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!

New publication: A sensitive method for the simultaneous UHPLC-MS/MS analysis of milrinone and dobutamine in blood plasma using NH4F as the eluent additive and ascorbic acid as a stabilizer

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.

Eurachem 2019 workshop: Validation of targeted and non-targeted methods of analysis

Our group is proud to be the leading organiser of the 2019 Eurachem General Assembly and the accompanying scientific workshop “Validation of targeted and non-targeted methods of analysis”!

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).

We are hoping to see many of you in Tartu in May!

 

Optimization of flow splitting and make‐up flow conditions in liquid chromatography/electrospray ionization mass spectrometry

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.

Comparing Electrospray Ionization Efficiencies of Derivatized and Non-Derivatized compounds

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

  1. 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.
  2. Measurement of derivatized compounds is possible from the mixture when using chromatographic separation technique.
  3. 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.

More information could be found in an article published recently at Journal of Mass Spectrometry.