Evaluation of Nontarget Long-Term LC-HRMS Time Series Data Using Multivariate Statistical Approaches.

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.

Identification of unknowns in industrial wastewater using offline 2D chromatography and non-target screening.

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.

Development and validation of an automated liquid-liquid extraction GC/MS method for the determination of THC, 11-OH-THC, and free THC-carboxylic acid (THC-COOH) from blood serum.

The analysis of Δ(9)-tetrahydrocannabinol (THC) and its metabolites 11-hydroxy-Δ(9)-tetrahydrocannabinol (11-OH-THC), and 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THC-COOH) from blood serum is a routine task in forensic toxicology laboratories. For examination of consumption habits, the concentration of the phase I metabolite THC-COOH is used. Recommendations for interpretation of analysis values in medical-psychological assessments (regranting of driver's licenses, Germany) include threshold values for the free, unconjugated THC-COOH. Using a fully automated two-step liquid-liquid extraction, THC, 11-OH-THC, and free, unconjugated THC-COOH were extracted from blood serum, silylated with N-methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA), and analyzed by GC/MS. The automation was carried out by an x-y-z sample robot equipped with modules for shaking, centrifugation, and solvent evaporation. This method was based on a previously developed manual sample preparation method. Validation guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh) were fulfilled for both methods, at which the focus of this article is the automated one. Limits of detection and quantification for THC were 0.3 and 0.6 µg/L, for 11-OH-THC were 0.1 and 0.8 µg/L, and for THC-COOH were 0.3 and 1.1 µg/L, when extracting only 0.5 mL of blood serum. Therefore, the required limit of quantification for THC of 1 µg/L in driving under the influence of cannabis cases in Germany (and other countries) can be reached and the method can be employed in that context. Real and external control samples were analyzed, and a round robin test was passed successfully. To date, the method is employed in the Institute of Legal Medicine in Giessen, Germany, in daily routine. Automation helps in avoiding errors during sample preparation and reduces the workload of the laboratory personnel. Due to its flexibility, the analysis system can be employed for other liquid-liquid extractions as well. To the best of our knowledge, this is the first publication on a comprehensively automated classical liquid-liquid extraction workflow in the field of forensic toxicological analysis. Graphical abstract GC/MS with MPS Dual Head at the Institute of Legal Medicine, Giessen, Germany. Modules from left to right: (quick) Mix (for LLE), wash station, tray 1 (vials for extracts), solvent reservoir, (m) VAP (for extract evaporation), Solvent Filling Station (solvent supply), cooled tray 2 (vials for serum samples), and centrifuge (for phase separation).