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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,
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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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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
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analyzed
by
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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
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interferences in complex matrices for the residue analysis.11-13 A dual SPE cartridge 3
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system was used as a pretreatment method for morpholine in pineapples by Gros et al,
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but it is time-consuming, laborious and costly.14 While a Quick, Easy, Cheap,
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Effective, Rugged, and Safe (QuEChERS)-based on dSPE method for sample cleanup
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has been developed to improve sample throughput for residue analysis,15,16 the
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purification effect is even worse than SPE.17 On the contrary, the dispersive micro
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solid phase extraction (DMSPE) can be categorized as a dSPE or SPE technique and
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exhibits some advantages over traditional dSPE (fewer matrix effects) and SPE (such
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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
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an extract and the cleanup procedure relies solely on shaking and centrifugation.
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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
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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
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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
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between variables and the non-linear relations with the responses into account.
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Therefore, the central composite design (CCD) approach was used for studying the
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optimal variables in this DMSPE cleanup method. 4
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MATERIALS AND METHODS
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Chemicals and reagents
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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).
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Morpholine
was
purchased
from
Supelco
(Bellefonte,
PA,
USA)
and
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d8–morpholine was obtained from Cambridge Isotope Laboratories (Tewksbury, MA,
110
USA). The purity of all these standards was no less than 98.0%.
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Standard stock solutions (1000 mg/L) were prepared by dissolving 10.0 mg of
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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 ℃.
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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 ℃.
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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
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into the sample. After adding 10 mL of 1% formic acid in acetonitrile/water (1:1 v/v),
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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
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the supernatant was transferred into 2 mL eppendorf tube with 30 mg PCX powder.
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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.
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The extracted solution was passed through the syringe and syringe filter manually and
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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.
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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)
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consisted of a mixture of 0.1% of formic acid and 4 mM ammonium formate in water
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and the organic phase (B) was acetonitrile with 0.1% formic acid. The gradient started
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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
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set to 300 µL/min with a resulting overall runtime of 9 min. The injection volume was
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10 µL.
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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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Scientific). The spray voltage was 3.5 kV for the positive mode. The temperature of
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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.
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The Q-Exactive detector was operated in targeted MS/MS (tMS/MS) mode. The
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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).
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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.
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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)
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factorial design; (2) center point (cp = 5); (3) two axial points on the axis of each
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design variable at a distance of α = 2.0 from the design center. The response Y (the 7
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efficiency of extraction) was established for the determination of optimum method
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conditions, and the recovery was selected as the indicator of the extraction efficiency.
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The optimal values of response Y were obtained by solving the regression equation
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and analyzing the three-dimensional response surface plot and contour plots.
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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.
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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
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of the internal standard was 10 µg/L for d8-morpholine.
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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
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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
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Matrix effect (ME) was determined by constructing calibration curves in blank
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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
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extract/slope of pure solvent standard.
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Stability of the analyte in matrix was determined by repeated analysis (n = 3) of
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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.
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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
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(2.1 mm × 100 mm, 1.7 µm), and HSS T3 (2.1 mm × 100 mm, 1.8 µm) were tested to
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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.
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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.
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Full scan mode is a full scan of all ions in the specified mass range. However, the 9
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precursor in the inclusion list (with retention times) can be selected in the quadrupole
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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.
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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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m/z for d8-morpholine).
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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
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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
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analysis data obtained from the response surface plots and the regression coefficient
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plots. The factor settings that maximize the efficiency of extraction were chosen in
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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
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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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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
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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
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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
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ultrasound-assisted
microextraction
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Figure captions
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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
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of extraction: (a) fixed C= 2.0 mL; (b) fixed B=2.55%; (c) fixed A=30 mg.
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Fig. 2 Extracted ion chromatograms for morpholine in the blank citrus (a), blank
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apple (b) and standard at 5 µg/L (c)
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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
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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
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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
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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