Journal of Separation Science

Determination of antibiotics in vegetables using QuEChERS

based method and liquid chromatography-quadrupole linear
ion trap mass spectrometry
Fo
Journal: Journal of Separation Science

Manuscript ID jssc.201701294

Wiley - Manuscript type: Original Paper

r

Date Submitted by the Author: 01-Nov-2017

Re

Complete List of Authors: He, Zeying; China Agricultural University, Department of Applied Chemistry
Wang, Yuehua; Agro-Environmental Protection Institute, Ministry of
Agriculture
Xu, Yaping; Agro-Environmental Protection Institute, Ministry of Agriculture
vi

Liu, Xiaowei; Agro-Environmental Protection Institute, Ministry of

Agriculture,
ew

Keywords: antibiotic, vegetable, QuEChERS, LC-QqLIT

On
ly

Wiley-VCH
Page 1 of 20 Journal of Separation Science

1 Determination of antibiotics in vegetables using QuEChERS based method and liquid

2 chromatography-quadrupole linear ion trap mass spectrometry

4 Zeying He, Yuehua Wang, Yaping Xu, Xiaowei Liu*

5 Key Laboratory of Environmental Factor Control for Agro-product Safety, MOA,

6 Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, P.R. China

Fo
7 *Corresponding author:

8 Xiaowei Liu, Key Laboratory of Environmental Factor Control for Agro-product Safety, MOA,
r

9 Agro-Environmental Protection Institute, Ministry of Agriculture, Tianjin 300191, P.R. China;

Re

10 Tel.: +86 022-23611006; Fax: +86 022-23611006;

vi

11 E-mail: hezeying222308@163.com

12
ew

13

14 Abbreviations:
On

15 d-SPE:dispersive solid phase extraction; GCB: grafted carbon black; LC-QLIT: liquid
ly

16 chromatography-quadrupole linear ion trap mass spectrometryPSA: primary secondary amine;

17

Wiley-VCH
 Journal of Separation Science Page 2 of 20

18 ABSTRACT

19 In this study, a simple and reliable method was developed for multiresidue analysis of 49

20 antibiotics in vegetables based on QuEChERS procedure and liquid chromatography-quadrupole

21 linear ion trap mass spectrometry determination. Premixed extraction salt packets were first used

22 to simplify the sample extraction procedures. Different buffer systems and other parameters were

23 optimized. MRM-IDA-EPI mode was used to acquire MS/MS spectra along with MRM survey
Fo
24 scan for library searching to prevent false positive/negative identification. The limits of

25 quantification (LOQs) were 2 and 5 µg/kg for the target antibiotics. Recoveries for most of
r

26 antibiotics ranged between 70 % and 100% with RSD < 20 %. Relative lower recoveries were
Re

27 observed for some of quinolones. The method was used to analyze 60 vegetable samples, and 33%
vi

28 of the samples were found to be contaminated with antibiotics. And false positive of

29 demeclocycline was avoided benefited from the application of MS/MS library searching.
ew

30

31 KEY WORDS: antibiotic, vegetable, QuEChERS, LC-QqLIT

On

32
ly

33 1. INTRODUCTION

34 Antibiotics are widely used for prevention and treatment of human and animal diseases, and

35 they are also added to animal feed as additives to promote animal growth [1]. China is the largest

36 producer and consumer of antibiotics in the world. According to the latest study, 16200 t of

37 antibiotics were used in China in 2013, and 52% of which were consumed by animals [2].

38 However, the antibiotics are weakly adsorbed and 30-90% are excreted without degradation

39 leading to high levels of antibiotics residues in pig, chicken and other manures [3]. The study of

Wiley-VCH
Page 3 of 20 Journal of Separation Science

40 Zhang et al. showed that as high as 416.8 mg/kg of oxytetracycline in chicken manure sample was

41 found from Shouguang, the largest vegetable-producing area in China [4]. Huge amount of

42 livestock manures were produced in China annually, make the antibiotics residue a big concern.

43 Manures are commonly applied to the field as organic fertilizer especially in protected

44 vegetable farming. It was estimated that 150 t/ha of organic fertilizers is applied annually in

45 protected vegetable farming, ten times of that applied in open field [5]. The high load of manures
Fo
46 leads to considerable antibiotics residues in agriculture fields and finally uptake by vegetables.

47 Boxall et al. found that carrots and lettuce can took up a wide range of antibiotics [6]. Pot
r

48 experiments showed that sulfamethoxazole and norfloxacin could be detected in radish and
Re

49 pakchoi with concentration in the range of 7.3-221.5 µg/kg [7]. Li et al. investigated the residues
vi

50 of three quinolones including ciprofloxacin, enrofloxacin and norfloxacin in 68 vegetable samples

51 from an important vegetable-growing region in Shandong Province of China [8]. The detection
ew

52 frequency for norfloxacin was 100% with concentrations ranged from 18.2-658.3 µg/kg. Therefore,

53 the antibiotic residues in vegetables, a potential threat to health, should be monitored and a simple,
On

54 robust and reliable analytical method is critical.

ly

55 The extraction and determination of multiple classes of antibiotics are challenging because of

56 the complexity of matrices and their various chemical characters including polarity, solubility, pKa

57 values and stability. Previous studies have mainly focused on the extraction and determination of

58 antibiotics in livestock tissues, manures, compost and environmental matrices such as soils,

59 sediment and sludge. The sample preparation generally involves extraction by ultrasonic, vortex or

60 vibration and followed by SPE clean-up [9-11]. Other extraction methods were also reported such

61 as pressurized liquid extraction (PLE), matrix solid phase extraction (MSPE) and stir bar sorption

Wiley-VCH
 Journal of Separation Science Page 4 of 20

62 extraction (SBSE). However, most of the methods need further clean-up by traditional SPE and

63 the whole extraction and SPE clean-up procedures are extremely time and solvent consuming. In

64 previous studies, the determination of antibiotics in vegetables was also conducted by complicated

65 procedures including ultrasonic extraction and SPE/LLE clean-up [7, 8, 12]. Therefore, new

66 analytical method should be developed to improve the analysis efficiency.

67 QuEChERS (quick, easy, cheap, effective, rugged, and safe) sample preparation method, first
Fo
68 introduced by Anastassiades et al., has thrived in the past years [13]. It was initially developed for

69 pesticides residue analysis in fruits and vegetables and has extended its application to various
r

70 matrices and pollutants like PCBs in soil [14]. In recent years, several works have been using
Re

71 QuEChERS procedures to determine antibiotics. Shendy et al. developed a modified QuEChERS

vi

72 method to determine 6 nitrofuran metabolites and nitroimidazole residues in honey [15]. Meng et

73 al. determined 20 antibiotics including 17 sulfonamides and 3 macrolides in soil using QuEChERS
ew

74 and liquid chromatography with tandem mass spectrometry [16]. Wang et al. determined 6

75 antibiotics in milk applying QuEChERS method [17]. Some other studies adopted the d-SPE
On

76 clean-up procedure in the determination of antibiotics in different matrices [18, 19]. However, the
ly

77 number of antibiotics and analytical performances of these modified QuEChERS methods were

78 limited. To our knowledge, QuEChERS method for determination of antibiotics in vegetables has

79 not been reported.

80 Currently, the determination of multiclass antibiotics are mainly conducted on triple quadrupole

81 based mass spectrometers in MRM mode coupled to liquid chromatography. However, existence

82 of interferences that have the same MRM transitions or weak response of the second transition at

83 low concentrations can lead to false positive or negative [20]. Quadruple linear ion trap mass

Wiley-VCH
Page 5 of 20 Journal of Separation Science

84 spectrometry (QqLIT-MS) combined a triple-quadruple (QqQ) scanning functionality with

85 sensitive linear ion trap scans [21]. In this hybrid MS system, MRM as survey scan and MS/MS

86 spectra in Enhanced Product Ion (EPI) mode triggered via information-dependent acquisition

87 (IDA) were acquired in a single run. Residue levels were quantified using MRM data, and at the

88 same time MS/MS spectra were searched against library for target identification to prevent false

89 positive/negative.
Fo
90 To improve the analytical efficiency and qualitative ability, a simple and reliable multiresidue

91 analysis method based on QuEChERS procedures and QqLIT determination was developed for
r

92 multiresidue analysis of antibiotics in vegetables. The extraction buffer system, d-SPE clean-up
Re

93 and other parameters were optimized. MRM-IDA-EPI mode was used in MS analysis to ensure
vi

94 unequivocal identification as well as accurate quantification.

95
ew

96 2. MATERIALS AND METHODS

97 2.1 Materials and reagent

On

98 The 49 targeted antibiotics including 17 sulfonamides, 16 quinolones, 6 macrolides, 5β

ly

99 -lactams and 5 tetracyclines were purchased from Dr. Ehrenstorfer (Ausberg, Germany) and

100 Toronto Research Chemicals Inc. (Canada). Seven isotope-labeled internal standards were used:

101 sulfadimidine-d4 and sulfamethoxazole-d4 for sulfonamides; ciprofloxacin-d8 for quinolones;

102 erythromycin-d6 for erythromycin, roxithromycin-d7 for other macrolides; penicillin V-d5 forβ

103 -lactams; and tetracycline-d6 for tetracyclines. All of the internal standards were purchased from

104 Toronto Research Cheimicals Inc. (Canada). Stock solutions of the standards were prepared by

105 dissolving each compound in methanol at 1000 mg/L or 100 mg/L. A standard mixture working

Wiley-VCH
 Journal of Separation Science Page 6 of 20

106 solution was prepared in 5 mg/L and diluted to an appropriate concentration before use. Internal

107 standard working solution at 5 mg/L was prepared in methanol.

108 Acetonitrile and methanol were HPLC grade and purchased from Fisher Scientific (Fair Lawn,

109 NJ). Water was purified by a Milli-Q (Millipore, Billerica, MA, USA) system. Buffer salts and

110 acids including citric acid, Na2HPO4, anhydrous MgSO4, KH2PO4, NaCl and H3PO4 were

111 purchased from Shanghai Macklin Biochemical Co., Ltd (Shanghai, China). Dispersive solid
Fo
112 phase extraction adsorbents (PSA, C18 and GCB), ceramic homogenizer and polypropylene

113 centrifuge tubes (50 mL for extraction and 2 mL for dSPE) were purchased from Agilent (Agilent
r

114 Technologies, Lake forest, CA).

Re

115 2.2 Instrument

vi

116 For antibiotics analysis, an ExionLC UPLC system (SCIEX, Redwood City, CA USA) equipped

117 with a C18 (HSS T3 column, 2.1 x 100 mm, 1.7 µm, Waters) column was used. The extract of 5
ew

118 µL was injected into the column. The column temperature was set at 40 ℃, and the flow rate was

119 0.3 mL/min. Mobile phase A was water containing 0.1% of formic acid and mobile B was
On

120 acetonitrile. The gradient was programed as follows: 0-1 min: 10% elute B, 1-8 min: gradient
ly

121 increase to 90 % elute B, 8-12 min: hold 90 % elute B, 12-12.1 min: 10 % elute B; 12.1-15: hold

122 10 % elute B.

123 Mass spectrometry detection was performed using a quadrupole linear ion trap mass

124 spectrometry (QTRAP 4500, SCIEX) in positive ionization mode with a DuoSpray ion source.

125 The source parameters were as follows: ion spray voltage floating (ISVF), 5500 V;

126 temperature, 500 ℃; nebulizing gas (GS1), 50 psi; heater gas (GS2), 50 psi; curtain gas, 30 psi.

127 MRM experiment was carried out to obtain the maximum sensitivity for the analysis of target

Wiley-VCH
Page 7 of 20 Journal of Separation Science

128 analytes. The specific MRM transitions for all the test antibiotics and other parameters are given in

129 Table 1. The second experiment was added in the Enhanced Product Ion (EPI) mode to acquire

130 MS/MS spectra. EPI spectra were acquired from 50 to 900 amu with a scan speed of 10,000 amu/s

131 and a Collision Energy (CE) of 35 eV with Collision Energy Spread (CES) of 15 eV after dynamic

132 background subtraction of the survey scan.

133 2.3 Sample preparation

Fo
134 A 10 g of minced vegetable sample was transferred into a 50 mL centrifuge tube and spiked

135 with 50 µL of isotope-labeled internal standard. The sample was vortexed for 30 s and allowed to
r

136 stand in the dark for 30 min. Acetonitrile (10 mL) was added into the centrifuge tube, followed by
Re

137 a ceramic homogenizer, and the QuEChERS extraction salt packet (anhydrous MgSO4, 4 g;
vi

138 sodium chloride, 1 g; citrate acid, 0.5 g; NaH2PO4, 0.049 g). The tube was immediately sealed and

139 vortex shaken for 3 min with a multi-position vortexer. The tube was centrifuged for 5 min at 4000
ew

140 rpm. The supernatant (1 mL) was transferred into the 2 mL centrifuge tube containing dispersive

141 solid phase extraction adsorbents (PSA 10 mg, C18 25 mg, GCB 2.5 mg and anhydrous MgSO4
On

142 150 mg). The extract was vortexed for 1 min and centrifuged for 5 min at 4000 rpm; Supernatant
ly

143 (0.5 mL) was transferred into another 2 mL centrifuge tube and 0.5 mL water containing 0.1%

144 formic acid was added. The mixture was filtered through a 0.22 µm PTEE filter for LC-QqLIT

145 analysis.

146 2.4 Method validation

147 Method accuracy and precision were evaluated by recovery studies using blank matrices at 5,

148 50 and 200 µg/kg. All recovery experiments were carried out with 5 replicates for each matrix.

149 The limits of quantification for each antibiotic was determined on the recovery results and defined

Wiley-VCH
 Journal of Separation Science Page 8 of 20

150 as the lowest concentration that can be quantified with acceptable accuracy and precision.

151 Matrix-matched and solvent-based calibration curves (1, 2.5, 5, 10, 25, 50, 100 and 200 µg/L)

152 were used to evaluate the linearity and matrix effects.

153 3. RESULTS AND DISCUSSION

154 3.1 Sample extraction

155 Buffer systems are used in typical QuEChERS procedures to maintain a certain pH during the
Fo
156 sample extraction to protect the analytes from degradation. Citrate buffer (pH 5-5.5) and acetate

157 buffer (pH 4.8) are used in official EN 15662 and AOAC 2007.1 QuEChERS methods, respectively
r

158 [22, 23] to protect pesticides that unstable at high pH values. The physicochemical property of
Re

159 antibiotics varies for different classes, therefore, pH values affect the extraction efficiency
vi

160 significantly. Extraction solvent with pH values in the range of 2.5 and 4.2 such as

161 methanol/acetonitrile with McIlvaine buffer (pH 2.5-4.2), potassium phosphate buffer (pH 3.0),
ew

162 citric acid buffer (pH 4.4) or acidic water (pH 2.5-3.0) were commonly used in previous studies

163 [19, 24-26]. In traditional methods, the buffer solutions were first prepared and mixed with
On

164 organic solvent to form the final extract solvent for sample extraction. In this study, the buffer
ly

165 salts along with anhydrous MgSO4 and NaCl were premixed to form extraction salt packets. The

166 extraction salt packets were added into the tubes before extraction to form buffer system.

167 Sample preparation efficiency was improved by simplify the procedures. Different buffer systems

168 including McIlvaine buffers (pH 2.4, 3.0 and 4.0), phosphate buffer (pH 3.0) packets and formic

169 acid (pH 2.7) were prepared and compared for their extract efficiencies. The extraction solvent

170 and buffer packets were prepared as listed in table S1 (see supporting information) and the

171 results are given in Fig. 1. As can be seen from the figure, better results were obtained using

Wiley-VCH
Page 9 of 20 Journal of Separation Science

172 phosphate buffer (pH 3.0) for sulfonamides, however, there were no significant differences

173 between these five buffers and all the recoveries were higher than 70 %. For β-lactams, better

174 recoveries were obtained using McIlvaine buffer (pH 3.0) compared to McIlvaine buffer (pH 2.4),

175 but the advantage was limited. For macrolides, the recoveries using McIlvaine buffer (pH 2.4)

176 were higher than other buffers for azithromycin, clarithromycin and tilmicosin. The recoveries of

177 quinolones and tetracyclines were much higher when using McIlvaine buffer (pH 2.4) compared
Fo
178 with other four buffers. Finally, McIlvaine buffer (pH 2.4) was selected as the extraction buffer.

179 Enough extraction time is required to get good recovery. In the QuEChERS method for pesticide
r

180 residue analysis in foods, 1 min of shaking is generally enough for good extraction [27, 28]. In this
Re

181 study, the extraction time of 1, 2, 3 and 4 min were evaluated for the extraction efficiency. The
vi

182 results are given in Fig. S1 (see supporting information). The extraction efficiency increased with

183 the prolonged time, and best results were obtained after 3 min of vertex for most of the
ew

184 antibiotics. Finally, 3 min of vortex were chosen as the extraction time.

185 3.2 Clean-up

On

186 Blank cabbage extract fortified with 50 µg/L of each antibiotic was cleaned up using different
ly

187 amount of PSA, C18 and GCB to evaluate the adsorption property. The recoveries after clean-up

188 for different adsorbents are given in Fig. S2-S4 (see supporting information). Good recoveries

189 were obtained for PSA, C18 and GCB clean-up at 10, 25, and 2.5 mg, respectively. However, with

190 the increase amount of d-SPE adsorbents, the recoveries decreased for some of the antibiotics. For

191 example, quinolones, macrolides, β-lactams and tetracyclines were significantly adsorbed when

192 25 and 50 mg of PSA were used. C18 did not show significant adsorption of different classes of

193 antibiotics, however, strong adsorptions of some quinolones were observed when 5 mg of GCB

Wiley-VCH
 Journal of Separation Science Page 10 of 20

194 was used. Since the pigment content is very high for some vegetables like leek and spinach, 2.5

195 mg of GCB was used. To get best clean-up effects, the combination of 10 mg PSA, 25 mg C18 and

196 2.5 mg GCB were finally selected as the d-SPE clean-up adsorbents.

197 3.3 Matrix effects

198 Matrix effects are commonly observed in LC-MS/MS analysis due to ionization competition

199 between co-eluting interfering compounds and analytes [29]. In this study, matrix effects was
Fo
200 calculated by the equation [30]:

 
% =  − 1 × 100

r

201 where Smatrix and Ssolvent are the calibration curve slopes of each antibiotic in matrix and solvent,
Re

202 respectively. Suppression or enhancement < 20 % are defined as softy matrix effects, which are
vi

203 negligible. However, when medium (suppression or enhancement of 20-50 %) and strong matrix

204 (suppression or enhancement > 50 %) effects were observed, certain measures should be taken
ew

205 to overcome the influence of matrix.

206 As can be seen from Fig. 2, matrix suppression was observed for the majority of the
On

207 sulfonamides and quinolones, and most of which exhibited medium matrix effects. Strong matrix
ly

208 enhancement were observed for spiramycin, azithromycin and tilmicosin, while, other three

209 macrolides exhibited matrix suppression effects. Forβ-lactams, very weak matrix effects were

210 observed except for penicillin G, which exhibited medium matrix enhancement. Strong matrix

211 enhancements were observed for all the tetracyclines.

212 3.4 Method validation

213 The proposed method was evaluated for the 49 antibiotics in three vegetable matrices

214 including cabbage, cucumber and tomato. Method parameters such as linearity, recovery,

10

Wiley-VCH
Page 11 of 20 Journal of Separation Science

215 accuracy and LOQ were calculated to evaluate the performance of the method.

216 Recovery study was carried out at three concentrations of 5, 50 and 200 µg/kg at replicates

217 (n=5) to determine the method accuracy and precision. The results are given in table S2-S4 in

218 supporting information. Good recoveries were obtained for sulfonamides, macrolides, β-lactams

219 and tetracyclines and the majority of these antibiotics have recoveries higher than 70 % with RSD

220 <20 %. However, for approximately half of the quinolones, lower recoveries were obtained
Fo
221 because of strong interactions between quinolones and the matrix [19]. And lower recoveries of

222 quinolones in different matrices were also observed in previous studies for both QuEChERS and
r

223 SPE methods [9, 11, 18]. For macrolides andβ-lactams, the RSDs were relatively higher than
Re

224 other classes of antibiotics, especially at low concentration of 5 µg/kg.

vi

225 The limit of quantification (LOQ) was determined based on recovery experiments and defined

226 as the lowest fortification levels that have acceptable recoveries and RSDs. As listed in table S5
ew

227 (see supporting information), the LOQs were 2 µg/kg for 36 out of the 49 antibiotics and 5 µg/kg

228 for the rest of the analytes. Linearity was determined over the range of 1-200 µg/L (internal
On

229 standard method was applied withe concentration of 25 µg/L). The coefficient of detection (r2), as
ly

230 presented in table S5, was higher than 0.995 for each antibiotic, which could guarantee accurate

231 quantification.

232 3.5 Method application

233 To further validate the feasibility of the proposed method and investigate the residue levels of

234 the selected antibiotics in vegetables, 60 real samples were analyzed. The samples were purchased

235 from local markets, including cabbage, cucumber, cauliflower, leek and other commonly

236 consumed vegetables. The samples were extracted as described in section 2.3 and data were

11

Wiley-VCH
 Journal of Separation Science Page 12 of 20

237 acquired using QqLIT in MRM-IDA-EPI mode to obtain MRM data and MS/MS spectra

238 simultaneously. As given in table S6 (see supporting information), 5 out of the 49 target

239 antibiotics were detected in 20 samples with concentration exceeded corresponding LOQs. There

240 were only one positive sample for sulfamethoxazole, enrofloxacin and chlorotetracycline,

241 respectively. However, 8 and 9 positive samples were detected for oxytetracycline and

242 doxycycline, respectively. The concentrations of sulfamethoxazole, enrofloxacin,

Fo
243 chlorotetracycline and doxycycline ranged between 2.0 µg/kg and 12.8 µg/kg, which were lower

244 compared to oxytetracycline with the highest concentration of 126 µg/kg detected in cabbage. To
r

245 guarantee the qualitative results, MS/MS spectra of antibiotics in positive samples were searched
Re

246 against library containing all the target analytes. As given in Fig. 3, the MS/MS spectrum of
vi

247 doxycycline was obtained in sample 2 at low concentration and matched well with the standard

248 spectrum in library. It should be noticed that, 2.2 µg/kg of demeclocycline was initially detected in
ew

249 sample 4 by MRM result with same retention time and acceptable ion ratio. However, the result

250 was proved to be false positive after library searching since the MS/MS spectrum in sample 4
On

251 could not match the standard spectrum of demeclocycline (Fig. S5, in supporting information).
ly

252 4. CONCLUDING REMARKS

253 Antibiotic residues in vegetables have been reported because of application of manures in

254 agricultural fields, leading to potential risk to human health. The residue levels should be

255 monitored for risk assessment. In this study, a simple QuEChERS method combined with

256 LC-QqLIT determination was proposed for multiclass analysis of antibiotics in vegetables.

257 QuEChERS extraction salt packets were first used to simplify the sample extraction procedures.

258 Good performance was obtained for the optimized method in the terms of LOQ, linearity,

12

Wiley-VCH
Page 13 of 20 Journal of Separation Science

259 accuracy and precision. False postive/ negative was successfully avoided because of the use of

260 MS/MS library searching under EPI mode. Overall, the proposed method exhibited good

261 sensitivity and reliability for accurate and unequivocal determination of antibiotic residues in

262 vegetables.

263

264 ACKNOWLEDGEMENTS
Fo
265 This work was supported by the subproject of the national key research and development plan

266 (grant number 2016YFD0201203)

r

267
Re

268 REFERENCES:

269 1. Schlüsener, M. P., Bester, K., Persistence of antibiotics such as macrolides, tiamulin and salinomycin
vi

270 in soil. Environ. Pollut. 2006, 143, 565-571.

271 2. Zhang, Q. Q., Ying, G. G., Pan, C. G., Liu, Y. S., Zhao, J. L., Comprehensive evaluation of antibiotics
ew

272 emission and fate in the river basins of China: source analysis, multimedia modeling, and linkage to
273 bacterial resistance. Environ. Sci. Technol. 2015, 49, 6772-6782.
274 3. Sarmah, A. K., Meyer, M. T., Boxall, A. B., A global perspective on the use, sales, exposure pathways,
275 occurrence, fate and effects of veterinary antibiotics (VAs) in the environment. Chemosphere 2006, 65,
On

276 725-759.
277 4. Zhang, H., Luo, Y., Wu, L., Huang, Y., Christie, P., Residues and potential ecological risks of veterinary
278 antibiotics in manures and composts associated with protected vegetable farming. Environ. Sci. Pollut.
279 R. 2015, 22, 5908-5918.
ly

280 5. Qiao, Q., Chen, J., Dong, Q. U., Yan, T., Rong, W., Advances and Characters of Fertilizer Application of
281 Protected Field Agriculture in China. Journal of Hubei Agricultural College 2002, 22, 373-376.
282 6. Boxall, A. B. A., Johnson, P., Smith, E. J., Sinclair, C. J., Stutt, E., Levy, L. S., Uptake of Veterinary
283 Medicines from Soils into Plants. J. Agr. Food Chem. 2006, 54, 2288-2297.
284 7. Wang, J., Lin, H., Sun, W., Xia, Y., Ma, J., Fu, J., Zhang, Z., Wu, H., Qian, M., Variations in the fate and
285 biological effects of sulfamethoxazole, norfloxacin and doxycycline in different vegetable–soil systems
286 following manure application. J. Hazard Mater. 2016, 304, 49-57.
287 8. Li, X. W., Xie, Y. F., Li, C. L., Zhao, H. N., Zhao, H., Wang, N., Wang, J. F., Investigation of residual
288 fluoroquinolones in a soil–vegetable system in an intensive vegetable cultivation area in Northern
289 China. Sci. Total Environ. 2014, 468-469, 258-264.
290 9. Zhang, Z., Li, X., Ding, S., Jiang, H., Shen, J., Xia, X., Multiresidue analysis of sulfonamides,
291 quinolones, and tetracyclines in animal tissues by ultra-high performance liquid
292 chromatography-tandem mass spectrometry. Food Chem. 2016, 204, 252-262.

13

Wiley-VCH
 Journal of Separation Science Page 14 of 20

293 10. Ho, Y. B., Zakaria, M. P., Latif, P. A., Saari, N., Simultaneous determination of veterinary antibiotics
294 and hormone in broiler manure, soil and manure compost by liquid chromatography-tandem mass
295 spectrometry. J. Chromatogr. A. 2012, 1262, 160-168.
296 11. Huang, Y., Cheng, M., Li, W., Wu, L., Chen, Y., Luo, Y., Christie, P., Zhang, H., Simultaneous extraction
297 of four classes of antibiotics in soil, manure and sewage sludge and analysis by liquid
298 chromatography-tandem mass spectrometry with the isotope-labelled internal standard method. Anal.
299 Methods-UK 2013, 5, 3721-3731.
300 12. Pan, M., Wong, C. K. C., Chu, L. M., Distribution of Antibiotics in Wastewater-Irrigated Soils and
301 Their Accumulation in Vegetable Crops in the Pearl River Delta, Southern China. J. Agr. Food Chem.
302 2014, 62, 11062-11069.
303 13. Anastassiades, M., Lehotay, S. J., Stajnbaher, D., Schenck, F. J., Fast and easy multiresidue method
304 employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the
305 determination of pesticide residues in produce. J. AOAC Int. 2003, 86, 412-431.
Fo
306 14. Zeying, H., Lu, W., Yi, P., Ming, L., Wenwen, W., Xiaowei, L., Determination of selected
307 polychlorinated biphenyls in soil and earthworm (Eisenia fetida) using a QuEChERS-based method and
308 gas chromatography with tandem MS. J. Sep. Sci. 2015, 38, 3766-3773.
r

309 15. Shendy, A. H., Alghobashy, M. A., Gad Alla, S. A., Lotfy, H. M., Development and validation of a
310 modified QuEChERS protocol coupled to LC-MS/MS for simultaneous determination of multi-class
Re

311 antibiotic residues in honey. Food Chem. 2016, 190, 982-989.

312 16. Meng, M., He, Z., Xu, Y., Wang, L., Peng, Y., Liu, X., Simultaneous extraction and determination of
313 antibiotics in soils using a method based on quick, easy, cheap, effective, rugged and safe extraction
vi

314 and liquid chromatography with tandem mass spectrometry. J. Sep. Sci. 2017, 40, 3214-3220.
315 17. Wang, Y., Guo, B., Liu, Z., Jing, R., Development of a Method for the Analysis of Multiclass
ew

316 Antibiotic Residues in Milk Using QuEChERS and Liquid Chromatography Tandem Mass Spectrometry.
317 Foodborn Pathog. Dis. 2015, 12, 693-703.
318 18. Bourdat-Deschamps, M., Leang, S., Bernet, N., Daudin, J. J., Nãlieu, S., Multi-residue analysis of
319 pharmaceuticals in aqueous environmental samples by online solid-phase
On

320 extraction-ultra-high-performance liquid chromatography-tandem mass spectrometry: optimisation

321 and matrix effects reduction by quick, easy, cheap, effective, rugged and safe extraction. J. Chromatogr.
322 A. 2014, 1349, 11-23.
323 19. Dorival-García, N., Labajo-Recio, C., Zafra-Gómez, A., Juárez-Jiménez, B., Vílchez, J. L., Improved
ly

324 sample treatment for the determination of 17 strong sorbed quinolone antibiotics from compost by
325 ultra high performance liquid chromatography tandem mass spectrometry. Talanta 2015, 138,
326 247-257.
327 20. Zhang, K., Wong, J. W., Yang, P., Hayward, D. G., Sakuma, T., Zou, Y., Schreiber, A., Borton, C.,
328 Nguyen, T. V., Kaushik, B., Protocol for an electrospray ionization tandem mass spectral product ion
329 library: development and application for identification of 240 pesticides in foods. Anal. Chem. 2012,
330 84, 5677-5684.
331 21. Bueno, M. J., Agüera, A., Gómez, M. J., Hernando, M. D., García-Reyes, J. F., Fernandez-Alba, A. R.,
332 Application of liquid chromatography/quadrupole-linear Ion trap mass spectrometry and
333 time-of-flight mass spectrometry to the determination of pharmaceuticals and related contaminants
334 in wastewater. Anal. Chem. 2007, 79, 9372-9384.
335 22. Payá, P., Anastassiades, M., Mack, D., Sigalova, I., Tasdelen, B., Oliva, J., Barba, A., Analysis of
336 pesticide residues using the Quick Easy Cheap Effective Rugged and Safe (QuEChERS) pesticide

14

Wiley-VCH
Page 15 of 20 Journal of Separation Science

337 multiresidue method in combination with gas and liquid chromatography and tandem mass
338 spectrometric detection. Anal. Bioanal. Chem. 2007, 389, 1697-1714.
339 23. Lehotay, S. J., Collaborators, M. O., Hans, A. V. G. M. C., Gnter, M. V. H. T. A., Mette, L. R. F. K. M.,
340 Hammack, E. P. A. B. W., Karen, J. M. C. L. A., Andr, L. M. G. M. H., de Kok, M. H. F. S., Parker, A. W. A.,
341 Determination of pesticide residues in foods by acetonitrile extraction and partitioning with
342 magnesium sulfate: collaborative study. J. AOAC Int. 2007, 90, 485-520.
343 24. Hu, W., Ma, L., Guo, C., Sha, J., Zhu, X., Wang, Y., Simultaneous extraction and determination of
344 fluoroquinolones, tetracyclines and sulfonamides antibiotics in soils using optimised solid phase
345 extraction chromatography-tandem mass spectrometry. Int. J. Environ. Anal. Chem. 2012, 92, 698-713.
346 25. Pan, M., Wong, C. K., Chu, L. M., Distribution of antibiotics in wastewater-irrigated soils and their
347 accumulation in vegetable crops in the Pearl River Delta, southern China. J. Agr. Food Chem. 2014, 62,
348 11062-11069.
349 26. Hou, J., Wan, W., Mao, D., Wang, C., Mu, Q., Qin, S., Luo, Y., Occurrence and distribution of
Fo
350 sulfonamides, tetracyclines, quinolones, macrolides, and nitrofurans in livestock manure and
351 amended soils of Northern China. Environ. Sci. Pollut. R. 2015, 22, 1-10.
352 27. He, Z., Wang, L., Peng, Y., Luo, M., Wang, W., Liu, X., Multiresidue analysis of over 200 pesticides in
r

353 cereals using a QuEChERS and gas chromatography-tandem mass spectrometry-based method. Food
354 Chem. 2015, 169, 372-380.
Re

355 28. He, Z., Chen, S., Wang, L., Peng, Y., Luo, M., Wang, W., Liu, X., Multiresidue analysis of 213
356 pesticides in leek and garlic using QuEChERS-based method and gas chromatography-triple
357 quadrupole mass spectrometry. Anal. Bioanal. Chem. 2015, 407, 2637-2643.
vi

358 29. Li, X. Q., Yang, Z., Zhang, Q. H., Li, H. M., Evaluation of matrix effect in isotope dilution mass
359 spectrometry based on quantitative analysis of chloramphenicol residues in milk powder. Anal. Chim.
ew

360 Acta 2014, 807, 75-83.

361 30. Rajski, Ł., Lozano, A., Uclés, A., Ferrer, C., Fernández-Alba, A. R., Determination of pesticide
362 residues in high oil vegetal commodities by using various multi-residue methods and clean-ups
363 followed by liquid chromatography tandem mass spectrometry. J. Chromatogr. A. 2013, 1304,
On

364 109-120.
365
366
ly

15

Wiley-VCH
 Journal of Separation Science Page 16 of 20

367

368 Figures

Fo
rR
ev
iew
On
ly
369

370 Fig. 1 Extraction efficiencies of different buffer systems

16

Wiley-VCH
Page 17 of 20 Journal of Separation Science

Fo
rR
ev
iew
On
ly
371

372 Fig. 2 Matrix effects of selected antibiotics in cabbage

373

17

Wiley-VCH
 Journal of Separation Science Page 18 of 20

Fo
rR
ev
iew
On
374

375 ly
376

377 Fig. 3 Identification of doxycycline in real sample by MS/MS library searching. (A) EPI spectrum of target doxycycline in vegetable sample and (B) EPI spectrum of

378 doxycycline standard in library

18

Wiley-VCH
Page 19 of 20 Journal of Separation Science

Table 1 Retention time and MRM parameters for selected antibiotics and internal standards (IS)
RT(min Precurso Product ion DP CE
) r ion
Sulfonamides Sulfacetamide 2.58 215 156/108 52 15/29
Sulfisomidine 2.55 279.1 124.1/186.1 80 30/23
Sulfadiazine 3.03 251.1 156/108.1 63 22/34
Sulfathiazole 3.38 256.1 156.1/108.1 60 22/35
Sulfapyridine 3.55 250.1 156/184.1 65 23/23
Sulfamerazine 3.81 265.1 156.1/172 73 24/24
Sulfameter 4.37 281.1 156.1/108.1 70 25/35
Sulfadimidine 4.32 279.1 186/124.1 75 25/35
Sulfamethizole 4.32 271 156.1/108 65 21/36
Sulfadoxin 5.24 311 156/108.1 80 26/37
Fo

Sulfamethoxazole 5.26 254.1 156/108 70 23/32

Sulfamoxole 5.50 268 108/113 70 35/25
Sulfamonomethoxine 4.77 281.1 156/126.2 75 25/30
r

Sulfisoxazole 4.12 268.1 156/113.1 70 21/23

Re

Sulfabenzamide 5.87 277.1 156/108.1 60 19/32

Sulfaquinoxaline 6.00 301.1 156.1/108 80 24/36
Sulfadimethoxine 5.99 311.3 156.1/218 100 28/28
vi

Sulfadimidine-d4 (IS) 4.30 283.3 186.2/124.2 70 25/32

Sulfamethoxazole-d4 (IS) 5.25 258.2 160/111.9 72 22/32
ew

Quinolones Ofloxacin 4.08 362.1 318.2/261.1 60 27/36

Danofloxacin 4.31 358.1 340/314 84 31/25
Enoxacin 3.90 321.1 303.4/232.2 45 35/48
Enrofloxacin 4.45 360.1 342.1/316.1 84 29/27
On

Difloxacin 4.93 400.1 382.1/356 91 31/27

Sarafloxacin 4.80 386.1 342/368 106 27/31
Sparfloxacin 4.87 393.1 349/292 45 30/38
Ciprofloxacin 4.13 332.4 288.3/245.3 45 25/33
ly

Norfloxacin 3.99 320.1 302/276.1 91 27/23

Orbifloxacin 4.55 396.2 352.1/295 102 25/33
Lomefloxacin 4.30 352.3 308.4/265.4 45 28/33
Cinoxacin 5.34 263.1 217.1/245 60 30/22
Fleroxacin 4.06 370.4 326.4/269.4 45 30/40
Nalidixic acid 6.73 233.3 215.2/187.4 45 24/36
Oxolinic acid 5.72 262.3 244.2/216.3 45 30/42
Flumequin 6.89 262.3 244.3/202.3 45 30/49
Ciprofloxacin-d8 (IS) 4.10 340.4 322.4/235.2 100 30/55
Macrolides Spiramycin 5.19 843.5 174.1/174 120 50/52
Erythromycin 6.43 734.4 158.3/576.3 100 30/27
Erythromycin-d6 (IS) 6.42 740.5 164.2/582.4 50 36/26
Roxithromycin 7.66 837.6 679.4/158 156 29/47

Wiley-VCH
 Journal of Separation Science Page 20 of 20

Azithromycin 5.31 749.9 83/158 80 100/49

Clarithromycin 7.55 748.8 590.3/558.5 135 44/44
Tilmicosin 6.09 870.1 174/88 100 57/124
Roxithromycin-d7 (IS) 7.63 844.5 686.5/158.1 100 30/38
β-lactams Cloxacillin 7.36 436 160/277 61 21/19
Penicillin G 6.32 335 176/160 120 18/15
amoxicillin 3.90 366 114/208 16 37/17
ampicillin 4.84 350.1 160/106 58 19/27
Penicillin V 6.83 351 160/114 35 17/47
Penicillin V-d5 (IS) 6.82 356 160/114 75 18/45
Tetracyclines Chlorotetracycline 5.43 479.1 444.1/154 41 27/37
Oxytetracycline 4.07 461.1 426.1/201.2 36 25/49
Fo
Tetracycline 4.39 445.1 410.1/427 36 25/19
Doxycycline 5.54 445.3 428.2/154.2 41 25/41
Demeclocycline 4.76 465.2 430/448 80 31/24
r
Tetracycline-d6 (IS) 4.38 451.2 416.2/160.1 80 25/35
Re
vi
ew
On
ly

Wiley-VCH