Rapid automated screening, identification and quantification of organic micro-contaminants and their main transformation products in wastewater and river waters using liquid chromatography–quadrupole-time-of-flight mass spectrometry with an accurate-mass database

Abstract

In this study we have developed and evaluated an analytical method for a rapid automated screening and confirmation of a large number of organic micro-contaminants (almost 400) and also the quantification of the positive findings in water samples of different types (surface and wastewaters) using liquid chromatography–electrospray quadrupole-time-of-flight mass spectrometry (LC–QTOFMS) based on the use of an accurate-mass database. The created database includes data not only on the accurate masses of the target ions but also on the characteristic in-source fragment ions, isotopic pattern and retention time data. This customized database was linked to commercially available software which extracted all the potential compounds of interest from the LC–QTOFMS raw data of each sample and matched them against the database to search for targeted compounds in the sample. The detailed fragmentation information has also been used as a powerful tool for the automatic identification of unknown compounds and/or transformation products with similar structures to those of known organic contaminants included in the database. The database can be continually enlarged. To confirm identification of compounds which have no fragment ions (or fragments with low intensity/relative abundance) from in-source CID fragmentation or isomers which are not distinguished within full single mass spectra, a “Targeted MS/MS” method is developed. Thereafter, these compounds can be further analyzed using the collision energy (CE) in QTOF-MS/MS mode. Linearity and limits of detection were studied. Method detection limits (MDLs) in effluent wastewater and river waters were, in most cases, lowers or equal to 5 and 2 ng/L, respectively. Only 15 compounds had MDLs between 5 and 50 ng/L in effluent wastewater matrix. We obtained a linearity of the calibration curves over two orders of magnitude. The method has been applied to real samples and the results obtained reveal that most of the pharmaceutically active compounds contained in the created database were present in the water samples with concentrations in the range of ng/L and μg/L levels and in most of the samples between 2 and 15 pesticides of the 300 contained in the database were also detected. In addition to the compounds included in the database, some degradation products were found, thus revealing the method as a useful tool for the analysis of organic micro-contaminants in waters.

Introduction

Contamination of water resources by micro-contaminant residues is one of the major challenges for the preservation and sustainability of the environment. Although anti-pollution measures taken over the past half-century [1] have dramatically reduced the presence of many known contaminants in water, the number of potentially hazardous chemicals that can reach the environment is very large and new substances are constantly being developed and released. The focus for water pollution research has recently been extended from “priority” contaminants to the so-called “emerging contaminants” or “new environmental contaminants”, many of which have been unknown until recently.

An important group of emerging contaminants are the pharmaceutically active substances. The relatively recent awareness of the impact of pharmaceutical products on the environment has been reflected in literature since the 1990s through the exponentially increasing number of studies concerning the emerging class of water pollutants [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]. This rising interest is not only concomitant with the widespread and growing use of these compounds consumption, but also with the improvements in analytical techniques allowing detecting traces (ng/L or less) in any type of water.

In addition to the analysis of emerging contaminants, analysis of the regulated contaminants must not be neglected. These have to be continuously monitored, as is the case with pesticides.

Because of the potentially adverse effects of the presence of organic micro-contaminants in the environment, data concerning the concentration, fate and behaviour of these contaminants in the environment is urgently needed. With this purpose in mind, analytical methods for a rapid, sensitive and selective determination of a broad range of compounds in complex environmental matrices are required. Multi-residue analytical methodologies are becoming the required tools, as they provide greater knowledge about the contamination of the waters [2], [3] and they reduce the overall analysis time, field sampling and cost. Papers related to multi-residue analytical methodologies have increased over recent years, although most of them are focused on target analysis method. The scope of such methods rarely exceeds several tens of analytes, and it is quite unusual to find analytical methods applied to more than 100 organic micro-contaminants. This means that a large number of compounds and their degradation products fall outside of any control. The analysis of the transformation/degradation products represents an important challenge for environmental analysts. There is great concern over these products, which can be even more toxic, more dangerous and be at higher concentrations than the initial compounds themselves [4], [5]. Regarding these, limited information is currently available.

Up to date, polar micro-contaminant residue analysis in waters has been accomplished by liquid chromatography–tandem mass spectrometry (LC–MS/MS) in the selected reaction monitoring (SRM) mode [2], [6], [7], [8]. This approach has a severe limitation – the number of compounds that can be screened in a single run [9], [10]. Up to 150–200 compounds (depending on the scan speed/dwell-time) can be analyzed in a run by LC–MS/MS in the SRM mode with a dedicated chromatographic method. In addition, when increasing the number of compounds included in the SRM method, the possibility of finding common or overlapped transitions for coeluting isobaric compounds rises. Besides, another major limitation of these SRM methods is that they are blind to compounds not defined in the SRM method (non-target analysis) so that no or scarce information on possible non-target/unknown organic micro-contaminants or their degradation products is available when using these techniques. The information provided by methods using the SRM mode is often insufficient in assessing the quality of wastewater and environmental waters, given that only a limited number of analytes are recorded. There is, therefore, a need for methods offering rapid and reliable screening of a large number of compounds.

Unlike gas chromatography/mass spectrometry (GC/MS) reverse-search methods, where library searching is possible (e.g. the large library of the National Institute for Standards and Testing (NIST)), one of the major shortcomings traditionally reported in the use of LC–MS is the unavailability of commercial libraries allowing a rapid screening of the samples as can be performed in GC/MS [11]. The universal applicability of mass spectral libraries has been hampered by the scarce reproducibility of in-source collision-induced dissociation (CID) spectra and the difficulty of interchanging spectra acquired with instruments from different manufacturers [9], [10]. In contrast, accurate mass measurements are almost specific and universal for every target analyte regardless of the instrumentation used. In this sense, liquid chromatography–electrospray-time-of-flight mass spectrometry (LC–TOFMS) is a cost-effective technique for performing routine accurate mass analysis based on target databases [13]. The main features of LC–TOFMS instruments are accurate mass analysis capabilities and high sensitivity in “full-scan” acquisition mode so that micro-contaminants can be detected in complex matrices at low nanogram levels. Unambiguous identification is accomplished by means of accurate mass measurements from (de)protonated molecules, in-source CID fragment ions, and isotope signature matching [13], [14], [15]. In addition, LC–TOFMS provides satisfactory analytical performance for quantitation purposes, as has been demonstrated so far in the literature [15], [16].

Since LC–TOFMS has the ability to record an unlimited number of compounds because it operates in full-scan mode, this technique is very convenient for the development of screening strategies based on the use of accurate-mass databases [13], [17]. When coupled to a quadrupole or ion trap mass filter, QTOFMS or IT-TOF-MS permit MS/MS or MSⁿ analysis with accurate mass measurements for both the precursor and product ion, which constitutes a higher order mass identification than those afforded by nominal mass measurements obtained by other types of mass analyzers.

This work reports the development and evaluation of a method for a rapid automated screening, identification and quantification of organic micro-contaminants in waters using LC–QTOFMS, based on the use of an accurate-mass database. The database created includes accurate masses of the target ions, their characteristic in-source fragment ions, isotopic signature information, and retention time data. This database was linked to software which extracts all the compounds of interest from the LC–QTOFMS raw data of each sample and matches them against the database to search for targeted compounds in the sample. The number of compounds that can be screened in a run can easily be upgraded (non-target capabilities), thus enabling the reevaluation of the recorder data. The detailed fragmentation information has also been used as a powerful tool for the automatic identification of unknown compounds and/or transformation products with similar structure to known organic contaminants included in the database.

While the LC–QTOF instrument used as a TOF-MS system provides screening and quantification of both unknown and targeted organic contaminants, LC–QTOF, working in MS/MS mode, was required to confirm identification of compounds which have no fragment ions (or fragments with low intensity/relative abundance) from in-source CID fragmentation and also isomers which were not distinguished with full single mass spectra – this was done thanks to the valuable information given by the full product ion spectra at accurate masses.