Novel Dispersive Micro-Solid-Phase Extraction Combined with

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A novel dispersive micro solid phase extraction combined with ultrahigh performance liquid chromatography-high resolution mass spectrometry to determine morpholine residue in citrus and apples Yunfeng Zhao J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 24 Dec 2014 Downloaded from http://pubs.acs.org on December 26, 2014

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A novel dispersive micro solid phase extraction combined with ultra-high performance liquid chromatography-high resolution mass spectrometry to determine morpholine residue in citrus and apples

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Journal of Agricultural and Food Chemistry jf-2014-041178.R1 Article 11-Dec-2014 Chen, Dawei; China National Center for Food Safety Risk Assessment, Miao, Hong; National Institution of Nutrition and food safety, Zou, Jianhong; The Second Artillery General Hospital, Cao, Pei; China National Center for Food Safety Risk Assessment, Ma, Ning; China National Center for Food Safety Risk Assessment, Zhao, Yunfeng; National Institution of Nutrition and food safety, Wu, Yongning; National Institution of Nutrition and food safety,

ACS Paragon Plus Environment

Page 1 of 28


1

A novel dispersive micro solid phase extraction combined with

2

ultra-high performance liquid chromatography-high resolution mass

3

spectrometry to determine morpholine residue in citrus and apples

4 5

Dawei Chen,1 Hong Miao,1 Jianhong Zou,2 Pei Cao,1 Ning Ma,1 Yunfeng Zhao,1*

6

Yongning Wu1

7 8

1

9

Center for Food Safety Risk Assessment, Beijing 100021, China

10

2

Key Laboratory of Food Safety Risk Assessment, Ministry of Health, China National

The Second Artillery General Hospital, Beijing 100088, China

11 12

*

13

E-mail: [email protected]

14

Tel: +86-10-67790051

15

Fax: +86-10-67770158

Correspondence: Yunfeng Zhao

16 17 18 19 20 21 22 23 24 25 1



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26

Abstract

27

This paper presents a new analytical method for the determination of morpholine

28

residue in citrus and apples using a novel dispersive micro solid phase extraction

29

(DMSPE) followed ultra-high performance liquid chromatography-high resolution

30

mass spectrometry (UHPLC–HRMS). Samples were extracted with 1% formic acid in

31

acetonitrile/water (1:1 v/v) and then cleaned up using DMSPE procedure. Morpholine

32

from the extract was adsorbed to a polymer cation exchange (PCX) sorbent and eluted

33

with ammonium hydroxide/acetonitrile (3:97 v/v) through the 1 mL syringe with a

34

0.22

35

UHPLC–HRMS/MS on a Waters Acquity BEH HILIC column using 0.1% formic

36

acid and 4 mM ammonium formate in water/acetonitrile as the mobile phase with

37

gradient elution. The method showed a good linearity (R2> 0.999) in the range of

38

1-100 µg/L for the analyte. The LOD and LOQ values of morpholine were 2 µg/kg

39

and 5 µg/kg respectively. The average recoveries of morpholine from the citrus and

40

apple samples spiked at three different concentrations (5, 20 and 100 µg/kg) were in a

41

range from 78.4 to 102.7%.

42

Keywords: dispersive micro solid phase extraction, fruit, morpholine, high resolution

43

mass spectrometry, design of experiments

µm

nylon

syringe

filter.

All

of

the

samples

44 45 46 47 48 49 50

2


were

analyzed

by

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51

INTRODUCTION

52

Morpholine, C4H9ON, has been widely used as accelerant in rubber manufacturing,

53

sulfuration agent, cleanser, descaling agent, surfactant, textile printing and fruit

54

preserving agent. It is added to some waxes as emulsifier used in the preparation of

55

wax coatings for fruits and vegetables.1 In the presence of excess nitrite, morpholine

56

can be chemically modified (nitrosated) to form N-nitrosomorpholine (NMOR) which

57

has been proved to be a genotoxic carcinogen in rodents.2

58

Although morpholine as a component of the coating in fresh fruits, is approved for

59

use in China,3 it is still under scrutiny by the European Union (EU) and has not been

60

approved for use in the EU.4 Based on a no observed adverse effect level (NOAEL:

61

96 mg/kg bw/day) in a chronic oral toxicity study for morpholine, an acceptable daily

62

intake (ADI) of 0.48 mg/kg bw/day was estimated by Health Canada.2 However, there

63

is no currently validated method to determine morpholine residue on waxed

64

commodities, which caused residue data and estimated exposure for morpholine in

65

citrus and apples fruits could not be obtained in China. It is well known that GC-TCD

66

and GC-MS or other methods (GLC-TEA and GLC-MS) could be used for analysis of

67

morpholine residue in various matrix samples,5-9 nevertheless, due to the high polarity

68

and low molecular weight of morpholine, its response and sensitivity are not good

69

enough for the residue analysis in complex matrices. Recently, an LC-MS method for

70

the determination of morpholine in citrus and apples has been published,10 but its

71

affiliated extraction and no cleanup process resulted in a >20% ion suppression. The

72

cleanup is considered to be the labor intensive but effective process to overcome ion

73

suppression. Currently, the cleanup methods including solid phase extraction (SPE)

74

and dispersive solid phase extraction (dSPE) are widely used to solve the matrix

75

interferences in complex matrices for the residue analysis.11-13 A dual SPE cartridge 3



76

system was used as a pretreatment method for morpholine in pineapples by Gros et al,

77

but it is time-consuming, laborious and costly.14 While a Quick, Easy, Cheap,

78

Effective, Rugged, and Safe (QuEChERS)-based on dSPE method for sample cleanup

79

has been developed to improve sample throughput for residue analysis,15,16 the

80

purification effect is even worse than SPE.17 On the contrary, the dispersive micro

81

solid phase extraction (DMSPE) can be categorized as a dSPE or SPE technique and

82

exhibits some advantages over traditional dSPE (fewer matrix effects) and SPE (such

83

as without complex equipment; short time requirement and less solvent

84

consumption).18-21 In DMSPE cleanup technique, the solid sorbent is added directly to

85

an extract and the cleanup procedure relies solely on shaking and centrifugation.

86

Graphene and carbon nanotubes (CNTs) are commonly used as solid sorbent materials

87

for application in the sorbent phase of such processes. PCX, as a high molecular

88

polymer, is a cation exchange sorbent material. PCX can adsorb the alkaline chemical

89

substances directly and provide an effective separation. In the present study, all those

90

parameters that were supposed to affect the extraction efficiency in DMSPE cleanup

91

method, such as the amount of PCX, the adsorption time, the type and volume of

92

eluent were carefully investigated and optimized. In analytical chemistry,

93

optimization is a critical stage to find out the value that each factor could be help to

94

reach the best possible response.22 Recently, the multivariate designs of experiments

95

(DOE) are accepted to obtain the optimal variable parameters. 23-26 The DOE designs

96

would not just significantly reduce the number of experiments which can take less

97

time, effort and resources than the univariate studies, but also take the interactions

98

between variables and the non-linear relations with the responses into account.

99

Therefore, the central composite design (CCD) approach was used for studying the

100

optimal variables in this DMSPE cleanup method. 4


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101

MATERIALS AND METHODS

102

Chemicals and reagents

103

Acetonitrile and methanol (HPLC grade) were obtained from Fisher Scientific (Fair

104

Lawn, NJ, USA). Formic acid and ammonium formate (HPLC grade) was purchased

105

from Tedia (Weston, America). Ultra-pure water was prepared from a Milli-Q reagent

106

water system (Millipore, Bedford, MA, USA). Cleanert® PCX powder was obtained

107

from Agela Technologies (Tianjing, China).

108

Morpholine

was

purchased

from

Supelco

(Bellefonte,

PA,

USA)

and

109

d8–morpholine was obtained from Cambridge Isotope Laboratories (Tewksbury, MA,

110

USA). The purity of all these standards was no less than 98.0%.

111

Standard stock solutions (1000 mg/L) were prepared by dissolving 10.0 mg of

112

individual compounds in a 10 ml volumetric flask with methanol. The stock solutions

113

were diluted in acetonitrile to prepare the intermediate standard solutions, the working

114

standard solutions of 10 mg/L and 1 mg/L respectively. All standard solutions were

115

made in amber volumetric flasks and stored at -20 ℃.

116

Samples collection and preparation

117

Apples and citrus were obtained from the local markets from Beijing in China.

118

Those apples were produced in Shandong and Gansu provinces in China as well as

119

imported from Chile and United States of America (USA). Those citrus were

120

transported from Jiangxi, Guangdong, Guangxi, Hubei, and Sichuan provinces in

121

China, as well as imported from South Africa and USA. All samples were cut into

122

small pieces and homogenized using an electric grinder. One sample from 1 kg

123

apples/citrus was prepared and kept in 4 ℃.

124

Ten grams of homogenized sample were weighed into a 50 mL polypropylene

125

centrifuge tube, and a total of 40 µL of the internal standard (10 mg/L) was spiked 5



126

into the sample. After adding 10 mL of 1% formic acid in acetonitrile/water (1:1 v/v),

127

the mixture was vortexed for 30 s, ultrasonicated for 10 min, and centrifuged at 8 000

128

rpm for 5 min. The supernatant was poured off into clean 50 mL polypropylene

129

centrifuge tube and distilled to 20 mL with acetonitrile for next purification. 1 mL of

130

the supernatant was transferred into 2 mL eppendorf tube with 30 mg PCX powder.

131

The mixture was vortexed for 30 s for interaction between the analytes and PCX

132

sorbent, and then poured off into a 1 mL syringe with a 0.22 µm nylon syringe filter.

133

The extracted solution was passed through the syringe and syringe filter manually and

134

was washed with 1 mL of acetonitrile again. Subsequently, the PCX enriched with the

135

analytes were eluted with 2 mL of ammonium hydroxide/acetonitrile (3:97 v/v). The

136

collected elution was prepared for UHPLC-MS analysis.

137

Chromatographic conditions

138

UHPLC analysis was performed on a UHPLC Ultimate 3000 system (Dionex),

139

using an Acquity BEH HILIC (2.1 mm × 100 mm, 1.7 µm particle size) analytical

140

column (Waters, USA) with temperature maintained at 40 ℃. The aqueous phase (A)

141

consisted of a mixture of 0.1% of formic acid and 4 mM ammonium formate in water

142

and the organic phase (B) was acetonitrile with 0.1% formic acid. The gradient started

143

at 90% B for 2 minutes, reduced to 40% B in the next 4 minutes and followed by

144

re-equilibration at 90% B for 3 minutes prior to the next injection. The flow rate was

145

set to 300 µL/min with a resulting overall runtime of 9 min. The injection volume was

146

10 µL.

147

Mass spectrometry conditions

148

Q-Exactive Mass Spectrometer (Thermo Fisher Scientific, Bremen, Germany) with

149

heated electrospray ionization (HESI) was operated in the positive (ESI+) electrospray

150

ionization modes. The system was controlled by Xcalibur 2.2 (Thermo Fisher 6


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151

Scientific). The spray voltage was 3.5 kV for the positive mode. The temperature of

152

ion transfer capillary, sheath gas, auxiliary gas, sweep gas and S-lens RF level were

153

set to 325 ℃, 30, 10, 0 (arbitrary units) and 55 V, respectively. The instrument was

154

calibrated in the positive mode every three days using the calibration solutions,

155

including caffeine, MRFA, and a mixture of fluorinated phosphazines ultramark 1621,

156

provided by the instrument manufacturer.

157

The Q-Exactive detector was operated in targeted MS/MS (tMS/MS) mode. The

158

tMS/MS scan mode is similar to the SRM scan mode used in the triple quadrupole

159

mass spectrometer. The mass spectrometer acquired a tMS/MS scan at a resolution of

160

70000 FWHM with 5.0e5 of Automatic Gain Control (AGC) target (the number of

161

ions to fill C-Trap) and 200 ms of maximum ion Injection Time (IT).

162

Design of experiments

163

The software Design-Expert (version 8.0.5b, Stat-Ease, Inc., Minneapolis, MN,

164

USA) was used for experimental design, data analysis and model building for

165

response surface methodology (RSM).

166

The central composite design (CCD) approach was employed to identify optimum

167

conditions in DMSPE cleanup procedure. CCD requires an experiment number to

168

meet N = 2k+2k+cp, where k is the factor number and cp is the replicate number of the

169

central point.22 In this study, the amount of PCX, the concentration of ammonium

170

hydroxide in acetonitrile and the volume of elute were set as three independent factors.

171

A five-level three-parameter experimental design of CCD approach was performed at

172

random order with 20 run experiments, as shown in Table 1 and Table S1 (Supporting

173

Information). The full CCD was described as follows: (1) a full two-level (-1 and +1)

174

factorial design; (2) center point (cp = 5); (3) two axial points on the axis of each

175

design variable at a distance of α = 2.0 from the design center. The response Y (the 7



Page 8 of 28

176

efficiency of extraction) was established for the determination of optimum method

177

conditions, and the recovery was selected as the indicator of the extraction efficiency.

178

The optimal values of response Y were obtained by solving the regression equation

179

and analyzing the three-dimensional response surface plot and contour plots.

180

Method validation

181 182

The selectivity of the method was performed by analyzing the blank samples and matrix interferences were checked close to the elution zone of the analyte.

183

Calibration curve was constructed using working standard solutions and by plotting

184

the peak area ratio of the quantitative ion of standard to internal standard at

185

concentrations of 1, 2, 5, 10, 25, 50 and 100 µg/L for morpholine. The concentration

186

of the internal standard was 10 µg/L for d8-morpholine.

187

LOD and LOQ were estimated for the signal-to-noise (S/N) ratio of more than 3

188

and 10 respectively from the chromatograms of samples spiked at the lowest

189

concentration

190

reproducibility, in terms of % RSDr and RSDR) and recovery were estimated by

191

spiked experiments in citrus and apple samples. Intra-day repeatability of the method

192

was evaluated by spiking the standard solutions to the six blank matrices at three

193

different concentration levels (5, 20 and 100 µg/kg) and analyzing in the same run of

194

the day on the LC-MS. For inter-day reproducibility, the three concentrations were

195

analyzed in three different days. Extraction recoveries of morpholine were measured

196

in citrus and apples which were fortified at three concentration levels (5, 20 and 100

197

µg/kg) with six replicates at each level. The recoveries were calculated from the

198

measured compared to the expected concentrations.

validated.

Precision

(intra-day

repeatability

and

inter-day

199

Matrix effect (ME) was determined by constructing calibration curves in blank

200

extract and in the pure solvent (n = 3). The effects were expressed in terms of signal 8


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suppression/enhancement (SSE) and calculated as follows: SSE = slope of spiked

202

extract/slope of pure solvent standard.

203

Stability of the analyte in matrix was determined by repeated analysis (n = 3) of

204

spiked citrus and apple samples (5 and 20 µg/kg) which were stored at -20 ℃. The

205

samples were extracted and analyzed after 7, 14 and 21 days.

206

RESULTS AND DISCUSSION

207

Optimization of chromatographic conditions

208

The effects of different liquid chromatographic columns on the target compound

209

separation and retention were initially studied. Three different types of analytical

210

columns, namely BEH C18 (2.1 mm × 100 mm, 1.7 µm) and BEH HILIC

211

(2.1 mm × 100 mm, 1.7 µm), and HSS T3 (2.1 mm × 100 mm, 1.8 µm) were tested to

212

their optimal elution conditions respectively, which were shown in Supporting

213

Information. Fig. S1 showed that morpholine had no good retention and weak MS

214

response in C18 and T3 columns due to its high polarity which led to its co-elution

215

with other impurities, while the HILIC column had a better retention effect and less

216

interference from other impurities. As for the mobile phase, acetonitrile and water

217

with a variety of modifiers were compared. A mixture of 4 mM ammonium formate

218

and 0.1% formic acid solution was selected as the most suitable component for the

219

mobile phase, in which a sufficiently good performance for ionization of morpholine

220

was achieved with good peak symmetry under the optimized linear gradient mode.

221

MS/MS optimization

222

Optimization of acquisition modes. Full scan, targeted single ion monitoring (tSIM)

223

and targeted MS/MS (tMS/MS) modes are three kinds of commonly used quantitative

224

models for Q Exactive and all were evaluated and optimized in the present study.

225

Full scan mode is a full scan of all ions in the specified mass range. However, the 9



226

precursor in the inclusion list (with retention times) can be selected in the quadrupole

227

with a particular isolation width by tSIM mode. In tMS/MS mode, the precursor

228

specified in the inclusion list is selected by quadrupole, and fragmented in a higher

229

energy collisional dissociation (HCD) cell with specific fragmentation energy. These

230

three acquired modes were evaluated by the signal to noise (S/N) ratios. The signals to

231

noise (S/N) ratios of morpholine (5 µg/L in blank citrus extract) in different modes

232

acquired within individual conditions were shown in Fig. S2 (Supporting Information).

233

This figure highlights the fact that tMS/MS is more selective and specific than tSIM

234

and Full scan modes due to complex matrices, which leads to better method detection

235

limits. Therefore, the tMS/MS mode was selected as the quantitative model for

236

morpholine in citrus and apples.

237

Optimization of MS condition in tMS/MS mode. Using the Q Exactive tune

238

application, the precursor ion was selected in the quadrupole and product ions were

239

found by increasing the Normalized Collision Energy (NCE) in tMS/MS mode. The

240

actual higher energy collisional dissociation (HCD) energy in eV is calculated on the

241

basis of the chosen NCE, mass and charge of the precursor ion.27 After choosing the

242

product ions, fragmentation energy scans were carried out to obtain the optimal NCE

243

for complete fragmentation of precursor ions. The optimal NCEs of the monitored

244

ions are shown in Table 2.

245

MS spectra of morpholine and d8-morpholine. Using tMS/MS mode, the parent

246

ions are selected in the quadruple (e.g., 88.0761 m/z for morpholine and 96.1259 m/z

247

for d8-morpholine) using a specified m/z window (4 Da) and subsequently fragmented

248

in the higher energy collision dissociation (HCD) cell. A full scan of all fragmented

249

ions originating from the parent ion was performed, and specific product ions were

250

used for data analysis (70.0658, 68.0502 m/z for morpholine and 78.1157, 76.1035 10


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251

m/z for d8-morpholine).

252

Optimization of the extraction procedure

253

1% acidic methanol has been used to extract morpholine with water (0-2 mL)

254

adding for aid in the extraction.10 Thus, the extraction efficiency was studied for the

255

mixture of 1% acidic acetonitrile/water used as the extraction solution with different

256

volume in this study. Fig. S3 (Supporting Information) showed that the extraction

257

efficiency for morpholine was better at 1% formic acid than that at acetonitrile/water

258

(1:1 v/v) and the 10 mL extraction solution was sufficient to extract the morpholine

259

completely. Moreover, ultrasound is of great help in the pretreatment of solid samples

260

as it facilitates and accelerates the dissolution, fusion and leaching.28 Thus, the

261

ultrasonic extraction was used to aid the extraction of morpholine from the citrus and

262

apples.

263

During DMSPE cleanup procedure, the parameters that affected the extraction

264

efficiency, like the amount of PCX, the adsorption time, the type and volume of eluent

265

were carefully studied. A one-factor analysis of variable preliminary experiment was

266

performed firstly and the preliminary study and results were shown in Supporting

267

Information. It was found that the adsorption of morpholine into PCX was a fast

268

process and the adsorption time greater than 30 s did not produce an extra

269

enhancement of adsorption efficiency. As a result, a 30 s adsorption time was chosen

270

for all subsequent experiments. As an analytical practice for SPE, it is well known

271

that alkaline substances are easily eluted from the strong cation exchange column in

272

alkaline condition. In addition, it is desirable to use a mobile phase for sample

273

preparation to inject sample solutions into an LC system. Therefore, ammonium

274

hydroxide in acetonitrile had been used to release morpholine from PCX sorbent

275

based on the ion exchange retention mechanism. Additionally, the detailed ion 11



276

exchange retention mechanism including the structure of morpholine and PCX was

277

provided in Fig. S4 (Supporting Information).

278

Design of experiments

279

The one-factor analysis indicated the amount of PCX, the concentration of

280

ammonium hydroxide in acetonitrile and the volume of eluent were the main effective

281

factors for DMSPE but did not take into account interactions between factors.

282

Therefore, the amount of PCX (10-50 mg, A), the concentration of ammonium

283

hydroxide in acetonitrile (0.1-5%, B) and the volume of eluent (1-3 mL, C) were

284

considered and optimized in central composite design (CCD). After 20 run

285

experiments described in the Table S1 (Supporting Information), the response Y was

286

calculated based on the efficiency of extraction for morpholine, and all statistical

287

analysis were performed by Design-Expert software. The analysis of variance

288

(ANOVA) for response surface quadratic model showed that the model was

289

significant with p-value of less than 0.0001. The lack of fit of the model relative to its

290

pure error showed a p-value of 0.1663 which indicated that the fitted model was

291

considered adequate to predict the efficiency of extraction under any sets of the

292

variables combination. Model coefficients for the response were shown in Table 3.

293

The final equation in terms of coded factors was: Response = 87.18 + 3.69 A + 11.81

294

B + 7.94 C - 2.87 AB + 0.13 AC - 3.12 BC - 3.41A2 - 6.16B2 - 3.28C2. The 3D surface

295

response plots and their related counters, obtained using the fitted model, were shown

296

in Fig. 1. As can be shown in Fig. 1, the concentration of ammonium hydroxide and

297

the volume of eluent were two important factors which had a significant positive

298

effect on the extraction efficiency. However, the amount of PCX affects the efficiency

299

of the extraction process with the increasing of PCX from 20-30 mg, but there was

300

almost unchanged above 30 mg. The optimum conditions were selected by the 12


Page 12 of 28

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301

analysis data obtained from the response surface plots and the regression coefficient

302

plots. The factor settings that maximize the efficiency of extraction were chosen in

303

response optimization. Among these settings, the most desirable factor levels ranged

304

as follows: 22-38 mg for the amount of PCX, 2.5-3.9% for the concentration of

305

ammonium hydroxide and 1.9-3 mL for the volume of eluent. In order to obtain the

306

maximum response (Y = 90.7%, desirability = 0.994), the best combination was found

307

to be 30 mg PCX and 2 mL 3% ammonium hydroxide in acetonitrile.

308

Under above optimized all of the PCX conditions for DMSPE cleanup method, the

309

pretreatment procedure was simple, fast and the cost of PCX for one sample is

310

approximately 0.30 US dollars. In addition, the total time required for the cleanup of

311

one sample was only approximately 3 min.

312

Method validation

313

Selectivity. With the high resolving power and accurate mass measurements of high

314

resolution mass spectrometry, no interfering peak at the retention time of morpholine

315

was observed from citrus and apple samples. Extracted ion chromatograms for

316

morpholine from the blank citrus, blank apple and standard at 5 µg/L were shown in

317

Fig. 2.

318

Linearity, LOD and LOQ. To obtain the internal calibration curves for the analyte,

319

the peak area ratio of the quantitative ion of morpholine to d8-morpholine was plotted

320

at the concentration of 1 to 100 µg/L (Table 2). The coefficients of determination (R2)

321

of the calibration curve for morpholine were above 0.999 which indicated good

322

linearity of the analytical range.

323

Here the limit of detection (LOD) and limit of quantification (LOQ) values of the

324

complete method were calculated as 3 and 10 times the signal/noise (S/N) ratio which

325

indicated 2 µg/kg and 5 µg/kg, respectively. 13



326

Recovery and precision. The results of the recovery, intra-day repeatability and

327

inter-day reproducibility experiment are presented in Table 4. Overall average

328

recoveries, repeatability, and reproducibility varied from 78.4 to 96.2%, from 1.8 to

329

6.4% (RSDr), and from 2.3 to 6.6% (RSDR), respectively for the citrus samples.

330

However, the mean recoveries, repeatability, and reproducibility ranged from 85.4 to

331

102.7%, with RSDr from 1.9 to 6.9% and RSDR from 3.2 to 8.4% for the apple

332

samples, which indicated that the established method was accurate enough and

333

suitable for the determination of morpholine in citrus and apple.

334

Comparison on matrix effects. To remove the interference and minimization of

335

matrix effect in LC/MS is the key to have an accurate, robust and sensitive

336

quantitative assay.29 In this study, matrix effects were evaluated by comparing slope

337

ratios of matrix-matched standard calibration slopes with solvent standard calibration

338

slopes (n = 3). It was found that slope ratios from the proposed method for citrus and

339

apple samples were 0.88±0.03 and 0.97±0.02 which were in the range Frenich et al.

340

(2011) had put forward to consider tolerable if the value was between 0.8 and 1.2.30 It

341

was also observed that there was a slight matrix effect for morpholine in citrus and

342

apple and the citrus had a stronger matrix effect than the apple. In order to

343

compensate the matrix effects and quantify accurately for morpholine in different

344

matrices, concentrations of morpholine in the samples should be calculated by the

345

internal calibration curves.

346

Stability. The stability tests were performed among spiked samples (5 and 20 µg/kg)

347

at different storage time (after 7, 14 and 21 days) and no obvious change for the

348

recoveries (83.6-95.3%) were observed.

349

Application to real samples

350

10 citrus and eight apples from local markets were analyzed for morpholine using 14


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351

the proposed method. 5 citrus and three apples were positive for morpholine residues

352

with the concentrations of 80.5-598.7 µg/kg for citrus and 43.4-328.2 µg/kg for apples.

353

Sen et al. (1988) had reported the contamination levels (0.25-7.7 mg/kg) of

354

morpholine in 17 batches of apples coated with liquid waxes. It indicated some citrus

355

and apples from the local markets might have been processed with coating wax using

356

morpholine. Further assessments should be conducted to determine whether the

357

presence of contamination levels of morpholine in these samples pose a potential

358

health hazard to human based on the ADI (0.48 mg/kg bw/day). Moreover, the

359

proposed DMSPE method can be compared with other reported methods for the

360

analysis of morpholine in citrus and apple samples. Table S2 compiles the comparison

361

of the analytical features of the selected references. Compared to the reported

362

methods, fewer matrix effects with similar or better detection levels for the analysis of

363

morpholine were achieved in this study.

364

In the present study, a new analytical method based on DMSPE cleanup procedure

365

was developed for the analysis of morpholine residue in citrus and apple samples. The

366

proposed method was validated with fortified samples and good recoveries were

367

obtained. The LOD and LOQ were low enough to efficiently quantify morpholine in

368

citrus and apple samples. To the best of our knowledge, this paper is the first to

369

introduce application of PCX as a sorbent in DMSPE for sample preparations of

370

alkaline substance and assumed to be widely used for alkaline contaminants at trace

371

levels in the future.

372

ASSOCIATED CONTENT

373

Supporting information

374

This materials is available free of charge via the Internet at http://pubs.acs.org.

375

Notes 15



376

The authors declare no competing financial interest.

377

Abbreviations used

378

DMSPE, dispersive micro solid phase extraction; UHPLC–HRMS, ultra-high

379

performance liquid chromatography-high resolution mass spectrometry; PCX,

380

polymer cation exchange; HILIC, hydrophilic interaction chromatography; SPE, solid

381

phase extraction; dSPE, dispersive solid phase extraction; LOD, limit of detection;

382

LOQ, limit of quantification; RSD, relative standard deviation; NOAEL, no observed

383

adverse effect level; ADI, acceptable daily intake; NMOR, N-nitrosomorpholine;

384

CCD, central composite design; GC-TCD, gas chromatography-thermal conductivity

385

detector; GLC-TEA, gas-liquid chromatography-thermal energy analyzer; GC-MS,

386

gas chromatography-mass spectrometry; HESI, heated electrospray ionization; HCD,

387

higher energy collisional dissociation; NCE, normalized collision energy.

388

Acknowledgements

389

This work was financially supported by the International Science and Technology

390

Cooperation Program of China (2011DFA-31770) and National Support Program for

391

Science and Technology (2012BAK01B01). The authors wish to thank Thermo Fisher

392

Scientific for technical support.

393 394 395 396 397 398 399 400

16


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Page 17 of 28


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nanomaterial

ultrasound-assisted

microextraction

499

20


method

for

Page 21 of 28


500

Figure captions

501

Fig. 1 Three-dimensional graphs of the effects of the amount of PCX (A), the

502

concentration of ammonium hydroxide (B) and the volume of elute (C) on efficiency

503

of extraction: (a) fixed C= 2.0 mL; (b) fixed B=2.55%; (c) fixed A=30 mg.

504

Fig. 2 Extracted ion chromatograms for morpholine in the blank citrus (a), blank

505

apple (b) and standard at 5 µg/L (c)

506

21



Page 22 of 28

Table 1 Variables and levels evaluated in the central composite design Coded level

Independent variables

Unit

Symbol

0

+1

mg

A

-α 10

-1

The amount of PCX The concentration of ammonium hydroxide

20

30

40

+α 50

%

B

0.1

1.32

2.55

3.77

5

The volume of eluent

mL

C

1

1.5

2

2.5

3

22


Page 23 of 28


Table 2 Calibration curve equations, correlation coefficients (R2), LOD, and LOQ for morpholine

Analyte

Precursor ion (m/z)

Fragment ion (m/z)

NCE (%)

Linear range (µg/L)

Linearity equation

R2

LOD/(µg/kg)

LOQ/(µg/kg)

morpholine d8-morpholine

88.0761 96.1259

70.0658*; 68.0502 78.1157*; 76.1035

75 75

1-100 /

Y = 0.10552X+0.022816 /

0.9997 /

2 /

5 /

*

quantitative ion

23



Page 24 of 28

Table 3 ANOVA for response surface quadratic model Source Model A B C AB AC BC A2 B2 C2 Lack of fit Pure error Corrected total SD CV

Sum of squares 4740.62 217.56 2232.56 1008.06 66.12 0.13 78.12 292.21 953.78 271.17 53.84 21.33 4815.80 2.74 3.57

df 9 1 1 1 1 1 1 1 1 1 5 5 19 R2 adj-R2

Mean square 526.74 217.56 2232.56 1008.06 66.12 0.13 78.12 292.21 953.78 271.17 10.77 4.27

24


0.9844 0.9703

F-value 70.07 28.94 296.98 134.09 8.80 0.017 10.39 38.87 126.87 36.07 2.52

P-value

Novel Dispersive Micro-Solid-Phase Extraction Combined with

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