drug and drug-a-like measurements in the biological matrices. And, last but not least, she has pulled together a large number of response factors from the literature and shown that these values are in good agreement with each other in spite the, sometimes observed, contradicting conclusions.

Piia with her supervisors Dr. Karl Kaupmees and Prof. Anneli Kruve and opponent Prof. Susan Richardson

Jaanus Liigand defended his PhD thesis on “Standard substance-free quantification for LC/ESI/MS analysis based on the predicted ionization efficiencies”. Prof. Dr. Jonathan Martin acted as an opponent of the defense. During his PhD studies, Jaanus has worked hard on understanding the mechanism of electrospray ionization in LC/ESI/MS; primarily understanding how the structure of the compound and the eluent used in the analysis influence the ionization efficiency. Jaanus has verified, based on the largest set of ionization efficiencies measured so far (roughly 400 compounds), that the more hydrophobic compounds and more basic compounds tend to have a higher response in ESI positive mode. From the mobile phase point of view, both organic solvent content, pH of the buffer, and buffer composition, influence the ionization efficiency in ESI/MS. In general, higher organic solvent content and lower pH result in higher ionization efficiency and, therefore, a higher response in positive mode ESI/MS. Also, he investigated how well are the ionization efficiency values measured on one instrument transferable to other instruments and found that with the aid of 5-6 common compounds the ionization efficiency values can be transferred from one instrument to another. Combining these promising results and machine learning approaches Jaanus has been able to develop a truly universal approach for applying ionization efficiency predictions for quantification in suspect and non-targeted LC/ESI/HRMS analysis.

Once more – congratulation to you both!

Short Courses for EU FT-ICR MS staff and students’ community: Ion-molecule reactions: fundamental and analytical aspects.

At the end of June, on 25-27th, two PhD fellows from our group Pilleriin and Eliise attended FT-ICR-MS short course, this time locating in Rome, Italy. Three-days course included both lectures from FT-ICR experts like Jevgeny Nikolaev, Maria Elisa Crestoni, Simonetta Fornarini, and hands-on lab practices. The central theme for the short-course was ion-molecule reactions. During these days, attendees were able to see how ion-molecule reaction experiments are conducted in the University of Sapienza, also get tutorial on how to process all of this extensive data from this process. The FT-ICR used in Sapienza is a 4.7 T Bruker Apex II instrument.

EU_FT-ICR-MS summer school and short course series is altogether a four-year-long project, which aim is to bring together all the scientist using FT-ICR all over Europe and promote the usage of this instrument. During this program 2018-2021, it is also possible to submit your project to use any of the FT-ICR instruments provided in this community.

48th International Symposium on High-Performance Liquid Phase Separations and Related Techniques

Between the 16th to 20th of June our group presented itself in HPLC 2019 in Milan. It was a 5-daylong and intense learning opportunity with more than 300 speakers and around 500 posters.

Topics ranged from fundamentals of HPLC, miniaturization to different omics, pharmaceutical analysis and innovative technologies (can HPLC have a FID as a detector?). For the first time, a whole section was dedicated to 3D printing technologies – a technique that is used to build 3 dimensional separation modules: for example, fascinating talks on using 3D printing to do liquid chromatography in 3 dimensions.

The conference gala dinner was held at the beautiful central courtyard of University of Milan under relieving cool evening sky opposing the hot temperatures of the day. HPLC 2019 also had two new additions that hopefully will become annual traditions: Separation Science Slam and HPLC Tube, offering an opportunity for scientists to express their love for their work in modern ways. The competitions were extremely creative and both the audience and participants were thoroughly enjoying the events. Participants from our analytical chemistry chair gave multiple contributions to the conference.

Ecstasy content in tablets is uneven

Max giving his talk

Max Hecht, MSc, presented an oral presentation on the evaluation of MDMA (also known as ecstasy and  ‘Molly’) content in 412 tablets and dissolution properties in 247 tablets, collected in the UK in the time period of 2001-2018. It was found that there are no physical tablet characteristics which correlate to dissolution rate classification, hence no way of users knowing a priori whether tablets were more likely to be fast or slow-releasing. Further, large within-batch variation in the dose and also dissolution rate was observed, giving the combined result of increasing significantly the danger of over-dosing.

Standard substance free quantification in LC/ESI/MS

Anneli giving her talk

Dr. Anneli Kruve presented the recent work of her group on standard substance free quantification of metabolites in green tea samples. In the metabolomics studies, the standard substances for all detected and identified metabolites are hardly ever available. The peak areas obtained from LC/HRMS analysis are also generally usable as different compounds ionize with vastly different ionization efficiencies; the differences may reach 100 million times. With the aid of ionization efficiency predictions, this shortcoming can be overcome and the absolute concentrations estimated. The current prediction accuracy for the green tea metabolites is 1.7 times, which allows comparison of different tea samples and also the identification of the samples with different origin. Importantly, the standard substance free quantitation allows transferring quantitative data from one lab to another. Anneli has also summarized the current status of standard substance free quantitation for the last issue of LCGC. You can find out more about it from kruvelab.com and quantem.co.

Novel eluent additives diversify analyst´s toolkit

Ruta giving her talk

Ruta Veigure, MSc, showed that fluoroalcohols, such as perfluoropinacol (PP) and 1,1,1,3,3,3-hexafluoro-2-methyl- 2-propanol (HFTB), are very useful alternatives to common eluent additives in RP HPLC-MS analysis, acting, among other effects, as weak ion-paring reagents. Novel eluent additives influenced elution of protonated bases by significantly improving analyte’s retention on C18 stationary phase as well as reduce the retention of acidic analytes, which are deprotonated. A comparison was performed to commonly used ammonium acetate and ammonium bicarbonate mobile phase additives. Her research will be rather influential for the analysis of pharmaceuticals, from whom the majority are basic.

Revolutionalizing pH measurements

Prof. Ivo Leito presented a poster introducing a conceptually new approach of measuring pH of mixed-solvent liquid chromatography (LC) mobile phases: the pHabs approach. The new approach is based on the recently introduced unified pH scale (pHabs scale), which enables direct comparison of acidities of solutions made in different solvents or solvents mixtures based on chemical potential of the proton in the solutions. The viewers praised the fact that real numerical values are now available showing how different the conventional pH values are from pHabs, as well as the educative aspect of the whole endeavour. Some visitors were eager to start immediately applying pHabs in their own work.

This work is part of a larger endeavor – to promote a wider usage the unified pH scale (pHabs) by the research and technology communities, which is currently in progress via the UnipHied. The UnipHied project is funded from the EMPIR programme (project 17FUN09) co-financed by the Participating States and from the European Union’s Horizon 2020 research and innovation programme.

  • Ivo presenting his poster
  • Asko presenting his poster

Automate what can be automated in method validation

Dr. Asko Laaniste introduced the progress on ValChrom, a software for the automation of chromatographic method validation. The development of ValChrom is ongoing and feedback is being gathered from potential users in different fields of chromatography in order to adapt to real needs (contact at valchrom@ut.ee). Feedback from viewers cemented the understanding of the problem that often validation is done in spreadsheets and textual software, that are prone to error. Viewers of the poster approved the endeavour for aiming to help small and medium-sized laboratories that do not have an affordable alternative. They were equally excited to promote the software further to their colleagues in the validation department.

67th ASMS Conference on Mass Spectrometry and Allied Topics

From 1st to 6th of June, two PhD fellows Piia Liigand and I presented our recent results at 67th ASMS annual conference in Atlanta, USA.

100% renewable energy utopia or reality?

The conference was kicked off with a plenary lecture by Prof. Mark Z. Jacobson on the topic of “Transitioning the World Energy for All Purposes to Stable Electricity Powered by 100% Wind, Water, and Sunlight”. He introduced how global, country wise, neighbourhood and individual energy production and preservation are feasible based on 100% renewable energy. I found fascinating the example of Prof. Jacobson himself as he has built his own house that uses and produces 100% renewable energy needed for maintaining the house and charging his electric car.

From simple ambient ionization techniques to “fishoil” from canola

The conference consisted of four days full of exciting talks which covered the fundamentals of mass spectrometry to application in diverse fields from cannabis analysis to proteomics. It was very encouraging to hear about the journey of Prof Dr Sarah Trimpin, this year Biemann medal awardee and her studies of very simple ionization techniques. I found exciting the results of a group from CSIRO, Australia. They applied mass spectrometry in food safety analysis, but even more fascinating was the genetically modified canola which can produce omega-3 fatty acids in large amounts to prevent overfishing to obtain omega-3-rich fish oil.

Quantem provides unification of ionization efficiencies from literature and standard substance free quantification in LC/ESI/MS

  • Piia presenting her poster
  • Jaanus presenting his poster

Piia presented her results on “Unifying ionization efficiencies: a quantitative comparison of diverse data sets and validation of prediction models” as a poster and there was a great interest in her topic and fruitful discussions about the findings retrievable from the unified datasets.

I presented the results on “Standard Substance Free Quantification of LC/ESI/MS on the Example of Pesticides in Cereal”. Although my poster was scheduled on the last day there was remarkable interest in our topic. Furthermore, future collaborations were arranged to test out our Quantem approach on datasets from different fields, metabolomics to the vaping industry.

The conference ended with the gala dinner in Georgia aquarium. The sea life, especially whale sharks and beluga whales, were astonishing. It was great to meet old friends and get to know new ones. We thank the ASMS team for a very nice conference in Atlanta and hope to meet next year in Houston at 68th ASMS annual conference. Additionally, we thank Graduate School of Functional materials and technologies and Kristjan Jaak scholarship for travel costs.

Eurachem 2019 workshop successfully finished!

Our group was the main organiser of the Eurachem 2019 Scientific workshop Validation of targeted and non-targeted methods of analysis, which took place in Dorpat Conference Centre in Tartu during May 20-21, 2019. This workshop also marks the 30th anniversary of Eurachem.

Approximately 160 people attended the workshop, which is the largest number of participants in the history of Eurachem workshops! The participants were from 42 countries of the member countries in Eurachem as well as Asia, North America, South America and Middle East. The farthest participants were from Fiji, the Philippines, Uruguay and Brazil.

The workshop was held with 13 oral presentations from established researchers, young scientists as well as industries. Together with 22 posters all presentations reflected the current and potential future developments related to methods validation. The workshop addressed 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). With the speaker permissions, all presentations will shortly be available at the Eurachem website. In addition to presentations, each day a Working Group session was organised with 3 topics in parallel (Image on the left: Welcome by Dr Marina Patriarca, the Eurachem chair).

Non-targeted methods are an especially noteworthy part of the programme, because their validation involves specific issues and their validation 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. (Image on the right: Prof. Jon Benskin from Stockholm University presenting an introduction to non-targeted analysis)

All sessions raised new issues and challenges, especially related to non-target method validation. The workshop clearly was also very inspirational for Eurachem from the point of view of preparing new guideline materials – especially the topics related to non-targeted analysis are still essentially not covered by official guidance documents.

Some example topics of the workshop: 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. Recent instrumental developments. Software tools for validation. (Image on the left: Dr. Koit Herodes presenting the ValChrom validation software)

The workshop certaily had a significant educational value and we are pleased by the large number of student participants: altogether close to 50! The international master’s programmes Excellence in Analytical Chemistry and Applied Measurement Science were both heavily represented: the majority of students of those programmes participated in the workshop (Image on the left: EACH and AMS students at the workshop).

The workshop was jointly organized by Eurachem and ECAC (University of Tartu, Tallinn University of Technology and the Estonian Environmental Research Centre).

 

Russian roulette with Ecstasy

PhD Fellow Max Hecht and Dr Karin Kipper with collaborators looked into the variability in content and dissolution profiles of MDMA tablets collected in the UK and published the results recently in Drug Testing and Analysis.

Ecstasy comes in all shapes, sizes and colours. 3,4-methylenedioxy-met(h)amf(/ph)etamine (MDMA) is the chemical name for a recreational drug taken for its empathogenic “high”, that alters the sensation of energy, empathy and pleasure. MDMA is mainly consumed in the form of tablets.

The trends in dose

To identify the risks associated with MDMA consumption, researchers from the UK and the University of Tartu analysed the drug dose of 412 tablets and, for the first time, studied the dissolution aspects of another 242 tablets collected in the UK in the time period between 2001-2018. Like in other European countries, the MDMA content in tablets is steadily increasing in the last decade, with some recent tablets containing unprecedented high doses.

The variability in dissolution

Dissolution testing is commonplace in the quality control and batch production of tablets in pharmaceutical laboratories and for bioequivalence testing. For MDMA, different dissolution rates were observed, ranging from fully dissolved in 30 min – the fast group, to up to 2 h until the whole content of the drug is completely released (slow group). No significant differences in a dose per tablet could be found between slow and fast releasing groups. As well as there was no association of colour, logo or shape with the dose or dissolution group. 

In conclusion, this means that, while it always is a gamble to take Ecstasy, the ever-present danger of overdosing is also on the rise. You are welcome to read our freshly published article for more details.

ESI outcompetes other ion sources in LC-MS trace analysis

Choosing the best possible ion source is a very important step in liquid chromatography-mass spectrometry (LC/MS) method development. In a recent paper,  ESI outcompetes other ion sources in LC-MS trace analysis Anal. Bioanal. Chem. 2019 Asko Laaniste from the UT Analytical chemistry group carried out a large scale survey of different LC/MS ion sources (and their different working modes) as applied to pesticide analysis. He compared electrospray ionisation (ESI) source, thermally focused/heated electrospray (HESI), atmospheric pressure photoionisation (APPI) source with and without dopant, and multimode source in ESI mode, atmospheric pressure chemical ionisation (APCI) mode, and combined mode using both ESI and APCI, i.e. altogether seven different ionisation modes for the analysis of 40 pesticides (see list below).

The lowest limits of detection (LoDs) were obtained by ESI and HESI. The widest linear ranges were observed with the conventional ESI source without heated nebulizer gas. ESI source was significantly less affected by matrix effect than the HESI source. APPI ranked second (after ESI) by not being influenced by matrix effect; therefore, it would be a good alternative to ESI if low LoDs are not required.

It was somewhat unexpected that ESI outperformed HESI. This may be caused by the instability of the additional heated gas (sheath gas) in HESI that is used in addition to the nebulising gas.

In conclusion, as a broad generalisation, Asko’s results show that although several new ion sources have been introduced during the last decade, the conventional ESI performs at least equally to these new ion sources in the case of polar to medium polarity pesticides. ESI with thermal focusing (HESI) performed more or less equally with ESI in terms of LoD. At the same time, HESI had significantly poorer intermediate precision of matrix effect values for most compounds. Thus, for trace analysis, ESI is still the ion source of choice.

(Full list of pesticides: pymetrozine, thiamethoxam, methiocarb-sulfoxide, chloridazon, imidacloprid, acetamiprid, methiocarb-sulfone, thiacloprid, imazalil, thiophanate-methyl, metribuzin, pyrimethanil, fenpropimorph, spiroxamine, propoxur, triasulfuron, bupirimate, paclobutrazol, methiocarb, azoxystrobin, epoxiconazole, myclobutanil, fenhexamid, fluquinconazole, flusilazole, mepanipyrim, bitertanol, propiconazole, triazophos, methoxychlor, ditalimfos, tebufenozide, benalaxyl, pyrazophos, buprofezin, indoxocarb, trifloxustrobin, quinoxyfen, pirimiphos-ethyl, hexythiazox)

 

(Image top left: Asko Laaniste preparing solutions; Image right: Comparison of accuracy of LC-MS analysis using different ion sources)

 

Standard-substance-free quantification in LC/ESI/MS is finally here

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

Let’s quant’em!