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May 1, 2017 - Liquid Chromatography−High Resolution Mass Spectrometry. Rosa Montes,* Josu Aguirre, Xandro Vidal, Rosario Rodil, Rafael Cela, and Jos...
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Screening for polar chemicals in water by trifunctional mixedmode liquid chromatography-high resolution mass spectrometry Rosa Montes, Josu Aguirre, Xandro Vidal, Rosario Rodil, Rafael Cela, and Jose Benito Quintana Environ. Sci. Technol., Just Accepted Manuscript • Publication Date (Web): 01 May 2017 Downloaded from http://pubs.acs.org on May 1, 2017

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Screening for polar chemicals in water by trifunctional mixedmode liquid chromatography-high resolution mass spectrometry

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Rosa Montes*, Josu Aguirre, Xandro Vidal, Rosario Rodil, Rafael Cela, José Benito Quintana*

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Department of Analytical Chemistry, Nutrition and Food Sciences, IIAA - Institute of Food Analysis and Research, Universidade de Santiago de Compostela. Constantino Candeira S/N, Santiago de Compostela, Spain

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*Corresponding authors:

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RM: [email protected]; Tel.: +34 881816035

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JBQ : [email protected]; Tel.: +34 881816035

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Abstract

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The presence of persistent and mobile organic contaminants (PMOC) in aquatic environments

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is a matter of high concern due to their capability of crossing through natural and anthropogenic

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barriers, even reaching the drinking water. Most analytical methods rely on reversed-phase

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liquid chromatography (RPLC), which is quite limited for the detection of very polar chemicals.

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Thus, many of these PMOCs may have not been recognized as water pollutants yet, due to the

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lack of analytical methods capable to detect them. Mixed-mode LC (MMLC), providing the

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combination of RP and ion-exchange functionalities is explored in this work with a trifunctional

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column, combining RPLC, anion and cation exchange, which allows the simultaneous

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determination of analytes with extremely different properties. A non-discriminant sample

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concentration step followed by a MMLC-high resolution mass spectrometry method was

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developed for a group of 37 very polar model chemicals with different acid/base functionalities.

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The overall method performance was satisfactory with a mean limit of detection of 50 ng/L,

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relative standard deviation lower than 20% and overall recoveries (including matrix effects)

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higher than 60% for the 54% of model compounds. Then, the method was applied to 15 real

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water samples, by a suspect screening approach. For those detected PMOC with standard

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available, a preliminary estimation of concentrations was also performed. Thus, 22 compounds

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were unequivocally identified in a range of expected concentrations from 6 ng/L to 540 µg/L.

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Some of them are well-known PMOC, such as acesulfame, perfluorobutanoic acid or metformin,

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but other novel pollutants were also identified, as for example di-o-tolylguanidine or

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trifluromethanesulfonic acid, which had not or were scarcely studied in water so far.

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Introduction

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High resolution mass spectrometry (HRMS) instrumentation and software has greatly improved

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during the last decade. Indeed, the increasing popularity of this technique has been a turning

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point in screening studies, since it allows the identification of unknowns without the need of

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initially having pure standards and post-targeted analysis approaches, because the full accurate

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spectrum is registered for each injection.1-3 Hence, the coupling of liquid chromatography (LC)

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to HRMS has been applied in several water screening studies.

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HRMS screening methods rely on reversed-phase LC (RPLC),5,

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detection of very polar chemicals, which usually exhibit poor retention in traditional C18

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columns.10 Thus, many very polar organic chemicals may have not been recognized as water

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pollutants of concern yet, because the analytical methods developed so far are unable to detect

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them.11 Since these polar pollutants are by definition highly mobile in water, many of them can

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spread in the water cycle and even reach drinking water if they are persistent, i.e. “persistent

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and mobile organic pollutants” (PMOC). Thus, it is necessary to explore new LC retention

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mechanisms which allow the determination of PMOC in water.

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The limitations of RPLC for polar analytes have been commonly avoided by the use of

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alternative chromatographic modes (e.g. ion-exchange or ion-pairing), but they present well-

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known intrinsic drawbacks in LC-MS (such as interface and spectrometer contamination).12

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Hydrophilic interaction liquid chromatography (HILIC)

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viable alternatives in the determination of such polar compounds.15 The use of HILIC has been

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described for the identification of polar compounds and isomers when retention problems using

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RPLC were observed.16 However, the main advantage of MMLC is that it provides more than

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one type of interaction between the stationary phase and the analytes, allowing the

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simultaneous determination of compounds with different physico-chemical properties (e.g. ionic,

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basic, acid and neutral chemicals, or hydrophilic and hydrophobic compounds) in one run.

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There are several types of MMLC columns available nowadays

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generations, recently, trimodal RP/anion exchange/cation exchange (RP/AX/CX) columns

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based on embedded double functionalized ligands on nanopolymeric silica hybrid technology

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have also been commercialized.

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However, most of these LC9

which is limited for the

and mixed-mode LC (MMLC) can be

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12, 17, 20-22

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, but among the newest

Several applications involving trimodal MMLC have

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been described in the pharmaceutical field

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ingredients can be measured in one run, but in environmental analysis applications are still rare.

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Thus, the aim of this work was to develop a new analytical methodology for PMOC screening by

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using trimodal MMLC combined to HRMS. To this end, a group of polar model chemicals with

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different acid/base functionalities was employed for exploring their retention behavior and

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method development, before final application in suspect screening studies of over 3000

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chemicals aiming to detect PMOC in a set of 15 water samples from different countries of

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Europe. To the best of our knowledge, this is the first application of MMLC in LC-HRMS

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environmental screening studies, despite its potential for effectively detecting chemicals of

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different physico-chemical properties.

, as for instance counterions and active

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Experimental

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Chemicals and reagents

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Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Acetic acid

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(≥99%) and amonium hydroxide solution (25%) were purchased from Sigma-Aldrich (Steinheim,

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Germany). For method development, a mixture of 45 model analytes, with logD values at pH 7.4

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ranging from -6 to 1.8 (average -2.3), was used, including acidic, basic, neutral and amphoteric

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substances. Detailed information regarding these model analytes, including physico-chemical

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properties and suppliers is given in Table S1.

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Samples and sample preparation

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The 15 analyzed samples were obtained from different locations in Spain, Germany, France,

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The Netherlands and Switzerland and included surface (5), ground (7), drinking (2) water and

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also effluent wastewater (1). The sampling campaign was programmed within the project

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PROMOTE - Protecting Water Resources from Mobile Trace Chemicals. 25

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These samples were submitted to a simple freeze-drying protocol that avoids discrimination of

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PMOC that could be produced e.g. by solid-phase extraction (SPE). Under final working 3

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conditions 100 mL of filtered and frozen water were completely freeze-dried and reconstituted

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using 15 mL (3x5mL) of methanol. The organic extract was filtered through 0.22 µm

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polypropylene (PP) filters and evaporated to dryness in a Büchi Syncore PolyVap (Flawil,

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Switzerland), following the supplier recommended conditions for methanol evaporation. Two

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hundred microliters of water:acetonitrile (9:1) were used to reconstitute samples, which were

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finally filtered through 0.22 µm PP filters and injected in the chromatographic system. Thus, the

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final concentration factor reached was 500 times.

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Determination conditions

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The MMLC columns used were the Acclaim Trinity P1 (2.6 µm particle size; 3 mm internal

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diameter, in both 50 and 100 mm length format), supplied by Dionex (Sunnyvale, CA. USA). A

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Varian (Walnut Creek, CA, USA) LC-tripe quadrupole-MS (LC-QQQ) system was used for

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investigating the parameters affecting MMLC retention. The Varian instrument comprises a

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ProStar 210 dual pump coupled to a Varian 320MS furnished with an ESI interface. Nitrogen

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was used as nebulizing (50 psi) and drying gas (200 °C, 19 psi) and Argon was employed as

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collision gas (2 mTorr). The ESI interface was operated both in the positive and negative modes

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and the voltage of the ESI needle was fixed at 5,000 V. Compounds were recorded in the

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multiple reaction monitoring (MRM) mode as detailed in Table S1.

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For the screening studies, an Agilent (Wilmington, DE, USA) 1200 Series LC was used. The LC

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system was interfaced to a quadrupole-time of flight (QTOF) HRMS model Agilent 6520

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equipped with a Dual electrospray (ESI) ion source. Nitrogen was used as nebulizing (40 psi)

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and drying gas (300 °C, 5 L min ) in the dual ESI source and also as collision gas in the

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MS/MS experiments. The QTOF instrument was operated in the high resolution 4 GHz mode.

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This mode provides a full width at half maximum (FWHM) resolution of ca. 10,600 at m/z

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118.0862 and ca. 16,900 at m/z 922.0097. A reference calibration solution, supplied by Agilent,

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was continuously sprayed in the source during the chromatographic run, providing the required

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accuracy of mass assignations.

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Several chromatographic conditions, in isocratic mode, were tested in order to establish

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adequate compromise elution conditions for the majority of model compounds, including mobile

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phase pH (3.5 and 5.5), concentration of buffer (ammonium acetate, 10-80 mM) and percentage

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of organic modifier (acetonitrile, 5-95%). The final LC method consisted of a simultaneous

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binary gradient from low organic content (2% ACN) and buffer (5 mM ammonium acetate, pH

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5.5) to high organic (80% ACN) and buffer (20 mM ammonium acetate, pH 5.5) in 10 min, with a

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final isocratic time of 15 min, with the 50 mm length column being used.

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Method performance evaluation

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The analytical parameters evaluated were limits of detection (LODs), repeatability and apparent

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recovery with the LC-QTOF instrument for the model compounds. Linearity of the method was

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not evaluated, since the quantification of samples was beyond the original goal of this

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methodology. The instrumental LODs (iLODs) were determined by the injection of standards, for

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a signal to noise ratio (S/N) of 3. The method LODs (mLODs) were calculated by spiking real

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river water samples at the 200 ng L level, submitting them to the entire protocol and calculating

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the S/N. Instrumental and full methodology repeatability was measured by the relative standard

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deviation (RSD) of three consecutive injections of either standards or extracts, and expressed

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as iRSD and mRSD, respectively. The apparent recovery was assessed by the addition of

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model analytes to real surface water samples (200 ng L ), and comparing the obtained

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response against pure standards. Blank samples were processed in order to evaluate the

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presence of these compounds in the selected matrix.

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Workflow for MMLC-QTOF suspect screening

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The scheme of the whole screening workflow is presented in Figure 1 (see Figure S1 for more

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detailed information). The accurate mass obtained results were processed using the Qualitative

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Analysis of MassHunter Workstation software B.07.00. The strategy used to analyze the data

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consisted on the use of the search algorithm “Find by Formula” (FBF), which analyzes each

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data file searching for the specified compounds, i.e those from a pre-selected database. In this

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work, two different databases were used: a) a database taken from Wode et al.26 containing

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over 2000 typical water micropollutants and b) a lab-developed library prepared within the

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PROMOTE consortium consisting of over 1000 polar compounds extracted from REACH

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(Registration, Evaluation, Authorization and restriction of Chemicals) database and fulfilling the

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criteria of being PMOCs and having a high emission score into the environment.27-28 Thus, FBF

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computes the neutral mass and the theoretical isotopic pattern corresponding to each specified

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-1

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formula, calculates the m/z of permitted ions (in this work set to protonation, Na+ K+ and NH4+

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adducts in positive mode; deprotonation only in negative) and searches for chromatographic

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peaks for such m/z values. Then, it extracts the spectrum and for each compound the software

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calculates a normalized score parameter (0-100) combining mass deviation, isotope abundance

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and spacing between ions. Thus a MS score of 100 implies an absolute match. As screening

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criteria, a maximum mass error (±5 ppm) and score>80 were set as cut-off values. Finally, the

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software provided a list of candidate compounds that were present in the databases and met

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the selected data processing tolerances.

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For those suspect compounds identified in the MS mode (scan range: 50-1100 m/z), MS/MS

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experiments (scanning from 40 m/z to 10 m/z units above the precursor ion) at two different

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collision energies (15 and 30 V) were carried out. Thus, the presence of some analytes was

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confirmed by their fragmentation pattern. Finally, the tentatively identified compounds that were

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commercially available were acquired and injected into the system in order to unequivocally

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confirm their presence. Thus, to give a positive identification the retention and at least two

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product ions in MS/MS spectrum should match 29.

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Finally, in order to obtain a tentative idea of the detected chemicals’ concentrations, the 10-90%

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percentile (P10-90) was calculated from the recovery evaluation of model analytes (see above

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and Figure S2). These percentile values were 15.4-96.3% apparent recoveries. Therefore, the

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final estimated range of concentration of the analytes in the extracts was calculated by

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comparison with the injection of standards and applying the P10-90 percentiles of recovery.

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Results and discussion

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Development of the MMLC–MS methodology

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Evaluation of chromatographic conditions: pH, ionic strength and organic modifier

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percentage

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In the first steps of this work, the retention mechanisms of model analytes were studied under

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isocratic conditions using a 100 mm Acclaim Trinity P1 column. The selected chromatographic

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parameters were evaluated taking into account the column supplier specifications, where the

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use of methanol as organic modifier and pH values outside the range 3-6 were not 6

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recommended. Thus, pH 3.5 and 5.5, different percentages of acetonitrile as organic modifier

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(5-95%) and 10 to 80 mM of ammonium acetate buffer were evaluated.

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Figure 2 shows the retention behavior for some model compounds, belonging to different

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chemical classes, i.e. acidics, neutrals, amphoterics, basics and cations. As shown, most of

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compounds eluted earlier at high pH (dotted lines) and buffer concentrations (round markers).

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Also, in general, the higher the acetonitrile percentage, the lower the retention time, as

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expectable because of the RPLC functionality of the column. However, for some model

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analytes, such as aspartame (Figure 2) retention is higher at either low or high acetonitrile

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content, i.e. there is RPLC retention at low organic and HILIC-like retention when this is

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increased sufficiently. Such behavior has been already described in the literature for some other

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chemicals

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perfluorobutanoic acid, presented acceptable retention at all the studied conditions, with a few

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compounds (e.g. monomethyl phthalate) showing the above mentioned dual HILIC-like and RP

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mechanisms depending on the percentage of organic modifier (data not shown). Neutral

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analytes, e.g. sucralose, showed low retention times at almost all studied conditions (see

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Figure 2), as the only available mechanism, RP, is not strong enough for these polar neutral

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compounds. Cationic and basic compounds were well-retained and the behavior of amphoteric

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chemicals was highly dependent on the conditions and the nature of each analyte. Thus,

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bipyridilium compounds (permanently positively charged), such as diquat and paraquat,

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phosphonic and amino acid group-containing compounds, e.g. glyphosate, and mono-

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substituted phosphates were too strongly retained at the studied conditions.

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In summary, evaluating the isocratic behavior of model analytes we observed that 1) most of

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compounds presented high retention at the lowest pH, i.e. 3.5; and 2) high buffer concentration

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could improve the elution of the most retained analytes (e.g. glyphosate)

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suppression in LC-MS. On the basis of the isocratic results, a gradient elution program was

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tested in order to obtain compromise elution conditions covering a wide range of model

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analytes. In this case, the pH value was fixed at 5.5 and a simultaneous gradient of buffer (from

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10 mM to 40 mM ammonium acetate) and organic modifier (from 2% to 80%) was programmed

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in 10 minutes, using a final isocratic step at maximum acetonitrile percentage of 15 min. The

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obtained retention factors are represented in Figure S3. More than 80% of model analytes elute

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. In terms of analytes’ properties, strongly acidic compounds, such as

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but led to strong ion

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in less than 15 min, with adequate peak shapes, showing a retention factor lower than 8. As

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previously mentioned, bipyridilium compounds, phosphonic and amino acid group-containing

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compounds and mono-substituted phosphates were too strongly retained and would need

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dedicated approaches. Thus, in further experiments, these 8 analytes were not further

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considered and since then, the model compounds mixture consisted of 37 compounds.

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Determination of those 8 strongly retained chemicals could be improved in the future by

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increasing the buffer concentration (not highly compatible with LC-MS), decreasing the column

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length bellow 5 cm or by selecting other MMLC column chemistries.

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Comparison of MMLC and RPLC

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The retention of the model compounds under optimal conditions in the MMLC column was

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compared to that obtained with a Waters Symmetry Shield RP18 (3.5 µm; 2.1 mm × 100 mm)

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column. For RP column a common water/acetonitrile (with 5 mM of ammonium acetate in both

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phases) gradient was selected. This gradient was programmed from 2% to 100% acetonitrile in

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10 min, with the same final isocratic time than the MM column. Figure 3 presents the obtained

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differences in the retention factor for both columns. From the model analytes, 62% of them

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showed better retention when the MMLC method was used, 16% presented similar and low

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retention in both approaches and the remaining 22 % model compounds were more efficiently

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retained in the RP column. Moreover 2 chemicals presented retention factors **) DW_003_pos.d Subtract 4.5 106.0650 4 3.5 3 116.0491 133.0755 2.5 2 91.0540 1.5 1 223.1223 0.5 0 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 Counts vs. Mass-to-Charge (m/z)

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(e) MS/MS spectrum C.E. 30V -> Standard x10 4 +ESI Product Ion (12.686-12.841 min, 11 Scans) Frag=100.0V [email protected] (240.1486[z=1] -> **) PM3_Q90.d Subtract 106.0651 1.6 1.4 133.0757 1.2 116.0495 1 0.8 91.0545 0.6 0.4 0.2 0

223.1228 60

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140 150 160 170 180 Counts vs. Mass-to-Charge (m/z)

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