Cristian Gómez-Canela

Liquid chromatography coupled to tandem mass spectrometry and high resolution mass spectrometry as analytical tools to characterize multi-class cytostatic compounds

Cytostatic compounds used in the treatment of cancer have emerged as a new generation of water contaminants due to the continuous amounts administered to patients and to the fact that a variable proportion is excreted unchanged. In this study, we have evaluated the performance of liquid-chromatography-tandem mass spectrometry (LC-MS/MS) and high resolution mass spectrometry using an Orbitrap analyzer (LC-HRMS) for the multiresidue determination of multi-class cytostatic compounds. In a first step, ionization conditions were tested in positive electrospray mode and optimum fragmentation patterns were determined. For LC-MS/MS, two selected reaction monitoring (SRM) transitions were optimized and for LC-HRMS, the molecular ion with 5 ppm error and two product ions were defined. Following, the chromatographic conditions were optimized considering that compounds analyzed have a very different chemical structure and chromatographic behavior. The best performance was obtained with a Luna C18 column, which permitted the separation of the 26 compounds in 15 min. Finally, the performance of LC-MS/MS and LC-HRMS was compared in terms of linearity, sensitivity, intra and inter-day precision and overall robustness. While LC-MS/MS provided good identification capabilities due to selective SRM transitions, LC-Orbitrap proved to be 100 times more sensitive. This study provides a comprehensive overview on the MS conditions to determine the outmost used cytostatic compounds and provides a spectral library to be used for the identification of these compounds in water or biological matrices.

Occurrence and sources of brominated and organophosphorus flame retardants in dust from different indoor environments in Barcelona, Spain

In this study, the simultaneous presence of eight polybrominated diphenyl ethers (PBDEs), nine new brominated flame retardants (NBFRs) and ten organophosphorus flame retardants (OPFRs) was investigated in dust samples collected from different indoor environments (homes, schools, theatres, a university and a Research Institute) in Barcelona, Spain. OPFRs were detected at the highest concentrations followed by PBDEs. ∑OPFRs ranged from 2053 to 72,090ngg(-1) and tris(2-chloroisopropyl) phosphate (TCIPP) was the most abundant compound. BDE-209 was the main PBDE congener detected (up to 14,990ngg(-1)), while other PBDEs ranged from 2.6 to 118ngg(-1). Among the studied NBFRs, decabromodiphenyl ethane (DBDPE - up to 4432ngg(-1)) followed by bis(2-ethylhexyl) tetrabromophthalate (BEH-TEBP - up to 508ngg(-1)) were detected at the highest concentration, whereas a lower detection frequency was observed for 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (EH-TBB), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), pentabromotoluene (PBT) and hexabromobenzene (HBB). The levels and profile of flame retardants (FRs) were characteristic of each environment, where theatres followed by homes presented the highest concentrations and schools had the lowest levels. Principal Component Analysis permitted to identify the main sources and distribution of all FRs, according to specific uses in each environment. The simultaneous presence of all FR families in indoor dust points to the need to monitor these compounds to minimize human exposure.

Predicting concentrations of cytostatic drugs in sewage effluents and surface waters of Catalonia (NE Spain)

Cytostatic drugs, used in chemotherapy, are excreted unchanged by urine and feces or modified as metabolites. Elimination of these drugs in wastewater treatment plants (WWTPs) is often incomplete and residues reach surface water. Their presence in the natural environment depends on consumption patterns, excretion fraction and the effectiveness of the wastewater treatment. This study compiled the total consumption of cytostatic drugs in Catalonia (NE Spain) and provides data on the occurrence and risk of anticancer drugs in the aquatic environment by calculating predicted environmental concentrations (PECs). PECs were estimated using publicly available consumption data in the period of 2010-2012, published or calculated excretion values and wastewater elimination rates for a suite of 132 compounds. This allows predicting the range of concentrations in effluent wastewaters and receiving waters. Out of the 132 cytostatics, mycophenolic acid and hydroxycarbamide had a PEC value higher than 10ngL(-1). PECs were compared with MECs (measured environmental concentrations) to evaluate the reliability of the estimation. A risk assessment was conducted to determine the potential adverse effects of cytostatics in the environment. All the risk quotients calculated using EC50 in Daphnia magna were below 1, showing no significant risk.

Identification of compounds bound to suspended solids causing sub-lethal toxic effects in Daphnia magna. A field study on re-suspended particles during river floods in Ebro River.

Identifying chemicals causing adverse effects in organisms present in water remains a challenge in environmental risk assessment. This study aimed to assess and identify toxic compounds bound to suspended solids re-suspended during a prolonged period of flushing flows in the lower part of Ebro River (NE, Spain). This area is contaminated with high amounts of organochlorine and mercury sediment wastes. Chemical characterization of suspended material was performed by solid phase extraction using a battery of non-polar and polar solvents and analyzed by GC-MS/MS and LC-MS/MS. Mercury content was also determined for all sites. Post-exposure feeding rates of Daphnia magna were used to assess toxic effects of whole and filtered water samples and of re-constituted laboratory water with re-suspended solid fractions. Organochlorine and mercury residues in the water samples increased from upstream to downstream locations. Conversely, toxic effects were greater at the upstream site than downstream of the superfund Flix reservoir. A further analysis of the suspended solid fraction identified a toxic component eluted within the 80:20 methanol:water fraction. Characterization of that toxic component fraction by LC-MS/MS identified the phytotoxin anatoxin-a, whose residue levels were correlated with observed feeding inhibition responses. Further feeding inhibition assays conducted in the lab using anatoxin-a produced from Planktothrix agardhii, a filamentous cyanobacteria, confirmed field results. This study provides evidence that in real field situation measured contaminant residues do not always agree with toxic effects.

Powerful combination of analytical and chemometric methods for the photodegradation of 5-Fluorouracil

&NA; The photodegradation of the antineoplastic drug 5‐fluorouracil (5‐FU) under the action of UV light was studied using UV–vis spectroscopy and High Performance Liquid Chromatography coupled to Diode Array Detector and High Resolution Mass Spectrometry (HPLC‐DAD‐HRMS). To analyze, integrate and interpret the degradation kinetics, the Multivariate Curve Resolution‐Alternating Least Squares (MCR‐ALS) chemometric method has been applied. The high complexity of the photodegradation process of this drug involving up to seven different photoproducts showed the advantages of the proposed approach which provides simultaneously mechanistic and structural information explaining the 5‐FU photodegradability. Moreover, this study provides a new tool to be used for elucidating the photodegradation kinetics at real time. Graphical abstract Figure. No caption available. HighlightsThe photodegradation of the antineoplastic drug 5‐fluorouracil (5‐FU) under the action of UV light was studied.UV–vis spectroscopy and HPLC‐DAD‐HRMS were the analytical techniques used for these purposes.To analyze, integrate and interpret the degradation kinetics, the MCR‐ALS chemometric method was applied.Up to seven photoproducts were detected and identified, as well as their kinetic, chromatographic, UV absorption and MS spectra profiles.

Comprehensive characterization of neurochemicals in three zebrafish chemical models of human acute organophosphorus poisoning using liquid chromatography-tandem mass spectrometry

There is a growing interest in biological models to investigate the effect of neurotransmitter dysregulation on the structure and function of the central nervous system (CNS) at different stages of development. Zebrafish, a vertebrate model increasingly used in neurobiology and neurotoxicology, shares the common neurotransmitter systems with mammals, including glutamate, GABA, glycine, dopamine, norepinephrine, epinephrine, serotonin, acetylcholine, and histamine. In this study, we have evaluated the performance of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the multiresidue determination of neurotransmitters and related metabolites. In a first step, ionization conditions were tested in positive electrospray mode and optimum fragmentation patterns were determined to optimize two selected reaction monitoring (SRM) transitions. Chromatographic conditions were optimized considering the chemical structure and chromatographic behavior of the analyzed compounds. The best performance was obtained with a Synergy Polar-RP column, which allowed the separation of the 38 compounds in 30 min. In addition, the performance of LC-MS/MS was studied in terms of linearity, sensitivity, intra- and inter-day precision, and overall robustness. The developed analytical method was able to quantify 27 of these neurochemicals in zebrafish chemical models for mild (P1), moderate (P2), and severe (P3) acute organophosphorus poisoning (OPP). The results show a general depression of synaptic-related neurochemicals, including the excitatory and inhibitory amino acids, as well as altered phospholipid metabolism, with specific neurochemical profiles associated to the different grades of severity. These results confirmed that the developed analytical method is a new tool for neurotoxicology research using the zebrafish model.

Analysis of neurobehavioural data by chemometric methods in ecotoxicological studies

Incorporation of chemometric tools in behavioural data management workflows allows for the early identification of most relevant endpoints complementarily to statistical confirmatory approaches. In this work, the effects of two model neurotoxicants, chlorpyrifos (CPF) and nicotine, exposures on behavioural profiles of adult zebrafish at three different times (2, 6 and 24h) were evaluated using open field test (OFT) paradigm experiments. Two chemometric methods like Principal Component Analysis (PCA) and Analysis of Variance-Simultaneous Component Analysis (ASCA) have been used to interpret the changes observed in the obtained behavioural data. A decreased of the locomotor activity, an anxiolytic effect and an altered exploratory behaviour were the most affected behavioural endpoints in the CPF exposures. However, an increase of the locomotor activity and an anxiogenic effect were observed in the nicotine exposures. Finally, an excellent correlation between the ASCA results and the results obtained using traditional statistical procedures for both compounds were encountered.

Modelling acrylamide acute neurotoxicity in zebrafish larvae

Acrylamide (ACR), a type-2 alkene, may lead to a synaptopathy characterized by ataxia, skeletal muscles weakness and numbness of the extremities in exposed human and laboratory animals. Currently, only the mildly affected patients undergo complete recovery, and identification of new molecules with therapeutic bioactivity against ACR acute neurotoxicity is urgently needed. Here, we have generated a zebrafish model for ACR neurotoxicity by exposing 5 days post-fertilization zebrafish larvae to 1 mM ACR for 3 days. Our results show that zebrafish mimics most of the pathophysiological processes described in humans and mammalian models. Motor function was altered, and specific effects were found on the presynaptic nerve terminals at the neuromuscular junction level, but not on the axonal tracts or myelin sheath integrity. Transcriptional markers of proteins involved in synaptic vesicle cycle were selectively altered, and the proteomic analysis showed that ACR-adducts were formed on cysteine residues of some synaptic proteins. Finally, analysis of neurotransmitters profile showed a significant effect on cholinergic and dopaminergic systems. These data support the suitability of the developed zebrafish model for screening of molecules with therapeutic value against this toxic neuropathy.

Acrylamide acute neurotoxicity in adult zebrafish

Acute exposure to acrylamide (ACR), a type-2 alkene, may lead to a ataxia, skeletal muscles weakness and numbness of the extremities in human and laboratory animals. In the present manuscript, ACR acute neurotoxicity has been characterized in adult zebrafish, a vertebrate model increasingly used in human neuropharmacology and toxicology research. At behavioral level, ACR-treated animals exhibited “depression-like” phenotype comorbid with anxiety behavior. At transcriptional level, ACR induced down-regulation of regeneration-associated genes and up-regulation of oligodendrocytes and reactive astrocytes markers, altering also the expression of genes involved in the presynaptic vesicle cycling. ACR induced also significant changes in zebrafish brain proteome and formed adducts with selected cysteine residues of specific proteins, some of them essential for the presynaptic function. Finally, the metabolomics analysis shows a depletion in the monoamine neurotransmitters, consistent with the comorbid depression and anxiety disorder, in the brain of the exposed fish.

Development of a vibrational startle response assay for screening environmental pollutants and drugs impairing predator avoidance

The present paper describes the vibrational startle response assay (VSRA), a new robust, simple and automated in vivo medium- to high-throughput procedure for assessment of the escape response and its habituation in zebrafish larvae. Such behaviors enable fish larvae to escape from predator strikes in aquatic ecosystems. The assay is based on measuring the distance moved by each larva during the startle response evoked by repetitive vibrational stimuli. The iterative reduction observed in the response to a series of tapping stimulus in VSRA met the main criteria of habituation. Subsequently, the analysis of concordance using a battery of neuroactive compounds modulating different neurotransmitter systems demonstrated that the results of VSRA are highly predictive of the effects on other vertebrates. Finally, as a proof of concept, VSRA was used to test two relevant environmental pollutants at different concentrations. The results demonstrated that VSRA is suitable for concentration-response analysis of environmental pollutants, opening the possibility to determine the potency and the associated hazard of impaired escape response for the different compounds. Therefore, we suggest that VSRA could be a valuable tool for screening of chemical compounds capable of compromising predator avoidance behavior.