To reduce the discharge of micropollutants, advanced wastewater treatment methods were investigated in the last years. Estrogenic effects were found to be reduced by ozonation. These activities are ...usually measured using genetically modified cell-based tests. As these bioassays are representing a sum parameter, also inhibitory effects such as antagonistic effects need to be further investigated as they are potentially reducing the detected activities. Therefore, a direct comparison of chemical target analysis and biological equivalent concentrations measured by bioassays is often difficult. To investigate the fate of antagonistic activities and their role in mixtures with agonistic activities, two hospital wastewater treatment plants were studied after different treatment steps. Thereby highly enriched samples were analyzed by a combination of bioassays with chemical target and non-target analyses. In order to achieve an in-depth characterization of the antagonistic activities a fractionation of the enriched samples was performed. To identify relevant compounds an effect directed identification approach was used by combining high-resolution mass spectrometry and bioassays. The results showed a high reduction for estrogene and androgene activities. However, a constant antagonistic activity after membrane bioreactor and ozone treatment was observed. A reduction of the antagonistic activity was observed after passing an activated carbon filter. The fractionation approach showed a specific finger-print of each sample of the different treatment steps. Hereby we could show that the composition of agonistic and antagonistic active compounds is changing after each treatment step while the overall measured activity stays the same. Using fractionation and the combination of bioassays the number of relevant features detected by chemical non-target screening could be reduced by >85%. As a result the phosphorous flame retardant TCEP could be identified as anti-estrogene active. Future research should be done to identify more antagonistic active compounds and potentially active transformation products after ozone treatment.
Display omitted
•Reduction of estrogene and androgene activity during Hospital wastewater treatment using ozone•Behaviour of agonistic and antagonistic endocrine activities during ozonation using Arxula Adeninivorans based yeast assays•Fractionation in combination with effect based analysis reduces the amount of relevant features•Reduction of antagonistic activities by granular activated carbon filter as post-treatment
The use of liquid chromatography coupled with high-resolution mass spectrometry (LC–HRMS) has steadily increased in many application fields ranging from metabolomics to environmental science. HRMS ...data are frequently used for nontarget screening (NTS), i.e., the search for compounds that are not previously known and where no reference substances are available. However, the large quantity of data produced by NTS analytical workflows makes data interpretation and time-dependent monitoring of samples very sophisticated and necessitates exploiting chemometric data processing techniques. Consequently, in this study, a prioritization method to handle time series in nontarget data was established. As proof of concept, industrial wastewater was investigated. As routine industrial wastewater analyses monitor the occurrence of a limited number of targeted water contaminants, NTS provides the opportunity to detect also unknown trace organic compounds (TrOCs) that are not in the focus of routine target analysis. The developed prioritization method enables reducing raw data and including identification of prioritized unknown contaminants. To that end, a five-month time series for industrial wastewaters was utilized, analyzed by liquid chromatography–time-of-flight mass spectrometry (LC–qTOF-MS), and evaluated by NTS. Following peak detection, alignment, grouping, and blank subtraction, 3303 features were obtained of wastewater treatment plant (WWTP) influent samples. Subsequently, two complementary ways for exploratory time trend detection and feature prioritization are proposed. Therefore, following a prefiltering step, featurewise principal component analysis (PCA) and groupwise PCA (GPCA) of the matrix (temporal wise) were used to annotate trends of relevant wastewater contaminants. With sparse factorization of data matrices using GPCA, groups of correlated features/mass fragments or adducts were detected, recovered, and prioritized. Similarities and differences in the chemical composition of wastewater samples were observed over time to reveal hidden factors accounting for the structure of the data. The detected features were reduced to 130 relevant time trends related to TrOCs for identification. Exemplarily, as proof of concept, one nontarget pollutant was identified as N-methylpyrrolidone. The developed chemometric strategies of this study are not only suitable for industrial wastewater but also could be efficiently employed for time trend exploration in other scientific fields.
Industrial wastewater is characterised by a complex composition of trace organic compounds (TrOC) in a difficult matrix. The identification of unknown pollutants is of high interest. On the one hand ...to ensure protection of the environment by elucidating contaminations and on the other hand to protect the biological treatment step in the wastewater treatment plant (WWTP). Due to the high variability of the matrix, the identification of compounds of interest is very time consuming and often unsuccessful. To overcome this limitation, a prioritisation method was developed to identify so called ‘known unknowns’, i.e. compounds frequently detected but not identified, as prioritised compounds in industrial wastewater. The method based on an offline two-dimensional (offline 2D) liquid chromatography (LC) approach with ultra violet (UV) detection in the first and high-resolution mass spectrometry (HRMS) in the second dimension. As a proof of concept, an identification process of one ‘known unknown’ is described. The compound was identified as a dichlorodinitrophenol isomer by retention time in two dimensions, UV spectrum, exact mass, mass fragmentation and 1H- NMR. As prioritisation method, the offline 2D LC in combination with non-target analysis provides a powerful workflow to determine tentative structures of unknown organic compounds in industrial wastewater.
Display omitted
•Frequently observed contaminants named ‘known unknowns’ serve as prioritised compounds.•2D offline LC-UV/LC-HRMS was used for identification.•1st dimension provides clean-up of interfering signals and UV absorption.•2nd dimension provides HRMS for elucidation of unknown wastewater compounds.
Unfortunately a second footnote (Published in the topical collection 24th International Symposium on Separation Sciences …) was added to the original submission.
For the identification of the optimal column combinations, a comparative orthogonality study of single columns and columns coupled in series for the first dimension of a microscale two-dimensional ...liquid chromatographic approach was performed. In total, eight columns or column combinations were chosen. For the assessment of the optimal column combination, the orthogonality value as well as the peak distributions across the first and second dimension was used. In total, three different methods of orthogonality calculation, namely the
Convex Hull
,
Bin Counting
, and
Asterisk
methods, were compared. Unfortunately, the first two methods do not provide any information of peak distribution. The third method provides this important information, but is not optimal when only a limited number of components are used for method development. Therefore, a new concept for peak distribution assessment across the separation space of two-dimensional chromatographic systems and clustering detection was developed. It could be shown that the
Bin Counting
method in combination with additionally calculated histograms for the respective dimensions is well suited for the evaluation of orthogonality and peak clustering. The newly developed method could be used generally in the assessment of 2D separations.
Graphical Abstract
ᅟ
The very fast separation and identification of isomeric small substances with a molecular weight under 800 Da is still a challenge for high-throughput analysis. Inadequate chromatographic or ...spectrometric separation hampers an unequivocal identification of isomers, which may be recorded as a sum parameter. Reversed-phase liquid chromatography can be considered a generic method for the separation of isomers. However, a separation is usually achieved within minutes and not milliseconds as is typical for ion mobility spectrometry. The aim of this study, therefore, was to investigate the potential of IMS to separate small isomeric compounds. Hence, 23 substances divided into 11 isomeric groups have been selected. Among them, cancer drugs, hormones, pain relievers and others are contained. Three ion mobility spectrometers with different separation principles were compared with respect to their resolving performance. These systems comprised a traveling wave ion mobility spectrometer, a differential ion mobility spectrometer and a differential mobility analyzer. For reference, the chromatographic resolution and peak capacity was determined by high-performance liquid chromatography using a reversed phase.
A novel multidimensional separation system based on online comprehensive two-dimensional liquid chromatography and hybrid high-resolution mass spectrometry has been developed for the qualitative ...screening analysis and characterization of complex samples. The core of the system is a consistently miniaturized two-dimensional liquid chromatography that makes the rapid second dimension compatible with mass spectrometry without the need for any flow split. Elevated temperature, ultrahigh pressure, and a superficially porous sub-3-μm stationary phase provide a fast second dimension separation and a sufficient sampling frequency without a first dimension flow stop. A highly loadable porous graphitic carbon stationary phase is employed in the first dimension to implement large volume injections that help countervailing dilution caused by the sampling process between the two dimensions. Exemplarily, separations of a 99-component standard mixture and a complex wastewater sample were used to demonstrate the performance of the dual-gradient system. In the second dimension, 30 s gradients at a cycle time of 1 min were employed. One multidimensional separation took 80–90 min (∼120 min including extended hold and re-equilibration in the first dimension). This approach represents a cost-efficient alternative to online LC × LC strategies working with conventionally sized columns in the rapid second dimension, as solvent consumption is drastically decreased and analytes still are detectable at environmentally relevant concentrations.
One of the biggest challenges in the field of water analysis is the determination of trace concentrations of emerging contaminants. These contaminants must therefore be enriched, typically by either ...off-line or online solid phase extraction. An alternative method is the direct large volume injection onto the separation column. In liquid chromatography, the limitation of the injection volume depends on the column dimensions, the analyte hydrophobicity, the injection solvent and the nature of the stationary phase. Exceeding of the maximum injection volume is indicated by a deterioration of peak shapes and a loss in signal intensity. By serial coupling of two stationary phases with different hydrophobicities, the focusing can be improved and the maximum injection volume can be increased. A suitable combination could be the hyphenation of a carbon-based phase like Hypercarb and a C-18 phase. This paper describes how to implement this concept based on nano-LC and UV detection. To cover a wide range of polarity, a polar, semi-polar and non-polar compound were used as model analytes. The recommended injection volume of 25 nL for a nanobore column could be successfully increased to 3,000 nL, without deterioration of peak shapes.
The interest in two-dimensional liquid chromatography separations is growing every year together with the number of open questions on the benefits of multidimensional systems in comparison to ...one-dimensional liquid chromatography. In order to solve some of these open questions this work presents a comparison of one-dimensional and microscale two-dimensional liquid chromatography coupled to high resolution mass spectrometry for targeted analysis in wastewater. The comparison is based on the evaluation of a reference standard mixture containing 99 compounds and a real wastewater sample. For the evaluation and compound identification three different criteria were chosen. At first, a deviation of ±5 ppm from the exact mass was defined as acceptable to include the compound for further evaluation. To eliminate false positive results, a maximum retention time deviation of less than 2.5% for each compound of the reference standard and the compounds detected in the wastewater sample was defined for a positive identification as a second criterion for 1D-LC and the second dimension of 2D-LC. In the third step, fragment information from MS/MS experiments was used for further identification of compounds in wastewater. Additionally, the influence of a higher mass accuracy of 1 ppm on the number of identified compounds in comparison to a mass accuracy of 5 ppm was investigated. The results showed that the number of identified compounds was higher by a factor of three in the wastewater sample when using the microscale 2D-LC approach. Moreover, a higher reliability for compound identification is obtained when using retention time and MS/MS information as identification criteria instead of only applying high mass accuracy of 1 or 5 ppm.
The interest in two-dimensional liquid chromatography separations is growing every year together with the number of open questions on the benefits.
The interest in two-dimensional liquid chromatography separations is growing every year together with the number of open questions on the benefits of multidimensional systems in comparison to ...one-dimensional liquid chromatography. In order to solve some of these open questions this work presents a comparison of one-dimensional and microscale two-dimensional liquid chromatography coupled to high resolution mass spectrometry for targeted analysis in wastewater. The comparison is based on the evaluation of a reference standard mixture containing 99 compounds and a real wastewater sample. For the evaluation and compound identification three different criteria were chosen. At first, a deviation of ±5 ppm from the exact mass was defined as acceptable to include the compound for further evaluation. To eliminate false positive results, a maximum retention time deviation of less than 2.5% for each compound of the reference standard and the compounds detected in the wastewater sample was defined for a positive identification as a second criterion for 1D-LC and the second dimension of 2D-LC. In the third step, fragment information from MS/MS experiments was used for further identification of compounds in wastewater. Additionally, the influence of a higher mass accuracy of 1 ppm on the number of identified compounds in comparison to a mass accuracy of 5 ppm was investigated. The results showed that the number of identified compounds was higher by a factor of three in the wastewater sample when using the microscale 2D-LC approach. Moreover, a higher reliability for compound identification is obtained when using retention time and MS/MS information as identification criteria instead of only applying high mass accuracy of 1 or 5 ppm.
PDF
