New publication – Comparison of derivatization methods for the quantitative gas chromatographic analysis of oils

Often paints contain oils that bind together the components (pigments, fillers, etc) of the paint. Analysis of these oils requires derivatization, but which derivatization method is the best for a quantitative approach?

The work on this topic began with the need of our cultural heritage group to have a routine method for the determination of fatty acid composition of oils with gas chromatography (GC). The analysis of oils with GC requires derivatization and for this a wide variety of procedures have been applied. It turned out there are only few comparisons of derivatization methods in the literature and all are quite limited by scope. For that reason, it was decided to explore this important topic comprehensively and, importantly, in the terms of absolute (not relative) quantification.

A member of our group – PhD student Eliise Tammekivi – implemented four common and well-known derivatization procedures to perform the absolute quantification of fatty acids with GC-MS and GC-FID. The four compared derivatization methods were: (1) methylation with m-(-trifluoromethyl)phenyltrimethylammonium hydroxide (TMTFTH), (2) two-step derivatization with sodium ethoxide (NaOEt) and N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), (3) two-step derivatization with KOH and BSTFA and (4) acid-catalyzed methylation (ACM).

This study has now been completed and the results have been published in the journal Analytical Methods by E. Tammekivi, S. Vahur, O. Kekišev, I. D. van der Werf, L. Toom, K. Herodes and I. Leito. In the publication, a comprehensive and wide-scale quantitative comparison of the four derivatization methods is presented. The results demonstrate that methylation with TMTFTH is the least work-intensive and most accurate derivatization method – both in terms of reproducibility and derivatization efficiency (yield).  For further information see: Analytical Methods, 2019, 11, 3514 – 3522

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.

TechnArt 2019 conference: Analytical techniques in cultural heritage

Anu, Signe, Pilleriin and Eliise

From 7th to 10th of May 2019 four members of our UT Analytical Chemistry group – Dr Signe Vahur, Dr Anu Teearu-Ojakäär, PhD students Pilleriin Peets and Eliise Tammekivi – attended the 7th international TechnArt conference in Bruges, Belgium.

The biggest conference of analytical techniques of cultural heritage

TechnArt is a place to present and discuss the newest results of the usage of analytical techniques in the field of cultural heritage. It is the biggest conference among its kind as it was also seen in TechnArt 2019, where the number of participants was about 400! The conference included three parallel oral presentation sessions, two poster sessions with 270 posters, a visit and dinner at the Halve Maan Brewery and an excursion with a boat trip in the historical city centre of Bruges, that has been listed as a UNESCO world heritage.

From Girl with a Pearl Earring to warship Mary Rose

Some of the most interesting talks included the presentation by Dr Abbie Vandivere from The Hague about the analysis of the painting Girl with a Pearl Earring (Johannes Vermeer, approx. 1665) and the difficult conservation of the remains of the warship Mary Rose (warship of the English navy under the command of King Henry VIII, sank in 1545) by Dr Eleanor Schofield from the Mary Rose Trust/Imperial College. Another interesting topic was addressed by Dr Lucia Toniolo who gave a talk on the conservation and monitoring issues of historical architecture, also addressing the hazard of climate change. However, with four days and three parallel oral sessions filled with presentations by the top scientists and conservators of the world, it is almost impossible to highlight all of the interesting and inspiring talks.

The highlights of our recent results

  • Anu presenting her poster
  • Eliise presenting her poster
  • Pilleriin presenting her poster

Also, TechnArt 2019 was the conference, where the attendance by our Cultural Heritage group members was the highest! Anu presented her poster „Analysis of resinous materials“, where ATR-FT-IR, SEM-EDS, GC-MS and ESI-FT-ICR-MS methods were combined for the analysis of the embalming materials obtained from two human mummies originating from Egypt and now exhibited at the University of Tartu Art Museum. Pilleriin presented her poster „Attenuated total reflectance and reflectance approaches for analysis of textile fibres with FT-IR spectroscopy“. This study showed, that both mentioned approaches are suitable and very useful methods for the identification of natural and synthetic fibres. Eliise presented her poster „Comparison of derivatization methods for the quantitative gas chromatographic analysis of oils“ where four widely used derivatization methods for the analysis of heritage samples were compared on the basis of absolute quantification.

Overall, TechnArt 2019 gave the members of our Cultural Heritage group the possibility to introduce their scientific work results, hear the inspiring lectures and have fruitful discussions in the magical historic city of Bruges.

Fluorescence spectroscopy course by EACH visiting scholar Todd Pagano

During Apr 22 to May 4 we had the pleasure to host visiting scholar, prof. Todd Pagano from the Rochester Institute of Technology (RIT), USA. He conducted, for the second time, a two week-intensive course Principles and applications of fluorescence spectroscopy.

In this course, students reviewed the principles of fluorescence spectroscopy, were introduced to the impact of photophysical phenomena on fluorescence data, and discussed new directions of fluorescence in analytical chemistry. Techniques in multidimensional fluorescence spectroscopy with chemometric analysis were highlighted, especially in the context of novel applications in environmental and related fields. The course consisted of lectures, seminars, tutorial sessions and two lab practicals. The latter were specifically set up for this course by prof. Pagano and were very much appreciated by students.

The first practical was about discovering the fundamental concepts of fluorescence (excitation and emission spectra, Stokes shift, Vavilov’s rule, fluorescence lifetime, etc). The second practical was about analysis of caffeine in beverages using salicylic acid as a fluorescent chemosensor. This analysis utilizes the ability of caffeine to quench the fluorescence of salicylic acid and introduces students to the fluorescence quenching, Stern-Volmer equation and inner filter effects.

Altogether 30 students (out of them 14 EACH students) participated in the course and their feedback was overwhelmingly positive.

 

Prof. Pagano is a passionate educator. He was the initiator of the Laboratory Science Technology program at Rochester Institute of Technology’s National Technical Institute for the Deaf, which is a unique science programme, specifically designed for deaf students. He was named “2012 U.S. Professor of the Year” by the Council for Advancement and Support of Education and the Carnegie Foundation for the Advancement of Teaching.

 

(Images: top left, group photo of prof. Pagano with course participants; right: prof. Pagano with students in lab)

 

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!

How to make maximum use of the available pKa data in non-aqueous solvents?

On 11.04.2019 Ivo Leito gives a presentation titled “How to make maximum use of the available pKa data in non-aqueous solvents?” at 26th Croatian Meeting of Chemists and Chemical Engineers (26HSKIKI), in Šibenik (Croatia). The presentation discusses the principles of acid-base behaviour in solvents and on how to estimate the pKa values of compounds in solvents where direct experimental data are not available.

 

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.