Stereoselective Determination of Tebuconazole in Water and

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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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Journal of Agricultural and Food Chemistry

1

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].

15 16 17 18 19 20 21 22 23 24 25 26 1

ACS Paragon Plus Environment


27

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

42 43 44 45 46 47 48 2


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49 50

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



71

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challenges.

72

Zebrafish (Danio renio) are a typical model organism that has been applied to

73

study biological processes with environmental and medical relevance.8 The

74

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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93

combination of SFC and mass spectrometry could improve the sensitivity using

94

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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115

MA). Analytical grade NaCl, MgSO4 and acetonitrile were purchased from Beihua

116

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

133

tebuconazole was a 150 mm × 4.6 mm i.d.,3 µm particle size, Chiralpak IA-3 (Daicel,

134

Japan).This

135

5-dimethylphenylcarbamate.Three additional chiral columns including a Chiralpak IA

136

[ amylose tris (3,5–dimethylphenycarbamate), 5 µm], Chiralpak IB-3 [cellulose tris

fluid

Chromatography/Tandem

column

was

coated

Triple

with

6


Quadrupole

amylose

Mass

tris-3,

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137

(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.

141

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.

145

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,

148

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.

150

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

154

50 L/h cone gas flow and a 900 L/h desolvation gas flow were employed. The

155

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.

158

Multiple reaction monitoring (MRM) mode was used for MS detection. The 7



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159

monitoring conditions were optimized for tebuconazole. A dwell time of 130 ms per

160

ion pair was used to maintain the high sensitivity of the analysis, and a number of data

161

points across the chromatographic peak were required. The typical conditions were as

162

follows: the cone voltage of tebuconazole was 39 V; m/z 308.1 was selected as the

163

precursor ion for tebuconazole, m/z 70.02 was selected for the product quantitative ion,

164

and m/z 125.03 was selected for the qualitative ion when the collision energy was set

165

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.

169

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

177

zebrafish were healthy. Each zebrafish was homogenized. Approximately 2.0 g of the

178

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

8


at

30

employed °C

on

a

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181

to allow the pesticide to distribute evenly. Two mL of acetonitrile were added, and the

182

tube was vortexed for 3 min. Then, 1g of NaCl was added to the mixtures. The tube

183

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



203

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.).

205

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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222

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.

224

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.

226

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).

229

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



244

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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266

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



288

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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310

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



332

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

343

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

347

spiked samples at the lowest spiked levels (10 µg/kg). The LOQs in the two sample

348

matrices were 1.0−1.32 µg/kg for the compound based on five replicates at 10 µg/kg.

349

Accuracy and Precision. The recoveries and RSDs of each enantiomers were

350

measured by spiking the blank samples with three different concentrations (i.e., 10,

351

100 and 1000 µg/kg) and then analyzing them in quintuplicate (see Table S3 of the

352

Supporting Information). All of the recoveries were determined from analyses of the

353

target compounds in the water and zebrafish samples. The precision of the method 16


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354

was determined using repeatability and reproducibility studies. The intra-day

355

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

357

precision (expressed as the RSDR) was determined by analyzing spiked samples from

358

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

360

experimental RSD values were less than 7.1% at the three fortified concentration

361

levels. In general, the intra-day (n = 5) and inter-day RSDs (n = 15) for the proposed

362

method ranged from 0.7–7.1% and 2.1–6.0%, respectively. Figure 4 showed typical

363

chromatograms of the water and zebrafish blanks and spiked samples. Based on the

364

results from the recovery studies, satisfactory precisions and accuracies were achieved

365

for the analysis of tebuconazole enantiomers in water and zebrafish samples. In

366

addition, the stabilities of tebuconazole were evaluated, and no significant difference

367

(P > 0.05) was observed from the solvent and matrix storage treatments, as previously

368

described.

369

Application to Authentic Samples. The effectiveness of this method for

370

measuring trace levels of rac-tebuconazole in water and zebrafish samples.

371

(+)-S-tebuconazole and (-)-R-tebuconazole in the water and zebrafish samples were

372

detected after 24 h at concentrations of 0.33 ± 0.05 and 0.35 ± 0.04, 3.67 ± 0.46 and

373

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



376

tebuconazole in water and zebrafish. Based on a modified QuEChERS method, the

377

target compounds were extracted and purified by acetonitrile and Florisil, respectively.

378

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

380

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

382

technical support for future studies investigating the bioaccumulation, metabolism and

383

fate of tebuconazole enantiomers in aquatic environments to minimize the risk posed

384

by tebuconazole to humans, animals and the ecosystem.

385

Abbreviations: SSE, signal suppression/enhancement; RT, retention time; ABPR,

386

auto back pressure regulator; UPC2, ultra performance convergence chromatography;

387

SFC, supercritical fluid chromatography; MRM, multiple reaction monitoring; MS,

388

mass spectrometry; ESI, electrospray ionization; CSP, chiral stationary phase

389

Acknowledgment

390

This work was financially supported by the National Natural Science Foundation

391

of China (31272071).

392

Supporting Information

393

Experimental parameters and SFC−MS/MS conditions for analysis of tebuconazole

394

(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

396

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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398

http://pubs.acs.org.

399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 19



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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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Stereoselective Determination of Tebuconazole in Water and

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