Evaluation of combined sewer overflow impacts on short-term pharmaceutical and illicit drug occurrence in a heavily urbanised tidal river catchment (London, UK).

The occurrence of pharmaceutical and illicit drug residues potentially arising from combined sewer overflows (CSOs) in the Central London portion of the Thames Estuary is presented. Approximately 39 million tonnes of untreated sewage enter the River Thames at 57 CSO points annually. Differential analysis of influents and effluents in a major wastewater treatment plant identified seven potential drug-related CSO markers based on removal rates. Three were present in influent at concentrations >1 µg L-1 (caffeine, cocaine and benzoylecgonine). During dry weather, analysis of hourly samples of river water revealed relatively consistent concentrations for most drugs, including CSO markers, over a tidal cycle. River water was monitored over a week in January and July and then daily across six consecutive weeks in November/December 2014. Out of 31 compounds monitored, 27 drug residues were determined in the River Thames and, combined, ranged between ~1000-3500 ng L-1. Total drug concentration generally declined during extended periods of drier weather. For CSO markers, short-term increases in caffeine, cocaine and benzoylecgonine concentration were observed ~24 h after CSO events (especially those occurring at low tide) and generally within one order of magnitude. Timings of elevated occurrence also correlated well with ammonium ion and dissolved oxygen data following CSOs. This work also represents an important study of pharmaceutical occurrence before a major 'Super Sewer' infrastructure upgrade in London aiming to reduce CSOs by 95%.

Screening new psychoactive substances in urban wastewater using high resolution mass spectrometry.

Analysis of drug residues in urban wastewater could complement epidemiological studies in detecting the use of new psychoactive substances (NPS), a continuously changing group of drugs hard to monitor by classical methods. We initially selected 52 NPS potentially used in Italy based on seizure data and consumption alerts provided by the Antidrug Police Department and the National Early Warning System. Using a linear ion trap-Orbitrap high resolution mass spectrometer, we designed a suspect screening and a target method approach and compared them for the analysis of 24 h wastewater samples collected at the treatment plant influents of four Italian cities. This highlighted the main limitations of these two approaches, so we could propose requirements for future research. A library of MS/MS spectra of 16 synthetic cathinones and 19 synthetic cannabinoids, for which analytical standards were acquired, was built at different collision energies and is available on request. The stability of synthetic cannabinoids was studied in analytical standards and wastewater, identifying the best analytical conditions for future studies. To the best of our knowledge, these are the first stability data on NPS. Few suspects were identified in Italian wastewater samples, in accordance with recent epidemiological data reporting a very low prevalence of use of NPS in Italy. This study outlines an analytical approach for NPS identification and measurement in urban wastewater and for estimating their use in the population.

Told through the wine: A liquid chromatography-mass spectrometry interplatform comparison reveals the influence of the global approach on the final annotated metabolites in non-targeted metabolomics.

This work focuses on the influence of the selected LC-HRMS platform on the final annotated compounds in non-targeted metabolomics. Two platforms that differed in columns, mobile phases, gradients, chromatographs, mass spectrometers (Orbitrap [Platform#1] and Q-TOF [Platform#2]), data processing and marker selection protocols were compared. A total of 42 wines samples from three different protected denomination of origin (PDO) were analyzed. At the feature level, good (O)PLS-DA models were obtained for both platforms (Q(2)[Platform#1]=0.89, 0.83 and 0.72; Q(2)[Platform#2]=0.86, 0.86 and 0.77 for Penedes, Ribera del Duero and Rioja wines respectively) with 100% correctly classified samples in all cases. At the annotated metabolite level, platforms proposed 9 and 8 annotated metabolites respectively which were identified by matching standards or the MS/MS spectra of the compounds. At this stage, there was no coincidence among platforms regarding the suggested metabolites. When screened on the raw data, 6 and 5 of these compounds were detected on the other platform with a similar trend. Some of the detected metabolites showed complimentary information when integrated on biological pathways. Through the use of some examples at the annotated metabolite level, possible explanations of this initial divergence on the results are presented. This work shows the complications that may arise on the comparison of non-targeted metabolomics platforms even when metabolite focused approaches are used in the identification.

Artificial neural network modelling of pharmaceutical residue retention times in wastewater extracts using gradient liquid chromatography-high resolution mass spectrometry data.

The modelling and prediction of reversed-phase chromatographic retention time (tR) under gradient elution conditions for 166 pharmaceuticals in wastewater extracts is presented using artificial neural networks for the first time. Radial basis function, multilayer perceptron and generalised regression neural networks were investigated and a comparison of their predictive ability for model solutions discussed. For real world application, the effect of matrix complexity on tR measurements is presented. Measured tR for some compounds in influent wastewater varied by >1min in comparison to tR in model solutions. Similarly, matrix impact on artificial neural network predictive ability was addressed towards developing a more robust approach for routine screening applications. Overall, the best neural network had a predictive accuracy of <1.3min at the 75th percentile of all measured tR data in wastewater samples (<10% of the total runtime). Coefficients of determination for 30 blind test compounds in wastewater matrices lay at or above R(2)=0.92. Finally, the model was evaluated for application to the semi-targeted identification of pharmaceutical residues during a weeklong wastewater sampling campaign. The model successfully identified native compounds at a rate of 83±4% and 73±5% in influent and effluent extracts, respectively. The use of an HRMS database and the optimised ANN model was also applied to shortlisting of 37 additional compounds in wastewater. Ultimately, this research will potentially enable faster identification of emerging contaminants in the environment through more efficient post-acquisition data mining.

An on-line solid phase extraction-liquid chromatography-tandem mass spectrometry method for the determination of perfluoroalkyl acids in drinking and surface waters.

An UHPLC-MS/MS multiresidue method based on an on-line solid phase extraction (SPE) procedure was developed for the simultaneous determination of 9 perfluorinated carboxylates (from 4 to 12 carbon atoms) and 3 perfluorinated sulphonates (from 4 to 8 carbon atoms). This work proposes using an on-line solid phase extraction before chromatographic separation and analysis to replace traditional methods of off-line SPE before direct injection to LC-MS/MS. Manual sample preparation was reduced to sample centrifugation and acidification, thus eliminating several procedural errors and significantly reducing time-consuming and costs. Ionization suppression between target perfluorinated analytes and their coeluting SIL-IS were detected for homologues with a number of carbon atoms less than 9, but the quantitation was not affected. Total matrix effect corrected by SIL-IS, inclusive of extraction efficacy, and of ionization efficiency, ranged between -34 and +39%. The percentage of recoveries, between 76 and 134%, calculated in different matrices (tap water and rivers impacted by different pollutions) was generally satisfactory. LODs and LOQs of this on-line SPE method, which also incorporate recovery losses, ranged from 0.2 to 5.0 ng/L and from 1 to 20 ng/L, respectively. Validated on-line SPE-LC/MS/MS method has been applied in a wide survey for the determination of perfluoroalkyl acids in Italian surface and ground waters.

Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry.

Humans can be exposed to mycotoxins through the food chain. Mycotoxins are mainly found as contaminants in food and could be subsequently excreted via biological fluids such as urine or human breast milk in native or metabolised form. Since breast milk is usually supposed as the only food for new-borns, the occurrence of mycotoxins in thirty-five human milk samples was evaluated by a newly developed method based on QuEChERS extraction and UHPLC-HRMS detection. The method described here allows the detection of target mycotoxins in order to determine the quality of this initial feeding. The method has been fully validated, with recoveries ranging from 64% to 93% and relative standard deviations (RSD, %) being lower than 20%. Using the method described, non-metabolised mycotoxins such as ZEA, NEO, NIV, ENA, ENA1, ENB, ENB1 and metabolites, such as ZEA metabolites, HT-2, DOM and T-2 triol were detected in human milk samples. Results obtained help to estimate the exposure of mothers and infants to mycotoxins. Moreover, to the best of our knowledge, this is the first work describing the simultaneous detection, quantification and screening of mycotoxins and their metabolites in human mature milk.

State-of-the-art in fast liquid chromatography-mass spectrometry for bio-analytical applications.

There is an increasing need of new bio-analytical methodologies with enough sensitivity, robustness and resolution to cope with the analysis of a large number of analytes in complex matrices in short analysis time. For this purpose, all steps included in any bio-analytical method (sampling, extraction, clean-up, chromatographic analysis and detection) must be taken into account to achieve good and reliable results with cost-effective methodologies. The purpose of this review is to describe the state-of-the-art of the most employed technologies in the period 2009-2012 to achieve fast analysis with liquid chromatography coupled to mass spectrometry (LC-MS) methodologies for bio-analytical applications. Current trends in fast liquid chromatography involve the use of several column technologies and this review will focus on the two most frequently applied: sub-2µm particle size packed columns to achieve ultra high pressure liquid chromatography (UHPLC) separations and porous-shell particle packed columns to attain high efficiency separations with reduced column back-pressures. Additionally, recent automated sample extraction and clean-up methodologies to reduce sample manipulation, variability and total analysis time in bio-analytical applications such as on-line solid phase extraction coupled to HPLC or UHPLC methods, or the use of other approaches such as molecularly imprinted polymers, restricted access materials, and turbulent flow chromatography will also be addressed. The use of mass spectrometry and high or even ultra-high resolution mass spectrometry to reduce sample manipulation and to solve ion suppression or ion enhancement and matrix effects will also be presented. The advantages and drawbacks of all these methodologies for fast and sensitive analysis of biological samples are going to be discussed by means of relevant applications.

Wide-range screening of banned veterinary drugs in urine by ultra high liquid chromatography coupled to high-resolution mass spectrometry.

In this work, an ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology is proposed for the multi-class multi-residue screening of banned and unauthorized veterinary drugs in bovine urine, using an Orbitrap Exactive™ analyzer working at a resolving power of 50,000 FWHM in full scan, both in positive and negative mode. The method currently covers 87 analytes belonging to different families such as steroid hormones, ß-agonists, resorcylic acid lactones (RAL), stilbens, tranquillizers, nitroimidazoles, corticosteroids, NSAIDs, amphenicoles, thyreostatics and other substances such as dapsone. A database including the elemental composition, the polarity of acquisition, retention time and expected adducts was built for the targeted analysis, and a high mass accuracy (<5 ppm) was set as one of the identification criteria. After comparing different sample preparation procedures, QuEChERS was selected as the most appropriate methodology. An efficient separation of analytes was achieved using ultra high performance liquid chromatography with a column packed with sub-2 µm particles. The performance of the method has been evaluated in accordance with the EU guidelines for the validation of screening methods for the analysis of veterinary drugs residues. The screening target concentrations were established between 0.2 µg/l and 20 µg/l, demonstrating the usefulness of UHPLC-HRMS as an ideal tool for compliance monitoring in regulatory laboratories.

Atmospheric pressure photoionization mass spectrometry of fullerenes.

Atmospheric pressure photoionization (APPI) was evaluated for the analysis of fullerenes. An important response improvement was found when using toluene mediated APPI in negative mode if compared with other atmospheric pressure ionization (API) sources (electrospray and atmospheric pressure chemical ionization). Fullerene APPI negative mass spectra were dominated by the isotopic cluster of the molecular ion, although isotopic patterns for M+1, M+2, and M+3 ions showed higher than expected relative abundances. These discrepancies are explained by the presence of two isobaric ions, one due to (13)C and the other due to the addition of hydrogen to a double bond of the fullerene structure. Triple quadrupole tandem mass spectrometry, ultrahigh resolution mass spectrometry, and accurate mass measurements were used to confirm these assignments. Additionally, cluster ions M+16 and M+32 were characterized following the same strategy. Ions due to the addition of oxygen and alkyl additions were attributed to the presence of methanol in the mobile phase. For the fast chromatographic separation of fullerenes (less than 3.5 min), a sub-2 µm C18 column and isocratic elution (toluene/methanol, 45:55 v/v) was used. Highly selective-selected ion monitoring (H-SIM) mode (mass resolving power, >12,500 fwhm) was proposed monitoring the two most intense isotope ions in the [M](-•) cluster. Method limits of quantitation down to 10 pg L(-1) for C(60) and C(70) fullerenes and between 0.75 and 5.0 ng L(-1) for larger fullerenes were obtained. Finally, the ultrahigh performance liquid chromatography (UHPLC)-APPI-MS method was used to analyze fullerenes in river and pond water samples.

New trends in fast liquid chromatography for food and environmental analysis.

There is an increasing need for applications in food and environmental areas able to cope with a large number of analytes in very complex matrices. The new analytical procedures demand sensitivity, robustness and high resolution within an acceptable analysis time. The purpose of this review is to describe new trends based on fast liquid chromatography applied to the food and environmental analysis. It includes different column technologies, such as monolithic, sub-2 µm, porous shell, as well as different stationary phases such as reversed phase (C8 and C18), hydrophilic interaction liquid chromatography (HILIC) and fluorinated columns. Additionally, recent sample extraction and clean-up methodologies applied to reduce sample manipulation and total analysis time in food and environmental analysis--QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), on line solid phase extraction coupled to ultrahigh pressure liquid chromatography (on line SPE-UHPLC), turbulent flow chromatography (TFC) and molecularly imprinted polymers (MIPs), were also addressed. The advantages and drawbacks of these methodologies applied to the fast and sensitive analyses of food and environmental samples are going to be discussed.

Fast liquid chromatography/tandem mass spectrometry (highly selective selected reaction monitoring) for the determination of toltrazuril and its metabolites in food.

In this work a fast liquid chromatography (LC)-tandem mass spectrometry (MS/MS) method was developed for the analysis of toltrazuril, a coccidiostatic drug, and its metabolites in meat food products. The applicability of atmospheric pressure chemical ionization (APCI) and heated electrospray ionization in both positive and negative modes was studied. APCI in negative mode provided the best results and the base peak originated from the loss of CF(3) (toltrazuril and toltrazuril sulfone) and CHF(3)* (toltrazuril sulfoxide) was used as the precursor ion in MS/MS. A fast LC separation on a C(18) Fused-Core column was used together with the APCI-MS/MS method developed using enhanced mass resolution mode (highly selective selected reaction monitoring, H-SRM) to improve the sensitivity and selectivity for the analysis of these compounds in food samples. A simple sample treatment based on an extraction with acetonitrile and a cleanup with a C(18) cartridge was used. The LC-MS/MS (H-SRM) method showed good precision (relative standard deviation lower than 10%), accuracy, and linearity and allowed the determination of these compounds in food samples down to the parts per billion level (limits of detection between 0.5 and 5 microg kg(-1)).

Fully automated screening of veterinary drugs in milk by turbulent flow chromatography and tandem mass spectrometry.

There is an increasing interest in screening methods for quick and sensitive analysis of various classes of veterinary drugs with limited sample pre-treatment. Turbulent flow chromatography in combination with tandem mass spectrometry has been applied for the first time as an efficient screening method in routine analysis of milk samples. Eight veterinary drugs, belonging to seven different classes were selected for this study. After developing and optimising the method, parameters such as linearity, repeatability, matrix effects and carry-over were studied. The screening method was then tested in the routine analysis of 12 raw milk samples. Even without internal standards, the linearity of the method was found to be good in the concentration range of 50 to 500 microg/L. Regarding repeatability, RSDs below 12% were obtained for all analytes, with only a few exceptions. The limits of detection were between 0.1 and 5.2 microg/L, far below the maximum residue levels for milk set by the EU regulations. While matrix effects--ion suppression or enhancement--are obtained for all the analytes the method has proved to be useful for screening purposes because of its sensitivity, linearity and repeatability. Furthermore, when performing the routine analysis of the raw milk samples, no false positive or negative results were obtained.

Characterisation of a proposed internet synthesis of N,N-dimethyltryptamine using liquid chromatography/electrospray ionisation tandem mass spectrometry.

The psychoactive properties of N,N-dimethyltryptamine (DMT) are known to induce altered states of consciousness in humans. These properties attract great interest from clinical, neuroscientific, clandestine and forensic communities. The Breath of Hope Synthesis was reported on an internet website as a convenient two-step methodology for the preparation of DMT. The analytical characterisation of the first stage was the subject of previous publications by the authors and involved the thermal decarboxylation of tryptophan and the formation of tryptamine. The present study reports on the characterisation of the second step of this procedure which was based on the methylation of tryptamine. This employed methyl iodide and benzyltriethylammonium chloride/sodium hydroxide as a phase transfer catalyst. The reaction product was characterised by liquid chromatography/electrospray ionisation tandem mass spectrometry and orthogonal acceleration time-of-flight mass spectrometry. Quantitative evaluation was carried out in positive multiple reaction monitoring mode (MRM), which included synthesis of the identified reaction products. MRM screening of the product did not lead to the detection of DMT. Instead, 11.1% tryptamine starting material, 21.0% N,N,N-trimethyltryptammonium iodide (TMT) and 47.4% 1-N-methyl-TMT were detected. A 0.5% trace of the monomethylated N-methyltryptamine was also detected. This study demonstrated the impact on product purity of doubtful synthetic methodologies discussed on the internet.

Fingerprint analysis of thermolytic decarboxylation of tryptophan to tryptamine catalyzed by natural oils.

A number of N,N-dialkylated tryptamines show psychoactive properties in man which resulted in a renewed interest in psychopharmacological research. Attempts to manufacture these derivatives are increasing within a clandestine environment, where literature procedures are adapted and information is exchanged on the Internet. One such example is based on the thermolytic decarboxylation of tryptophan to tryptamine as the precursor to psychoactive derivatives. This procedure was proposed to make use of household solvents such as turpentine substitute and white spirit to facilitate decarboxylation. Discussions on websites also suggested the catalytic use of natural oils in order to accelerate these reactions. In this research, the analytical characterization of this preparation procedure was carried out using gas chromatography-ion trap single and tandem stage mass spectrometry in electron and chemical ionization mode that led to the identification of previously unreported 1-mono and 1,1-disubstituted tetrahydro-beta-carboline (THBCs) by-products. The tryptamine product and several THBC by-products were determined quantitatively and a "fingerprint" analysis of the crude products allowed for the differentiation between the essential oil catalysts involved as indicated by the presence of tetrahydro-beta-carbolines and their imine intermediates.

Halogenated solvent interactions with N,N-dimethyltryptamine: formation of quaternary ammonium salts and their artificially induced rearrangements during analysis.

The psychoactive properties of N,N-dimethyltryptamine (DMT) 1a are known to induce altered states of consciousness in humans. This particular attribute attracts great interest from a variety of scientific and also clandestine communities. Our recent research has confirmed that DMT reacts with dichloromethane (DCM), either as a result of work-up or storage to give a quaternary N-chloromethyl ammonium salt 2a. Furthermore, this was observed to undergo rearrangement during analysis using gas chromatography-mass spectrometry (GC-MS) with products including 3-(2-chloroethyl)indole 3 and 2-methyltetrahydro-beta-carboline 4 (2-Me-THBC). This study further investigates this so far unexplored area of solvent interactions by the exposure of DMT to other halogenated solvents including dibromomethane and 1,2-dichloroethane (DCE). The N-bromomethyl- and N-chloroethyl quaternary ammonium derivatives were subsequently characterised by ion trap GC-MS in electron and chemical ionisation tandem MS mode and by NMR spectroscopy. The DCE-derived derivative formed at least six rearrangement products in the total ion chromatogram. Identification of mass spectrometry generated by-products was verified by conventional or microwave-accelerated synthesis. The use of deuterated DCM and deuterated DMT 1b provided insights into the mechanism of the rearrangements. The presence of potentially characteristic marker molecules may allow the identification of solvents used during the manufacture of controlled substances, which is often neglected since these are considered inert.

N,N-Dimethyltryptamine and dichloromethane: rearrangement of quaternary ammonium salt product during GC-EI and CI-MS-MS analysis.

N,N-Dimethyltryptamine (DMT) 1 is a simple tryptamine derivative with powerful psychoactive properties. It is abundant in nature and easily accessible through a variety of synthetic routes. Most work-up procedures require the use of organic solvents and halogenated representatives are often employed. DMT was found to be reactive towards dichloromethane, either during work-up or long term storage therein, which led to the formation of the quaternary ammonium salt N-chloromethyl-DMT chloride 2. Analysis of this side-product by gas chromatography ion trap mass spectrometry (GC-MS), both in electron and chemical ionisation tandem MS modes, gave only degradation products. For example, 2 could not be detected but appeared to have rearranged to 3-(2-chloroethyl)indole 3 and 2-methyltetrahydro-beta-carboline 4, whereas HPLC analysis enabled the detection of 2. GC-MS is a standard tool for the fingerprinting of drug products. The identification of a particular synthetic route is based on the analysis of impurities, provided these side products can be established to be route-specific. The in situ detection of both 3 and 4 within a DMT sample may have led to erroneous conclusions with regards to the identification of the synthetic route.