Rapid Trace Analysis of Multiresidue Contaminants

Mira Petrovic from the Catalan Institute for Water Research (ICRA) in Girona, Spain, reveals the advantages and practical applications of a novel method she developed for the multiresidue trace analysis of pharmaceutical compounds and their corresponding metabolites and transformation products using dual-column liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS).

Interview by Alasdair Matheson, Editor-in-Chief, LCGC Europe

Q. Your group investigates emerging contaminants in the environment at trace level. What analytes have you recently studied and why?

A: Our group investigates the fate and behaviour of emerging contaminants in natural and engineered systems. The compounds of interest include pharmaceuticals and their metabolites and transformation products (TPs). A method we recently published in Talanta in 2016 (1) has been developed and validated to analyze 12 pharmaceuticals from different therapeutic classes, including diclofenac, acetaminophen, sulfamethoxazole, sulfapyridine, sulfamethazine, sulfadiazine, venlafaxine, diazepam, carbamazepine, fluoxetine, metoprolol, verapamil, and their 20 metabolites and transformation products in different types of water, including influent and effluent wastewaters and surface water.

These pharmaceuticals were selected for both their high consumption rates and environmental relevance (a high occurrence in the environment). Their metabolites and TPs were selected depending on their commercial availability and also considering the amount of information available regarding their environmental presence.

Q. What problems were associated with the methods traditionally used to detect these analytes?

A: Currently, multiresidue analytical methods have become the preferred tool for tracing different pharmaceuticals. Offline solid-phase extraction (SPE) followed by liquid chromatography (LC) coupled to tandem triple quadrupole mass spectrometry (LC–QqQ-MS) is the usual method of choice for quantitative analysis. However, the challenge for trace analysis of pharmaceuticals in water matrices has shifted from reaching enough sensitivity and selectivity for their detection, to the reduction of the analysis time, manipulation of the samples in a minimum number of steps, and the reduced use of solvents. Offline SPE involves a certain number of steps that imply several hours of preparation, and also requires sample volumes of 100−1000 mL to obtain the desired sensitivity, and the use of significant amounts of solvents. There is therefore a need for methods involving less sample manipulation by the analyst (lower probability of error), reduced sample volume, reduced time and solvents, as well as improved throughput. In this context, online preconcentration has become one of the most suitable sample preparation approaches available.

Q. The method you devised applied automated online solid-phase extraction dual-column liquid chromatography coupled to tandem mass spectrometry. What is novel about this approach?

A: The method allows simultaneous trace analysis of selected pharmaceuticals and their metabolites and TPs, all belonging to different families and with different structures and properties, with a total run time of 14 min and with reduced sample pretreatment (only filtration), which was a significant improvement compared with the methods reported previously. The use of a dual-column LC system reduced the interferences and improved the analysis, resulting in a robust, fast, and high‑throughput method.

Q. What benefits does this method offer compared to previous methods?A: Ordinary online SPE has been improved by using a dual-column LC switching system because only one preconcentration column is used for the whole set of samples, rather than one SPE cartridge for each sample. The analytical method is highly selective, sensitive, and accurate. It allows for a very efficient preconcentration and clean-up of the samples, requiring minimum manipulation and pretreatment, and also a very low volume of the sample (1 mL to 5 mL depending on the matrix or injection). The new method allows the simultaneous analysis in both negative ion (NI) and positive ion (PI) mode without compromising the sensitivity of the analysis, obtaining limits of detection (LODs) in the low ng/L range for most of the compounds. Matrix effects were also reduced by means of online LC cleanup.

Q. Were there any particular challenges you encountered developing this method?A: Online techniques have some advantages, such as the reduction of solvent consumption, the reuse of SPE columns, time savings, and less exposure to hazardous solvents, but also some disadvantages because the system is less flexible in the choice and combination of extraction solid phases. It is also not possible to add internal standards after the extraction step. Consequently, the challenge of developing an SPE method lies in the requirements for careful optimization of preconcentration and elution operating parameters, such as the selection of a preconcentration column, sample volume, sample pH, transfer time, elution time, and mobile phase composition, to achieve a satisfactory result in a single run for a wide number of compounds, including classes of compounds with different physicochemical characteristics.

Q.Have you used this method for “real life” samples?A: The method was successfully applied to study the presence of the target analytes in different wastewater and surface water samples collected near the city of Girona (Catalonia, Spain). The results demonstrated the widespread presence of the different metabolites and TPs in all the water matrices studied, at similar or even higher levels than the corresponding parent compounds. The presence of the TPs of venlafaxine in all the samples analyzed, generally at concentrations higher than those of the parent compound, should be emphasized. Similar results were obtained for TPs of sulfapyridine and for fluoxetine in wastewaters. The TP diclofenac amide was detected only in effluent wastewaters, suggesting the formation of this product during wastewater treatment. These results reinforce the need for including metabolites and TPs within the scope of future monitoring studies because these data lead to a better understanding of biodegradation and attenuation processes of these pharmaceutical compounds once discharged into the environment.

Q. Have you any tips for analysts considering developing this technique for other analytes?

A: The optimization process of an online SPE method can be a long and troublesome procedure because the effects of the elution gradient and sample volume as well as matrix modifications should be investigated. However, it is of crucial importance to understand how all these factors impact the performance of an SPE method and each matrix should be treated with dedicated attention and the matrix effect carefully evaluated. In addition, minimal sample pretreatment can lead to the injection of environmental samples with a heavy load of particles and matrix components, which can be accumulated and eluted from the front of the extraction column when the elution is performed in the common backflush mode. Therefore, depending on the complexity of environmental samples, an additional clean-up step may be added to the enrichment step, that is, clean-up based on restricted access media (RAM) or turbulent flow chromatography (TFC).

Q. Are there any other areas where you think this approach could be applied?

A: This approach could be used to study biotic and abiotic transformation processes in the environment, such as biodegradation and photodegradation. Similarly, it could be used to investigate the fate and behaviour of selected compounds in engineered water systems, where the understanding of transformation processes is of crucial importance. Furthermore, the approach could be useful in any study requiring simultaneous trace-level quantification of parent pharmaceutical compounds and their metabolites and TPs, such as food analysis, metabolic studies, or pharmacological studies.

Q. What other projects are your group working on at the moment?

A: Within the H2020 TreatREC project we are working on the development and application of a nontargeted analysis of wastewater dissolved organic matter (DOM) using LC–high resolution mass spectrometry (HRMS). Our objective is to integrate the latest advances in HRMS and statistical analysis of data to develop and optimize a smart methodology (workflow) to assess the overall quality of wastewater treatment. The method considers the entire available data (typically, 103–105 of mostly unknown signals) obtained by LC with orbital trap MS detection. The approach is a step towards a more comprehensive monitoring of dissolved organics in wastewater and contributes to the understanding of the current treatment technologies. Current approaches to analyze organic micro‑contaminants in wastewater often do not consider the entire chemical picture of the system and concentrate on a few “hand-picked” pollutants. The group is also involved in the development and application of advanced LC–MS analytical methods for a variety of relevant antibiotics. The work is done within the frame of the H2020 project RESOURCE, which is aimed at broadening knowledge about the possible environmental risks associated with swine manure reuse as a fertilizer in agriculture.

The project has a special focus on assessing the occurrence of veterinary antibiotics and pharmaceuticals in manure, evaluating the performance of on-site manure treatment techniques to remove target antibiotics and pharmaceuticals, and studying their persistence and fate in manure amended soils and transport to groundwater bodies of hot-spot areas in Catalonia with intensive livestock activities.

Reference

• M.J. García-Galán, M. Petrovic, S. Rodríguez-Mozaz, and D. Barceló, Talanta158, 330–341 (2016).

Mira Petrovic is a Catalan Institution for Research and Advanced Studies (ICREA) research professor and is currently a senior researcher at the Catalan Institute for Water Research (ICRA) in Girona, Spain. She joined ICRA in 2011 to research pollutants in wastewater. From 1999–2011 she was a research scientist at the Department of Environmental Chemistry, Institute for Environmental Assessment and Water Studies (IDAEA-CSIC) in Barcelona, Spain. Before that, after receiving her Ph.D. in chemistry in 1995 from the Faculty of Chemical Engineering and Technology at the University of Zagreb in Croatia, she worked at the same university as an assistant professor.