Amphibian skin secretion provides the primary defense for frogs against various threats, including predators and pathogens. The composition of this secretion, which is rich in peptides, is unique to each amphibian species and may serve as a taxonomic biomarker, not only for frog species but also for their populations. In this study, we sequenced the components of the skin peptidome of frogs from the Novosibirsk population of Rana arvalis using a range of tandem mass spectrometry techniques, including CID, HCD, ETD, and EThcD. For the first time, we applied an alternative method for collecting secretion in field conditions, which involved manual stimulation to release the secretion. We also evaluated the applicability of the Novor.Cloud program for automatic peptide sequencing in Ranid frogs, focusing on the Novosibirsk population of R. arvalis. The skin peptidomes of the Novosibirsk, Moscow, and Central Slovenian populations of moor frogs were compared. Brevinin 1AVa, brevinin 1AVb, and the melittin-related peptide FQ-22-1, found in the skin secretion of all three populations, may serve as biomarkers for the moor frog, Rana arvalis.

With the aid of radical and non-radical reactive species (RS), advanced oxidation processes can efficiently degrade emerging organic contaminants including antibiotics but may generate toxic transformation products (TPs). However, the detoxification capacity of popular RS has not been well elucidated. This study compared the detoxification of enrofloxacin (ENR) with three RS-dominated systems: 1O2, SO4•-+•OH, •OH. The toxicity of ENR TPs generated from those systems was evaluated with multiple methods. It was found that the 1O2-dominated system detoxified ENR more effectively than the other systems in terms of microbial respiratory inhibition, developmental toxicity in zebrafish embryos, and three typical molecular biomarkers, including reactive oxygen species (ROS), and lactate dehydrogenase (LDH), and glutathione S-transferase (GST). Based on their chemical structures of ENR TPs projected with UPLC-QTOF-MS/MS, the toxicity prediction tool (T.E.S.T) revealed that the 1O2-dominated system led to more harmless TPs than the others. The results of this study underscore the great potential of 1O2-dominated system in the detoxification of organic contaminants.

Mass spectrometry contributed significantly to the creation of proteomics as a science and is now the principal analytical tool of proteomics. Although the bottom-up approach remains the most popular, more and more studies apply the top-down method for sequencing. Novel tandem mass spectrometry methods resolved numerous sequencing problems and allowed for the analysis of intact peptides and even proteins. Natural frog peptides are convenient models to develop mass spectrometry methods for de novo sequencing of intact biomolecules. Here we demonstrate the ability of ExD tandem mass spectrometry to sequence intact peptides from skin secretions of Pelophylax ridibundus from the Rostov (Russia) population. Studying that secretion, we detected an array of mono- and diformylated skin peptides at various sites of the backbone. The main goal of the study dealt with the determination of the formylation sites of these up to 46-mer long peptides. ExD reliably solved the problem without any additional mass spectrometry experiments or preliminary chemical derivatization. We propose a mechanism for the observed formylation and a biochemical and experimental rationale.

Ornidazole (ONZ), a nitroimidazole antibiotic detected in water bodies, may negatively impact the aquatic ecosystem. Its reaction kinetics during ozonation which is a feasible and applicable technology to control the contamination of emerging contaminants, however, has not been reported in literature. In this study, we measured the apparent second-order kinetic constant of ONZ with ozone molecules via the excessive ozone method and the competing method which led to an average value of 103.8 ± 2.7 M−1 s−1 at pH 7. The apparent second-order kinetic constant of ONZ with HO• was calculated to be 4.65 × 109 M−1 s−1 with the concept of Rct measured via para-chlorobenzoic acid as a probe. The transformation products (TPs) of ONZ during ozonation at pH 3 and pH 11 were separately analyzed with HPLC-MS/MS and some unique products were found at pH 11, reflecting the influence of HO•. The toxicity of individual TPs was predicted with the tool of T.E.S.T. It was found that 62% of 21 identified TPs could be more toxic than ONZ in terms of at least one acute toxicity endpoint, including chlorinated amines and N-oxides. The analysis with a respirometer further revealed that the toxicity of mixing TPs generated at HO• rich conditions was slightly lower than O3 dominated conditions. In general, this study provides the basic kinetic data for designing ozonation processes to eliminate ONZ and the important reference for understanding the toxicity evolution of ONZ during ozonation.

Bromhexine and ambroxol are among the mucolytic drugs most widely used to treat acute and chronic respiratory diseases. Entering the municipal wastewater and undergoing transformations during disinfection with active chlorine, these compounds can produce nitrogen- and bromine-containing disinfection by-products (DBPs) that are dangerous for aquatic ecosystems. In the present study, primary and deep degradation products of ambroxol and bromhexine obtained in model aquatic chlorination experiments were studied via the combination of high-performance liquid and gas chromatography with high-resolution mass spectrometry. It was shown that at the initial stages, the reactions of cyclization, hydroxylation, chlorination, electrophilic ipso-substitution of bromine atoms with chlorine, and oxidative N-dealkylation occur. Along with known metabolites, a number of novel primary DBPs were tentatively identified based on their elemental compositions and tandem mass spectra. Deep degradation of bromhexine and ambroxol gives twenty-four identified volatile and semi-volatile compounds of six classes, among which trihalomethanes account for more than 50%. The specific class of bromhexine- and ambroxol-related DBPs are bromine-containing haloanilines. Seven of them, including methoxy derivatives, were first discovered in the present study. One more novel class of DBPs associated with bromhexine and ambroxol is represented by halogenated indazoles formed through dealkylation of the primary transformation products containing pyrazoline or tetrahydropyrimidine cycle in their structure.

De novo sequencing of any novel peptide/protein is a difficult task. Full sequence coverage, isomeric amino acid residues, inter- and intramolecular S-S bonds, and numerous other post-translational modifications make the investigators employ various chemical modifications, providing a variety of specific fragmentation MSn patterns. The chemical processes are time-consuming, and their yields never reach 100%, while the subsequent purification often leads to the loss of minor components of the initial peptide mixture. Here, we present the advantages of the EThcD method that enables establishing the full sequence of natural intact peptides of ranid frogs in de novo top-down mode without any chemical modifications. The method provides complete sequence coverage, including the cyclic disulfide section, and reliable identification of isomeric leucine/isoleucine residues. The proposed approach demonstrated its efficiency in the analysis of peptidomes of ranid frogs from several populations of Rana arvalis, Rana temporaria, and Pelophylax esculentus complexes.

The microdroplet chemical reactor prototype based on multiplexed electrospray was fabricated. The apparent acceleration factor value for the reaction of phenylhydrazine with cyclohexanone exceeds 83 million, and the productivity can reach grams/hour.

Natural peptides secreted under stress conditions by many organisms are bioactive molecules with a broad spectrum of activities. These molecules could become potential models for novel pharmaceuticals, to which bacteria, according to modern scientific concepts, do not have and cannot develop resistance. Taking this into consideration, it is necessary to clarify the amino acid sequences of such peptides. Here we describe our approach to de novo sequencing of amphibians' skin secretion peptides.

Among numerous disinfection by-products (DBP) forming during aqueous chlorination nitrogen containing species are of special concern due to their toxicological properties. Nevertheless, corresponding reaction products of these natural and anthropogenic compounds are not sufficiently studied so far. An interesting reaction involves dealkylation of the substituted amine moiety. Here we present the results of the comparative study of one-electron oxidation and aqueous chlorination of several aliphatic and aromatic amines. The reaction products were reliably identified with gas chromatography - high resolution mass spectrometry (GC-HRMS), high pressure liquid chromatography - electrospray ionization high resolution mass spectrometry HPLC-ESI/HRMS), and electrochemistry - electrospray ionization high resolution mass spectrometry (EC-ESI/HRMS). Certain similarities dealing with the formation of the corresponding aldehydes and substitution of alkyl groups at the nitrogen atom for hydrogen were shown for the studied processes. The mechanism of the substituted amines' aqueous chlorination involving one-electron oxidation is proposed and confirmed by the array of the observed reaction products. Alternative reactions taking place in conditions of aqueous chlorination, i.e. aromatic electrophilic substitution, may successfully compete with dealkylation and produce major products.

Recent studies have found the presence of surface-bound radicals (Surf-R) during the heterogeneously catalyzed peroxymonosulfate (PMS), especially when the catalyst carriers possess a high adsorption capacity. However, the effects of Surf-R on the toxicity of transformation products (TPs) are still not well elucidated. In this study, Co and Mn were loaded onto commonly used carriers: activated carbon and alumina (CoMn@AC vs CoMn@Al2O3) to create comparable catalytic centers but distinct adsorption capacity, which was proved by material characterization (SEM, EDS, BET, XPS), adsorption and catalytic tests. Both images and quantitative analysis with 3D excitation-emission matrix fluorescence spectroscopy demonstrated that the reaction solutions of enrofloxacin from two catalysts composed differently when a same degradation efficiency was achieved. It was further found that the respiratory inhibition of CoMn@AC catalyzed samples was only 7~36% of the counterpart. 50 TPs were identified with UPLC-MS/MS, among which 31 TPs were possibly more toxic than enrofloxacin according to the calculation with the Toxicity Estimation Software Tool. CoMn@Al2O3 produced 14 unique toxic TPs while CoMn@AC only 4. The analysis with EPR revealed that both catalysts generated SO4•-, •OH, and 1O2; quenching tests proved a lot of Surf-R with CoMn@AC but not with CoMn@Al2O3. This study demonstrates the high potential of Surf-R to enhance the detoxification of organic contaminants during PMS oxidation and thus provides a reference for designing novel catalysts to achieve efficient contaminant degradation and detoxification.

Modern gas chromatography-mass spectrometry (GC–MS) allows for the analysis of complex samples, such as fragrances. However, identifying all the constituents in natural fragrance mixtures, especially allergens that need to be listed on product labels, is a significant challenge. This is primarily due to the high complexity of the sample and the fact that electron ionization, the most commonly used ionization method in GC–MS, produces numerous nonspecific fragment ions, often resulting in the absence or very low abundance of the molecular ion. These factors affect confidence in assigning the analyte. In this study, we demonstrate that the combination of GC × GC separation, with high mass resolution and accurate mass measurements, as well as chemical ionization in addition to traditional electron ionization, becomes an efficient tool for reliable qualitative analysis of a mixture containing 100 fragrance allergens, even when many of them are closely related species or isomers. The proposed approach expands the applicability of the comprehensive GC × GC-HRMS method, which includes complementary ionization techniques, from studies on anthropogenic priority pollutants and emerging contaminants to the analysis of natural products. Although targeted qualitative and quantitative analysis of allergens in the modern laboratories is well organized, GC × GC-HRMS, being a useful complement to routine quality control of volatile allergens in fragrances, definitely gives an additional contribution to the analytical cases when conventional 1D-GC–MS faces some problems or uncertainties.

The skin of amphibians is a rich source of peptides with a wide range of biological activities. They are stored in secretory granules in an inactive form. Upon stimulation, they are secreted together with proteases into the skin. Once activated, they rapidly exert their biological effects, including fighting microorganisms and predators, while their excess is immediately destroyed by the released proteases. To keep bioactive peptides in their initial form, it is necessary to inhibit these enzymes. Several inhibitors for this purpose have previously been mentioned; however, there has not been any reliable comparison of their efficiency so far. Here, we studied the efficiency of methanol and hydrochloric and formic acids, as well as phenylmethylsulfonyl fluoride, in the inhibition of nine frog peptides with the known sequence, belonging to five families in the secretion of Pelophylax esculentus. The results demonstrated that methanol had the highest inhibitory efficiency, while phenylmethylsulfonyl fluoride was the least efficient, probably due to its instability in aqueous media. Possible cleavages between certain amino acid residues in the sequence were established for each of the inhibitors. These results may be helpful for future studies on the nature of proteases and on prediction of the possible cleavage sites in novel peptides.

The purity of drinking water is an important issue of the human life quality. Water disinfection has saved millions people from the diseases spread with water. However, that procedure has a certain drawback due to formation of toxic organic disinfection products. Establishing the structures of these products and the mechanisms of their formation and diminishing their levels in drinking water represent an important task for chemistry and medicine, while mass spectrometry is the most efficient tool for the corresponding studies. The current review throws light upon natural and anthropogenic sources of the formation of disinfection by-products (DBPs) and the mechanisms of their formation related to the structural peculiarities and the presence of functional groups. In addition to chlorination, bromination is discussed since it is used quite often as an alternative method of disinfection, particularly, for the purification of swimming pool water. The benefits of the contemporary GC/MS and LC/MS methods for the elucidation of DBP structures and study of the mechanisms of their formation are discussed. The reactions characteristic for various functional groups and directions of transformation of certain classes of organic compounds in conditions of aqueous chlorination/bromination are also covered in the review.

An approach to the highly sensitive and selective determination of the bromine-containing antiviral drug umifenovir (Arbidol) and its metabolites in natural and waste water, activated sludge, and bottom sediments by chromatography-mass spectrometry based on a combination of two techniques– inductively coupled plasma mass spectrometry (ICP MS) and electrospray ionization high-resolution mass spectrometry (ESI HRMS) is developed. Reversed-phase chromatographic separation and detection based on ESI HRMS provide the reliable detection and identification of analytes in complex matrices, while the use of ICP-MS with 79 Br signal detection makes it possible to exclude matrix effects and use a single analytical standard for quantitative analysis. The use of solid-phase extraction and pressurized liquid extraction as sample preparation methods made it possible to achieve limits of detection at a level of 0.2 ng/L and 2 µg/kg for liquid and solid samples, respectively. The developed approach was successfully tested in the analysis of real samples. It is shown that the concentrations of analytes in urban wastewaters are in the range 4.4–260 ng/L. The highest concentration (up to 3.7 mg/kg) is characteristic of activated sludge, which acts as an effective sorbent for umifenovir and its transformation products.

State-of-the-art technologies based on advanced oxidation processes (AOPs) are currently being developed for the removal of environmental pollutants such as pharmaceuticals from water matrices. Such studies are very important and should include the impact of inorganic and organic matrix on the degradation process of pollutants. One of the AOPs that is in focus of investigations nowadays, is ionizing radiation. Doxazosin (DOX) is a widely used pharmaceutical for the treatment of hypertension, which can be potentially harmful due to its occurrence in the aquatic environment. This study reports the radiation-induced degradation of DOX in aqueous solution. Removal of 10 mg L-1 DOX reached almost 100 % at irradiation doses of 200 Gy regardless of the dose rate used. The effect of saturated solutions with N2, N2O, air and the addition of radical scavengers such as 2PrOH and thiourea on DOX degradation was investigated. The efficiency of degradation increased in the order: thiourea < 2-PrOH < N2 < air < N2O. The effects of pH, various inorganic ions and water matrix on DOX degradation were also studied. DOX degradation was lower in underground water than in ultrapure water. Under prolonged irradiation, mineralization of about 60 % of DOX solutions was observed based on the dissolved organic carbon (DOC) evaluation. Toxicity tests with V. fischeri luminescent bacteria showed higher toxicity of samples irradiated at 500 Gy. The main degradation products were identified using LC-HRMS and degradation pathways were proposed. Overall, irradiation technology could be a promising technique for the removal of micropollutants, such as pharmaceuticals in real water matrices.

Amphibians are one of the oldest classes of vertebrates inhabiting the Earth. Meanwhile, the only weapon their majority possess involves exclusively peptide cocktail secreted by their skin glands in stress conditions. These peptides are considered potential pharmaceuticals of future generations as the mechanism of their interaction with pathogenic microbiota differs greatly from that of the modern antibiotics. High resolution tandem mass spectrometry showed itself as the most powerful tool to uncover the primary structure (sequence) of peptides. In the present study that analytical tool was used to establish skin peptidome of the Rana temporaria frog population from Arkhangelsk, Russia. Thirty-four peptides were sequenced while five of them turned out to be novel. The established peptidome was compared with those of studied earlier populations of Rana temporaria specie (Moscow, central Slovenian, and Italian). Temporins were proposed to be the most variable peptide family responsible for the adaptation of Rana temporaria frog to the conditions of their habitat. The EThcD MS3 method of fragmentation demonstrated its excellent ability to deal with intact disulfide-containing peptides, short peptides capable of cyclizing in conditions of MS/MS experiment, and isomeric Leu/Ile residues.

A large variety of 1,2,3-thiadiazoles and 1,2,3-triazoles are used extensively in modern pure and applied organic chemistry as important structural blocks of numerous valuable products. Creation of new methods of synthesis of these isomeric compounds requires the development of reliable analytical tools to reveal the structural characteristics of these novel compounds, which are able to distinguish between isomers. Mass spectrometry (MS) is a clear choice for this task due to its selectivity, sensitivity, informational capacity, and reliability. Here, the application of electrospray ionization (ESI) with ion detection in positive and negative modes was demonstrated to be useful in structural studies. Additionally, interconversion of isomeric 4,5-functionalized 1,2,3-triazoles and 1,2,3-thiadiazoles was demonstrated. Application of accurate mass measurements and tandem mass spectrometry in MS2 and MS3 modes indicated the occurrence of gas-phase rearrangement of 1,2,3-triazoles into 1,2,3-thiadiazoles under (+)ESI-MS/MS conditions, independent of the nature of substituents, in line with the reaction in the condensed phase. Infrared multiple photon dissociation (IRMPD) spectroscopy enabled the establishment of structures of some of the most crucial common fragment ions, including [M+H-N2]+ and [M+H-N2-RSO2]+ species. The (−)ESI-MS/MS experiments were significantly more informative for the sulfonyl alkyl derivatives compared to the sulfonyl aryl ones. However, there was insufficient evidence to confirm the solution-phase transformation of 1,2,3-thiadiazoles into the corresponding 1,2,3-triazoles.

Peptides released on frogs’ skin in a stress situation represent their only weapon against micro-organisms and predators. Every species and even population of frog possesses its own peptidome being appropriate for their habitat. Skin peptides are considered potential pharmaceuticals, while the whole peptidome may be treated as a taxonomic characteristic of each particular population. Continuing the studies on frog peptides, here we report the peptidome composition of the Central Slovenian agile frog Rana dalmatina population. The detection and top-down de novo sequencing of the corresponding peptides was conducted exclusively by tandem mass spectrometry without using any chemical derivatization procedures. Collision-induced dissociation (CID), higher energy collision-induced dissociation (HCD), electron transfer dissociation (ETD) and combined MS3 method EThcD with stepwise increase of HCD energy were used for that purpose. MS/MS revealed the whole sequence of the detected peptides including differentiation between isomeric Leu/Ile, and the sequence portion hidden in the disulfide cycle. The array of the discovered peptide families (brevinins 1 and 2, melittin-related peptides (MRPs), temporins and bradykinin-related peptides (BRPs)) is quite similar to that of R. temporaria. Since the genome of this frog remains unknown, the obtained results were compared with the recently published transcriptome of R. dalmatina.

This work summarizes our research on synthesis, characterization and toxicity of selected UV-A filters and their antioxidant shield in commercial formulation - resveratrol. Benzophenone type of UV filters react under disinfection conditions with chlorine and form different mono- and dichlorinated products, while dibenzoylmethane derivatives, such as avobenzone, react with chlorine and form two main bridge chlorinated products followed by numerous chlorinated species at the advanced stages of the process. Resveratrol showed three main susceptible centers to chlorination, starting from the electrophilic addition to the double bond and continuing with the chlorination of the phenolic moieties. Several experiments conducted under different disinfection conditions (pool/sea water, addition of salts, irradiation) showed basically similar chlorination patters with some variations in terms of product formation. The results of toxicity assessment using different test organisms (Vibrio fischeri, microalgae, daphnids) have shown different sensitivity of testing organisms to the parent UV filters in comparison with chlorinated products as well as different toxicity for specific UV filter in comparison to the others. As the closing loop of all experiments in the laboratory, an up-scaling to the real human skin is presented.