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Analysis of Diquat and Paraquat Using UHPLC Orbitrap MS – Method Development, Matrix Effects and Performance
Overview
The purpose of this work was to investigate the effect of ultrahigh-performance liquid
chromatography (UHPLC) mobile phases and operational parameters of a UHPLC-
Orbitrap™ mass spectrometry system used in the analysis of quaternary ammonium
herbicides paraquat (PQ) and diquat (DQ). UHPLC mobile phases of different pH
values were evaluated to achieve optimum separation of PQ and DQ on a Thermo
Scientific™ Acclaim™ Trinity™ Q1 column which was specifically designed for this
application, as well as to observe the relative intensity changes of mass spectral
peaks. The signal-to-noise ratio (SNR) of extracted ion chromatograms (XIC) obtained
from different
m/z
at different pH values and declustering potential (corona voltage) of
electrospray ionization (ESI) source were evaluated. Based on results obtained from
this study, a method was developed for the unambiguous identification of PQ and DQ
in environmental water samples with the ability to deliver analytical data with superior
SNR, high precision and accuracy.
Introduction
Paraquat (PQ, 1,1
′
-dimethyl-4,4
′
-bipyridylium dichloride, C
12
H
14
N
2
Cl
2
) and diquat (DQ,
1,1
′
-ethylene-2,2
′
-bipyridilium dibromide, C
12
H
12
N
2
Br
2
) are quaternary amines widely
used as non-selective and non-systematic herbicides for both terrestrial and aquatic
plant control. Both PQ and DQ are toxic by contact and/or ingestion. The Ontario
Drinking Water Quality Standards (Ontario Regulation 169/03) has a standard of 70
and 10
µ
g/L, respectively for diquat and paraquat. Diquat is also regulated by the
United States (U.S.) Environmental Protection Agency (EPA) at a maximum
contaminant limit (MCL) of 20
μ
g/L in drinking water, while PQ is unregulated by the
U.S. EPA. The European Union has a drinking water MCL of 0.1 µg/L for any individual
pesticide and a combined 0.5 µg/L MCL for all pesticides. Different data quality
objectives (DQO) derived from these regulations dictate the need for a
reliable/versatile method with a superior analytical sensitivity (i.e. <0.1 µg/L or better)
to meet different regulatory requirements.
Methods commonly used for PQ and DQ analysis include the separation by ion-pairing
liquid chromatography, capillary electrophoresis, hydrophilic interaction liquid
chromatography or ion-exchange chromatography using either ultraviolet (UV) or mass
spectrometry for detection. Depending on the technology, method detection limits
(MDL) have been established in the low
μ
g/L for PQ and high ng/L for DQ. A 2012 U.S.
Geological Survey report showed that about 3 million and 150,000 pounds of PQ and
DQ were used annually in the United States (Ref 1).
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an ESI
interface has been the method of choice for PQ and DQ analysis since late 1990s.
Depending on the pH of LC mobile phase and ESI source used, the deprotonated
cation [M – H]
+
(
m/z
183 for DQ and
m/z
185 for PQ), the singly charged radical ion
[M]
+
.
(
m/z
184 for DQ and
m/z
186 for PQ) and, to a less extent, the doubly charged
quasi molecular ion M
2+
(
m/z
92 for DQ and
m/z
93 for PQ) have been observed in the
ESI mass spectra. The multiple reaction monitoring (MRM) transitions used in the
analysis varied depending on the instrument and mobile phase. Commonly used
precursor ions are the singly charged radical ion [M]
+ .
and deprotonated cation [M –
H]
+
with a limited mentioning on the use of the doubly charged quasi molecular ion M
2+
(Ref 2). Many product ions have been used in the MRM transitions for PQ and DQ
analysis. These can be, for example, from the loss of masses 15 ([M – CH
3
]
+
,
m/z
170)
or 27 ([(M – H) – HCN]
+
,
m/z
158) for PQ; while those at
m/z
168 ([(M – H) – CH
3
]
+
)
and
m/z
157 ([(M – H) – C
2
H
2
]
+
) for DQ analysis (Ref. 3). Product ions resulted from
the loss of masses 16 ([(M – H) – CH
3
– H]
+
,
m/z
169) or 42 ([(M – H) – CH
3
– HCN]
+
,
m/z
143) for PQ; and at
m/z
130 ([(M – H) – C
2
H
2
– HCN]
+
) for DQ analysis (Ref. 4). A
literature review showed more than 10 different MRM transitions may be used in the
analysis of these two pesticides.
With the three available precursor ions from PQ (
m/z
93, 185 and 186) and DQ (
m/z
92, 183, 184), products ions of PQ and DQ may be differentiated by 1 amu. As the DQ
13
C-isotopic mass at
m/z
185 would overlap with the [M – H]
+
of PQ, one might expect
interference in the analysis of PQ and DQ with inferior LC separation and MS data
collected with unit mass resolution. Diquat has been known to have high ionization
efficiency, with about 13% intensity of the native mass spectral peak of DQ contributing
to PQ through the
13
C-isotopic peak, quantitative results obtained for PQ might be
biased high. We report in this poster the relationship between pH of mobile phase and
the population of the three possible molecular formations of PQ DQ, the root cause of
analytical interference and a direct injection UHPLC-Orbitrap MS method for the
analysis of PQ and DQ that meets the regulatory need of different jurisdictions.
Methods
Sample Preparation and Ch
Individual stock solutions of P
Analytical Solutions (Brockville
labelled PQ (D
8
-PQ) and DQ (
Claire, QC, Canada). Native a
prepared by mixing the corres
analytical standard solutions
nanopure water (pure water, g
Thermo Scientific™ Barnstead
ON, Canada). Due to the high
silanized glassware were use
ACS reagent grade ammoniu
hydrochloric acid (HCl) were p
HPLC grade acetonitrile (CH
3
Canada). The current method
preparation. Environmental sa
and refrigerated at 5 3 ºC unti
while surface water samples
aliquot of each sample was tra
with 10
µ
L of 500
µ
g/L, D-labe
vortexed and stored under refr
Ultra High Performance Liq
The Thermo Scientific™ Dion
consisted of a HRG-3400RS b
column compartment. Separat
Trinity Q1 column (2.1 × 50 m
acetonitrile:100 mM, pH5.0 a
The column oven was set at 3
Mobile phases used in the pH
analysis but prepared at pH of
using 0.013 mm i.d. x 100 cm
and four different pH levels to
UHPLC Orbitrap MS analysis.
Mass Spectrometry
The UHPLC was interfaced to
using a HESI II probe interfac
positive mode by infusion of st
(>99%) was used in the ESI s
dissociation (HCD) cell, enabli
without precursor ion selection
was done by using normalized
rate of 0.45 mL/min and colum
seconds. Mass spectrometric
equivalent of declustering pot
140,000 (defined by the full-wi
resulting a scanning rate of >
C-trap inject time of 50 msec.
accurately define each XIC ch
The effect of SV on the formati
DQ was also studied by differe
Data Analysis
Analytical data collected were
ExactFinder™ and TraceFind
Xcalibur was used to process
and TraceFinder softwares we
data, a mass extraction windo
sides of the base peak were u
were exported to Microsoft
®
E