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.

Guests from Mülheim learning our pKa measurement method

Starting from last week the Analytical chemistry group of University of Tartu has the pleasure to host Dr Monika Lindner and Hendrik van Thienen from the group of professor Benjamin List – a worldwide known guru in the field of strongly acidic catalysts working at the Max-Planck-Institut für Kohlenforschung (Mülheim, Germany).

The purpose of their stay is to learn our pKa measurement method and set it up in Mülheim. This is the logical continuation of our group’s collaboration with the List group – the pKa values of a number of their catalysts have been measured here at Tartu (see Nature Chemistry 2018, 10, 888-894 and Angew. Chem. Int. Ed. 2017, 56, 1411-1415) – and we are glad that our pKa measurement method thereby gains even wider acceptance than it has now. We wish Monika and Hendrik all the success in learning the measurements!

(On the photo, from left: Märt Lõkov, Monika Lindner and Hendrik van Thienen)

 

UT Analytical chemistry at Suprachem 2019: From receptor synthesis to ion-selective electrode

During 24-26.02.2019 the Suprachem 2019 conference took place at University of Würzburg (Germany). The UT Analytical chemistry chair was represented by two posters: Optimization of the reaction pathway for the synthesis of substituted 1,3-bis(carbazolyl)urea anion receptors by Alo Rüütel and Mihkel Ilisson and From receptor-anion binding ladder to ion-selective electrode by Kerli Martin, Sandip A. Kadam, Ulriika Mattinen, Johan Bobacka and Ivo Leito.

Synthesis of 1,3-bis(carbazolyl)urea based anion receptors

The presentation from Alo and Mihkel focused on optimization of the synthesis route of anion receptors (ionophores) based on the 1,3-bis(carbazolyl)urea building block, augmented with additional amide functionalities (see the poster). The developed synthesis route is very valuable for the synthesis of a wide variety of analogous receptors (ionophores) that have previously been demonstrated to bind carboxylates with high affinity (see K. Martin et al, Eur. J. Org. chem. 2017, 5231-5237).

First solid contact acetate electrodes using receptor molecules

Kerli Martin et al present the first practical application of such receptors: a solid-contact acetate-selective electrodes. For acetate anion the electrodes show linearity over the activity range of 10-4.50 – 10-1.10 with a sub-Nernstian slope of -51.3 mV per decade and a detection limit of 10-5.00. The anion-selectivity pattern of these electrodes deviates markedly from the pattern found in DMSO solution and from the Hofmeister pattern. The selectivity coefficients of SCN–, I–, NO3– and Br– decrease by 3-5 orders of magnitude when adding the studied ionophore to the membrane. The selectivity coefficients of hydrophilic anions such as Cl–, F–, HPO42–, and SO42– are significantly lower than in case of the ionophore-free control membrane. All in all, it is clear that a lot of work is still needed for improving the selectivity of the electrode.
This work has been accepted for publication: K. Martin et al Electroanalysis 2019 (the link will work soon).

The combined contributions from our group stood out from the majority (the conference altogether had 27 oral and 111 poster presentations) by a clear line of development from fundamental research (receptor design) to highly practical application (ion-selective electrode).

(Photos: above left: Mihkel Ilisson explaining his work to Dr Diane Smith from Wiley; right: Poster by Kerli Martin et al)

 

New Publication: Determination of pKa values of fluorocompounds in water using 19F NMR

Our group is well-known for pKa measurements in different solvents. So far, mostly UV-Vis spectrophotometric method has been used. Recently Elisabeth Parman (on the photo) from our group has been developing a method for measuring pKa values of fluorocompounds, using 19F NMR. NMR measurements to determine pKa values have several advantages compared to UV-Vis spectrophotometry. For example, accurate concentrations are not needed, the method is much more tolerant to impurities and several compounds can be measured simultaneously. The newly developed method has now been published in the Journal of Physical Organic Chemistry: Determination of pKa values of fluorocompounds in water using 19F NMR by E. Parman, L. Toom, S. Selberg and I. Leito.
In this work, the developed method was applied to 19 fluorocompounds – fluorinated phenols, carboxylic acids and sulfonamides, hexafluoroisopropanol (HFIP), perfluoropinacol, etc – with the aim of determining their pKa values in water, in the range from about 3.5 to 10. Several advantages of the 19F NMR method were demonstrated in pKa measurement. In particular, using multiple relative measurements against several reference compounds with known acidities it is possible to obtain highly reliable pKa values. Elisabeth convincingly demonstrated that the literature pKa values of two of the investigated compounds were inaccurate and provided more reliable values for them.

New type of pKa – biphasic pKa in octanol water system

It is not frequent that a new quantitative measure of some physicochemical characteristic is introduced. Recently our group managed to do exactly that by introducing a novel approach – termed as biphasic pKa values – of measuring the acidity/basicity of lipophilic compounds embedded in water-immiscible media (membranes or solutions) at equilibrium with aqueous solution.

In this approach, the H+ ion activity is determined in the aqueous phase, while the concentration ratio of protonated and deprotonated forms of acid/base is measured in the lipophilic phase with any suitable technique. The approach has been demonstrated on the example of biphasic pKa values of some lipophilic indicator bases in the octanol: water system, measured using UV-Vis spectrophotometry. The counterion identity and concentration, as well as the overall ionic strength in the aqueous phase, are included in the standard state definition.

This work has been published S. Selberg, S. Tshepelevitsh, I. Leito Croat. Chem. Acta 2018, 91, (4) and will form a part of the PhD thesis of Sigrid Selberg.

Biphasic pKa values are envisaged to be useful for characterizing lipophilic indicators, synthetic receptors, phase transfer catalysts and other implements that are used in hydrophobic media (e.g. in sensor membranes) in contact with water.

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.

(On photo: Sigrid Selberg)

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.

IRUG 13 Conference on 5-7 December in Sydney, Australia

13th biennial conference of Infrared & Raman User Group, shortly IRUG, was for the first time ever held in Sydney, Australia in a beautiful Art Gallery of New South Wales. IRUG conferences are gathering people using IR and Raman spectroscopy for the analysis of cultural heritage, architecture, and forensic materials. This year 101 people were attending, including Dr Signe Vahur and PhD student Pilleriin Peets from our group. Pilleriin was also introducing her results in analyzing textile fibres with IR spectroscopy in an oral presentation. Development of methods for the analysis of various textiles using ATR- and reflectance FT-IR spectroscopy turned out to be very relevant: throughout all three days, many people approached to discuss the topic, ask questions and advice or to make contacts for later cooperation. We were also very glad that conference participants were interested in our ATR-FT-IR spectral database of cultural heritage and conservation materials, available here.
Three days were filled with a wide range of interesting presentations from analysis of Aboriginal Australian pigments and identification of contents from the coffin of a 2500-year-old Egyptian mummy Mer-Neith-it-es to investigations of cultural heritage materials using neutron techniques. Besides the analysis of traditional pigments, organic pigments, especially synthetic organic pigments and their use in art were discussed (oral presentation by Dr Suzanne Lomax and Dr Steven Saverwyns). Another very interesting and thought-provoking presentation was done by Dr Gregory Smith from Indianapolis Museum of Art. He discussed the difficulties in getting accurate standard reference materials in the field of cultural heritage analysis. His results showing how many false materials can be found on the market (and not only vendors like Kremer Pigmente, but also providers like Sigma Aldrich) were very surprising.
All in all, IRUG 13 conference offered three full days of interesting presentations, a lot of knowledge and useful tips for further research and a pleasant atmosphere with excellent company.