Estrogens determination exploiting a SIA-LOV system prior in-port derivatization-large volume injection-programmable temperature vaporization-gas chromatography

Highlights

• First method for estrogens using automated pretreatment with on-line derivatization is proposed.

• Automatic microsolid phase extraction gives good precision and accuracy.

• In-port derivatization leads to a safer method while decreasing reagents consumption and time.

• Good precision and recoveries have been obtained in seawater samples.

Abstract

In this work, we present a method for the clean-up, preconcentration and quantification of the four most widely found estrogens (estrone E1, estradiol E2, estriol E3 and ethynyl estradiol EE2) in seawater samples. A sequential injection analysis-lab on valve system (SIA-LOV) has been developed to perform the microsolid phase extraction (µSPE) of the analytes in a fully automated way. After testing different resins and solvents, C18 resin with acetonitrile (ACN) as eluent have been chosen as they provided the best results. Several parameters affecting the extraction have been studied and optimized. Besides, extraction column lifetime has also been checked as it is indicative of the number of consecutive analysis that the column is able to perform before replacing it. Results showed that the same column can be used up to 50 times. Then, the derivatization of the extracts has been performed unattended by exploiting an in-port derivatization of the analytes with N-methyltrimethylsilyltrifluoroacetamide (BSTFA) prior their quantification using large volume injection with programmable temperature vaporization gas chromatography (LVI-PTV-GC-MS).

Introduction

During recent years the presence of estrogens in the aquatic environment has raised great concern. Some natural estrogens, as estrone (E1), 17β-estradiol (E2), and estriol (E2), and synthetic ones, like 17α-ethynylestradiol (EE2), have been catalogued as Contaminants of Emerging Concern by the Environmental Protection Agency of the United States (US-EPA) [1] since they have endocrine-disrupting properties and can cause adverse health effects in organisms or its progeny, as consequence of changes in their endocrine function [2], [3].

Many techniques have been used for the determination of estrogens, i.e. enzyme-linked immunosorbent essay (ELISA) [4], radioimmunoassay [5], and chromatography [6], [7]. Among them, the combination of gas chromatography-mass spectrometry (GC-MS) is one of the preferred strategies as it allows the simultaneous separation and identification of both natural and synthetic estrogenic steroids [8]. However, before the injection in the chromatograph a sample clean-up and analytes preconcentration is mandatory in order to attain matrix removal and also a preconcentration of the analytes, which are expected to be in very low concentrations in the environment. Liquid–liquid extraction (LLE) and solid phase extraction (SPE) are widely used for this purpose. Usually, LLE methods require the use of large volumes of volatile organic solvents, while SPE is faster and more environmentally friendly. For this reason, SPE is preferred over LLE. Both off-line [9] and on-line [10], [11] SPE methods have been reported for the determination of estrogens in water samples. Furthermore, the combination of SPE with flow analysis techniques offers important advantages as the minimizing of the sample and reagents consumption (green chemistry) and also the improvement in the precision of the method. Besides, a significant decrease of time and cost per analysis is achieved. Within this context, a lab on valve system (SIA-LOV) has been developed to carry out he µSPE of the studied analytes. LOV technique is especially suitable when dealing with SPE pretreatment, because it permits the use of smaller amounts of resin (compared with conventional SPE) and its automated transport in the system to renew the microcolumn when necessary with a high degree of repeatability [12].

As it is widely known, estrogens are non-volatile compounds; hence derivatization is needed for their quantification using gas chromatography. Typically, derivatization reactions are carried out off-line, requiring additional time before sample analysis and leading to experimental errors such contamination of the samples or a loss of sample due to evaporation and re-suspension steps [13]. On-line derivatization [14] can reduce the time needed to process the sample, decrease the amount of reagents used and increase the efficiency and speed of the analysis [15]. In-port derivatization is one of these on-line approaches, which involves introducing both the derivatization reagent and the sample directly into the GC inlet, where the reaction takes place in gas-phase. The reagent and sample can be injected manually, what requires the presence of the analyst to start each analysis [16], or using a software-controlled autosampler that allows the sequential injection of the sample and reagents, giving better repeatability results since no analyst intervention is needed [13]. In the present work, the second option has been exploited. Moreover, in order to study the variables affecting the derivatization reaction, the injection has been carried out by using programmable temperature vaporization technique (PTV-GC-MS) [17]. During the injection, the inlet is kept cool while solvent vaporization occurs. Solvent vapours are separated from analytes through venting of the vapour in the liner, and then the inlet is heated up quickly to transfer the sample into the column using a temperature controlled program. Besides, this technique enables injecting up to hundreds of microliters of the extract (Large Volume Injection, LVI) instead to the conventional 1–2 µL, increasing method sensitivity.

Therefore, the aim of this work was to accomplish a µSPE exploiting a SIA-LOV system prior in-port derivatization-LVI-PTV-GC-MS analysis to carry out the extraction, preconcentration and determination of estrogens in seawater samples. Several parameters affecting both the µSPE and the chromatographic analysis have been studied and optimized. The applicability of the proposed method to seawater samples has been evaluated.