Liquid Chromatography Mass Spectrometry

Contact: Assoc. prof. Koit Herodes, koit.herodes@ut.ee

We study LC/MS on a fundamental level as well as in applications. Our research interests are processes occurring in ambient ionization sources (ESI, APCI, sponge spray), as well as studying pharmacokinetics, distribution coefficients, the composition of archaeological materials and crude oils with LC/MS; and LC/MS method development. We also carry out synthesis in ESI droplets and design novel derivatization reagents for MS. Recently, we have launched a MOOC and published tutorial reviews on LC/MS validation and on the estimation of LoD as well as a more general MOOC on uncertainty estimation.

Ionization efficiencies

Our aim is to quantitatively predict the concentration of substances in the sample based on the LC/MS response and molecular structure. This is complicated, as ionization efficiencies of compounds range over at least 7 orders of magnitude. This means that two compounds may yield 10 million times different signal at the same concentration. We have modelled ionization efficiencies for: ESI positive mode, ESI negative mode, and APCI positive mode.

Altogether more than 3000 ionization efficiency values have been measured. The compounds cover a wide range of properties and we have incorporated various solvents, (biological) matrices, and instruments. Compound choice includes pesticides, pharmaceuticals, oligopeptides as well as other compounds with various functional groups and properties.

The best models lead to an average misprediction of only 3.5 times, which is several orders of magnitude better than using just peak areas for quantity estimation.

Tutorial video about calculating ionization efficiencies.

Matrix effects

Matrix effect is the suppression (or sometimes enhancement) of the analyte signal in the ESI source caused by the matrix component. We have studied the origin of matrix effects and described its core properties. We have also proposed several measures to reduce or account for matrix effect. For example, the extrapolative dilution approach has proven to be very useful in diagnosing the presence of matrix effect for real samples. It also helps to estimate the analyte concentration even if the matrix effect cannot be overcome.

Derivatization reagents and novel eluent additives

Directly linked to the ionization efficiency topic is the development of derivatization reagents for LC/MS analysis, as well as novel eluent additives, specifically hexafluoro isopropyl alcohol (HFIP) as a mobile phase additive in LC/MS. Some aims of the new derivatization reagent designs include matrix effect free analysis, targeted and non-targeted analysis as well as semi-quantitative analysis. Additionally, commercially available derivatization reagents are characterized based on properties such as ionization efficiency, matrix effects, fragmentation etc. for ESI as well as APCI. Recent works also include investigation of how derivatization influences the ionization efficiencies of amino acids. The benefit of using fluoroalcohols for separating acidic and basic compounds (such as drug-like molecules and drugs) includes significant MS signal enhancement and alternative selectivity in reversed phase chromatography.

Ambient ionization – sponge and paper spray ionization

Ambient ionization is a relatively new technique of sample introduction to the mass spectrometer, without extensive sample preparation or chromatographic separation. An entrance to this field was made with paper spray, which proved the possibility of generating ions directly from a paper triangle. Fingerprints, swaps, and blood spots have been analyzed this way. Our group has measured pesticides directly from surfaces of different fruits. Unfortunately, this technique is rather a qualitative approach, with reproducibility issues, due to the natural paper fibre composition.

Sponge spray ionization improves on the idea of paper spray by using a volumetric absorption microsampling device, with the inherent property of taking up a fixed amount of sample. This brings ambient ionization one step closer to quantitative measurements.

Bioanalytics and pharmacokinetic studies

We have developed numerous bioanalytical LC/MS assays to support pharmacokinetic studies and clinical trials. For example, a simultaneous assay for clonidine, midazolam, morphine and their metabolites using HFIP as eluent additive to boost the sensitivity and achieve LoQ of 50 pg/mL in human plasma. Signal enhancement for steroid-like molecules in human plasma in positive ionization mode is achieved by using NH₄F.

A range of pharmacokinetic and pharmacodynamic studies such as (and not limited to) penicillin G in neonates, meropenem in neonates and very-low-birth-weight neonates, doripenem and piperacillin/tazobactam in patients with septic shock, ertapenem in obese adults have been supported by our bioanalytical work.

During the bioanalytical method development and validation, the LoD is not required to be established and the drug concentrations measured below LoQ during the pharmacokinetic studies will be reported as ‘BLQ’. We have proposed a way forward to allow a bioanalytical laboratory to release the BLQ data by conducting an additional validation exercise and evaluating the data variability BLQ.

Distribution coefficients

Lipophilicity of the substance (commonly expressed as a partition or distribution coefficient in a certain solvent pair, most often octanol/water) has a decisive influence on its ADMET properties (absorption, distribution, metabolism, excretion, and toxicity), environmental fate and technological applicability. We have experimentally determined partition and distribution coefficients for a variety of compounds in a range of solvent pairs, both as a part of compound characterization and for the development or assessment of prediction methods. The experimental methodology combining shake-flask liquid-liquid extraction and HPLC analysis does not require calibration with standard substances and therefore allows logD determinations for compounds with unknown structure and components of complex mixtures. Recently, a proof-of-concept model for predicting the logD values for unidentified compounds, intended to facilitate optimization of liquid-liquid extraction procedures, was developed.

LogD₇ and logP values between water and various organic solvents can be found here.

Other applications

In our cultural heritage group LC/MS, GC/MS, also direct HRMS (MALDI- and ESI-FT-ICR) methods are used for the analysis of natural textile dyes and resins (and their products, e.g. tars, varnishes). Currently, a method combining HPLC with ESI-FT-ICR-MS is being set up for resinous samples to obtain more information about these complex substances. For the analysis of paint binders, as well as archaeological lipid residue, GC/MS methods have been developed. For the analysis of archaeological lipid residues also MALDI-FT-ICR-MS has proven an invaluable tool. Read more about studies of cultural heritage objects here.

LC/ESI/MS/MS has also been used for the determination of neonicotinoids in honey. Neonicotinoids are infamous pesticides associated with the declination of bee populations). It was shown that residues of some neonicotinoids remain for over 5 years in honey. Also, the amount of one neonicotinoid, thiacloprid, in honey was in correlation with the import data of thiacloprid.

MOOC and tutorial review on LC/MS validation

We launched a MOOC on LC/MS validation and published a tutorial review part I and part II on validation of LC/MS methods. The course runs each autumn semester and we have places for only 500 students. You can check out the current availability of the course here! We have also published a tutorial review part I and part II on the estimation of LoD in LC/MS/MS analysis.