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.

7th EuCheMS 2018 Liverpool, UK

This week two PhD students of our group, Rūta Veigure and Max Hecht had a chance to present their research in the 7th European Association for Chemical and Molecular Sciences (EuCheMS) Congress in Liverpool, organized by the European chemistry society. The conference offered a 5 day packed programme and celebrated the organization of different chemists in societies all over the world. For example, the Estonian chemical society is turning 100 years this year.

Rūta Veigure presented a poster on “Widening the range of eluent additives for LC-MS analysis to improve the retention of drug-like molecules”, while Max Hecht delivered a talk on the “The Evolution of Sponge spray for direct Sampling and Analysis by MS”.

Liverpool itself offered a variety of sights and enjoyable experiences, and as being the home-town of Beatles, there was even a chance to enjoy the tribute band “The Bootleg Beatles” during one of the Congress’ evenings.

Prediction of ionization efficiencies in biological matrices

In the recent years, we have made significant progress in ionization efficiency studies. In our previous publications, we have shown that our approach can be applied in both electrospray positive and negative mode (we have recently also enabled comparing the logIE values measured in both ESI modes numerically), that our approach is transferable between different instruments with various ESI source geometries from all major mass spectrometric systems vendors, and that it is also transferable between different eluent compositions.

The logIE approach is already applicable in nice clean matrices such as neat solvents. We wanted to see if our approach also works with more difficult matrices, such as bodily fluids and tissues. The results were recently published in Analytica Chimica Acta.

In this study, we took a representative set of 10 compounds, including drugs, e.g., naproxen and lincomycin. As matrices, we used blood, plasma, urine, cerebrospinal fluid, brain and liver tissue homogenates and neat solvent to compare with. We carried out a simple and robust sample pretreatment of protein precipitation. We measured the IEs in a worst-case scenario in flow injection mode without any chromatographic separation.

As with different instrumental setups and solvents, the IEs vary between biological matrices, but the order of the compounds remains roughly the same. These variations between different matrices and variations between a certain biological matrix and neat solvent demonstrate that matrix affects ionization efficiencies and also the prediction models of ionization efficiencies. This, in turn, shows that matrix affects the importance of properties of compounds in the prediction model.

Even though the effects are big and matrix effect is strong we were happy to see that the correlations between IEs measured in the neat solvent and IEs measured in a biological matrix are in good correlation (R2 from 0.7 to 0.9). These good correlations were a promising start to predict IEs in biological matrices similarly to previous predictions in the neat solvent. The most accurate model was obtained for the solvent with a mismatch of 2 times which was also expected since it is the cleanest matrix. But also for liver and brain tissues the mismatch of the model is only 3-fold.

The correlation between predicted and calculated IEs is good with the average mismatch over all biological matrices of 8 times. This means that the accuracy of standard substance free quantitation has been improved by more than an order of magnitude for the set of compounds used in this study.

More details can be found in the paper published in Analytica Chimica Acta. Piia Liigand also gave a talk on the topic in ASMS which can be found here. More papers by our group on the topic of ionization efficiencies can be found here.

Sponge Spray – New Approach for Direct Sampling and Analysis by MS

DSC_0294Mass spectrometry is currently probably the No 1 technique for determining trace-level components in complex (especially biomed-related) mixtures. The key issues in such applications are sample preparation, sample introduction to MS and ionization of components of interest (analytes). Big efforts are continually made to improve any of them.

In his recent development – Sponge spray – Max Hecht, an AMS graduate, now PhD student at UT attempts improvements in all of the above issues. The elegant approach proposed by Max utilizes a volumetric sampling device – a hydrophilic sponge, which after absorbing a predetermined amount of sample (e.g. blood or urine), can be directly used for sample introduction to MS and ionizing the analytes.

The seriousness of the work has been demonstrated by the fact that it was accepted for publication by Analytical Chemistry, the top journal in the field. The published article Sponge Spray – Reaching New Dimensions of Direct Sampling and Analysis by MS is now available from the journal website.

Further developments of this approach may lead e.g. to fast medical diagnosis MS methods that, contrary to the current situation with MS in medicine, could be applied as “bed-side” diagnosis tools in hospitals.

(Photo: Max Hecht working with the sponge spray ion source)