Development and validation of an ultra-high performance liquid chromatographic high resolution Q-Orbitrap mass spectrometric method for the simultaneous determination of steroidal endocrine disrupting compounds in aquatic matrices
Graphical abstract
Introduction
The abundance of endocrine disrupting compounds (EDCs) in water is detrimental to the aquatic environment and its ecological health [1], [2], resulting in severe consequences such as loss of animal habitats, reduction in biodiversity and intoxication - both acute and chronic - of organisms. For example, in case of fish, such as cyprinids and zebra fish, several negative effects have been reported. Not only do EDCs, such as the synthetic oestrogen 17α-ethinylestradiol or the androgen trenbolone, influence the fertility and reproduction capabilities of fish, they also impact the gender distribution [3], [4], [5], [6]. In spite of the plethora of adverse effects that have been observed for many EDCs, only 17β-estradiol and 17β-ethinylestradiol have been included in the European watch list for water quality monitoring so far [7]. In order to further improve environmental quality standards, other EDCs, which have received little attention in the aquatic environment, need to be monitored as was recently recommended by Fent et al. (2015) [8].
EDCs mainly include steroidal and non-steroidal compounds. The non-steroidal EDCs comprise contaminants such as phthalates, phenols, antibiotics and polychlorinated biphenyls, whereas the steroidal compound group consists of androgens, oestrogens, progestins, and corticosteroids [9]. Due to the fact that steroidal compounds are the most potent endocrine disruptors in aquatic systems [10], [11], these were the main focus within this work. The first steroidal subgroup concerns the androgens, which are the most abundant hormones found in effluents of wastewater treatment plants. These hormones originate from urinary excretion of humans and animals, whereby their presence is due to their usage for therapy and growth treatment [12]. The second steroidal subgroup, i.e. the oestrogens, and in particularly 17α-ethinylestradiol, are widely consumed as oral and non-oral contraceptives [13]. The third steroidal subgroup, i.e. the progestins, are extensively used for contraception and medical treatments and are consumed more than androgens and oestrogens. The last subgroup, i.e. the corticosteroids, are used to treat a variety of diseases, such as asthma, rheumatism, allergies and inflammation [14].
Up to now, studies mainly report on the occurrence of EDCs and related compounds in freshwater environments, whereas data for marine environments are relatively scarce [15], [16]. The fresh water environments that have mainly been monitored for EDCs are riverine water, groundwater, drinking water, and wastewater [17], [18], [19], [20], [21]. These four major freshwater bodies suffer from contamination primarily due to local anthropogenic activities. As a result, only information on the occurrences of local EDCs is available [22]. As all water eventually ends up in the marine environment and in light of the above-mentioned effects, it is of utter importance to map the contamination status of marine waters as well. However, seawater analysis is complicated by the fact that EDCs prevail in the marine environment in the low ng L−1 range and that each EDC can occur in one or more of the following forms: parent EDCs, metabolites, transformation products, and or degradation products [8], [23], [24]. So far, only three EDCs, namely estrone, 17β-estradiol and 17α-estradiol, have been studied in the marine environment [16].
In this study, a method is presented that allows quantifying 70 target EDCs leaves also the possibility to screen for a virtually unlimited number of (un)known compounds in the marine environment. To realise this, an appropriate extraction and ultra-high performance liquid chromatographic high resolution Q-orbitrap™ mass spectrometric method (UHPLC-HR-Q-orbitrap™-MS) was developed for EDCs in marine waters. The UHPLC-part enables fast simultaneous separation of oestrogens, androgens, progestins and corticosteroids. Furthermore, the HRMS allows a reliable, selective and accurate target detection of the various EDC classes. The analytical method was validated according to CD 2002/657/EC [25], CD 2009/90/EC [26], Eurachem guidelines [27] and review articles [28], [29] and eventually applied on real environmental samples.
Section snippets
Chemicals and reagents
In this study, 70 steroidal EDCs were included (Table 1 and Table A.1.), which were purchased at Steraloids Inc (Newport, RI, USA) and Sigma Aldrich (St. Louis, MO, USA). The selected EDCs were based on relevant literature [15], [17], [18], [19], [20], [30], and covered 4 classes, i.e. 33 androgens, 14 oestrogens, 12 progestins and 11 corticosteroids. The selected deuterated internal standards for each class were purchased at Steraloids (Newport, RI, USA) and Sigma Aldrich (St. Louis, MO, USA)
Liquid chromatography
Given the superior performance of UHPLC in terms of chromatographic resolution compared to conventional HPLC [39], the UHPLC separation strategy was selected in this work for multi-EDC profiling. Optimal conditions, relating to the stationary phase, flow rate, mobile phase composition, additives, column temperature, and injection volume, were determined by studying their impacts on the inter-linked resolution, chromatographic peak shape, and interfering background for the 70 targeted analytes.
Conclusions
A new analytical UHPLC-HR-Q-Orbitrap-MS multi-residue method was developed and successfully validated for the simultaneous quantification of 70 EDCs in sea and fresh water samples. The empirical MQLs in aquatic matrices for the androgens, oestrogens, progestins, and corticosteroids ranged respectively between 0.13 and 5.00 ng L−1, 0.25–5.00 ng L−1, 0.13–2.50 ng L−1, and 0.50–5.00 ng L−1. These low MQLs have shown to be necessary during the environmental application, due to the low concentration
Acknowledgments
This work fits within the NewSTHEPS project (BR/143/A2/NEWSTHEPS), supported by the Belgian Science Policy (BELSPO). More information can be found on the NewSTHEPS website; www.newstheps.be. The authors wish to thank Mieke Naessens for her technical contribution to the manuscript.
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2022, Molecular and Cellular EndocrinologyCitation Excerpt :Liquid chromatography coupled with mass spectrometry (LC-MS) has been a traditional approach to identify chemical structure of chemicals in environmental samples. Samples are prepared in a manner similar to other assays; however, after solid-phase extraction and evaporation, the concentrated sample is resuspended in a solvent that is compatible with the solvent system used by the LC-MS instrument (Huysman et al., 2017). While robust, this method fails to identify environmentally modified estrogenic and other hormonal mimetics that are physiologically active but whose structures have not been previously reported Stavreva et al., 2012b.