Targeted and non-targeted liquid chromatography-mass spectrometric workflows for identification of transformation products of emerging pollutants in the aquatic environment

https://doi.org/10.1016/j.trac.2014.11.009Get rights and content

Highlights

  • Biotic and abiotic formation of transformation products of emerging pollutants.

  • Comprehensive database on transformation products of emerging pollutants.

  • Workflows for suspect and non-target screening.

  • The use of high-resolution mass spectrometry to identify transformation products.

  • Gaps and future outlook for identification using high-resolution mass spectrometry.

Abstract

Identification of transformation products (TPs) of emerging pollutants is challenging, due to the vast number of compounds, mostly unknown, the complexity of the matrices and their often low concentrations, requiring highly selective, highly sensitive techniques. We compile background information on biotic and abiotic formation of TPs and analytical developments over the past five years. We present a database of biotic or abiotic TPs compiled from those identified in recent years. We discuss mass spectrometric (MS) techniques and workflows for target, suspect and non-target screening of TPs with emphasis on liquid chromatography coupled to MS (LC-MS). Both low- and high-resolution (HR) mass analyzers have been applied, but HR-MS is the technique of choice, due to its high confirmatory capabilities, derived from the high resolving power and the mass accuracy in MS and MS/MS modes, and the sophisticated software developed.

Introduction

The term “emerging pollutants” (EPs) [or “emerging contaminants” (ECs)] refers to compounds and their metabolites that are not currently covered by existing water-quality regulations, have not been studied often, are overlooked and are thought to be potential threats to environmental ecosystems and human health and safety. According to NORMAN (Network of reference laboratories, research centers and related organizations for monitoring emerging environmental substances), they are compounds that are not included in routine environmental monitoring programs and may be candidates for future legislation due to their adverse effects and/or persistency (http://www.norman-network.net/). Most regulating and implementation bodies, responsible for water and wastewater treatment, are working on the assumption that the so-called priority pollutants are responsible for the most significant share of environmental, human health and economic risk, even though they represent a minor fraction of the universe of known and yet-to be identified chemicals [1].

EPs encompass a diverse group of compounds, including pharmaceuticals and personal-care products (PPCPs), drugs of abuse (DoAs) and their metabolites, steroids and hormones, endocrine-disrupting compounds, surfactants, perfluorinated compounds, phosphoric ester flame retardants, industrial additives and agents (e.g., benzotriazoles and benzothiazoles), siloxanes, artificial sweeteners, and gasoline additives.

Once released into the environment, EPs are subject to biotic and abiotic transformation processes that are responsible for their transformation and/or elimination, according to their persistence, transport, and ultimate destination. Various transformations can take place, producing compounds that, to some extent, differ in their environmental behavior and ecotoxicological profile from the parent compound. Formation of transformation products (TPs) occurs mainly through oxidation, hydroxylation, hydrolysis, conjugation, cleavage, dealkylation, methylation and demethylation. EPs and their TPs can move vertically through the soil profile to groundwater and away from the source site with mobile groundwater. They also have the potential to reach surface water when they travel laterally as surface run-off or through sub-soil tile drains, entering streams, major rivers, reservoirs, and ultimately estuaries and oceans [2].

Since there is a gap in the information on the occurrence and the toxicity of TPs in the environment, we are unable to evaluate their significance in risk assessment [3], [4]. Standardized toxicity tests can provide quantitative information on the toxicity of the TP, compared to its parent compound, but these studies are limited [5], [6], [7]. In general, TPs are less toxic and more polar than the parent compounds. However, in some cases, they may be more persistent or exhibit higher toxicity or be present at much higher concentrations [8].

Although there is legislation regulating chemicals [e.g., pesticides, veterinary drugs, and persistent organic pollutants (POPs)], there is little mention of their TPs. Concerns over the TPs of pesticides in plants have been expressed since 1991 (European Directive 91/414/EEC), while the term “metabolite” appears in Regulation (EC) 1107/2009, concerning plant-protection products, and in Directives 2001/82/EC and 98/8/EC, concerning veterinary medical and biocidal products, respectively. European Medicines Agency (EMEA, 2006) also referred to the need for assessment of potential environmental risks of human medicinal products. However, in all these documents, there is no clarification on the determination, limits and toxicological effects of metabolites or TPs.

In OECD guidelines, concerning the Aerobic and Anaerobic Transformation in Aquatic Sediment Systems, adopted in 2002, it is claimed that TPs detected at ≥10% of the applied radioactivity should be identified. Meanwhile, EU Regulation 1907/2006 (REACH) requires identification of major TPs and degradation products for the registration of the substance. In the Regulation (EC) 850/2004 on POPs, a reference to their transformation processes also exists.

There is therefore a clear need to reveal the qualitative and quantitative occurrence of TPs in the environment, but this is only possible with continual development of instrumental analysis. Thereby, the range of identifiable chemicals is extended, and the quantification limits are lowered. With respect to obtaining a holistic view of risk, target-based environmental monitoring should be accompanied by non-target analysis using high-resolution (HR) hybrid mass spectrometers. The development of these highly resolved, accurate, hybrid, tandem mass spectrometers, and improved sophisticated software, has enabled more reliable, selective target analysis of highly polar compounds, and screening for unknown pollutants. The major benefit of full-scan and HR, accurate MS is that, within a single analytical run, target, suspect and non-target compounds can be analyzed or identified.

In the analysis of EPs, HR mass spectrometry (HR-MS) has been widely reported [3], [9], [10], [11], [12]. Moreover, for identification of TPs in environmental, food and biological samples, hybrid HR mass analyzers [e.g., linear ion trap Orbitrap MS and quadrupole time-of-flight MS (Q-TOF)] have been used, following specific workflows [12], [13], [14], [15], [16]. More specifically, human and microbial metabolites, oxidation and photodegradation TPs of pharmaceuticals have been discussed often [17], [18], [19], [20], [21]. Similarly, TPs of pesticides in biological (human metabolism, phase II), food and environmental samples have been reviewed [22], [23]. Furthermore, the TPs of anthelmintics [24], UV filters in the environment [25], [26] and steroidal compounds in biological samples [27] are included in recent review papers. An interesting fact concerning the analysis of EPs and their TPs is enantioselective biotransformation. Chiral EPs or chiral TPs formed may have enantioselective activity or toxicity, making chiral chromatography indispensable [28].

Achievements, future trends and new developments in the analysis of EPs and their TPs were summarized by Farré et al. [29] and Fisher et al. [30]. Recently, highly sophisticated, comprehensive, step-wise workflows were also presented by Moschet et al. [31] and Hug et al. [32] for suspect and non-target screening of pesticides and EPs, including TPs in their suspect lists. However, it is still challenging to profile TPs in environment samples, since they are formed through many possible reactions, automatic workflows for the identification are not readily available, so manual data inspection is necessary, though time consuming, and, finally, there are no standards available.

The aim of this review is to compile the recent information regarding the background of (biotic and abiotic) transformation of EPs. We provide a brief overview of existing literature on transformation studies under biotic and abiotic conditions in recent years and we compile a list of all the EPs studied and comprehensive information for researchers in the field. We briefly summarize target analysis, since the development of accurate mass instruments and sophisticated computer tools has led to suspect and non-target analysis, even though all three procedures are indispensable parts of an integrated approach to determination of EPs and their TPs. We present the design of laboratory studies to facilitate identification of TPs by LC-MS and appropriate sample preparation. We thoroughly discuss target, suspect and non-target workflows using HR-MS/MS to identify new TPs.

Section snippets

Classification of transformation products (TPs)

TPs occurring in the environment can be classified into two main categories: biotransformation products formed by biotic or abiotic processes. This classification has subcategories that we describe in detail below, emphasizing the aquatic environment. The biotransformation products include human, animal and microbial metabolites in engineered and natural systems. The abiotic TPs are the outcome of hydrolysis, photolytic and photocatalytic degradation in the natural environment and

Identification approaches – laboratory studies

Simulation of the transformation processes in batch experiments under well-defined conditions with appropriate controls is a very common first approach for the identification of TPs. Batch experiments can be applied under biotic and abiotic conditions at high concentrations of the parent EPs.

For biodegradation experiments, samples can be provided directly from a wastewater-treatment plant (WWTP) or a pilot-scale WWTP (ps-WWTP) or from natural waters [33], [34], [51]. Moreover, the ability of

Identification approaches – analytical techniques

Nowadays, liquid chromatography (LC) coupled to MS (LC-MS) using a variety of mass analyzers is the technique of choice for the investigation of EPs and TPs in environmental samples.

LC is a suitable chromatographic technique for polar, thermo-labile compounds, thus for the identification of TPs, which are generally more polar than their parent molecules.

Mass analyzers commonly employed are triple quadrupole (QqQ), time-of-flight (TOF), ion-trap (IT), Orbitrap and hybrid [e.g., quadrupole

Future needs and trends

Development of generic and retrospective analytical techniques should permit the simultaneous determination of parent compounds and their TPs, within a single run. In the identification of TPs, the future lies in tiered approaches, which employ HR-MS, complementary techniques and advanced software tools. HR-MS outperforms LR-MS, regarding the level of identification of an unknown compound. Moreover, identification by HR-MS analysis has different levels of confidence, regarding the supporting

Acknowledgments

This project was implemented under the Greek Operational Program «Education and Lifelong Learning» and funded by the European Union (European Social Fund) and Greek National Resources (ARISTEIA 624).

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