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Stereoselective Determination of Tebuconazole in Water and Zebrafish by Supercritical Fluid Chromatography tandem mass spectrometry Na Liu, Fengshou Dong, Jun Xu, Xingang Liu, Zenglong Chen, Yan Tao, Xinglu Pan, XiXi Chen, and Yongquan Zheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02450 • Publication Date (Web): 30 Jun 2015 Downloaded from http://pubs.acs.org on July 6, 2015
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Stereoselective Determination of Tebuconazole in Water and Zebrafish by
2
Supercritical Fluid Chromatography Tandem Mass Spectrometry
3 †,‡
, Fengshou Dong ‡,Jun Xu ‡, Xingang Liu ‡, Zenglong Chen‡, Yan Tao‡,
4
Na Liu
5
Xinglu Pan‡, XiXi Chen‡, Yongquan Zheng *,‡
6 7
†
8
University, Shenyang, 110866, P. R. China
9
‡
Department of Pesticide Science, College of Plant Protection, Shenyang Agricultural
Institute of Plant Protection, Chinese Academy of Agricultural Sciences, State Key
10
Laboratory for Biology of Plant Diseases and Insect Pests, Beijing, 100193, P. R.
11
China
12 13
* Corresponding Author: Tel: +86-01-62815908, Fax: +86-01-62815908; E-mail:
14
[email protected].
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ABSTRACT: A simple and sensitive method for the enantioselective determination
28
of tebuconazole enantiomers in water and zebrafish has been established using
29
supercritical fluid chromatography (SFC)-MS/MS. The effects of the chiral stationary
30
phases, mobile phase, auto back pressure regulator (ABPR) pressure, column
31
temperature, flow rate of the mobile phase and the compensation pump solvent were
32
evaluated. Finally, the optimal SFC-MS/MS working conditions were determined to
33
include a CO2/MeOH mobile phase (87/13, v/v), 2.0 mL/min flow rate, 2200 psi
34
ABPR and 30 ºC column temperature using a Chiralpak IA-3 chiral column under
35
electrospray ionization positive mode. The modified QuEChERS method was applied
36
to water and zebrafish samples. The mean recoveries for the tebuconazole
37
enantiomers were 79.8% to 108.4% with RSDs ≤ 7.0% in both matrices. The LOQs
38
ranged from 0.24 to 1.20 µg/kg. The developed analytical method was further
39
validated by application to the analysis of authentic samples.
40
KEYWORDS: tebuconazole; chiral separation; Supercritical Fluid Chromatography
41
tandem mass spectrometry; zebrafish
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INTRODUCTION Tebuconazole,
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[(R,S)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] is
52
a broad-spectrum chiral triazole fungicide that is used to control many plant diseases.
53
Tebuconazole is one of the most widely sold fungicides in the world and widely used
54
on agricultural crops. However, tebuconazole is considered to be toxic to aquatic
55
organisms and can lead to long-term adverse effects on the aquatic environment.1
56
Environmental monitoring has indicated that tebuconazole is ubiquitous in water.2
57
The concentration of tebuconazole has continued to increase, especially in the
58
stream.2 For example, one study reported that its concentration in surface water has
59
reached 175 – 200 µg/L.3 Pesticides in an aquatic ecosystem can be transferred
60
through phytoplankton to fish and ultimately to humans.4 A previous study
61
determined that lipid and carbohydrate metabolism as well as some enzymatic
62
activities of zebrafish were affected by exposure to tebuconazole.5Therefore, the
63
environmental safety of tebuconazole has received increasing attention in recent years.
64
In particular, tebuconazole consists of two enantiomers due to the existence of a chiral
65
center in the structure. The bioactivity of (-)-R-tebuconazole is greater than that of
66
(+)-S-tebuconazole,6 and (-)-R-tebuconazole also exhibits high toxicity to aquatic
67
non-target organisms.7 However, traditional risk assessment of chiral pesticides does
68
not discriminate the difference between the enantiomers, leading to the
69
underestimation of the environmental effect. The development of an effective
70
analytical method to evaluate tebuconazole at an enantiomeric level remains a 3
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challenges.
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Zebrafish (Danio renio) are a typical model organism that has been applied to
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study biological processes with environmental and medical relevance.8 The
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development of a method to monitor tebuconazole enantiomers in zebrafish as well as
75
its living environment is required, to provide a comprehensive understanding of the
76
enantioselective transformation and bioaccumulation process.
77
Numerous chiral separation methods have been developed for tebuconazole in
78
water, soil, fruit and vegetable samples using normal-phase high-performance liquid
79
chromatography (NP-HPLC),9,10 capillary electrophoresis (CE),11reverse-phase
80
high-performance liquid chromatography (RP-HPLC),12 liquid chromatography-mass
81
spectrometry/mass
82
chromatography.13 However, these methods have several disadvantages including
83
poor separation and/or long retention time. For aquatic environmental samples, the
84
determination of rac-tebuconazole has been reported using matrix solid-phase
85
dispersion (MSPD) in fish liver and crab hepatopancreas by GC-MS14 and
86
liquid-liquid extraction in zebrafish by GC-MS.15 However, to the best of our
87
knowledge, no method is available for the determination of tebuconazole enantiomers
88
in aquatic organism samples.
spectrometry
(LC-MS/MS)7
and
supercritical
fluid
89
In this study, supercritical fluid chromatography (SFC) was employed for the
90
analysis. This method reduced the analytical time and the amount of organic solvent,
91
which makes its more attractive for routine or aquatic environment analysis.16 CO2 has
92
many advantages such as non-toxic, non-flammable and easily purified. In addition, a 4
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combination of SFC and mass spectrometry could improve the sensitivity using
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post-column polar solvent compensation technology. Furthermore, SFC-MS/MS may
95
be easier to achieve than LC-MS due to the high proportion of volatile CO2, which
96
enhances the evaporation step during the ionization process.17 This green technique is
97
becoming more popular. In addition, QuEChERS (Quick, Easy, Cheap, Effective,
98
Rugged, and Safe) sample preparation approaches have been proposed for the analysis
99
of aquatic environmental samples. The procedure for dispersive solid-phase extraction
100
in the QuEChERS method is easy to perform. In comparison to the traditional SPE
101
approach, dispersive-SPE saves time, labor, and solvent by using a much smaller
102
quantity of sorbent.18 Therefore, we established a method with low cost, faster
103
separation and better resolution for tebuconazole using SFC-MS/MS.
104
To the best of our knowledge, this report provides the first enantioselective
105
analysis of tebuconazole in water and zebrafish samples using SFC-MS/MS. The
106
results from this study will provide a new reference for the development of green
107
chromatographic separation of chiral compound, as well as offering an important
108
foundation for future aquatic safety and accurate risk assessment.
109
MATERIALS AND METHODS
110
Chemicals and Reagents. Racemic tebuconazole (98.7% purity) was obtained
111
from the China Standard Material Center (Beijing, China). High-purity CO2 (≥
112
99.999%) and N2(≥99.999%) was acquired from Haike Yuanchang Gas (Beijing,
113
China). HPLC-grade acetonitrile and methanol were purchased from Fisher Scientific
114
(Shanghai, China). Ultra-pure water was obtained from a Milli-Q system (Bedford, 5
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MA). Analytical grade NaCl, MgSO4 and acetonitrile were purchased from Beihua
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Fine-Chemicals Co. (Beijing, China). The sorbents including PSA (primary secondary
117
amine)(40 µm), C18 (40 µm), and Florisil (120-400 mesh size) were obtained from
118
Agela Technologies Inc. (Newark, DE). The mobile phase solvents were distilled and
119
filtered through a 0.22 µm pore size filter membrane (Tengda, Tianjin, China) prior to
120
the determination.
121
The standard stock solutions (100 mg/L) of racemic tebuconazole were prepared in
122
pure acetonitrile. The standard working solutions of rac-tebuconazole at 0.01, 0.05,
123
0.1, 0.5, 1.0 and 5.0 mg/L were prepared in pure acetonitrile from the stock solution
124
by serial dilution. The concentrations of each enantiomer were 0.005, 0.025, 0.05,
125
0.25, 0.5 and 2.5 mg/L. All of the solutions were protected from light using aluminum
126
foil and stored in a refrigerator at 4 ºC prior to analysis. The working standard
127
solutions exhibited no degradation for 3 months.
128
Supercritical
129
Spectrometry (SFC-MS/MS). An ACQUITY UPC2 system (Waters, Milford, MA)
130
which was equipped with a binary solvent manager, column manager, convergence
131
manager, sample manager-FL, and Waters 515 compensation pump was used for the
132
separation of the analytes. The column used for the separation of the stereoisomers of
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tebuconazole was a 150 mm × 4.6 mm i.d.,3 µm particle size, Chiralpak IA-3 (Daicel,
134
Japan).This
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5-dimethylphenylcarbamate.Three additional chiral columns including a Chiralpak IA
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[ amylose tris (3,5–dimethylphenycarbamate), 5 µm], Chiralpak IB-3 [cellulose tris
fluid
Chromatography/Tandem
column
was
coated
Triple
with
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Quadrupole
amylose
Mass
tris-3,
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(3,5-dimethylphenylcarbamate),
3
µm]
and
Chiralpak
IC-3
[cellulose
138
tris(3,5-dichlophenylcarbamate), 3 µm] were employed. The separation was carried
139
out using an isocratic elution with solvent A (CO2) and solvent B (methanol) ratio of
140
87:13 (v: v) at a flow rate of 2.0 mL/min for 5 min. The ABPR 2200 psi was selected.
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The flow rate of 515 compensation pump was 0.15 ml/min which used 0.1%
142
FA-MeOH (v/v) as a postcolumn additive. The temperature of the column and sample
143
manager were maintained at 30 °C and 25 °C, respectively. In each run, 1µLof the
144
sample was injected.
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The parameters including the capacity factor (k), separation factor (α),and
146
resolution (Rs), were calculated from the formula k = (t −t0)/t0, α=k2/k1,Rs=2(t2
147
−t1)/(W1 +W2), where t0 was the void time at the given conditions (t0 = 0.85 min,
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determined using 1,3,5- tri-tert-butylbenzene),t was the retention time, k was the
149
retention factor, and W was the baseline peak width.
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A triple quadrupole Xevo-TQD mass spectrometer (Waters Inc., Milford, MA)
151
equipped with an electrospray ionization source (ESI) was used to quantify the
152
tebuconazole stereoisomers. The analyses were performed in ESI+ with a 3.5 kV
153
capillary voltage, 150 °C source temperature, and 500 °C desolvation temperature. A
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50 L/h cone gas flow and a 900 L/h desolvation gas flow were employed. The
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nebulizer gas was 99.95% N2, and the collision gas was 99.99% Ar at a pressure of
156
2×10−3 mbar in the T-Wave cell. MassLynx NT v. 4.1 software (Waters, U.S.) was
157
used to collect and analyze the obtained data.
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Multiple reaction monitoring (MRM) mode was used for MS detection. The 7
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monitoring conditions were optimized for tebuconazole. A dwell time of 130 ms per
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ion pair was used to maintain the high sensitivity of the analysis, and a number of data
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points across the chromatographic peak were required. The typical conditions were as
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follows: the cone voltage of tebuconazole was 39 V; m/z 308.1 was selected as the
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precursor ion for tebuconazole, m/z 70.02 was selected for the product quantitative ion,
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and m/z 125.03 was selected for the qualitative ion when the collision energy was set
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to 23 and 37 V (see Table S1 of the Supporting Information). Under the conditions,
166
the retention times of (+)-S-tebuconazole and (-)-R-tebuconazole were approximately
167
3.26 and 3.60 min, respectively (Figure 1). These settings were utilized for all
168
subsequent studies.
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The micro-preparation of the stereoisomers was achieved using an Agilent 1100
170
series HPLC system (Agilent Technology, Waldbronn, Germany) coupled with an
171
on-line
172
n-hexane/isopropanol
173
CHIRALPAK AS-H column. It was a 250 mm ×4.6 mm i.d., 5µm column (Daicel,
174
Japan) which was coated with tris (S)-1-phenylethylcarbamate.
175
Sample Preparation. The water and zebrafish samples (total length of 2.0 ± 1.0 cm,
176
weight of 0.1 ± 0.05 g) were not contaminated by the target analyte. All of the
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zebrafish were healthy. Each zebrafish was homogenized. Approximately 2.0 g of the
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blank zebrafish samples (2.0 mL water) were weighed into a 10 mL PTFE centrifuge
179
tube. Suitable concentrations of tebuconazole standard solutions were added to the
180
tube and vortexed for 30 s and followed by equilibration for 2 h at room temperature
OR-2090
detector
(Jasco,
(90:10,v/v)
as
Japan). the
mobile
The
separation
phase
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30
employed °C
on
a
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to allow the pesticide to distribute evenly. Two mL of acetonitrile were added, and the
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tube was vortexed for 3 min. Then, 1g of NaCl was added to the mixtures. The tube
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was vortexed again for 1 min followed by centrifugation for 5 min at a relative
184
centrifugal force (RCF) of 2599 × g. Next, 1.0 mL of the upper layer was transferred
185
into a single-use 2 mL centrifuge tube containing 150 mg of Florisil and 150 mg of
186
anhydrous MgSO4 (for the water samples, 1.0 mL of the upper layer was filtered
187
directly using a 0.22-µm nylon syringe filter for SFC-MS/MS injection).The tubes
188
were vortexed for 30 s and centrifuged for 5 min at RCF 2400 × g. Finally, the
189
resulting supernatant (acetonitrile) was filtered using a 0.22-µm nylon syringe filter
190
for SFC-MS/MS injection.
191
For the SPE, the extraction volume was 6 mL, and the clean-up procedure involved
192
transferring 3 mL of the upper layer into rotary evaporation bottles to evaporate to
193
dryness followed by redissolving in a 1 mL mixture (acetone: n-hexane=1:9,v:v).The
194
1 mL solution was passed slowly through a Florisil SPE cartridge at a flow rate of
195
approximately1 mL/min. The cartridges were pre-conditioned using 5 mL of acetone:
196
n-hexane=1:9 (v:v) followed by 5 mL of n-hexane. Next, a 10 mL mixed solution
197
(acetone: n-hexane=1:9, v:v) was added to elute the target analytes. Then, the organic
198
solvent was evaporated to dryness using a rotary evaporator (30 °C, 0.09 MPa). The
199
resulting residue was redissolved in 1 mL of ACN and filtered using a 0.22 µm nylon
200
syringe filter for chromatographic injection.
201
For the determination of the authentic samples, the water and zebrafish samples
202
were obtained from a bioaccumulation test (Beijing, China, collected 24 h after an 9
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exposure period, the spiked solutions were prepared by adding rac-tebuconazole
204
dissolved in acetone to deionized water for a final concentration of 1.0 mg/L.).
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Method Validation. The following parameters were used to evaluate the performance
206
of the developed method: specificity, linearity, limit of detection (LOD), limit of
207
quantitation (LOQ), matrix effect, accuracy, precision, and stability.
208
Blank samples (water and zebrafish) were analyzed by monitoring the
209
characteristics of selected ion chromatograms. The linearity of the method was
210
determined by analyzing the standard solutions and three matrices in triplicate at five
211
concentrations ranging from 5 to 500 µg/kg for each enantiomer.
212
The matrix-dependent LOD and LOQ were determined using the blank and
213
calibration standards consisting of water and zebrafish matrices. The LOD for the
214
enantiomers of the chiral pesticide was regarded as the concentration with a
215
signal-to-noise (S/N) ratio of 3, and the LOQ was defined as the concentrations that
216
produced a signal-to-noise ratio of 10. These values were estimated from the
217
chromatogram corresponding to the lowest point used in the matrix-matched
218
calibration. The matrix effect can be computed as follows: , % =
slope of calibration curves in matrix − slope of calibration curves in solvent slope of calibration curves in solvent
× 100% 219
The recovery assays were carried out to investigate the accuracy and precision of
220
the method. Five replicates of the spiked samples at different levels (i.e., 10, 100 and
221
1000 µg/kg) for water and zebrafish were prepared on three different days. The 10
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precision under these conditions for repeatability, which is expressed as the relative
223
standard deviation (RSD), was determined by the intra- and inter-day assays.
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The stability was determined in acetonitrile and in the matrix. The stability of the
225
stock solution was tested monthly by injection of a newly prepared working solution.
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The stability of the spiked samples (100 mg/kg) with tebuconazole was evaluated
227
monthly, and all of the samples used in the stability test were stored at -20 °C. The
228
results were analyzed using Student’s t-test (P < 0.05).
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RESULTS AND DISCUSSION
230
Optimization of Enantioseparation Conditions. Selection of the chiral column.
231
Four chiral columns (i.e., Chiralpak IA-3, Chiralpak IA, Chiralpak IB-3, and
232
Chiralpak IC-3) were tested for their ability to separate the tebuconazole enantiomers
233
under the same chromatographic conditions. The stationary phase of the four columns
234
was polysaccharide chiral stationary phase. These chiral stationary phases exhibited
235
good feasibility due to their individual chiral carbohydrate monomers and long-range
236
helical secondary structure, which affect the separations. Among the four tested
237
columns, the best chromatographic separation of the two tebuconazole enantiomers
238
was achieved with Chiralpak IA-3 and IA (Figure 2). Chiralpak IB-3 and IC-3
239
exhibited insufficient discrimination ability for the tebuconazole stereoisomers. Due
240
to the particle size of the columns and the retention time, Chiralpak IA-3 was chosen.
241
However, Chiralpak IB-3 and IC-3 were filled with derivatives of cellulose chiral
242
stationary phase. In addition, Chiralpak IA-3 and IA were filled with derivatives of an
243
amylose chiral stationary phase. The polar carbamate groups of CDMPC were thought 11
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to be the main chiral recognition regions because those can physically interact with
245
the analytes via hydrogen bonds with the NH groups and dipole-dipole interactions
246
with the C=O groups.19 The derivatives of amylose exhibited higher recognition
247
abilities for tebuconazole. Flutriafol has a similar structure to tebuconazole, which has
248
also been well separated on a Chiralpak IA-3 column using SFC-MS/MS.20
249
In comparison to the 5-µm particles in the Chiralpak IA chiral column, Chiralpak
250
IA-3 has 3-µm particles, and this column exhibited a highly effective chiral separation
251
capacity for tebuconazole. Based on the solvent consumption, peak shape, and
252
retention time, the Chiralpak IA-3 column was the optimal choice. The benefits of
253
using columns with smaller particle sizes for chiral analysis has been previously
254
discussed.21
255
Composition of the Mobile phase .Different compositions of the mobile phase
256
were investigated to achieve good separation of the tebuconazole enantiomers on the
257
Chiralpak IA-3 column. Comparative analysis assays were conducted using methanol,
258
ethanol, 2-propanol, 1-butanol and acetonitrile. The results indicated that
259
tebuconazole enantiomers were well separated when methanol was used as the
260
modifier. In contrast, lower Rs were obtained when ethanol and 1-butanol were
261
employed. However, the two enantiomers cannot be separated using acetonitrile or
262
2-propanol as the modifier.
263
Different ratios of the modifier (methanol) were also tested (Figure 3A). The
264
retention time of the enantiomers was shorter as the proportion of methanol increased.
265
This result may be due to the strong elution ability of methanol. Finally, 12
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CO2/methanol (83:17, v/v) was chosen as the mobile phase due to the relatively better
267
resolution (2.75) and shorter retention time (3.26, 3.60 min).
268
Effect of the auto back pressure regulator (ABPR) pressure. The ABPR settings
269
can affect the retention time by changing the density of the mobile phase prior to the
270
release of pressure.
271
from 1600 psi to 4000 psi (Figure 3B). However, the instrument system pressure
272
would be beyond this range at higher ABPR pressures. In addition, higher ABPR
273
pressure is disadvantageous for the lifetime of the chiral columns. Therefore, an
274
ABPR pressure of 2200 psi was the optimal choice.
275
20
The retention time decreased as the ABPR pressure increased
Effect of column temperature. Temperature plays an important role in chiral 22
276
separation.
The column temperature affects the selectivity and retention in
277
SFC-MS/MS. In this study, the column temperature was evaluated from 25 to 40 °C
278
during the chiral separation. The results for the retention time and separation factor
279
indicated that the temperature exhibited only a slight influence on the chiral
280
separation. The two enantiomers were both well separated at all of the tested
281
temperatures. The column temperature was ultimately set to 30 °C. The results were
282
similar to those reported by Li et al.6
283
Flow Rate of the Mobile Phase. Different flow rates of the mobile phase were
284
investigated based on a mobile phase consisting of CO2/methanol (83:17, v/v).As
285
shown in Figure 3C, the retention time was shorter as the flow rate of the mobile
286
phase increased. Methanol has a strong elution ability. The volume of methanol
287
increased with the high flow rates. Therefore, the elution ability increased due to the 13
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high velocity. Two mL/min was selected for better resolution (2.75) and a relatively
289
suitable retention time (3.26, 3.60 min).
290
Flow Rate of the Compensation Pump Solvent. The compensation pump was
291
used to provide the compensation solution, which could improve the ionization
292
efficiency of ESI-MS/MS. 0.1% formic acid/methanol is often selected as a
293
compensation solvent. In this study, different flow rates of the compensation pump
294
solvent in a range of 0.15-0.45 mL/min were examined. The results indicated that the
295
flow rate of the compensation pump solvent had no affect on the retention time and
296
resolution. However, this flow rate influenced the peak area (Figure 3D). For
297
comprehensive consideration, the optimal choice was 0.15 mL/min, which provided
298
with the high peak areas for the tebuconazole enantiomers.
299
Elution Order of Tebuconazole Enantiomers. The relationships between the
300
absolute configurations and ORs (optical rotations) of the tebuconazole enantiomers
301
were determined in a previous study.23 Each single isomer and racemic tebuconazole
302
were individually subjected to SFC-MS/MS determination under the same
303
chromatographic conditions mentioned above. In this study, the enantiomer that eluted
304
first was (+)-S-tebuconazole, followed by (-)-R-tebuconazole. The results were similar
305
to the separation using LC-MS/MS reported by Li et al.7
306
Optimization of extraction and clean up procedure. The extraction and clean-
307
up procedures are important for residue analysis. The water samples were relatively
308
clean with few impurities. Therefore, the water samples were directly extracted with
309
acetonitrile without purification. For the zebrafish samples, different extraction 14
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solvents (acetonitrile and acetonitrile (saturated with n-hexane)) were investigated at
311
first (Figure 4).The results indicated that acetonitrile extraction performed the best. A
312
comparison between dispersive and solid-phase extraction for the clean-up step was
313
carried out for the zebrafish samples. The dispersive clean-up procedure in the
314
QuEChERS method was simple. Florisil, which is a normal-phase sorbent, can
315
effectively remove lipid interferences. The results indicated that the recovery was
316
high with 150 mg of Florisil. D-SPE (dispersive solid-phase extraction) exhibited
317
more interaction between the sample and the sorbent, which contributed to better
318
recoveries, improved the clean-up by removing the interferences and generated less
319
waste due to the smaller volume of organic solvent employed in the sample
320
preparation.24
321
The results indicated that the recoveries were less than 80% with SPE.
322
Therefore, the QuEChERS method was the optimal choice, and this approach saved
323
time, money and solvents compared to traditional SPE.
324
Method Validation. Specificity, Linearity, and Matrix Effect. The blank samples
325
(water and zebrafish) were analyzed to evaluate the specificity of the previously
326
mentioned method, and no interference was detected at the retention time of each
327
enantiomer. Linear regression analysis was performed in a concentration range of
328
5.0-500 µg/kg for each enantiomer. Table S2 in the Supporting Information provides
329
the standard solution and matrix-matched calibration curves (acetonitrile, water and
330
zebrafish) for each enantiomer which including the slopes and coefficients of
331
determination (R2).The results indicated that a mean R2 higher than 0.9933 was 15
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obtained for each enantiomer.
333
The matrix effects result from co-eluting matrix components that affect the
334
ionization of the target analyte, resulting either in ion suppression or ion enhancement
335
in some cases.25 Matrix effects can be highly variable and difficult to control or
336
predict. In this study, the matrix effects were investigated by comparing the standards
337
in the solvent with the matrix-matched standards. As shown in Table S2 in the
338
Supporting Information, signal suppression was observed for tebuconazole in the
339
matrices, based on the slope ratios of the matrix/acetonitrile in the range of
340
0.007−0.010, and the slope values were 99.0% and 99.3%. In general, the suppression
341
or enhancement effect originates from the inadequate removal of endogenous
342
compounds, such as phospholipids, fatty acids, saccharides, phenols and pigments.26
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Therefore, external matrix-matched standards were selected to obtain more accurate
344
quantification results.
345
LODs and LOQs. In this study, the LODs for each enantiomer were estimated to
346
be 0.3−0.4 µg/kg and obtained from all five replicated extractions and analyses of
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spiked samples at the lowest spiked levels (10 µg/kg). The LOQs in the two sample
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matrices were 1.0−1.32 µg/kg for the compound based on five replicates at 10 µg/kg.
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Accuracy and Precision. The recoveries and RSDs of each enantiomers were
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measured by spiking the blank samples with three different concentrations (i.e., 10,
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100 and 1000 µg/kg) and then analyzing them in quintuplicate (see Table S3 of the
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Supporting Information). All of the recoveries were determined from analyses of the
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target compounds in the water and zebrafish samples. The precision of the method 16
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was determined using repeatability and reproducibility studies. The intra-day
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precision (expressed as the RSDr) was measured by comparing the standard deviation
356
of the recovery percentages of the spiked samples run on the same day. The inter-day
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precision (expressed as the RSDR) was determined by analyzing spiked samples from
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three different days. As shown in Table S3 in the Supporting Information, satisfactory
359
mean recovery values (79.8–108.4%) and precisions were obtained. All of the
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experimental RSD values were less than 7.1% at the three fortified concentration
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levels. In general, the intra-day (n = 5) and inter-day RSDs (n = 15) for the proposed
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method ranged from 0.7–7.1% and 2.1–6.0%, respectively. Figure 4 showed typical
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chromatograms of the water and zebrafish blanks and spiked samples. Based on the
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results from the recovery studies, satisfactory precisions and accuracies were achieved
365
for the analysis of tebuconazole enantiomers in water and zebrafish samples. In
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addition, the stabilities of tebuconazole were evaluated, and no significant difference
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(P > 0.05) was observed from the solvent and matrix storage treatments, as previously
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described.
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Application to Authentic Samples. The effectiveness of this method for
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measuring trace levels of rac-tebuconazole in water and zebrafish samples.
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(+)-S-tebuconazole and (-)-R-tebuconazole in the water and zebrafish samples were
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detected after 24 h at concentrations of 0.33 ± 0.05 and 0.35 ± 0.04, 3.67 ± 0.46 and
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3.06 ± 0.42 mg/kg, respectively.
374
In the current study, a simple and reliable method using SFC-ESI-MS/MS has
375
been successfully established and validated for the stereoselective determination of 17
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tebuconazole in water and zebrafish. Based on a modified QuEChERS method, the
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target compounds were extracted and purified by acetonitrile and Florisil, respectively.
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This approach is simple and rapid with good mean recoveries and excellent linearities
379
compared to the traditional SPE method. Satisfactory results were obtained for real
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samples. The current study developed an analytical method to detect tebuconazole
381
enantiomers in water and zebrafish samples using SFC-MS/MS, but also to provide a
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technical support for future studies investigating the bioaccumulation, metabolism and
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fate of tebuconazole enantiomers in aquatic environments to minimize the risk posed
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by tebuconazole to humans, animals and the ecosystem.
385
Abbreviations: SSE, signal suppression/enhancement; RT, retention time; ABPR,
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auto back pressure regulator; UPC2, ultra performance convergence chromatography;
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SFC, supercritical fluid chromatography; MRM, multiple reaction monitoring; MS,
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mass spectrometry; ESI, electrospray ionization; CSP, chiral stationary phase
389
Acknowledgment
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This work was financially supported by the National Natural Science Foundation
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of China (31272071).
392
Supporting Information
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Experimental parameters and SFC−MS/MS conditions for analysis of tebuconazole
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(Table S1), Comparison of matrix-matched calibration and solvent calibration with all
395
of the range at 5- 500 µg/kg for each enantiomer (Table S2), and accuracy and
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precision of the proposed method in the two studied matrices at three spiked levelsa
397
(Table S3).This material is available free of charge via the Internet at 18
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http://pubs.acs.org.
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mass
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FIGURE CAPTIONS Figure.1 Chemical structure of tebuconazole stereoisomers. Figure.2 SFC-MS/MS chromatograms for the separation of tebuconazole enantiomers on an IA-3 chiral column: A, chromatogram of racemic tebuconazole; B,SFC-MS/MS chromatogram
of
(+)-S-tebuconazole;
C,SFC-MS/MS
chromatogram
of
(-)-R-tebuconazole. Figure.3 Typical SFC-MS/MS (MRM) chromatograms of tebuconazole on four columns —— A, Chiralpak IA-3; B, Chiralpak IC-3; C, Chiralpak IB-3; D, Chiralpak IA. Figure.4 Comparison of effects of different parameters (A, ratio of CO2/Methanol; B, ABPR; C, flow rate of mobile phase; D, flow rate of compensation solvent ) on the enantioseparation of tebuconazole enantiomers. Figure.5 Effect of different kinds of sorbents and extraction solvents for tebuconazole enantiomers in zebrafish at 100 µg/kg level (n=5).
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