Tandem Mass Spectrometry Experiments Research Articles

Photocatalytic proximity labelling of MCL-1 by a BH3 ligand

Ligand-directed protein labelling allows the introduction of diverse chemical functionalities onto proteins without the need for genetically encoded tags. Here we report a method for the rapid labelling of a protein using a ruthenium-bipyridyl (Ru(II)(bpy)3)-modified peptide designed to mimic an interacting BH3 ligand within a BCL-2 family protein-protein interactions. Using sub-stoichiometric quantities of (Ru(II)(bpy)3)-modified NOXA-B and irradiation with visible light for 1 min, the anti-apoptotic protein MCL-1 can be photolabelled with a variety of functional tags. In contrast with previous reports on Ru(II)(bpy)3-mediated photolabelling, tandem mass spectrometry experiments reveal that the labelling site is a cysteine residue of MCL-1. MCL-1 can be labelled selectively in mixtures with other proteins, including the structurally related BCL-2 member, BCL-xL. These results demonstrate that proximity-induced photolabelling is applicable to interfaces that mediate protein-protein interactions, and pave the way towards future use of ligand-directed proximity labelling for dynamic analysis of the interactome of BCL-2 family proteins.

Heterocyclic Product Formation in Aqueous Brown Carbon Systems

Brown carbon in aerosol remains a significant source of error in global climate modeling due to its complex nature and limited product characterization. Though significant efforts have been made in the previous decade to identify the major light-absorbing brown carbon chromophores formed through the reactions of carbonyl-containing compounds with ammonium, substantial work is still required to identify the main absorbing species resulting from reactions of glyoxal, glycolaldehyde, and hydroxyacetone with ammonium sulfate (AS). Using tandem mass spectrometry and 15N experiments to confirm proposed structures and support their mechanistic pathways, compelling evidence is provided for the formation of pyrazines and imidazoles in the glyoxal + AS, glycolaldehyde + AS, and hydroxyacetone + AS systems. Through density functional theory calculations, the N-containing oligomers and aromatic heterocycles formed within these reaction systems are shown to contribute to brown carbon light absorption, thus holding sig...

Combination of Isotope Labeling and Molecular Networking of Tandem Mass Spectrometry Data To Reveal 69 Unknown Metabolites Produced by Penicillium nordicum.

The secondary metabolome of Penicillium nordicum is poorly documented despite its frequent detection on contaminated food and its capacity to produce toxic metabolites such as ochratoxin A. To characterize metabolites produced by this fungi, we combined a full stable isotopes labeling with the dereplication of tandem mass spectrometry (MS/MS) data by molecular networking. First, the untargeted metabolomic analysis by high-resolution mass spectrometry of a double stable isotope labeling of P. nordicum enabled the specific detection of its metabolites and the unambiguous determination of their elemental composition. Analyses showed that infection of substrate by P. nordicum lead to the production of at least 92 metabolites and that 69 of them were completely unknown. Then, curated molecular networks of MS/MS data were generated with GNPS and MetGem, specifically on the features of interest, which allowed highlighting 13 fungisporin-related metabolites that had not previously been identified in this fungus and 8 that had never been observed in any fungus. The structures of the unknown compounds, namely, a native fungisporin and seven linear peptides, were characterized by tandem mass spectrometry experiments. The analysis of P. nordicum growing on its natural substrates, i.e. pork ham, turkey ham, and cheese, demonstrated that 10 of the known fungisporin-related metabolites and three of the new metabolites were also synthesized. Thus, the curation of data for molecular networking using a specific detection of metabolites of interest with stable isotopes labeling allowed the discovery of new metabolites produced by the food contaminant P. nordicum.

A comprehensive study on rearrangement reactions in collision-induced dissociation mass spectrometric fragmentation of protonated diphenyl and phenyl pyridyl ethers.

Recently, we have reported a forced degradation study of a pharmaceutical drug regorafenib which contains a phenyl pyridyl ether derivative as building block. We observed interesting rearrangements in two of its degradation products in tandem mass spectrometry (MS/MS) experiments. As diphenyl ether derivatives are also molecular building blocks of biological importance and used as herbicides and flame retardants, we decided to investigate specifically the fragmentation behavior of these compounds along with phenyl pyridyl derivatives in detail using high-resolution electrospray ionization (ESI) MS/MS. To understand the fragmentation reactions of protonated substituted diphenyl ethers and phenyl pyridyl ethers, ESI-MS/MS experiments were performed using a quadrupole time-of-flight (QTOF) mass spectrometer. In contrast to radical cations of diphenyl ether derivatives which do not eliminate CO, the [M + H]+ ions of substituted diphenyl ethers undergo rearrangement reactions after loss of neutral molecules (H2 O, HCl, etc.) to form a bicyclic structure containing a keto group and do eliminate CO. Similar rearrangement followed by fragmentation was observed for protonated phenyl pyridyl ethers and the degradation products formed from regorafenib and sorafenib. The protonated ions of substituted diphenyl ethers and phenyl pyridyl ethers on collision-induced dissociation have exhibited interesting rearrangement reactions, despite the nature of the substituent on both the aryl moieties. The proposed fragmentation patterns of these compounds give an insight into the understanding of gas-phase reactions in mass spectrometric studies of diphenyl ether and phenyl pyridyl ether derivatives.

Compositional characterization of complex protopeptide libraries via triboelectric nanogenerator Orbitrap mass spectrometry.

Understanding of the molecular processes that led to the first biomolecules on Earth is one of the key aspects of origins-of-life research. Depsipeptides, or polymers with mixed amide and ester backbones, have been proposed as plausible prebiotic precursors for peptide formation. Chemical characterization of depsipeptides in complex prebiotic-like mixtures should benefit from more efficient ion sources and ultrahigh-resolution mass spectrometry (UHR-MS) for elemental composition elucidation. A sliding freestanding (SF) Triboelectric Nanogenerator (TENG) was coupled to glass nanoelectrospray emitters for the analysis of a depsipeptide library created using 11 amino acids and 3 alpha-hydroxy acids subjected to environmentally driven polymerization. The TENG nanoelectrospray ionization (nanoESI) source was coupled to an UHR Orbitrap mass spectrometer operated at 1,000,000 resolution for detecting depsipeptides and oligoesters in such libraries. Tandem mass spectrometry (MS/MS) experiments were performed on an Orbitrap Q-Exactive mass spectrometer. Our previous proteomics-like approach to depsipeptide library characterization showed the enormous complexity of these dynamic combinatorial systems. Here, direct infusion UHR-MS along with de novo sequencing enabled the identification of 524 sequences corresponding to 320 different depsipeptide compositions. Van Krevelen and mass defect diagrams enabled better visualization of the chemical diversity in these synthetic libraries. TENG nanoESI coupled to UHR-MS is a powerful method for depsipeptide library characterization in an origins-of-life context.

Abstract 389: Role of her3 signaling pathways in er+ and her2+ breast cancers

Abstract HER3 is an important molecule that may regulate oncogenic activity in estrogen receptor (ER)+ breast cancers, which accounts for about 70-80% of all breast cancers. HER3 gene expression is highest in ER+ or luminal tumors. Treatment with the ER degrader fulvestrant induces protein expression of HER3 in ER+ clinical breast tumors, suggesting that HER3 may have therapeutic value in luminal breast cancers. About 20% of all breast cancers diagnosed are HER2 positive (+). HER3 is as essential as HER2 for maintaining cell viability in HER2-overexpressing breast cancer cells. We aim to identify proteins that bind to HER3 upon therapeutic inhibition of HER2 or anti-endocrine therapy. We have found that ERα is upregulated in ER+ T47D and MCF-7 breast cancer cell lines expressing the activating hotspot T355I HER3 mutation compared to HER3 wild-type (WT) cells. We noted that ERα co-immunoprecipitated to HER3-T355I but not to HER3-WT T47D cells. Knocking down HER3 resulted in decreased ERα expression in both HER3-WT and HER3-T355I expressing T47D cells. We are currently analyzing the nuclear expression of endogenous HER3 in MCF-7, T47D, BT474, MDA-MB-361, MDA-MB-453 and SK-BR3 breast cancer cells. We are determining if stimulation with the HER3 ligand heregulin alters the subcellular location of HER3 or the ability to co-immunoprecipitate with ERα in cells expressing exogenous HER3-WT, exogenous HER3-T355I or endogenous HER3. We are using a proteomics approach to identify HER3-interacting proteins to determine how HER2 inhibition affects the repertoire of HER3 binding partners in HER2+ breast cancer cells. We immunopurified HER3 from BT474 cells treated with DMSO or with neratinib (24 hour treatment). We then identified enrichment of specific proteins by mass spectrometry. MALDI tandem mass spectrometry experiments identified non-muscle myosin-II heavy chain from HER3 immunoprecipitates treated 24 hours with neratinib treatment but not 24 hours of DMSO treatment in BT474 cells. Furthermore, our immunoprecipitation data indicated that there is more myosin-IIa bound to HER3 in response to neratinib treatment. Myosin-IIa has a key role in cytokinesis, cell migration, polarization, adhesion and metastasis. Thus, we aim to decipher the mechanism of action by which myosin-IIa interacts with HER3 and determining if downstream myosin/actin signaling could be a possible target in HER2 over-expressing breast cancer patients who show resistance to HER2 targeted therapy. In conclusion, we have identified two potential novel HER3 binding partners in breast cancer: ERα and myosin-IIa. Deciphering the function of HER3 binding partners has potential implication for understanding the invasive properties of ER+ and HER2+ breast cancers. Citation Format: Rosalin Mishra, Samar Alanazi, Hima Patel, Long Yuan, Joan T. Garrett. Role of her3 signaling pathways in er+ and her2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 389.

Identification of Protonated Primary Carbamates by Using Gas-Phase Ion–Molecule Reactions Followed by Collision-Activated Dissociation in Tandem Mass Spectrometry Experiments

The levels of potentially mutagenic impurities (PMIs) in active pharmaceutical ingredients are highly regulated and must be below a critical safety threshold. One class of PMIs is primary carbamates, which are formed during drug manufacturing and formulation. To comply with safety regulations, it is critically important to develop analytical techniques that enable the identification of primary carbamates during the drug development process. In this study, tandem mass spectrometry combined with gas-phase ion–molecule reactions as well as collision-activated dissociation (CAD) is demonstrated to enable the identification of protonated primary carbamates. Primary carbamates were protonated via atmospheric pressure chemical ionization (APCI) in a linear quadrupole ion trap mass spectrometer, isolated, and allowed to react with trimethoxymethylsilane (TMMS) introduced into the ion trap via an external reagent mixing manifold. Protonated primary carbamates reacted with TMMS to form an adduct ion, [M + H + TMMS]...

Collision-induced dissociation of stable nitroxides: A combined tandem mass spectrometry and computational study of TEMPO• and SG1•.

The dissociation behavior of two stable nitroxides, namely 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO•) and N-tert-butyl-1-diethylphosphono-2,2-dimethylpropyl nitroxide (SG1•), subjected as protonated molecules to collisional activation was investigated using a combination of different mass spectrometry experiments and theoretical calculations. Elemental composition of reaction products was derived from accurate mass data measured in high resolution tandem mass spectrometry experiments, primary fragments were distinguished from secondary ions based on both breakdown curves and MS3 data, and H/D exchange experiments were performed to support proposed structures. Postulated fragmentation pathways were then studied in terms of energetic, using the standard B3LYP/6-31G(d) method. While protonation of TEMPO• mainly occurred on the oxygen atom of the nitroxyl function, a series of protomers were found for SG1• with the adducted proton preferentially located onto the P=O group of this phosphorylated species. For both protonated nitroxides, major product ions measured in tandem mass spectrometry arose from reactions occurring at low energy costs via elimination of radical species. Formation of secondary fragments that were detected with low abundance when raising the activation level of both precursor ions could be rationalized with pathways proceeding via high energy transition states.

Imaging Mass Spectrometry–Based Lipidomic Approach to Classification of Architectural Features in Nevi

Imaging Mass Spectrometry–Based Lipidomic Approach to Classification of Architectural Features in Nevi

Electrochemical simulation of metabolism for antitumor-active imidazoacridinone C-1311 and in silico prediction of drug metabolic reactions

The metabolism of antitumor-active 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) has been investigated widely over the last decade but some aspects of molecular mechanisms of its metabolic transformation are still not explained. In the current work, we have reported a direct and rapid analytical tool for better prediction of C-1311 metabolism which is based on electrochemistry (EC) coupled on-line with electrospray ionization mass spectrometry (ESI-MS). Simulation of the oxidative phase I metabolism of the compound was achieved in a simple electrochemical thin-layer cell consisting of three electrodes (ROXY™, Antec Leyden, the Netherlands). We demonstrated that the formation of the products of N-dealkylation reactions can be easily simulated using purely instrumental approach. Newly reported products of oxidative transformations like hydroxylated or oxygenated derivatives become accessible. Structures of the electrochemically generated metabolites were elucidated on the basis of accurate mass ion data and tandem mass spectrometry experiments. In silico prediction of main sites of C-1311 metabolism was performed using MetaSite software. The compound was evaluated for cytochrome P450 1A2-, 3A4-, and 2D6-mediated reactions. The results obtained by EC were also compared and correlated with those of reported earlier for conventional in vitro enzymatic studies in the presence of liver microsomes and in the model peroxidase system. The in vitro experimental approach and the in silico metabolism findings showed a quite good agreement with the data from EC/ESI-MS analysis. Thus, we conclude here that the electrochemical technique provides the promising platform for the simple evaluation of drug metabolism and the reaction mechanism studies, giving first clues to the metabolic transformation of pharmaceuticals in the human body.

Carbohydrate isomer resolution via multi-site derivatization cyclic ion mobility-mass spectrometry.

Oligosaccharides serve many roles in extant life and may have had a significant role in prebiotic chemistry on the early Earth. In both these contexts, the structural and isomeric diversity among carbohydrates presents analytical challenges necessitating improved separations. Here, we showcase a chemical derivatization approach, where 3-carboxy-5-nitrophenylboronic acid (3C5NBA) is used to label vicinal hydroxyl groups, amplifying the structural difference between isomers. We explore the applicability of state-of-the-art ion mobility - mass spectrometry (IM-MS) instrumentation in the analysis of derivatized carbohydrates. In particular we focus on the resolving power required for IM separation of derivatized isomers. A recently developed cyclic ion mobility (cIM) mass spectrometer (MS) was chosen for this study as it allows for multi-pass IM separations, with variable resolving power (Rp). Three passes around the cIM (Rp ∼ 120) enabled separation of all possible pairs of four monosaccharide standards, and all but two pairs of eight disaccharide standards. Combining cIM methodology with tandem mass spectrometry (MS/MS) experiments allowed for the major products of each of the 3C5NBA carbohydrate derivatization reactions to be resolved and unequivocally identified.

Structure and Performance of Polygonal Electrode Linear Ion Trap

Abstract The polygonal electrode linear ion trap (PeLIT) can produce quadrupolar electric field plus some higher order field, which balances the relationship between mass resolution and electrode manufacturing difficulties. The electrodes of PeLIT are relatively simple, but have a good mass resolving power. This study investigated the relationship between the electric field distribution and the ion trap structures, and the performances of PeLIT through theoretical simulation and experimental study. Research results of simulation showed that the polygonal electrode linear ion traps with different structures had different electric field distributions and mass analysis performances. The negative decapole field distorted the performances significantly. The experimental results showed that the mass resolution of reserpine ions (m/z 609) was more than 2500 using a polygonal electrode ion trap. At the same time, mass selective excitation and tandem mass spectrometry experiments were also carried.

Direct quantitation and characterization of fatty acids in salmon tissue by condensed phase membrane introduction mass spectrometry (CP-MIMS) using a modified donor phase.

Existing mass spectrometric methods for the analysis of fatty acids often require derivatization, chromatographic separations, and/or extensive sample preparation. Direct mass spectrometry strategies can avoid these requirements, but may also suffer from poor quantitation and/or lack of sensitivity. Condensed phase-membrane introduction mass spectrometry (CP-MIMS) provides direct quantitative measurements of analytes in complex samples with little or no sample preparation. CP-MIMS uses a semipermeable membrane to transfer neutral, hydrophobic compounds from real-world samples to a mass spectrometer. The results presented utilize aqueous/organic sample solvent (donor) mixtures to allow for the sensitive (pptr) detection of a range of fatty acids. The relative sensitivity across a homologous series of fatty acids is observed to change, favoring short- or long-chain fatty acids, depending on the amount of miscible co-solvent added to the donor phase. Further, lithium acetate added online via the acceptor phase was used in tandem mass spectrometry experiments to determine the location of double bonds in polyunsaturated fatty acids (PUFAs). The method was applied to direct measurements and structural determinations for selected PUFAs in salmon tissue samples. Standard addition was employed to quantify the amount of PUFAs in a variety of salmon samples, yielding 0.27-0.42 and 0.40-0.84 w/w % for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), respectively, for Sockeye and Chinook salmon, in good agreement with the literature. This work presents, to our knowledge, the first use of CP-MIMS for the direct analysis of fatty acids in oily foodstuff samples. Graphical abstract ᅟ.

Internal Standard Quantification Using Tandem Mass Spectrometry of a Tryptic Peptide in the Presence of an Isobaric Interference.

Model mixtures of isobaric peptides were studied to evaluate the possibility, using tandem mass spectrometry experiments, for internal standard quantification of a tryptic peptide in the presence of an isobaric interference. To this end, direct injection electrospray ionization-tandem mass spectrometry (ESI-MS/MS) experiments were performed on an ion trap instrument using a large mass-selection window (15 m/ z) encompassing the isobaric mixture and the internal standard; MS/MS experiments were carried out to remove completely the interference from the mixture by fragmenting it. This allowed for the correct intensity assignment for the protonated peptide peak and, thus, for the analyte to be quantified through the relative intensity estimate of this peak with respect to the internal standard. This was done by monitoring the 15 m/ z mass-selection window only and without the necessity for careful inspection of any fragment ions peaks. The interference removal was assessed by determining an excitation voltage large enough for the analyte/internal standard ratio to remain constant ensuring correct quantification despite isobaric contamination. A calibration curve was obtained to predict reference samples and compared to reference samples purposely spiked with the interference using the proposed methodology; internal standard quantification of the analyte was made possible with ∼1% deviation despite the isobaric contamination.

Silver ion induced folding of alkylamines observed by ion mobility experiments

Recent studies point to an additional stability of metal-complexed functionalized hydrocarbon chains. This additional stabilization is suggested to arise from a bidentate complexation of the metal cation with both the functional group and aliphatic chain, leading to more folded structures. In the present work, this issue is verified for silver complexed alkylamines by tandem mass spectrometry and ion mobility experiments combined with quantum-chemical calculations. Upon increasing the length of the hydrocarbon chains, the alkylamine/silver complexes undergo a structural transition to reach a folded structure in which the silver cation interacts with both the amine group and hydrogen atoms located at the chain end. This folding is further examined by monitoring the loss of AgH upon collision-induced dissociation experiments performed on alkylamines with different chain lengths. Ion mobility experiments performed on the fragment ions indicate that the removal of the silver cation induces the unfolding of the chain.

MetWork: a web server for natural products anticipation.

The annotation of natural products and more generally small molecules is one of the major drawbacks in untargeted mass spectrometry analysis. Molecular networking has emerged as a structured way to organize and mine data from untargeted tandem mass spectrometry (MS/MS) experiments. Despite the great potential of this tool, the annotation is usually performed manually by the expert as only few spectral libraries are available. Herein we propose a web server of in silico metabolization of metabolites that represents a full implementation of the metabolome consistency concept. The workflow is based on MS/MS data, organized in molecular network using the Global Natural Products Social Molecular Networking (GNPS) platform, a collaborative library of reactions and a MS/MS spectra prediction module. Having one node identified in the molecular network, the server generates putative structures and predict the associated MS/MS spectra when the exact mass is detected in the network. A similarity comparison between the MS/MS spectra is then performed in order to annotate the node. The web server is available at: https://metwork.pharmacie.parisdescartes.fr.

Detection of Neutral CO Lost During Ionic Dissociation Using Atmospheric Pressure Thermal Dissociation Mass Spectrometry (APTD-MS).

Elucidation of ion dissociation patterns is particularly important to structural analysis by mass spectrometry (MS). However, typically, only the charged fragments from an ion dissociation event are detected in tandem MS experiments; neutrals are not identified. In recent years, we have developed an atmospheric pressure thermal dissociation (APTD) technique that can be applied to dissociate ions at atmosphere pressure and thus provide one way to characterize neutral fragments. In this paper, we focus on the detection of neutral CO resulting from amino acid and peptide ion dissociation. In the first set of experiments, several protonated amino acids (e.g., + 1 ion of phenylalanine) were found to undergo loss of a neutral (s) of total mass 46Da, a process leading to iminium ion formation. We successfully detected the neutral species CO by using a CO sensor, UV-Vis and MS analysis following selective CO trapping with a rhodium complex. The capture of CO from dissociation of protonated amino acids supports the assignment of the loss of 46Da to neutral losses of CO and H2O, rather than loss of formaldehyde or dihydroxycarbene, other possible fragmentation pathways that have been subject of debate for a long time. In a second experiment, we used the APTD method in combination with the CO detection technique, to demonstrate the formation of CO in the conversion of b ions to a ions during peptide ion dissociations. These results showed the potential of APTD in the elucidation of ion dissociation mechanisms, using simple home-built apparatus. Graphical Abstract ᅟ.

Perspectives on Liquid Chromatography-High-Resolution Mass Spectrometry for Pesticide Screening in Foods.

This perspective discusses the use of liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) for multiresidue analysis of pesticides in foods and agricultural commodities. HRMS has the important distinction and advantage of mass-resolving power and, therefore, requires different concepts, experiments, and guidance for screening, identification, and quantitation of pesticides in complex food matrices over triple quadrupole mass spectrometry. HRMS approaches for pesticide screening, including full-scan experiments in conjunction with tandem mass spectrometry (MS/MS) experiments, are described. This approach results in the generation of chromatographic retention times and high-resolution mass spectra with accurate mass measurements that can be used to create compound databases. New data processing tools can create an efficient and optimized screening approach that can speed the analysis and identification of compounds, reduce the need for chemical standards, and harmonize pesticide analytical procedures.

Gas-phase stability of sandwich complexes of crown ethers with metal cations - as studied by collision induced dissociation tandem mass spectrometry.

The gas-phase stabilities of the sandwich complexes formed by 12-crown-4 or 15-crown-5 with metal cations (Na+ , K+ , Rb+ , Tl+ , Ag+ , Ca2+ , Sr2+ , Ba2+ , Pb2+ , Hg2+ , Cd2+ ) are compared with the stability of the sandwich complex formed by benzo-12-crown-4 or benzo-15-crown-5 with the cations. It is interesting to check if the possible cation-π interactions increase the gas-phase stabilities of the sandwich complexes of benzo-crown ethers with metal cations. The sandwich complexes were generated in the gas phase by electrospray ionization (ESI) and then they were subjected to the collision induced dissociation tandem mass spectrometry experiments (CID-MS/MS). On the basis of the obtained product ion spectra, the respective breakdown plots of the ion abundances ratios against collision energy expressed in the terms of center-of-mass were made. The gas-phase stabilities of [(B12C4)2 +Tl]+ , [(B15C5)2 +SrNO3 ]+ , [(B15C5)2 +PbNO3 ]+ and [(B15C5)2 +SrCl]+ were higher than those of [(12C4)2 +Tl]+ , [(15C5)2 +SrNO3 ]+ , [(15C5)2 +PbNO3 ]+ and [(15C5)2 +SrCl]+ , respectively. For the other sandwich complexes the stabilities of the complexes with benzo-crown ethers were not higher than those of the complexes of simple crown ethers. It is reasonable to assume that the cation-π interaction increases the stability of the sandwich complex of B12C4 with Tl+ and the stabilities of sandwich complexes of B15C5 with Sr2+ and Pb2+ .

Identification of Pharmaceuticals in The Aquatic Environment Using HPLC-ESI-Q-TOF-MS and Elimination of Erythromycin Through Photo-Induced Degradation.

Monitoring pharmaceuticals throughout the water cycle is becoming increasingly important for the aquatic environment and eventually for human health. Targeted and non-targeted analysis are today's means of choice. Although targeted analysis usually conducted with the help of a triple quadrupole mass spectrometer may be more sensitive, only compounds previously selected can be identified. The most powerful non-targeted analysis is performed through time of flight mass spectrometers (TOF-MS) extended by a quadrupole mass analyzer (Q), as used in this study. Preceded by solid phase extraction and high-performance liquid chromatography (HPLC), the non-targeted approach allows to detect all ionizable substances with high sensitivity and selectivity. Taking full advantage of the Q-TOF-MS instrument, tandem mass spectrometry (MS/MS) experiments accelerate and facilitate the identification while a targeted MS method enhances the sensitivity but relies on reference standards for identification purposes. The identification of four pharmaceuticals from Rhine river water is demonstrated. The Rhine river originates in Tomasee, Graubünden, Switzerland and flows into the North Sea, near Southern Bight, The Netherlands. Its length amounts to 1232.7 km. Since it is of prime interest to effectively eliminate pharmaceuticals from the water cycle, the effect UV-C irradiation is demonstrated on a laboratory scale. This method allows fast degradation of pharmaceuticals, which is exemplarily shown for the macrolide antibiotic erythromycin. Using the above HPLC-Q-TOF-MS method, concentration-time diagrams are obtained for the parent drug and their photodegradation products. After establishing the equations for first-order sequential reactions, computational fitting allows the determination of kinetic parameters, which might help to predict irradiation times and conditions when potentially considered as fourth stage within wastewater treatment.