Our on-line LC-MS Method Validation Course 2020-2021: Registration is open!

Validation_of_LC-MS_Methods_Online_CourseWe are glad to announce that the fifth edition of the online course LC-MS Method Validation created and organised by our Analytical Chemistry group is open for registration at the address https://sisu.ut.ee/lcms_method_validation/ !

The course will be offered as a Massive Open On-line Course (MOOC) during Nov 24, 2020 to Feb 05, 2021.

This is a practice-oriented on-line course on validation of analytical methods, specifically using liquid chromatography-mass spectrometry (LC-MS) as technique, mostly (but not limited to) using the electrospray (ESI) ion source. The course will also be of interest to chromatographists using other detector types. The course introduces the main concepts and mathematical apparatus of validation, covers the most important method performance parameters and ways of estimating them. The course is largely based on the two-part tutorial review:

The course materials assembled by the members of our group include video lectures, texts, tables, schemes, practical exercises, and numerous tests for self-testing. In spite of being introductory, the course intends to offer sufficient knowledge and mathematical skills for carrying out validation for most of the common LC-MS analyses in routine laboratory environment. The real-life analysis situations for which there are either examples or self-tests are for example determination of pesticides in fruits and vegetables, perfluoroalkyl acids in water, antibiotics in blood serum, glyphosate, and AMPA in surface water, etc. It is important to stress, that for successfully carrying out validation, practical experience – both in analytical chemistry as such and also specifically in validation – is crucial and this can be acquired only through hands-on laboratory work, not via an on-line course.

Participation in the course is free of charge. Receiving a digital certificate (in the case of successful completion) is also free of charge. Printed certificate (to be sent by post) is available for a fee of 60 EUR. Registration is possible until the start of the course. The course material is available from the above address all the time and can be used via the web by anyone who wishes to improve the knowledge and skills in analytical method validation (especially when using LC-ESI-MS).

We are happy to introduce – Dr. Ruta Hecht and Dr. Max Hecht

Today, on August 28, 2020, Ruta Hecht and Max Hecht defended successfully their PhD theses!

The dissertation by Ruta was titled “Novel eluent additives for LC-MS based bioanalytical methods”. In this thesis, novel fluoroalcoholic eluent additives that showed to have a positive influence on analyte ionisation in the MS source were studied. Additionally, two practical applications employing novel eluent additives were developed and validated for several pharmaceuticals. The two bioanalytical methods were used to obtain data for pharmacokinetic and pharmacodynamic studies in paediatric patients. Both methods reached exceptionally low limits of quantification, with minimal sample amount used due to the positive influence of novel eluent additives on analyte ionisation.

The PhD thesis by Max titled “Advances in the development of a point-of-care mass spectrometer test“. In this work, an on-site testing method was developed and evaluated for medical examination. For example, this method enabled to determine the concentration of an antibiotic and an opioid directly in blood, plasma, or urine in as little as 5 minutes.  Furthermore, traditional laboratory-based methods were developed. These included the diagnose of the rare MNGIE-disease and a 36-second test for the analysis of ecstasy tablets. To investigate potentially harmful drugs added to ecstasy tablets, a large panel of over 100 drugs was measured within a single analysis run on a miniaturised mass spectrometer. 

Congratulations to you both! We wish you all the best for the future!

Dr. Artur Gornischeff – PhD thesis on the study of ionization efficiencies and standard free quantification

On the 18th of August, 2020, Artur Gornischeff defended his PhD thesis titled Study of ionization efficiencies for derivatized compounds in LC/ESI/MS and their application for targeted analysis

In the thesis by Artur, a method that allowed to measure and evaluate the ionization efficiencies of important constituents (amino acids, biogenic amines) in different foodstuffs and beverages was developed. The developed useful method was used to estimate analyte concentrations without standard substances in different matrices (beer, wine, and tea). In addition, the effect of derivatization on ionization efficiencies and the problem of how to choose the suitable ion source and eluent components were addressed. One benefit of the overall results is the possibility to help with distinguishing counterfeit drinks from the original ones. 

Congratulations, Artur! We wish you all the best for your future.  

 

 

 

 

 

Our LC-MS Method Validation e-course received quality label from HITSA!

Each year the Estonian Information Technology Foundation for Education (HITSA) recognizes the e-courses crested by Estonian educational institutions, that have demonstrated high quality in their online teaching. The main aim of this award is to improve the level of e-courses and to endorse those, that have shown excellence in their teaching.

The awarded quality label certifies the high level of the e-course and recognizes the authors for achieving excellent results in the implementation of e-learning to their teaching process. The importance of e-courses grows year by year. This was especially seen this spring when the majority of teaching and studying had to be done via the web because of the Covid-19 situation. 

This year our LC-MS Method Validation web course was honored to receive the quality label. Altogether 511 people from 77 countries registered to the course last autumn. The LC-MS Method Validation MOOC will start again in autumn 2020.

Congratulations on this well-deserved recognition to Ivo, Anneli, Riin, Maarja-Liisa, Hanno, Koit, Karin, Irja, and Asko from our analytical chemistry group!

Jaanus Liigand was awarded the 1st prize in Estonian National Contest for University Students for his doctoral thesis

Jaanus Liigand was awarded the 1st prize in category of natural sciences and technology in the Estonian National Contest for University Students supported by Estonian Research Council for his doctoral thesis.

He defended his PhD thesis on “Standard substance-free quantification for LC/ESI/MS analysis based on the predicted ionization efficiencies”. 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 contentpH 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.

Dr. Piia Liigand and Dr. Jaanus Liigand

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.

He is continuing his research at University of Alberta in Canada in Prof. David Wishart research group to further improve mass spectrometric analysis with machine learning.

Congratulations to you, Jaanus, for the well-deserved acknowledgment!

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.

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.

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.