Comparative study of analytical methods involving gas chromatography–mass spectrometry after derivatization and gas chromatography–tandem mass spectrometry for the determination of selected endocrine disrupting compounds in wastewaters

Abstract

Two GC–MS methods, based on the application of N,O-bis(trimethylsilyl)trifluoroacetamide-derivatization–GC–MS (selected-ion monitoring) and GC–MS–MS without derivatization, respectively, were optimised and applied to the determination of a group of five selected endocrine disrupting compounds (EDCs) in wastewaters. Both methods included solid-phase extraction with Oasis HLB cartridges allowing an enrichment factor for wastewater samples of 100-fold. The investigated EDCs were estrone, 17β-estradiol, 17α-ethynylestradiol, 4-tert-octylphenol and bisphenol A. Results obtained from the validation studies yielded comparable results in both cases. Recoveries in spiked wastewaters at 50 ng/l were higher than 90% for all the compounds, except for 4-tert-octylphenol (75%). Repeatability and reproducibility were adequate, varying from 1.6 to 14%, except for estrone which reproducibility was 28% when the derivatization–GC–MS method was applied. Limits of detection calculated ranged from 2.5 to 27.5 ng/l with differences between both methods from 1.1 (estrone) to 10.4 (bisphenol A) times. Both methods were successfully applied to the analysis of the target compounds in sewage treatment plant influents and effluents. Traces of bisphenol A, 4-tert-octylphenol, estrone and 17β-estradiol were detected at concentration levels ranging from 13.3 to 1105.2 ng/l.

Introduction

Increasingly, evidence that endocrine disrupting compounds (EDCs) can have harmful effects on the aquatic organisms has emerged. Some of the compounds with highest estrogenic capacity include both natural (e.g. 17β-estradiol, estrone) and synthetic estrogens (17α-ethynylestradiol). Apart from these, chemicals from household or industrial processes, such as bisphenol A, or alkylphenol polyethoxylates (APEO_n, e.g. 4-nonylphenol or 4-tert-octylphenol) can exert endocrine disruption by different mechanisms by mimicking or antagonising the effects of hormones, by altering the synthesis and metabolism of hormones, and by modifying hormone receptor levels [1].

The EDCs may be released directly or indirectly to the aquatic environment. Wastewater treatment plants appear to be one of the major sources of pollution because these compounds are not totally removed or degraded by biological treatments. They have been detected in wastewaters and surface waters at concentration levels of ng/l [2], [3]. However, the exposition of aquatic organisms, even at these very low concentration levels can induce estrogenic responses. Reported studies have observed the vitellogenin production (feminisation processes) in male fish exposed to low ng/l levels of EDCs [4], [5].

APEOn [nonylphenol, octylphenol and alkylphenols (4-p-nonylphenol, 4-p-tert-octylphenol] have been recently included as priority substances in the field of water policy and octylphenols will be subject to a review for identification as possible “priority hazardous substance” (Decision No. 2455/2001/EC) [6].

Different analytical methods have been developed for analysing EDCs from wastewater samples. The most common are liquid or gas chromatography coupled with mass spectrometry (LC–MS or GC–MS). LC–MS enables the determination of APEOn using electrospray ionization (ESI) in both positive and negative mode at μg/l level [7]. Few papers reporting extremely high sensitivity (<0.1–5.0 ng/l) have been published using LC–MS with ESI or atmospheric pressure chemical ionization (APCI) detection [8], or LC–tandem MS [7], [9], [10]. However, important signal suppression effects are frequently observed when LC–atmospheric pressure ionization (API) MS is applied [11]. Low concentrations (ng/l) of EDCs are generally determined by GC–MS [7], [12], [13], [14], [15], [16], [17]. In our knowledge, all the analytical methods proposed in the literature apply derivatization procedures before GC–MS analysis. Different reagents have been used to derivatize EDCs, including pentafluorobenzyl (PFBr), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) or N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) that lead to the formation of TMS and TBS derivatives. These are often chosen because they are stable during approximately 30 min and allow improving the sensitivity [18], [19], [20], [21]. However, derivatization processes can make the sample preparation laborious and time consuming, and can increase the possibility of contamination as consequence of undesirable reactions with the matrix.

This paper proposes avoiding this tedious and critical step performing SPE and direct analysis of the extracts by GC–MS–MS. This method is compared with a well-established method that uses BSTFA as derivatizating agent by using the same fortified samples. So, a comparison between the two methods has been performed. The obtaining of similar results in both cases will allow demonstrate that the elimination of the derivatization step is feasible.