High resolution quadrupole time of flight mass spectrometry in pharmaceutical bioanalysis

Abstract

This cumulative PhD thesis put forward several aspects of the analysis of biomolecules employing high resolution quadrupole time of flight mass spectrometry (HR-QTOF-MS). Particular analytical challenges in the context of the analysis of various classes of analytes, i.e. amino acids and peptides, oligonucleotide complexes, triterpenoid esters, intact proteins have been addressed and new analytical solutions by either liquid chromatographic separation or HR-QTOF-MS, respectively, their hyphenation have been suggested. This PhD-Thesis is comprised of four thematically distinct parts:

The first part dealt with the stereoselective analysis of amino acids and peptides. In one study, the complete stereoconfiguration of an antimicrobial active lipopeptide, poaeamide, was determined. Lipopeptides are typically synthesized by a non-ribosomal enzymatic peptide synthesis machinery. As result, they frequently contain several D-amino acids providing hydrolysis resistance towards target organism peptidases. As lipopeptides are of general interest for research on and development of antimicrobial compounds, complete structural elucidation is essential, which encompasses also determination of the absolute configurations of the amino acids constituting the respective peptide, which is presented in Publication II. The analysis strategy enveloped the incomplete hydrolysis of the peptide yielding overlapping sequence fragments, micro-scale preparative liquid chromatography and stereoconfiguration analysis of hydrolysis fragments by chiral GC-MS, ultimately providing determination of the stereoconfigurations of its comprising amino acids enantiomers. As the latter cannot be distinguished by mass spectrometry alone, HR-QTOFMS has been hyphenated with appropriate enantioselective chromatography using cinchonan carbamate based chiral stationary phases. This work involved the optimization of the chromatographic and MS conditions and the demonstration of the feasibility of aforementioned phases in providing complementary chromatographic selectivity when compared to RP and HILIC type phases, in detail emphasized in Publication VIII. A major challenge was the determination of configurations of amino acids with more than one stereogenic centers (Thr/allo-Thr, Ile/allo-Ile) and those also present as constitutional isomers (Leu/Ile).

A particular intricacy solved by the analysis strategy of a combined LC and GC approach employing reversed phase (RP), hydrophilic interaction (HILIC) and enantioselective stationary phases was the localization of D-Leu discovered in the peptide, as its position was disguised by presence of several Leu residues in the lipopeptide. To advance insight into the enantioselective interaction between the employed LC stationary phases and amino acids and derivatives thereof, Publication I reported the application of quantitative structure-retention and structure-enantioselectivity relationships to quantitatively study enantioselective molecular recognition mechanisms. By employing a Free-Wilson type generalized linear modelling approach, Publication I validated hypotheses that describe binding energy contribution of individual molecular moieties as being linear independent of each other. Major contribution to retention of the analytes could be attributed to pi-interacting derivatization groups, a finding that stands in congruence to experimental findings reported amongst others by Publication II.

With mass spectrometry today representing the chromatographic detection method of choice and consequently entailing the desideratum of stationary phases compatible with this technology, the first part of this thesis was concluded by Publication VII enhancing mass spectrometric compatibility of the employed chiral stationary phases. With a hydrolysis stable crosslinked methylpolysiloxane type surface chemistry, also providing a scaffold for various surface ligand modifications by the employed thiol-ene click chemistry, significant enhancement of mass spectrometric compatibility could be demonstrated. Using the enantioselective cinchonan carbamate based chiral stationary phases as an example ligand, ameliorated phase stability and resultant enhancement in mass spectrometric sensitivity was assessed and confirmed by high resolution quadrupole time of flight mass spectrometry. In the second part, challenging analysis, both from chromatographic and mass spectrometric perspective, of regioisomers of pentacyclic triterpenoid fatty acid esters instable even under soft ionizing conditions was addressed by Publication V. Novel esters of triterpenoids with anti-inflammatory potential, amongst them mixed esters of faradiol, myristic and palmitic acid could be confirmed to be present in extracts of by employment of orthogonal analysis methods, namely NMR, GC-MS and LC-HR-QTOF-MS. In order to address the challenging liquid chromatographic separation of mixed regioisomeric diesters, molecular shape selective chromatography was employed using C30-type RP-stationary phases tailored for the task. Chromatographic and mass spectrometric requirements, the latter stemming from the astonishing instability of the analytes during ionization in the presence of water, even under the soft ionization conditions encountered in electrospray (ESI) or atmospheric pressure chemical ionization (APCI), could be harmonized by application of a non-aqueous binary eluent system, cold LC column temperatures facilitating entropic optimization of regioisomer separation and adequate application and tuning of parameters of APCI-QTOFMS for sensitivity, mass accuracy and resolution.

The third part was dedicated to intact protein mass spectrometric analysis (Publications IV IX X and XI). Employing the Sciex 5600+ TripleTOFs capability in mass accuracy, mass resolution and sensitivity even for large molecular species by adequate mass spectrometric and chromatographic method development, analytical questions revolving around analysis of intact proteins could be addressed, including antibody characterization and, in a straightforward approach demonstrating mass accuracy and resolution of QTOF, direct confirmation of attachment and correct target location of covalent kinase inhibitors with sub kDa molecular weight to >40kDa protein targets.

Finally, the fourth part of this thesis includes two studies (Publications III and XII) that examine interaction between G4-DNA-selective ligands and G4-DNA-quadruplexes, the latter representing an in vivo form of a DNA that is of oncological research interest as it is frequently encountered in promoter regions of oncogenes. Ligands specifically binding to this DNA form is subject of research aimed at cancer imaging or potential anticancer drugs. Study of such non-covalent complexes in solution is preferably performed by NMR. However, NMR spectra interpretation is both regularly and in case of Publication III and XII severely hampered by extensive peak broadening and overlapping as consequence of fast to intermediate exchange rates relative to the NMR chemical shift timescale of ligands occupying different binding sites Fluorescence titration, employed as orthogonal method in

both studies, also could not unequivocally unveil the stoichiometry of the complexes studied in the two publications. The author’s contribution was the development of a native electrospray ionization high resolution quadrupole time of flight mass spectrometry (ESI-HRQTOF) method to elucidate stoichiometry and binding mode of aforementioned complexes. The intricacy to address for both studies was the provision of mass spectrometric method capable of mapping non-covalent complex stoichiometries and properties from solution to the vacuum of the mass spectrometric ion path without distortion for example of secondary structure or ligand binding by the ionization process, by the atmosphere to vacuum transition or flight through the ion path. The native-ESI-QTOF-MS method developed was capable of providing these requirements for noncovalent DNA-ligand complexes of several kDa molecular mass, yet still allowing to quantitively monitor specific binding of very low molecular (e.g. ammonium NH4+) species to the complex.

Overall, the studies summarized in this Thesis, demonstrated the great utility and wide area of application of high-resolution quadrupole time of flight mass spectrometry, either in its hyphenated form with liquid chromatography or as direct infusion-MS, to solve challenging analytical questions in the context of (bio)pharmaceutical analysis.