Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)
Article
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
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Environmental Science & Technology is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 24
Environmental Science & Technology
3
Screening for polar chemicals in water by trifunctional mixedmode liquid chromatography-high resolution mass spectrometry
4
Rosa Montes*, Josu Aguirre, Xandro Vidal, Rosario Rodil, Rafael Cela, José Benito Quintana*
5 6 7
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
1 2
8
*Corresponding authors:
9
RM:
[email protected]; Tel.: +34 881816035
10
JBQ :
[email protected]; Tel.: +34 881816035
11
Abstract
12
The presence of persistent and mobile organic contaminants (PMOC) in aquatic environments
13
is a matter of high concern due to their capability of crossing through natural and anthropogenic
14
barriers, even reaching the drinking water. Most analytical methods rely on reversed-phase
15
liquid chromatography (RPLC), which is quite limited for the detection of very polar chemicals.
16
Thus, many of these PMOCs may have not been recognized as water pollutants yet, due to the
17
lack of analytical methods capable to detect them. Mixed-mode LC (MMLC), providing the
18
combination of RP and ion-exchange functionalities is explored in this work with a trifunctional
19
column, combining RPLC, anion and cation exchange, which allows the simultaneous
20
determination of analytes with extremely different properties. A non-discriminant sample
21
concentration step followed by a MMLC-high resolution mass spectrometry method was
22
developed for a group of 37 very polar model chemicals with different acid/base functionalities.
23
The overall method performance was satisfactory with a mean limit of detection of 50 ng/L,
24
relative standard deviation lower than 20% and overall recoveries (including matrix effects)
25
higher than 60% for the 54% of model compounds. Then, the method was applied to 15 real
26
water samples, by a suspect screening approach. For those detected PMOC with standard
27
available, a preliminary estimation of concentrations was also performed. Thus, 22 compounds
28
were unequivocally identified in a range of expected concentrations from 6 ng/L to 540 µg/L.
29
Some of them are well-known PMOC, such as acesulfame, perfluorobutanoic acid or metformin,
30
but other novel pollutants were also identified, as for example di-o-tolylguanidine or
31
trifluromethanesulfonic acid, which had not or were scarcely studied in water so far.
1
ACS Paragon Plus Environment
Environmental Science & Technology
Page 2 of 24
32
Introduction
33
High resolution mass spectrometry (HRMS) instrumentation and software has greatly improved
34
during the last decade. Indeed, the increasing popularity of this technique has been a turning
35
point in screening studies, since it allows the identification of unknowns without the need of
36
initially having pure standards and post-targeted analysis approaches, because the full accurate
37
spectrum is registered for each injection.1-3 Hence, the coupling of liquid chromatography (LC)
38
to HRMS has been applied in several water screening studies.
39
HRMS screening methods rely on reversed-phase LC (RPLC),5,
40
detection of very polar chemicals, which usually exhibit poor retention in traditional C18
41
columns.10 Thus, many very polar organic chemicals may have not been recognized as water
42
pollutants of concern yet, because the analytical methods developed so far are unable to detect
43
them.11 Since these polar pollutants are by definition highly mobile in water, many of them can
44
spread in the water cycle and even reach drinking water if they are persistent, i.e. “persistent
45
and mobile organic pollutants” (PMOC). Thus, it is necessary to explore new LC retention
46
mechanisms which allow the determination of PMOC in water.
47
The limitations of RPLC for polar analytes have been commonly avoided by the use of
48
alternative chromatographic modes (e.g. ion-exchange or ion-pairing), but they present well-
49
known intrinsic drawbacks in LC-MS (such as interface and spectrometer contamination).12
50
Hydrophilic interaction liquid chromatography (HILIC)
51
viable alternatives in the determination of such polar compounds.15 The use of HILIC has been
52
described for the identification of polar compounds and isomers when retention problems using
53
RPLC were observed.16 However, the main advantage of MMLC is that it provides more than
54
one type of interaction between the stationary phase and the analytes, allowing the
55
simultaneous determination of compounds with different physico-chemical properties (e.g. ionic,
56
basic, acid and neutral chemicals, or hydrophilic and hydrophobic compounds) in one run.
57
There are several types of MMLC columns available nowadays
58
generations, recently, trimodal RP/anion exchange/cation exchange (RP/AX/CX) columns
59
based on embedded double functionalized ligands on nanopolymeric silica hybrid technology
60
have also been commercialized.
4-8
13-14
However, most of these LC9
which is limited for the
and mixed-mode LC (MMLC) can be
17-18
12, 17, 20-22
19
, but among the newest
Several applications involving trimodal MMLC have
2
ACS Paragon Plus Environment
Page 3 of 24
Environmental Science & Technology
17, 21, 23
61
been described in the pharmaceutical field
62
ingredients can be measured in one run, but in environmental analysis applications are still rare.
63
24
64
Thus, the aim of this work was to develop a new analytical methodology for PMOC screening by
65
using trimodal MMLC combined to HRMS. To this end, a group of polar model chemicals with
66
different acid/base functionalities was employed for exploring their retention behavior and
67
method development, before final application in suspect screening studies of over 3000
68
chemicals aiming to detect PMOC in a set of 15 water samples from different countries of
69
Europe. To the best of our knowledge, this is the first application of MMLC in LC-HRMS
70
environmental screening studies, despite its potential for effectively detecting chemicals of
71
different physico-chemical properties.
, as for instance counterions and active
72
73
Experimental
74
Chemicals and reagents
75
Acetonitrile (HPLC grade) was purchased from Merck (Darmstadt, Germany). Acetic acid
76
(≥99%) and amonium hydroxide solution (25%) were purchased from Sigma-Aldrich (Steinheim,
77
Germany). For method development, a mixture of 45 model analytes, with logD values at pH 7.4
78
ranging from -6 to 1.8 (average -2.3), was used, including acidic, basic, neutral and amphoteric
79
substances. Detailed information regarding these model analytes, including physico-chemical
80
properties and suppliers is given in Table S1.
81
Samples and sample preparation
82
The 15 analyzed samples were obtained from different locations in Spain, Germany, France,
83
The Netherlands and Switzerland and included surface (5), ground (7), drinking (2) water and
84
also effluent wastewater (1). The sampling campaign was programmed within the project
85
PROMOTE - Protecting Water Resources from Mobile Trace Chemicals. 25
86
These samples were submitted to a simple freeze-drying protocol that avoids discrimination of
87
PMOC that could be produced e.g. by solid-phase extraction (SPE). Under final working 3
ACS Paragon Plus Environment
Environmental Science & Technology
Page 4 of 24
88
conditions 100 mL of filtered and frozen water were completely freeze-dried and reconstituted
89
using 15 mL (3x5mL) of methanol. The organic extract was filtered through 0.22 µm
90
polypropylene (PP) filters and evaporated to dryness in a Büchi Syncore PolyVap (Flawil,
91
Switzerland), following the supplier recommended conditions for methanol evaporation. Two
92
hundred microliters of water:acetonitrile (9:1) were used to reconstitute samples, which were
93
finally filtered through 0.22 µm PP filters and injected in the chromatographic system. Thus, the
94
final concentration factor reached was 500 times.
95
Determination conditions
96
The MMLC columns used were the Acclaim Trinity P1 (2.6 µm particle size; 3 mm internal
97
diameter, in both 50 and 100 mm length format), supplied by Dionex (Sunnyvale, CA. USA). A
98
Varian (Walnut Creek, CA, USA) LC-tripe quadrupole-MS (LC-QQQ) system was used for
99
investigating the parameters affecting MMLC retention. The Varian instrument comprises a
100
ProStar 210 dual pump coupled to a Varian 320MS furnished with an ESI interface. Nitrogen
101
was used as nebulizing (50 psi) and drying gas (200 °C, 19 psi) and Argon was employed as
102
collision gas (2 mTorr). The ESI interface was operated both in the positive and negative modes
103
and the voltage of the ESI needle was fixed at 5,000 V. Compounds were recorded in the
104
multiple reaction monitoring (MRM) mode as detailed in Table S1.
105
For the screening studies, an Agilent (Wilmington, DE, USA) 1200 Series LC was used. The LC
106
system was interfaced to a quadrupole-time of flight (QTOF) HRMS model Agilent 6520
107
equipped with a Dual electrospray (ESI) ion source. Nitrogen was used as nebulizing (40 psi)
108
and drying gas (300 °C, 5 L min ) in the dual ESI source and also as collision gas in the
109
MS/MS experiments. The QTOF instrument was operated in the high resolution 4 GHz mode.
110
This mode provides a full width at half maximum (FWHM) resolution of ca. 10,600 at m/z
111
118.0862 and ca. 16,900 at m/z 922.0097. A reference calibration solution, supplied by Agilent,
112
was continuously sprayed in the source during the chromatographic run, providing the required
113
accuracy of mass assignations.
114
Several chromatographic conditions, in isocratic mode, were tested in order to establish
115
adequate compromise elution conditions for the majority of model compounds, including mobile
116
phase pH (3.5 and 5.5), concentration of buffer (ammonium acetate, 10-80 mM) and percentage
−1
4
ACS Paragon Plus Environment
Page 5 of 24
Environmental Science & Technology
117
of organic modifier (acetonitrile, 5-95%). The final LC method consisted of a simultaneous
118
binary gradient from low organic content (2% ACN) and buffer (5 mM ammonium acetate, pH
119
5.5) to high organic (80% ACN) and buffer (20 mM ammonium acetate, pH 5.5) in 10 min, with a
120
final isocratic time of 15 min, with the 50 mm length column being used.
121
Method performance evaluation
122
The analytical parameters evaluated were limits of detection (LODs), repeatability and apparent
123
recovery with the LC-QTOF instrument for the model compounds. Linearity of the method was
124
not evaluated, since the quantification of samples was beyond the original goal of this
125
methodology. The instrumental LODs (iLODs) were determined by the injection of standards, for
126
a signal to noise ratio (S/N) of 3. The method LODs (mLODs) were calculated by spiking real
127
river water samples at the 200 ng L level, submitting them to the entire protocol and calculating
128
the S/N. Instrumental and full methodology repeatability was measured by the relative standard
129
deviation (RSD) of three consecutive injections of either standards or extracts, and expressed
130
as iRSD and mRSD, respectively. The apparent recovery was assessed by the addition of
131
model analytes to real surface water samples (200 ng L ), and comparing the obtained
132
response against pure standards. Blank samples were processed in order to evaluate the
133
presence of these compounds in the selected matrix.
134
Workflow for MMLC-QTOF suspect screening
135
The scheme of the whole screening workflow is presented in Figure 1 (see Figure S1 for more
136
detailed information). The accurate mass obtained results were processed using the Qualitative
137
Analysis of MassHunter Workstation software B.07.00. The strategy used to analyze the data
138
consisted on the use of the search algorithm “Find by Formula” (FBF), which analyzes each
139
data file searching for the specified compounds, i.e those from a pre-selected database. In this
140
work, two different databases were used: a) a database taken from Wode et al.26 containing
141
over 2000 typical water micropollutants and b) a lab-developed library prepared within the
142
PROMOTE consortium consisting of over 1000 polar compounds extracted from REACH
143
(Registration, Evaluation, Authorization and restriction of Chemicals) database and fulfilling the
144
criteria of being PMOCs and having a high emission score into the environment.27-28 Thus, FBF
145
computes the neutral mass and the theoretical isotopic pattern corresponding to each specified
-1
-1
5
ACS Paragon Plus Environment
Environmental Science & Technology
Page 6 of 24
146
formula, calculates the m/z of permitted ions (in this work set to protonation, Na+ K+ and NH4+
147
adducts in positive mode; deprotonation only in negative) and searches for chromatographic
148
peaks for such m/z values. Then, it extracts the spectrum and for each compound the software
149
calculates a normalized score parameter (0-100) combining mass deviation, isotope abundance
150
and spacing between ions. Thus a MS score of 100 implies an absolute match. As screening
151
criteria, a maximum mass error (±5 ppm) and score>80 were set as cut-off values. Finally, the
152
software provided a list of candidate compounds that were present in the databases and met
153
the selected data processing tolerances.
154
For those suspect compounds identified in the MS mode (scan range: 50-1100 m/z), MS/MS
155
experiments (scanning from 40 m/z to 10 m/z units above the precursor ion) at two different
156
collision energies (15 and 30 V) were carried out. Thus, the presence of some analytes was
157
confirmed by their fragmentation pattern. Finally, the tentatively identified compounds that were
158
commercially available were acquired and injected into the system in order to unequivocally
159
confirm their presence. Thus, to give a positive identification the retention and at least two
160
product ions in MS/MS spectrum should match 29.
161
Finally, in order to obtain a tentative idea of the detected chemicals’ concentrations, the 10-90%
162
percentile (P10-90) was calculated from the recovery evaluation of model analytes (see above
163
and Figure S2). These percentile values were 15.4-96.3% apparent recoveries. Therefore, the
164
final estimated range of concentration of the analytes in the extracts was calculated by
165
comparison with the injection of standards and applying the P10-90 percentiles of recovery.
166
Results and discussion
167
Development of the MMLC–MS methodology
168
Evaluation of chromatographic conditions: pH, ionic strength and organic modifier
169
percentage
170
In the first steps of this work, the retention mechanisms of model analytes were studied under
171
isocratic conditions using a 100 mm Acclaim Trinity P1 column. The selected chromatographic
172
parameters were evaluated taking into account the column supplier specifications, where the
173
use of methanol as organic modifier and pH values outside the range 3-6 were not 6
ACS Paragon Plus Environment
Page 7 of 24
Environmental Science & Technology
174
recommended. Thus, pH 3.5 and 5.5, different percentages of acetonitrile as organic modifier
175
(5-95%) and 10 to 80 mM of ammonium acetate buffer were evaluated.
176
Figure 2 shows the retention behavior for some model compounds, belonging to different
177
chemical classes, i.e. acidics, neutrals, amphoterics, basics and cations. As shown, most of
178
compounds eluted earlier at high pH (dotted lines) and buffer concentrations (round markers).
179
Also, in general, the higher the acetonitrile percentage, the lower the retention time, as
180
expectable because of the RPLC functionality of the column. However, for some model
181
analytes, such as aspartame (Figure 2) retention is higher at either low or high acetonitrile
182
content, i.e. there is RPLC retention at low organic and HILIC-like retention when this is
183
increased sufficiently. Such behavior has been already described in the literature for some other
184
chemicals
185
perfluorobutanoic acid, presented acceptable retention at all the studied conditions, with a few
186
compounds (e.g. monomethyl phthalate) showing the above mentioned dual HILIC-like and RP
187
mechanisms depending on the percentage of organic modifier (data not shown). Neutral
188
analytes, e.g. sucralose, showed low retention times at almost all studied conditions (see
189
Figure 2), as the only available mechanism, RP, is not strong enough for these polar neutral
190
compounds. Cationic and basic compounds were well-retained and the behavior of amphoteric
191
chemicals was highly dependent on the conditions and the nature of each analyte. Thus,
192
bipyridilium compounds (permanently positively charged), such as diquat and paraquat,
193
phosphonic and amino acid group-containing compounds, e.g. glyphosate, and mono-
194
substituted phosphates were too strongly retained at the studied conditions.
195
In summary, evaluating the isocratic behavior of model analytes we observed that 1) most of
196
compounds presented high retention at the lowest pH, i.e. 3.5; and 2) high buffer concentration
197
could improve the elution of the most retained analytes (e.g. glyphosate)
198
suppression in LC-MS. On the basis of the isocratic results, a gradient elution program was
199
tested in order to obtain compromise elution conditions covering a wide range of model
200
analytes. In this case, the pH value was fixed at 5.5 and a simultaneous gradient of buffer (from
201
10 mM to 40 mM ammonium acetate) and organic modifier (from 2% to 80%) was programmed
202
in 10 minutes, using a final isocratic step at maximum acetonitrile percentage of 15 min. The
203
obtained retention factors are represented in Figure S3. More than 80% of model analytes elute
13
. In terms of analytes’ properties, strongly acidic compounds, such as
30
but led to strong ion
7
ACS Paragon Plus Environment
Environmental Science & Technology
Page 8 of 24
204
in less than 15 min, with adequate peak shapes, showing a retention factor lower than 8. As
205
previously mentioned, bipyridilium compounds, phosphonic and amino acid group-containing
206
compounds and mono-substituted phosphates were too strongly retained and would need
207
dedicated approaches. Thus, in further experiments, these 8 analytes were not further
208
considered and since then, the model compounds mixture consisted of 37 compounds.
209
Determination of those 8 strongly retained chemicals could be improved in the future by
210
increasing the buffer concentration (not highly compatible with LC-MS), decreasing the column
211
length bellow 5 cm or by selecting other MMLC column chemistries.
212
Comparison of MMLC and RPLC
213
The retention of the model compounds under optimal conditions in the MMLC column was
214
compared to that obtained with a Waters Symmetry Shield RP18 (3.5 µm; 2.1 mm × 100 mm)
215
column. For RP column a common water/acetonitrile (with 5 mM of ammonium acetate in both
216
phases) gradient was selected. This gradient was programmed from 2% to 100% acetonitrile in
217
10 min, with the same final isocratic time than the MM column. Figure 3 presents the obtained
218
differences in the retention factor for both columns. From the model analytes, 62% of them
219
showed better retention when the MMLC method was used, 16% presented similar and low
220
retention in both approaches and the remaining 22 % model compounds were more efficiently
221
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)
240
250
260
270
(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
70
80
90
100
110
120
130
140 150 160 170 180 Counts vs. Mass-to-Charge (m/z)
190
200
210
220
230
240
250
260
Montes et al. Fig. 4 ACS Paragon Plus Environment
