Tandem mass spectrometry of small-molecule antiviral drugs: 1. HIV-related antivirals.

Antiviral drugs are a class of compounds developed specifically for the treatment of viral infections. In the development and subsequent application of antiviral drugs, like for any other class of drugs, quantitative analysis in biological matrix is important, e.g., to establish bioavailability, to study pharmacokinetics, and later on possibly for therapeutic drug monitoring. Liquid chromatography-mass spectrometry (LC-MS) with tandem mass spectrometry (MS-MS) operated in selected-reaction monitoring (SRM) mode is the method of choice in quantitative bioanalysis. As information of the fragmentation of antiviral drugs in MS-MS is very much scattered in the scientific literature, it was decided to collect this information and to review it, not only to understand which product ions are actually used in SRM, but also to assist in other studies, e.g., in the identification of drug metabolites or (forced) degradation products. In this first study, attention is paid to antiviral agents used against HIV infection. The review provides fragmentation schemes of ca. 40 antiviral agents as well as several phosphorylated anabolites. The identity of the product ions used in SRM, i.e., elemental composition and exact-m/z, is tabulated, and more detailed fragmentation schemes are provided.

Tandem mass spectrometry of small-molecule antiviral drugs: 3. antiviral agents against herpes, influenza and other viral infections.

For the treatment of various viral infections, antiviral drugs may be used. Liquid chromatography-mass spectrometry (LC-MS) with tandem mass spectrometry (MS-MS) operated in selected-reaction monitoring (SRM) mode is the method of choice in quantitative bioanalysis of drugs, e.g., to establish bioavailability, to study pharmacokinetics, and later on possibly for therapeutic drug monitoring. In this study, the fragmentation in MS-MS of small-molecule antiviral drugs against herpes and influenza viruses is reviewed. In this way, insight is gained on the identity of the product ions used in SRM. Fragmentation schemes of antiviral agents are also relevant in the identification of drug metabolites or (forced) degradation products. As information of the fragmentation of antiviral drugs in MS-MS and the identity of the product ions is very much scattered in the scientific literature, it was decided to collect this information and to review it. In this third study, attention is paid to small-molecule antiviral agents used against herpes and influenza virus infections. In addition, some attention is paid to broad-spectrum antiviral agents, that are investigated with respect to their efficacy in challenging virus infections of this century, e.g., involving Ebola, Zika and corona viruses, like SARS-CoV-2, which is causing a world-wide pandemic at this very moment. The review provides fragmentation schemes of ca. 35 antiviral agents. The identity of the product ions used in SRM, i.e., elemental composition and exact-m/z, is tabulated, and more detailed fragmentation schemes are provided.

Fragmentation of toxicologically relevant drugs in negative-ion liquid chromatography-tandem mass spectrometry.

Negative-ion LC-MS analysis of drugs is applied far less frequently than positive-ion LC-MS. Data on the interpretation of negative-ion MS-MS spectra are even more scarce. Therefore, following the recent review on the class-specific fragmentation of toxicologically relevant compounds in positive-ion MS-MS, it was decided to perform a similar study in negative-ion MS-MS. To this end, a set of over 500 negative-ion MS-MS spectra was collected from three libraries applied in toxicological general unknown screening and systematic toxicological analysis. The compounds involved were classified by chemical and therapeutic class. The MS-MS spectra were manually interpreted and relevant interpretation data were searched for in the scientific literature. The emphasis in the discussion is on class-specific fragmentation, because discussing fragmentation of all individual compounds would take far too much space. Negative-ion MS-MS fragmentation is discussed for a wide variety of toxicologically relevant compounds, including dihydropyridine calcium channel blockers, diuretics, barbiturates, anti-inflammatory drugs, anti-diabetics, sulfonamide and betalactam antibiotics, and a number of classes of pesticides.

Fragmentation of toxicologically relevant drugs in positive-ion liquid chromatography-tandem mass spectrometry.

The identification of drugs and related compounds by LC-MS-MS is an important analytical challenge in several application areas, including clinical and forensic toxicology, doping control analysis, and environmental analysis. Although target-compound based analytical strategies are most frequently applied, at some point the information content of the MS-MS spectra becomes relevant. In this article, the positive-ion MS-MS spectra of a wide variety of drugs and related substances are discussed. Starting point was an MS-MS mass spectral library of toxicologically relevant compounds, available on the internet. The positive-ion MS-MS spectra of ∼570 compounds were interpreted by chemical and therapeutic class, thus involving a wide variety of drug compound classes, such benzodiazepines, beta-blockers, angiotensin-converting enzyme inhibitors, phenothiazines, dihydropyridine calcium channel blockers, diuretics, local anesthetics, vasodilators, as well as various subclasses of anti-diabetic, antidepressant, analgesic, and antihistaminic drugs. In addition, the scientific literature was searched for available MS-MS data of these compound classes and the interpretation thereof. The results of this elaborate study are presented in this article. For each individual compound class, the emphasis is on class-specific fragmentation, as discussing fragmentation of all individual compounds would take far too much space. The recognition of class-specific fragmentation may be quite informative in determining the compound class of a specific unknown, which may further help in the identification. In addition, knowledge on (class-specific) fragmentation may further help in the optimization of the selectivity in targeted analytical approaches of compounds of one particular class.

Tandem mass spectrometry of small-molecule signal transduction inhibitors: Accurate-m/z data to adapt structure proposals of product ions.

Protein kinases inhibitors or, more generally, signal transduction inhibitors (STIs) can be used to treat diseases in which deregulation of the protein kinase activity plays a role, such as in cancer. A wide variety of drugs has been developed and/or is under investigation to act as protein kinase inhibitors, especially in tyrosine kinase inhibition. The bioanalysis of STIs has received considerable attention in the past 20 years. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) in selected-reaction monitoring (SRM) mode is the method-of-choice in such studies. In several of these studies from us and others, structures are proposed for the product ions applied in SRM. A critical review of these proposed structures is presented using accurate-m/z data, which we have now generated with a linear-ion-trap-Orbitrap hybrid mass spectrometer. This led to adaptation and new structural proposals of 18 product ions for 13 STIs. Our investigation endorses the power of accurate-m/z analysis in structure elucidation of product ions in bioanalytical LC-MS-MS studies and for which the SRM mode in tandem-quadrupole instruments is apparently less suitable.

Identification of very long-chain (>C24) fatty acid methyl esters using gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with atmospheric pressure chemical ionization source.

Gas chromatography coupled to a quadrupole time-of-flight mass analyzer (QTOF) with an atmospheric pressure chemical ionization (APCI) source has been tested to study the ionization and mass spectrometric behavior of long-chain and very long-chain polyunsaturated fatty acids (LC-PUFAs, C18-24; VLC-PUFAs, >C24). The protonated molecule ([M+H]+), measured at accurate mass, became the base peak of the spectrum for all the studied compounds and was promoted by the addition of water into the source. This fact overcame the existing difficulties for the identification of VLC-PUFAs when using an electron ionization source (EI). The extensive fragmentation of PUFAs in this source is the main drawback due to the fact that since reference standards are not commercially available, final identification relies on retention time estimation. The application of GC-APCI-QTOF to the screening of lipid extracts from the eyes of different fish species added confidence to the identification of several VLC-PUFAs. Further investigation of ion ratios allowed to predict the position of key double bonds enabling the classification of VLC-PUFAs as ω3 or ω6 compounds. VLC-PUFAs spectra found in biological samples were compared to those obtained from corresponding peaks found in heterologous expression experiments of fish's Elovl4.

Metal-complex formation in continuous-flow ligand-exchange reactors studied by electrospray mass spectrometry.

Electrospray ionization mass spectrometry was used to investigate complex formation of different metal complexes in a continuous-flow ligand-exchange reactor. A computer program was developed based on normal equilibrium calculations to predict the formation of various metal-ligand complexes. Corresponding to these calculations, infusion electrospray mass spectrometric experiments were performed to investigate the actual complex formation in solution. The data clearly show good correlation between the theoretically calculated formation of metal-ligand complexes and the experimental mass spectrometric data. Moreover, the approach demonstrates that the influence of the pH can be investigated using a similar approach. Indirectly, these infusion experiments provide information on relative binding constants of different ligands towards a metal-ion. To demonstrate this, a continuous-flow ligand-exchange detection system with mass spectrometric detection was developed. Injection of ligands, with different affinity for the metal-ion, into the reactor shows good correlation between binding constants and the response in the ligand-exchange detection system. Additional information on the introduced ligand, and the complexes formed after introduction of the ligand, can be obtained from interpretation of the mass spectra.

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry.

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.

Screening for metal ligands by liquid chromatography--ligand-exchange--electrospray mass spectrometry.

Electrospray ionization mass spectrometry is applied for the selective detection of metal ligands after a post-column continuous-flow ligand-exchange reaction. The detection is based on the specific release of a reporter ligand from a metal-reporter ligand complex by a high affinity ligand. Constant infusion and direct-injection experiments are performed to optimize the method. The on-line coupling of a liquid chromatographic separation prior to the continuous flow ligand-exchange reaction enables the screening for high affinity ligands in complex samples. The feasibility of the method is demonstrated by using several ligands with a different affinity for either Cu(II) or Zn(II) ions. The selectivity of the ligand-exchange detection method can be tuned by the choice of the reporter ligand. This is demonstrated by using either 2,2'-bipyridyl or 5-methyl-1,10-phenanthroline as reporter ligands.

Amino acid analysis using chromatography-mass spectrometry: An inter platform comparison study.

The analysis of amino acids has become a central task in many aspects. While amino acid analysis has traditionally mainly been carried out using either gas chromatography (GC) in combination with flame ionization detection or liquid chromatography (LC) with either post-column derivatization using ninhydrin or pre-column derivatization using o-phthalaldehyde, many of today's analysis platforms are based on chromatography in combination with mass spectrometry (MS). While derivatization is mandatory for the GC-based analysis of amino acids, several LC platforms have emerged, particularly in the dawn of targeted metabolite profiling using hydrophilic interaction liquid chromatography (HILIC) coupled to MS, allowing the analysis of underivatized amino acids. Among the numerous analytical platforms available for amino acid analysis today, we here compare three prominent approaches, being GC-MS and LC-MS after amino acid derivatization using chloroformate and HILIC-MS of underivatized amino acids. We compare and discuss practical issues as well as performance characteristics, e.g., the use of (13)C-labeled internal standards, of the different platforms and present data on their practical implementation in our laboratory. Finally, we compare the real-life applicability of all three platforms for a complex biological sample. While all three platforms are very-well suited for the analysis of complex biological samples they all show advantages and disadvantages for some analytes as discussed in detail in this manuscript.

Progress in liquid chromatography-mass spectrometry instrumentation and its impact on high-throughput screening.

In the past 10 years, liquid chromatography-mass spectrometry (LC-MS) has rapidly matured to become a very powerful and useful analytical tool that is widely applied in many areas of chemistry, pharmaceutical sciences and biochemistry. In this paper, recent instrumental developments in LC-MS-related interfacing, ionization and mass analysis are reviewed from the perspective of the application of LC-MS in high-throughput screening of combinatorial libraries and the related high-throughput quantitative bioanalysis in early drug-discovery studies, such as early adsorption, distribution, metabolism and excretion studies.

Trace-level determination of pesticides in water by means of liquid and gas chromatography.

The trace-level determination of pesticides and their transformation products (TPs) in water by means of liquid and gas chromatography (LC and GC) is reviewed. Special attention is given to the use of (tandem) mass spectrometry for identification and confirmation purposes. The complementarity of LC- and GC-based techniques and the potential of comprehensive GCXGC are discussed, and also the impressive performance of time-of-flight mass spectrometry. It is also indicated that, in the near future, the TPs rather than the parent compounds should receive most attention--with a better understanding of matrix effects and eluent composition on the ionization efficiency of analytes being urgently required. Finally, the merits of using much shorter LC columns, or even no column at all (flow-injection analysis) in target analysis are shown, and a more cost-efficient and sophisticated strategy for monitoring programmes is briefly introduced.

Identification of new omeprazole metabolites in wastewaters and surface waters.

Omeprazole is one of the world-wide most consumed pharmaceuticals for treatment of gastric diseases. As opposed to other frequently used pharmaceuticals, omeprazole is scarcely detected in urban wastewaters and environmental waters. This was corroborated in a previous research, where parent omeprazole was not detected while four transformation products (TPs), mainly resulting from hydrolysis, were found in effluent wastewaters and surface waters. However, the low abundance of omeprazole TPs in the water samples together with the fact that omeprazole suffers an extensive metabolism, with a wide range of excretion rates (between 0.01 and 30%), suggests that human urinary metabolites should be investigated in the water environment. In this work, the results obtained in excretion tests after administration of a 40 mg omeprazole dose in three healthy volunteers are reported. Analysis by liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (LC-QTOF MS) reported low concentrations of omeprazole in urine. Up to twenty-four omeprazole metabolites (OMs) were detected and tentatively elucidated. The most relevant OM was an omeprazole isomer, which obviously presented the same exact mass (m/z 346.1225), but also shared a major common fragment at m/z 198.0589. Subsequent analyses of surface water and effluent wastewater samples by both LC-QTOF MS and LC-MS/MS with triple quadrupole revealed that this metabolite (named as OM10) was the compound most frequently detected in water samples, followed by OM14a and OM14b. Up to our knowledge, OM10 had not been used before as urinary biomarker of omeprazole in waters. On the contrary, parent omeprazole was never detected in any of the water samples. After this research, it seems clear that monitoring the presence of omeprazole in the aquatic environment should be focused on the OMs suggested in this article instead of the parent compound.

Tandem mass spectrometry study of p38α kinase inhibitors and related substances.

The p38 mitogen-activated protein kinase α (p38α) is an important drug target widely investigated for therapy of chronic inflammatory diseases. Its inhibitors are rather lipophilic and as such not very favourable lead compounds in drug discovery. Therefore, we explored various approaches to access new chemical space, create diversity, and generate lead libraries with improved solubility and reduced lipophilicity, based on known p38α inhibitors, e.g., BIRB796 and TAK-715. Compound modification strategies include incubation with human liver microsomes and bacterial cytochrome P450 mutants from Bacillus megaterium and treatment by electrochemical oxidation, H2O2, and intense light irradiation. The MS/MS fragmentation pathways of p38α inhibitors and their conversion products have been studied in an ion-trap-time-of-flight MS(n) instrument. Interpretation of accurate mass MS(n) data for four sets of related compounds revealed unexpected and peculiar fragmentation pathways that are discussed in detail. Emphasis is put on the usefulness of HRMS(n)-based structure elucidation in a screening setting and on peculiarities of the fragmentation with regard to the analytes and the MS instrument. In one example, an intramolecular rearrangement reaction accompanied by the loss of a bulky group is observed. For BIRB796, the double-charge precursor ion is used in MS(2), providing a wider range of fragment ions in our instrument. For TAK-715, a number of related compounds could be produced in a large-scale incubation with a Bacillus megaterium mutant, thus enabling comparison of the structure elucidation by (1)H NMR and MS(n). A surprisingly large number of homolytic cleavages are observed. Competition between two fragmentation pathways involving either the loss of CH3(•) or OH(•) radicals was observed for SB203580 and its conversion products.

Investigating the presence of omeprazole in waters by liquid chromatography coupled to low and high resolution mass spectrometry: degradation experiments.

Omeprazole is one of the most consumed pharmaceuticals around the world. However, this compound is scarcely detected in urban wastewater and surface water. The absence of this pharmaceutical in the aquatic ecosystem might be due to its degradation in wastewater treatment plants, as well as in receiving water. In this work, different laboratory-controlled degradation experiments have been carried out on surface water in order to elucidate generated omeprazole transformation products (TPs). Surface water spiked with omeprazole was subjected to hydrolysis, photo-degradation under both sunlight and ultraviolet radiation and chlorination. Analyses by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF MS) permitted identification of up to 17 omeprazole TPs. In a subsequent step, the TPs identified were sought in surface water and urban wastewater by LC-QTOF MS and by LC coupled to tandem mass spectrometry with triple quadrupole. The parent omeprazole was not detected in any of the samples, but four TPs were found in several water samples. The most frequently detected compound was OTP 5 (omeprazole sulfide), which might be a reasonable candidate to be included in monitoring programs rather than the parent omeprazole.

Identification and quantification of drug-albumin adducts in serum samples from a drug exposure study in mice.

The formation of drug-protein adducts following the bioactivation of drugs to reactive metabolites has been linked to adverse drug reactions (ADRs) and is a major complication in drug discovery and development. Identification and quantification of drug-protein adducts in vivo may lead to a better understanding of drug toxicity, but is challenging due to their low abundance in the complex biological samples. Human serum albumin (HSA) is a well-known target of reactive drug metabolites due to the free cysteine on position 34 and is often the first target to be investigated in covalent drug binding studies. Presented here is an optimized strategy for targeted analysis of low-level drug-albumin adducts in serum. This strategy is based on selective extraction of albumin from serum through affinity chromatography, efficient sample treatment and clean-up using gel filtration chromatography followed by tryptic digestion and LC-MS analysis. Quantification of the level of albumin modification was performed through a comparison of non-modified and drug-modified protein based on the relative peak area of the tryptic peptide containing the free cysteine residue. The analysis strategy was applied to serum samples resulting from a drug exposure experiment in mice, which was designed to study the effects of different acetaminophen (APAP) treatments on drug toxicity. APAP is bioactivated to N-acetyl-p-benzoquinoneimine (NAPQI) in both humans and mice and is known to bind to cysteine 34 (cys34) of HSA. Analysis of the mouse serum samples revealed the presence of extremely low-level NAPQI-albumin adducts of approximately 0.2% of the total mouse serum albumin (MSA), regardless of the length of drug exposure. Due to the targeted nature of the strategy, the NAPQI-adduct formation on cys34 could be confirmed while adducts to the second free cysteine on position 579 of MSA were not detected.

Derivatization of the tricarboxylic acid cycle intermediates and analysis by online solid-phase extraction-liquid chromatography-mass spectrometry with positive-ion electrospray ionization.

The analysis of cellular metabolic processes is of fundamental biological interest. Cellular metabolites, such as the intermediates of the tricarboxylic acid (TCA) cycle, provide essential information about the metabolic state of the cell. Not only is the TCA cycle a key factor in the energy regulation within aerobic cells, it possibly also plays a role in cell signaling. This paper describes a novel derivatization strategy, using the empirically selected N-methyl-2-phenylethanamine as derivatization reagent with a carbodiimide as co-reagent, for the selective derivatization of carboxylic acids, such as the di- and tri-carboxylic acids of the TCA cycle. Optimization of the derivatization protocol is described. This procedure enables analysis of the derivatives using on-line solid-phase extraction and reversed-phase liquid chromatography in combination with sensitive positive-ion electrospray ionization mass spectrometry. The complete procedure, involving the use of core-shell silica column material, allows the rapid analysis of TCA cycle intermediates in sample matrices, here shown for pig heart tissue extracts, with a good linearity over 3-4 orders of magnitude. Detection limits range from 12 to 1000 nM, depending on the analyte.

High-resolution metabolic profiling towards G protein-coupled receptors: rapid and comprehensive screening of histamine H4 receptor ligands.

In the past years we developed high-resolution screening platforms involving separation of bioactive mixtures and on-line or at-line bioassays for a wide variety of biological targets with parallel mass spectrometric detection and identification. In the current research, we make a major step forward in the development of at-line bioassays by implementation of radioligand receptor binding and functional cellular assays to evaluate bioactvity and selectivity. We demonstrate a new platform for high-resolution metabolic profiling of lead compounds for functional activity and selectivity toward the human histamine H(4) receptor (hH(4)R), a member of the large family of membrane-bound G protein-coupled receptors. In this platform analytical chemistry, cell biology and pharmacology are merged. The methodology is based on chromatographic separation of metabolic mixtures by HPLC coupled to high-resolution fractionation onto (multiple) microtiter well plates for complementary assaying. The methodology was used for efficient and rapid metabolic profiling of the drug clozapine and three selective hH(4)R lead compounds. With this new platform metabolites with undesired alterations in target selectivity profiles can be readily identified. Moreover, the parallel identification of metabolite structures, with accurate-mass measurements and MS/MS, allowed identification of liable metabolic 'hotspots' for further lead optimization and plays a central role in the workflow and in this study. The methodology can be easily adapted for use with other receptor screening formats. The efficient combination of pharmacological assays with analytical techniques by leveraging high-resolution at-line fractionation as a linking technology will allow implementation of comprehensive metabolic profiling in an early phase of the drug discovery process.

Protein digestion optimization for characterization of drug-protein adducts using response surface modeling.

The formation of drug-protein adducts in vivo may have important clinical and toxicological implications. Consequently, there is a great interest in the detection of these adducts and the elucidation of their role in the processes leading to adverse and idiosyncratic drug reactions. Enzymatic digestion is a crucial step in bottom-up proteomics strategies for the analysis of drug-protein adducts. The chosen proteolytic enzyme and digestion conditions have a large influence on the protein coverage of the modified protein and identification of its modification site. In this work, the enzymatic digestion conditions (pH, temperature and time) of trypsin and thermolysin were optimized specifically for the characterization of Human Serum Albumin (HSA) adducts. Using a Design of Experiments (DOE), it was found that of the three optimized parameters mainly pH and temperature showed strong effects on both responses. The optimized digestion conditions were different from those obtained from the suppliers or literature. Their application to HSA adducts resulted in improved protein coverage and signal intensity regarding the peptide containing the modification site, thereby highlighting the importance of a detailed optimization of digestion conditions.

Derivatization of carboxylic acids with 4-APEBA for detection by positive-ion LC-ESI-MS(/MS) applied for the analysis of prostanoids and NSAID in urine.

In order to develop a generic positive ionization ESI LC-MS method for a variety of interesting substance classes, a new derivatization strategy for carboxylic acids was developed. The carboxylic acid group is labeled with the bromine containing 4-APEBA reagent based on carbodiimide chemistry. The derivatization reaction can be carried out under aqueous conditions, thereby greatly simplifying sample preparation. In this paper, the derivatization of carboxylic acids is exemplified for the determination of prostanoids and non-steroidal anti-inflammatory drugs (NSAID). Optimization of the derivatization conditions was studied. In order to prove the applicability of the presented approach, we applied the described protocol to urine samples from complex regional pain syndrome (CRPS) patients and were able to detect several prostanoids not visible in the urine of healthy volunteers. Further, the determination of the non-steroidal anti-inflammatory drug ibuprofen in a urine sample was possible.