Untargeted metabolomics in doping control: detection of new markers of testosterone misuse by ultrahigh performance liquid chromatography coupled to high-resolution mass spectrometry.

The use of untargeted metabolomics for the discovery of markers is a promising and virtually unexplored tool in the doping control field. Hybrid quadrupole time-of-flight (QTOF) and hybrid quadrupole Orbitrap (Q Exactive) mass spectrometers, coupled to ultrahigh pressure liquid chromatography, are excellent tools for this purpose. In the present work, QTOF and Q Exactive have been used to look for markers for testosterone cypionate misuse by means of untargeted metabolomics. Two different groups of urine samples were analyzed, collected before and after the intramuscular administration of testosterone cypionate. In order to avoid analyte losses in the sample treatment, samples were just 2-fold diluted with water and directly injected into the chromatographic system. Samples were analyzed in both positive and negative ionization modes. Data from both systems were treated under untargeted metabolomic strategies using XCMS application and multivariate analysis. Results from the two mass spectrometers differed in the number of detected features, but both led to the same potential marker for the particular testosterone ester misuse. The in-depth study of the MS and MS/MS behavior of this marker allowed for the establishment of 1-cyclopentenoylglycine as a feasible structure. The putative structure was confirmed by comparison with synthesized material. This potential marker seems to come from the metabolism of the cypionic acid release after hydrolysis of the administered ester. Its suitability for doping control has been evaluated.

Understanding associations between antimicrobial agents usage and antimicrobial resistance genes prevalence at the community level using wastewater-based epidemiology: A Spanish pilot study.

Understanding the development and spread of antimicrobial resistance (AMR) is important for combating this global threat for public health. Wastewater-based epidemiology (WBE) is a complementary approach to current surveillance programs that minimizes some of the existing limitations. The aim of the present study is to explore WBE for monitoring antibiotics and antibiotic resistance genes (ARGs) in wastewater samples collected during 2021/2022 from the city of Castellon (Spain). Eighteen commonly prescribed antibiotics have been selected and measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with triple quadrupole mass analysers. Moreover, qPCR for specific ARGs has been performed to obtain information of these genes in co-presence with antibiotics. All selected ARGs, along with a total of 11 antibiotics, were identified. The highest population-normalized daily loads were observed for the macrolide azithromycin, followed by the quinolones ciprofloxacin and levofloxacin. Subsequently, daily consumption estimates based on wastewater data were compared with prescription data of antibiotics. Statistical analyses were conducted to explore if there is correlation between antibiotics and ARGs. While no correlations were found between antibiotics and their corresponding ARGs, certain correlations (p < 0.05) were identified among non-corresponding ARGs. In addition, a strong positive correlation was found between the sum of all antibiotics and the intl1 gene. Moreover, population-normalized ARG loads significantly correlate with the 16S rRNA-normalized ARG loads, serving as an indicator for population size. Results provide a baseline for future work and a proof-of-concept emphasising the need for future work and long-term surveillance, and highlight the need of similar programs at a regional and global levels worldwide.

Comprehensive study on the potential environmental risk of temporal antibiotic usage through wastewater discharges.

Antibiotic residues can reach aquatic ecosystems through urban wastewater discharges, posing an ecotoxicological risk for aquatic organisms and favoring the development of bacterial resistance. To assess the emission rate and hazardousness of these compounds, it is important to carry out periodic chemical monitoring campaigns that provide information regarding the actual performance of wastewater treatment plants (WWTPs) and the potential impact of the treated wastewater in the aquatic environment. In this study, 18 of the most widely consumed antibiotics in Spain were determined by liquid chromatography-tandem mass spectrometry in both influent (IWW) and effluent wastewater (EWW) samples collected over four seasons along 2021-2022. Eleven antibiotics were detected in EWW with azithromycin, ciprofloxacin and levofloxacin showing the highest concentration levels (around 2 µg L-1 of azithromycin and 0.4 µg L-1 of quinolone compounds). Data showed that only 4 out of the 11 compounds were removed by more than 50 % in the WWTP, with sulfamethoxazole standing out with an average removal efficiency >80 %. The risk that treated water could pose to the aquatic environment was also assessed, with 6 compounds indicating a potential environmental risk by exceeding established ecotoxicological and resistance thresholds. Based on the risk assessment, the WWTP removal efficiency required to reduce such risk for antibiotics was estimated. In addition, pooled wastewater samples were screened by LC coupled to high resolution mass spectrometry with ion mobility separation, searching for metabolites and transformation products of the antibiotics investigated to widen future research. Studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and risk prevention monitoring programs.

Application of catalytic ozonation using Y zeolite in the elimination of pharmaceuticals in effluents from municipal wastewater treatment plants.

Catalytic ozonation using faujasite-type Y zeolite with two different SiO2/Al2O3 molar ratios (60 and 12) was evaluated for the first time in the removal of 25 pharmaceutical compounds (PhCs) present in real effluents from two municipal wastewater treatment plants both located in the Mediterranean coast of Spain. Additionally, control experiments including adsorption and direct ozonation, were conducted to better understand the fundamental aspects of the different individual systems in wastewater samples. Commercial zeolites were used in sodium form (NaY). The results showed that the simultaneous use of ozone and NaY zeolites significantly improved the micropollutants degradation rate, able to degrade 95 % of the total mixture of PhCs within the early 9 min using the zeolite NaY-12 (24.4 mg O3 L-1 consumed), while 12 min of reaction with the zeolite NaY-60 (31 mg O3 L-1 consumed). In the case of individual experiments, ozonation removed 95 % of the total mixture of PhCs after 25 min (46.2 mg O3 L-1 consumed), while the direct adsorption, after 60 min of contact time, eliminated 30 % and 44 % using the NaY-12 and NaY-60 zeolites, respectively. Results showed that the Brønsted acid sites seemed to play an important role in the effectiveness of the treatment with ozone. Finally, the environmental assessment showed that the total risk quotients of pharmaceuticals were reduced between 87 %-99 % after ozonation in the presence of NaY-60 and NaY-12 zeolites. The results of this study demonstrate that catalytic ozonation using NaY zeolites as catalysts is a promising alternative for micropollutant elimination in real-world wastewater matrices.

Analytical key issues and challenges in the LC-MS/MS determination of antibiotics in wastewater.

The research on antibiotics occurrence in the aquatic environment has become a hot topic in the last years due to their potential negative effects, associated to possible bacterial antibiotic-resistance, after continuous exposure to these compounds. Most of antibiotic residues are not completely removed in the wastewater treatment plants (WWTPs) and end up in the aquatic environment through treated wastewater (WW). The development of reliable analytical methodologies for the determination of antibiotics in influent (IWW) and effluent wastewater (EWW) is needed with different purposes, among others: monitoring their occurrence in the aquatic environment, performing environmental risk assessment, estimating removal efficiencies of WWTPs, or estimating the consumption of these compounds. In this paper, we perform an in-depth investigation on analytical key issues that pose difficulties in the determination of antibiotics in complex matrices, such as WW, and we identify challenges to be properly addressed for successful analysis. The analytical technique selected was liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), as it is the most powerful and widely applied at present for antibiotic residues determination. The mass spectrometric behavior of 18 selected antibiotics, the chromatographic performance, ion ratio variations associated to the sample matrix when using different precursor ions or protomers, and the macrolides adsorption to glass vial, were some of the issues studied in this work. On the basis of the detailed study performed, an analytical LC-MS/MS method based on sample direct injection has been developed for quantification of 18 antibiotics in IWW and EWW, allowing their determination at low ng L-1 levels.

Use of soft and hard ionization techniques for elucidation of unknown compounds by gas chromatography/time-of-flight mass spectrometry.

Investigation of trace-level non-target compounds by gas chromatography/mass spectrometry (GC/MS) often is a challenging task that requires powerful software tools to detect the unknown components, to obtain the deconvoluted mass spectra, and to interpret the data if no acceptable library match is obtained. In this paper, the complementary use of electron ionization (EI) and chemical ionization (CI) is investigated in combination with GC/time-of-flight (TOF) MS for the elucidation of organic non-target (micro)contaminants in water samples. Based on accurate mass measurement of the molecular and fragment ions from the TOF MS, empirical formulae were calculated. Isotopic patterns, carbon number prediction filter and nitrogen rule were used to reduce the number of possible formulae. The candidate formulae were searched in databases to find possible chemical structures. Selection from possible structure candidates was achieved using information on substructures and observed neutral losses derived from the fragment ions. Four typical examples (bifenazate, boscalid, epoxiconazole, and fenhexamid) are used to illustrate the methodology applied and the various difficulties encountered in this process. Our results indicate that elucidation of unknowns cannot be achieved by following a standardized procedure, as both expertise and creativity are necessary in the process.

Rapid and sensitive analytical method for the determination of amoxicillin and related compounds in water meeting the requirements of the European union watch list.

The presence of antibiotics in the aquatic environment is becoming one of the main research focus of scientists and policy makers. Proof of that is the inclusion of four antibiotics, amongst which is amoxicillin, in the EU Watch List (WL) (Decision 2020/1161/EU)) of substances for water monitoring. The accurate quantification of amoxicillin in water at the sub-ppb levels required by the WL is troublesome due to its physicochemical properties. In this work, the analytical challenges related to the determination of amoxicillin, and six related penicillins (ampicillin, cloxacillin, dicloxacillin, penicillin G, penicillin V and oxacillin), have been carefully addressed, including sample treatment, sample stability, chromatographic analysis and mass spectrometric detection by triple quadrupole. Given the low recoveries obtained using different solid-phase extraction cartridges, we applied the direct injection of water samples using a reversed-phase chromatographic column that allowed working with 100% aqueous mobile phase. Matrix effects were evaluated and corrected using the isotopically labelled internal standard or correction factors based on signal suppression observed in the analysis of spiked samples. The methodology developed was satisfactorily validated at 50 and 500 ng L - 1 for the seven penicillins studied, and it was applied to different types of water matrices, revealing the presence of ampicillin in one surface water sample and cloxacillin in three effluent wastewater samples.

How Lineage Tracing Studies Can Unveil Tumor Heterogeneity in Breast Cancer.

Lineage tracing studies have become a well-suited approach to reveal cellular hierarchies and tumor heterogeneity. Cellular heterogeneity, particularly in breast cancer, is still one of the main concerns regarding tumor progression and resistance to anti-cancer therapies. Here, we review the current knowledge about lineage tracing analyses that have contributed to an improved comprehension of the complexity of mammary tumors, highlighting how targeting different mammary epithelial cells and tracing their progeny can be useful to explore the intra- and inter-heterogeneity observed in breast cancer. In addition, we examine the strategies used to identify the cell of origin in different breast cancer subtypes and summarize how cellular plasticity plays an important role during tumorigenesis. Finally, we evaluate the clinical implications of lineage tracing studies and the challenges remaining to address tumor heterogeneity in breast cancer.

The relevant role of ion mobility separation in LC-HRMS based screening strategies for contaminants of emerging concern in the aquatic environment.

Ion mobility separation (IMS) coupled to high resolution mass spectrometry (IMS-HRMS) is a promising technique for (non-)target/suspect analysis of micropollutants in complex matrices. IMS separates ionized compounds based on their charge, shape and size facilitating the removal of co-eluting isomeric/isobaric species. Additionally, IMS data can be translated into collision cross-section (CCS) values, which can be used to increase the identification reliability. However, IMS-HRMS for the screening of contaminants of emerging concern (CECs) have been scarcely explored. In this study, the role of IMS-HRMS for the identification of CECs in complex matrices is highlighted, with emphasis on when and with which purpose is of use. The utilization of IMS can result in much cleaner mass spectra, which considerably facilitates data interpretation and the obtaining of reliable identifications. Furthermore, the robustness of IMS measurements across matrices permits the use of CCS as an additional relevant parameter during the identification step even when reference standards are not available. Moreover, an effect on the number of true and false identifications could be demonstrated by including IMS restrictions within the identification workflow. Data shown in this work is of special interest for environmental researchers dealing with the detection of CECs with state-of-the-art IMS-HRMS instruments.

Ecological risk assessment of pesticides in the Mijares River (eastern Spain) impacted by citrus production using wide-scope screening and target quantitative analysis.

The widespread use of pesticides, especially in agricultural areas, makes necessary to control their presence in surrounding surface waters. The current study was designed to investigate the occurrence and ecological risks of pesticides and their transformation products in a Mediterranean river basin impacted by citrus agricultural production. Nineteen sites were monitored in three campaigns distributed over three different seasons. After a qualitative screening, 24 compounds was selected for subsequent quantitative analysis. As expected, the lower section of the river was most contaminated, with total concentration >5 µg/L in two sites near to the discharge area of wastewater treatment plants. The highest concentrations were found in September, after agricultural applications and when the river flow is reduced. Ecological risks were calculated using two mixture toxicity approaches (Toxic Unit and multi-substance Potentially Affected Fraction), which revealed high acute and chronic risks of imidacloprid to invertebrates, moderate-to-high risks of diuron, simazine and 2,4-D for primary producers, and moderate-to-high risks of thiabendazole for invertebrates and fish. This study shows that intensive agricultural production and the discharge of wastewater effluents containing pesticide residues from post-harvest citrus processing plants are threatening freshwater biodiversity. Further actions are recommended to control pesticide use and to reduce emissions.

The embodiment of wastewater data for the estimation of illicit drug consumption in Spain.

Data obtained from wastewater analysis can provide rapid and complementary insights in illicit drug consumption at community level. Within Europe, Spain is an important country of transit of both cocaine and cannabis. The quantity of seized drugs and prevalence of their use rank Spain at the top of Europe. Hence, the implementation of a wastewater monitoring program at national level would help to get better understanding of spatial differences and trends in use of illicit drugs. In this study, a national wastewater campaign was performed for the first time to get more insight on the consumption of illicit drugs within Spain. The 13 Spanish cities monitored cover approximately 6 million inhabitants (12.8% of the Spanish population). Untreated wastewater samples were analyzed for urinary biomarkers of amphetamine, methamphetamine, MDMA, cocaine, and cannabis. In addition, weekend samples were monitored for 17 new psychoactive substances. Cannabis and cocaine are the most consumed drugs in Spain, but geographical variations showed, for instance, comparatively higher levels of methamphetamine in Barcelona and amphetamine in Bilbao, with about 1-fold higher consumption of these two substances in such metropolitan areas. For amphetamine, an enantiomeric profiling was performed in order to assure the results were due to consumption and not to illegal dumping of production residues. Furthermore, different correction factors for the excretion of cannabis were used to compare consumption estimations. All wastewater results were compared with previously reported data, national seizure data and general population survey data, were a reasonable agreement was found. Daily and yearly drug consumption were extrapolated to the entire Spanish population with due precautions because of the uncertainty associated. These data was further used to estimate the retail drug market, where for instance cocaine illicit consumption alone was calculated to contribute to 0.2-0.5% of the Spanish gross domestic product (ca. 3000-6000 million Euro/year).

Occurrence and ecological risks of pharmaceuticals in a Mediterranean river in Eastern Spain.

Pharmaceuticals are biologically active molecules that may exert toxic effects to a wide range of aquatic organisms. They are considered contaminants of emerging concern due to their common presence in wastewaters and in the receiving surface waters, and the lack of specific regulations to monitor their environmental occurrence and risks. In this work, the environmental exposure and risks of pharmaceuticals have been studied in the Mijares River, Eastern Mediterranean coast (Spain). A total of 57 surface water samples from 19 sampling points were collected in three monitoring campaigns between June 2018 and February 2019. A list of 40 compounds was investigated using a quantitative target UHPLC-MS/MS method. In order to complement the data obtained, a wide-scope screening of pharmaceuticals and metabolites was also performed by UHPLC-HRMS. The ecological risks posed by the pharmaceutical mixtures were evaluated using species sensitivity distributions built with chronic toxicity data for aquatic organisms. In this study, up to 69 pharmaceuticals and 9 metabolites were identified, out of which 35 compounds were assessed using the quantitative method. The highest concentrations in water corresponded to acetaminophen, gabapentin, venlafaxine, valsartan, ciprofloxacin and diclofenac. The compounds that were found to exert the highest toxic pressure on the aquatic ecosystems were principally analgesic/anti-inflammatory drugs and antibiotics. These were: phenazone > azithromycin > diclofenac, and to a lower extent norfloxacin > ciprofloxacin > clarithromycin. The monitored pharmaceutical mixtures are expected to exert severe ecological risks in areas downstream of WWTP discharges, with the percentage of aquatic species affected ranging between 65% and 82% in 3 out of the 19 evaluated sites. In addition, five antibiotics were found to exceed antibiotic resistance thresholds, thus potentially contributing to resistance gene enrichment in environmental bacteria. This work illustrates the wide use and impact of pharmaceuticals in the area under study, and the vulnerability of surface waters if only conventional wastewater treatments are applied. Several compounds included in this study should be incorporated in future water monitoring programs to help in the development of future regulations, due to their potential risk to the aquatic environment.

Searching for anthropogenic contaminants in human breast adipose tissues using gas chromatography-time-of-flight mass spectrometry.

The potential of gas chromatography-time-of-flight mass spectrometry (GC-TOF MS) for screening anthropogenic organic contaminants in human breast adipose tissues has been investigated. Initially a target screening was performed for a list of 125 compounds which included persistent halogen pollutants [organochlorine (OC) pesticides, polychlorinated biphenylss (PCBs), polybrominated diphenyl ethers (PBDEs)], polyaromatic hydrocarbons (PAHs), alkylphenols, and a notable number of pesticides from the different fungicide, herbicide and insecticide families. Searching for target pollutants was done by evaluating the presence of up to five representative ions for every analyte, all measured at accurate mass (20-mDa mass window). The experimental ion abundance ratios were then compared to those of reference standards for confirmation. Sample treatment consisted of an extraction with hexane and subsequent normal-phase (NP) High performance liquid chromatography (HPLC) or SPE cleanup. The fat-free LC fractions were then investigated by GC-TOF MS.Full-spectral acquisition and accurate mass data generated by GC-TOF MS also allowed the investigation of nontarget compounds using appropriate processing software to manage MS data. Identification was initially based on library fit using commercial nominal mass libraries. This was followed by comparing the experimental accurate masses of the most relevant ions with the theoretical exact masses with calculations made using the elemental composition calculator included in the software.The application of both target and nontarget approaches to around 40 real samples allowed the detection and confirmation of several target pollutants including p,p'-DDE, hexachlorobenzene (HCB), and some polychlorinated biphenyls (PCBs) and polyaromatic hydrocarbons (PAHs). Several nontarget compounds that could be considered anthropogenic pollutants were also detected. These included 3,5-di-tert-butyl-4-hydroxy-toluene (BHT) and its metabolite 3,5-di-tert-butyl-4-hydroxybenzaldehyde (BHT-CHO), dibenzylamine, N-butyl benzenesulfonamide (N-BBSA), some naphthalene-related compounds and several PCBs isomers not included in the target list. As some of the compounds detected are xenoestrogens, the methodology developed in this paper could be useful in human breast cancer research.

GC-MS/MS multi-residue method for the determination of organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers in human breast tissues.

In this work, a multiresidue method for the quantification and confirmation of around 30 organohalogenated compounds in human breast tissue samples has been developed. Analytes tested included organochlorine (OC) (pesticides and polychlorinated biphenyls) and organobromine (OBr) (polybrominated diphenyl ether) compounds. The approach is based on a simple extraction with hexane, followed by a SPE clean-up using silica cartridges and final measurement by GC coupled to triple quadrupole MS. Analyses were performed in both ionizations, electron impact (EI) (selected reaction monitoring (SRM) mode) and negative chemical ionization (NCI) (selected ion recording (SIR) mode). Three isotopically labeled standards were added before extraction and used as surrogates: HCB-13C6, lindane-D6 and p,p'-DDE-D8. The method was validated in terms of accuracy, precision, LOQ and LOD and confirmation reliability, using breast tissue spiked at three concentration levels in the range 1-100 ng/g for OC compounds and at two levels 0.1 and 10 ng/g for OBr compounds (0.5 and 50 ng/g for BDE 209). The usefulness of the developed method was tested by the analysis of real human samples, giving as a result the detection of several OC and OBr compounds in different samples analyzed. The acquisition of at least two SRM transitions (in EI) or ions (in NCI) per analyte allowed positive findings to be confirmed by accomplishment of ion ratios between the quantification and the confirmation transitions or ions.

Methodical approach for the use of GC-TOF MS for screening and confirmation of organic pollutants in environmental water.

The potential of gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF MS) for the screening of organic pollutants in water was explored. After a conventional SPE step with C(18) cartridges, the comparison of spectra with available libraries together with an evaluation of the mass accuracy was the first approach used for the screening and confirmation of target analytes. However, at low analyte concentrations (i.e. below 0.1 microg/l), this procedure was not feasible and the use of the application manager TargetLynx was evaluated. This application allows the selection of up to five representative ions per analyte, measured with high mass accuracy, and their intensity ratio evaluation. Ion selection, extraction mass window and concentration levels were found to be the critical parameters. The reference compound used as 'lock mass' was also found to affect to the quality of information obtained in some particular cases. Full spectral acquisition data generated by the TOF MS analyzer allowed investigation of the presence of several analytes in samples in a post-target style, without the need of reanalyze the water samples.Finally, a methodical approach was established for the reliable screening and confirmation of organic pollutants (PAHs, pesticides, octyl/nonyl phenols) in real-world samples, which led to satisfactory results of approximately 0.1 microg/l.

Study of different atmospheric-pressure interfaces for LC-MS/MS determination of acrylamide in water at sub-ppb levels.

A rapid, sensitive and selective method based on LC-MS/MS has been developed for the direct determination of acrylamide residues in water in compliance with the current European Union (EU) 98/83 Drinking Water Directive. Given the high polarity of acrylamide, the application of a rapid on-line solid phase extraction step, commonly used for preconcentrating low analyte levels, was not found to be completely satisfactory. Therefore, an alternative approach based on the use of direct large-volume injection into the LC-MS/MS system has been used. Three atmospheric-pressure interfaces (ESI, APCI and Ion Sabre APCI) were checked to reach the required sensitivity (0.1 microg/l). All three interfaces were tested by analysis of six different water samples (surface water, groundwater, drinking water and three treated water samples) spiked at three concentration levels each (0.1, 1 and 10 microg/l). When using ESI, poor sensitivity and high matrix effects were observed. This situation improved when APCI was used as the interface because no matrix effect was found, although sensitivity was not completely satisfactory. The best results were obtained by interfacing the Ion Sabre APCI; its higher sensitivity for acrylamide (LOD 0.03 microg/l) and the absence of matrix effects recommended its selection. Using this approach, satisfactory recoveries (90-97%) and precision (<12%) were obtained for all water samples studied. Besides, the acquisition of two different MS/MS transitions allowed not only the quantification but also the confirmation of acrylamide in water at concentration levels around 0.1 microg/l.

Efficient approach for the reliable quantification and confirmation of antibiotics in water using on-line solid-phase extraction liquid chromatography/tandem mass spectrometry.

The potential of solid-phase extraction coupled on-line to liquid chromatography/electrospray tandem mass spectrometry (SPE-LC-ESI-MS/MS) has been investigated in this paper for the efficient sensitive quantification and confirmation of 16 antibiotics in water. The list of targeted analytes included 10 quinolones (oxolinic acid (OXO), nalidixic acid (NAL), flumequine (FLU), marbofloxacine (MAR), ofloxacine (OFLO), enrofloxacine (ENR), pefloxacine (PEF), ciprofloxacine (CIP), pipemidic acid (PIPE), norfloxacine (NOR)) and 6 penicillins (penicillin G (PEN), oxacillin (OXA), dicloxacillin (DIC), piperacillin (PIP), cloxacillin (CLO) and ampicillin (AMP)) that were determined in ground and surface water. The procedure is based on the injection of 9.8 mL of sample into the SPE-LC-MS/MS system and the measurement of antibiotics by selected reaction monitoring mode, using a triple quadrupole analyser. The method has been validated at realistic low concentrations that might be present in environmental water, i.e. 10 and 100 ng L(-1), obtaining recoveries between 74% and 123% with relative standard deviation lower than 14%. Matrix effects were not relevant in most of cases, except for ampicillin in surface water, where notable signal suppression was observed. The limits of detection were as low as 0.4-4.3 ng L(-1). The method developed allows the rapid screening and quantification of all the analytes selected by acquiring one MS/MS transition (normally the most sensitive) for each compound. It was applied to a number of actual surface and groundwater samples with several compounds being detected, mainly quinolones, at low ng L(-1) levels. Special attention was given to the confirmation of compounds detected in water due to the difficulties of obtaining confident confirmation at low ng L(-1). This matter has been of growing concern in the last few years as reflected by recent papers and correspondence. The acquisition of several MS/MS transitions for each compound detected in a second independent analysis allowed the unequivocal confirmation of identity, avoiding reporting false-positives. Finally, the potential of QTOF instruments to confirm positive samples has also been evaluated and compared with triple quadrupole analysers.

Comprehensive monitoring of organic micro-pollutants in surface and groundwater in the surrounding of a solid-waste treatment plant of Castellón, Spain.

The solid-waste treatment plant of RECIPLASA is located in the municipality of Onda (Castellón province), which is an important agricultural area of Spain, with predominance of citrus crops. In this plant, all urban solid wastes from the town of Castellón (around 200,000 inhabitants) and other smaller towns as Almassora, Benicàssim, Betxí, Borriana, L'Alcora, Onda and Vila-Real are treated. In order to evaluate the potential impact of this plant on the surrounding water, both surface and groundwater, a comprehensive monitoring of organic pollutants has been carried out along 2011, 2012 and 2013. To this aim, an advanced analytical strategy was applied for wide-scope screening, consisting on the complementary use of liquid chromatography (LC) and gas chromatography (GC) coupled to mass spectrometry (MS) with quadrupole (Q)-time of flight analyser (TOF). A generic solid-phase extraction with Oasis HLB cartridges was applied prior to the chromatographic analysis. The screening included more than 1500 organic pollutants as target compounds, such as pesticides, pharmaceuticals, veterinary drugs, drugs of abuse, UV-filters, polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), among others. Pesticides, mainly herbicides, were the compounds more frequently detected. Other compounds as antioxidants, cosmetics, drugs of abuse, PAHs, pharmaceuticals and UV filters, were also identified in the screening though at much lower frequency. Once the screening was made, quantitative analysis focused on the compounds more frequently detected was subsequently applied using LC coupled to tandem MS with triple quadrupole analyser. In this way, up to 24 pesticides and transformation products (TPs), 7 pharmaceuticals, one drug of abuse and its metabolite could be quantified at sub-ppb concentrations. Along the three years of study, ten compounds were found at concentrations higher than 0.1µg/L. Most of them were pesticides and TPs, a fact that illustrates that the main source of pollution seems to be the agricultural activities in this area.

Identification of substances migrating from plastic baby bottles using a combination of low-resolution and high-resolution mass spectrometric analysers coupled to gas and liquid chromatography.

This work presents a strategy for elucidation of unknown migrants from plastic food contact materials (baby bottles) using a combination of analytical techniques in an untargeted approach. First, gas chromatography (GC) coupled to mass spectrometry (MS) in electron ionisation mode was used to identify migrants through spectral library matching. When no acceptable match was obtained, a second analysis by GC-(electron ionisation) high resolution mass spectrometry time of flight (TOF) was applied to obtain accurate mass fragmentation spectra and isotopic patterns. Databases were then searched to find a possible elemental composition for the unknown compounds. Finally, a GC hybrid quadrupole-TOF-MS with an atmospheric pressure chemical ionisation source was used to obtain the molecular ion or the protonated molecule. Accurate mass data also provided additional information on the fragmentation behaviour as two acquisition functions with different collision energies were available (MS(E) approach). In the low-energy function, limited fragmentation took place, whereas for the high-energy function, fragmentation was enhanced. For less volatile unknowns, ultra-high pressure liquid chromatography-quadrupole-TOF-MS was additionally applied. Using a home-made database containing common migrating compounds and plastic additives, tentative identification was made for several positive findings based on accurate mass of the (de)protonated molecule, product ion fragments and characteristic isotopic ions. Six illustrative examples are shown to demonstrate the modus operandi and the difficulties encountered during identification. The combination of these techniques was proven to be a powerful tool for the elucidation of unknown migrating compounds from plastic baby bottles.

Potential of capillary-column-switching liquid chromatography-tandem mass spectrometry for the quantitative trace analysis of small molecules. Application to the on-line screening of drugs in water.

We have investigated the potential of capillary-column-switching liquid chromatography coupled to tandem mass spectrometry (cLC-MS-MS) for the quantitative on-line trace analysis of target compounds in aqueous solutions. The technical design of the nano-scale cLC system developed at our Institute for peptide and protein identification has been tested and evaluated for the direct trace analysis of drugs in water samples. Sulphametoxazole, bezafibrate, metoprolol, carbamazepine and bisoprolol occurring frequently in Dutch waters, were selected as test compounds. Adequate conditions for trapping, elution and MS-MS detection were investigated by employing laboratory made 200 microm i.d. capillary columns packed with 5 microm aqua C18 material. In the final cLC-MS-MS conditions, a 1 cm length trapping column and a 4 cm length analytical column were selected. Under these conditions, the target compounds could be directly determined in water down to a level of around 50 ng/l employing only 25 microl of water sample. Validation was done by recovery experiments in ground-, surface- and drinking-water matrices as well as by the analysis of water samples with incurred residues and previously analyzed with a conventional procedure involving off-line solid-phase extraction and narrow-bore LC with MS-MS detection. The new methodology provided recoveries (50-500 ng/l level) between 50 and 114% with RSDs (n = 3, each level) below 20% for most of the compounds. Despite the somewhat less analytical performance in comparison to the conventional procedure, the on-line approach of the new methodology is very suitable for screening of drugs in aqueous samples.