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Development of a Method for Rapid Determination of Morpholine in Juices and Drugs by Gas Chromatography-Mass Spectrometry

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A reliable derivatization method has been developed to detect and quantify morpholine in apple juices and ibuprofen with gas chromatography-mass spectrometry. Morpholine can react with sodium nitrite under acidic condition to produce stable and volatile N-nitrosomorpholine derivative. In this experiment, various factors affecting the derivatization and extraction process were optimized, including volume and concentration of hydrochloric acid, quantity of sodium nitrite, derivatization temperature, derivatization time, extraction reagents, and extraction time. The derivative was extracted with dichloromethane and determined by gas chromatography-mass spectrometry. The linearity range of morpholine was 10–500 μ g·L ⁻¹ with good correlation, and limits of detection (LOD) and limits of quantification (LOQ) were 7.3 μ g·L ⁻¹ and 24.4 μ g·L ⁻¹ , respectively. Low, medium, and high concentrations of morpholine were added in apple juices and ibuprofen samples to evaluate standard recovery rate and relative standard deviation. The spiked recovery rate ranged from 94.3% to 109.0%, and the intraday repeatability and interday reproducibility were 2.0%–4.4% and 3.3%–7.0%, respectively. The developed method has good accuracy and precision. This quantitative method for morpholine is simple, sensitive, rapid, and low cost and can successfully be applied to analyze the residual morpholine in apple juices and drug samples.
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Research Article
Development of a Method for Rapid Determination of
Morpholine in Juices and Drugs by Gas
Chromatography-Mass Spectrometry
Mengsi Cao,
1
Pingping Zhang,
2
Yanru Feng,
1
Huayin Zhang,
1
Huaijiao Zhu,
1
Kaoqi Lian ,
1
,
3
and Weijun Kang
1
1
School of Public Health, Hebei Medical University, Shijiazhuang 050017, China
2
Department of Reproductive Genetic Family, Hebei General Hospital, Shijiazhuang 050017, China
3
Hebei Province Key Laboratory of Environment and Human Health, Shijiazhuang 050017, China
Correspondence should be addressed to Kaoqi Lian; liankq@hebmu.edu.cn and Weijun Kang; kangwj_hebmu@126.com
Received 30 July 2017; Revised 18 October 2017; Accepted 7 November 2017; Published 26 April 2018
Academic Editor: Serban C. Moldoveanu
Copyright ©2018 Mengsi Cao et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
A reliable derivatization method has been developed to detect and quantify morpholine in apple juices and ibuprofen with gas
chromatography-mass spectrometry. Morpholine can react with sodium nitrite under acidic condition to produce stable and
volatile N-nitrosomorpholine derivative. In this experiment, various factors affecting the derivatization and extraction process
were optimized, including volume and concentration of hydrochloric acid, quantity of sodium nitrite, derivatization temperature,
derivatization time, extraction reagents, and extraction time. e derivative was extracted with dichloromethane and determined
by gas chromatography-mass spectrometry. e linearity range of morpholine was 10–500 μg·L
1
with good correlation, and limits
of detection (LOD) and limits of quantification (LOQ) were 7.3 μg·L
1
and 24.4 μg·L
1
, respectively. Low, medium, and high
concentrations of morpholine were added in apple juices and ibuprofen samples to evaluate standard recovery rate and relative
standard deviation. e spiked recovery rate ranged from 94.3% to 109.0%, and the intraday repeatability and interday re-
producibility were 2.0%–4.4% and 3.3%–7.0%, respectively. e developed method has good accuracy and precision. is
quantitative method for morpholine is simple, sensitive, rapid, and low cost and can successfully be applied to analyze the residual
morpholine in apple juices and drug samples.
1. Introduction
Morpholine (tetrahydro-2H-1,4-oxazine), a heterocyclic
secondary amine, is a colorless, hygroscopic, alkaline, oily
liquid at normal temperature and pressure with an am-
moniacal odor and is miscible with water and organic
solvents in any ratio [1]. Morpholine is used as an emulsifier
for protective wax coating on apples and other fruits to keep
them fresh and storable [2–4]. Nowadays, more and more
people like to drink fresh juice instead of fresh fruit, and
some manufacturers produce fresh juice together with the
pericarp to improve dietary fiber in fruit juice and economic
benefits, thereby increasing the residual content of mor-
pholine in the juice, such as apple juice. e compound also
effectively suppresses the hatching process of the eggs of
golden apple snails, a known pest of the rice crops in Asia,
and thereby controls the reproduction of those snails to
protect the rice crops [5]. Being a cyclic amine, morpholine
is commonly used in pharmaceutical industries for synthesis
of different active pharmaceutical substances, such as
morinidazole [6], and to increase aqueous solubility of
gefitinib [7]. Morpholine has been used for preparing a series
of new antimicrobial and antiviral diphenyl diselenides [8].
It is also used as a reagent to prepare the morpholine de-
rivative, 4-(2-aminoethyl) morpholine, also called AEM [9].
AME, triethylamine, and methacryloyl chloride are used to
synthesize N-ethyl morpholine methacrylamide (EMA) [10].
EMA is a pH-sensitive polymer hydrogel which is used to
Hindawi
Journal of Analytical Methods in Chemistry
Volume 2018, Article ID 9670481, 8 pages
https://doi.org/10.1155/2018/9670481
prevent crystallization of ibuprofen [11]. Consequently,
morpholine residues may be present in the production of
ibuprofen. Morpholine causes irritation of eye, skin, and
digestive tract and may be absorbed in the body through skin
contact, inhalation, and ingestion [1]. As a result, the use of
morpholine has been prohibited as an emulsifier in pro-
tective wax coating on citrus fruits, apples, and cosmetic
preparations in the European Union (EU) [1, 12]. As per
Health Canada Monograph [13], the no-observed-adverse-
effect level (NOAEL) of morpholine is 96 mg·kg
1
of body
weight (bw) day
1
and the acceptable daily intake (ADI) is
0.48 mg·kg
1
of bw day
1
[14]. erefore, establishing a
rapid and effective method to detect and quantify mor-
pholine in fruit juices and pharmaceuticals is of primary
importance.
In recent years, numerous studies have reported various
analytical methods for qualitative and quantitative estima-
tion of morpholine. ese analytical methods employed
various available analytical techniques, such as gas chro-
matography (GC) [15–17], gas chromatography-mass
spectrometry (GC-MS) [18], gas-liquid chromatography-
high resolution mass spectrometry (GLC-MS) [19], liquid
chromatography (LC) [20], ultra performance liquid chro-
matography (UPLC) [21], hydrophilic interaction liquid
chromatography with electrospray ionization and tandem
mass spectrometry (HILIC-ESI-MS/MS) [22], and ultrahigh
performance liquid chromatography-high resolution mass
spectrometry (UHPLC-HRMS) [14]. However, these pub-
lished methods have different disadvantages, such as tedious
operation steps [14] and high cost [22]. Applying the de-
rivatization method with 2,4-dinitrofluorobenzene
(2,4-DNFB) by GC-MS to detect morpholine has better
sensitivity, but has low stability [18].
is experiment was based on some of the secondary
amines that could react with sodium nitrite to produce
volatile N-nitrosamines (NAms) under acidic conditions
[23]. We found that morpholine as a cyclic secondary amine
can generate N-nitrosomorpholine (NMOR) by using so-
dium nitrite as the derivatization reagent under acidic
condition, and NMOR which is stable and volatile can be
determined by GC-MS. Our team used to establish a method
to determine ketamine in urine and plasma by this derivative
method and obtained good experimental results [24]. We
have extensive experience about this derivatization reaction.
erefore, various factors affecting derivatization process
and extraction efficiency can be optimized to develop a re-
liable method for rapid determination of morpholine in
apple juice and drug granules through GC-MS. Compared
with other existing derivatization methods, sodium nitrite
and hydrochloric acid as derivatization reagents are cheap
and obtained easily in this experiment. e samples only
needed centrifugate and filter without complicated sample
pretreatment process and was analysed rapidly by GC-MS.
e consumption of organic solvents was very small in the
whole test process, thereby reducing the pollution of the
environment. is study established a rapid, sensitive,
simple, low-cost, and reliable method to determine mor-
pholine in apple juice and drugs, and had highly realistic
application value.
2. Experimental
2.1. Chemicals. All chemicals and reagents were of ana-
lytical grade unless otherwise stated. Standard morpholine
was purchased from Aladdin Reagent Co., Ltd. (Shanghai,
China). e derivatization reagents of sodium nitrite
(NaNO
2
) and hydrochloric acid (HCl) were purchased
from Henan Jiaozuo ree Chemical Plant (Jiaozuo, China)
and Shijiazhuang Reagent Factory (Shijiazhuang, China),
respectively. Dichloromethane, ethyl acetate, chloroform,
n-hexane, and carbon disulfide from Xilong Chemical
Factory (Shantou, China) or Tianjin General Chemical
Reagent Factory (Tianjin, China) were tested to select the
most optimal extraction reagent. Pure water (18.2 MΩ/cm)
was obtained from Heal Force SMART-N ultrapure water
system (Hong Kong).
2.2. Quantitative Methods and Quality Control Samples.Stock
standard solution of morpholine (50 mg·L
1
) was prepared
in pure water. Working calibrators at 10, 25, 50, 100, 200,
300, 400, and 500 µg·L
1
were prepared by diluting in pure
water, and the calibration curve was fitted by linear re-
gression method through the measurement of the peak areas
corresponding to the concentrations. e acceptance cri-
terion for the calibration curve is a correlation coefficient of
0.99 or better. Quality control (QC) samples were prepared
by freshly spiking the appropriate working solution into
blank apple juice and ibuprofen samples to prepare con-
centrations of 50, 200, and 400 μg·L
1
for morpholine. e
series of standard solution and QC samples were freshly
prepared before use.
2.3. Pretreatment of Samples. e apple juices were obtained
from a local supermarket and filtered with 0.22 µm mem-
brane filter. Ibuprofen granules were purchased from a local
pharmacy and dissolved in purified water and centrifuged
(10,000 rpm for 15 min) after mixing. e supernatant liquid
was filtered with 0.22 µm membrane filter. All the samples
were stored at 4°C.
2.4. Derivatization and Liquid-Liquid Extraction. A certain
amount of morpholine stock standard solution was added to
20 mL of apple juice or ibuprofen solution in a 50 mL dis-
posable sample pretreatment tube. e samples were
centrifuged and filtered as described in the Section 2.3. To
2.0 mL of pretreated apple juice or ibuprofen solution,
200 μL of 0.05 mol·L
1
HCl and 200 μL of saturated NaNO
2
were added and vortex-mixed. e resultant solution was
placed in a 10 mL glass test tube and mixed thoroughly. e
mixture was heated at 40°C for 5 min on a heating block.
After cooling, 0.5 mL of dichloromethane was added, and
the mixture was vortex-mixed for 1 min and allowed to stand
for 10 min to extract the derivative. en, 200 μL of organic
layer was transferred with a micropipette to a tipped glass
tube and placed in an ice bath to prevent the volatilization of
dichloromethane and the impact on experiment results.
2Journal of Analytical Methods in Chemistry
en, 1 μL of this organic layer was injected into the GC-MS
with a 10 μL syringe (from Agilent).
2.5. GC-MS Analysis. An Agilent Technologies (Little Falls,
DE, USA) gas chromatograph 7890 equipped with an
electronically controlled split/splitless injection port, an
inert 5975C mass selective detector with electron impact (EI)
ionization chamber, and a 7683B series injector/autosampler
were employed for identification and quantification of
N-nitrosomorpholine that was the derivative of morpholine.
e GC separation was conducted with a TM-1701
30 m ×0.32 mm I.D., 0.5 μm film thickness column (Tech-
comp, China). e carrier gas was helium with a constant
flow rate of 2 mL·min
1
. e injection volume was 1 μL and
was vaporized at 250°C with a 1 : 7 split ratio. e GC oven
was operated with the following temperature program:
initial temperature 100°C held for 4 min and programmed to
120°C at a rate of 10°C min
1
and held for 3 min, and then
ramped at 20°C min
1
–250°C and held for 5 min. e total
run time was 18 min.
Two different ions were selected to detect and quantify
N-nitrosomorpholine (86.1, 116.1) at the selected ion-
monitoring (SIM) mode. Ionization was performed by
electron impact (EI) mode at 70 eV energy. e tempera-
tures used were 280°C for the transfer line, 230°C for the ion
source, and 150°C for the MS quadrupole. e solvent delay
was 4.5 min.
3. Results and Discussion
3.1. Principles of Derivatization and Identification of
Derivative. Morpholine, as a secondary amine, reacts with
sodium nitrite under acidic conditions to produce stable and
volatile NMOR which can be determined by GC-MS. e
reaction is shown in Figure 1. 2.0 mL of 400 μg·L
1
mor-
pholine standard solution was used to verify the de-
rivatization reaction. e total ion current chromatogram
and mass spectra of the NMOR derivative are shown in
Figure 2. Analyses of mass spectra and MS data of the
derived sample proved that the derivative was NMOR.
3.2. Optimization of Derivatization and Extraction. A rapid
and low-cost derivatization technique has been developed
for detection and determination of morpholine. e de-
rivatization process of morpholine has been described in the
Section 2.4. Various factors associated with derivatization
and extraction process were optimized, which included
concentration and dosage of hydrochloric acid (HCl), the
amount of saturated sodium nitrite (NaNO
2
), derivatization
temperature, derivatization time, the extraction reagents,
and extraction time.
3.2.1. Concentration and Quantity of Hydrochloric
Acid.e derivatization process was affected by the con-
centration and quantity of hydrochloric acid. e concentration
H
NN
N
O
NaNO2
Acidic condition
Figure 1: e derivatization reaction of morpholine.
5.00 6.00 7.00 8.00
1000
2000
3000
4000
5000
6000
0 102030405060708090100110120130
56.1
116.1
86.1
28.1
42.1
70.0
m/z
Time (
min)
Abudance
Figure 2: e total ion current chromatogram and mass spectra of the N-nitrosomorpholine.
Journal of Analytical Methods in Chemistry 3
of HCl was optimized as the first step. e effects of adding
200 μL of HCl with different concentrations between 0.01
and 0.06 mol·L
1
are shown in Figure 3(a). e derivatization
rate was found to increase with the increasing concentration
of HCl in the range from 0.01 to 0.05 mol·L
1
and then
became stable. us, the best result was obtained when 200 μL
of 0.06 mol·L
1
HCl was added during the process of
derivatization.
3.2.2. e Amount of Saturation Solution of Sodium
Nitrite. Optimum quantity of saturation solution of sodium
nitrite required for the derivatization process was de-
termined (Figure 3(b)) by varying the addition of saturation
solution of sodium nitrite in the range of 50–300 μL. e
derivatization yields increased with the addition of satura-
tion solution of sodium nitrite up to 200 μL and then became
stable. erefore, the optimum volume of saturation solu-
tion of sodium nitrite for derivatization was 200 μL.
3.2.3. Derivatization Temperature and Derivatization
Time. e effects of derivatization temperature and time
were tested in this experiment. e effect of temperature
(0°C (ice-bath), 4°C (refrigeration), 25°C (room tempera-
ture), 40°C, 60°C, and 80°C) on derivatization was in-
vestigated. e rate of derivatization increased with reaction
temperature and then became stable at 40°C (Figure 3(c)).
erefore, 40°C was selected as the optimum temperature for
this experiment. Moreover, the effect of reaction time on
derivatization process was investigated; the reaction time
was varied between 1 and 30 min. e derivatization leveled
off at 5 min (Figure 3(d)), suggesting the optimum reaction
time to be 5 min.
3.2.4. Extraction Reagents and Extraction Time. Selection of
suitable solvent is an important criterion for extraction of
the derivative. e extraction efficiencies of n-hexane,
dichloromethane, chloroform, carbon disulfide, and ethyl
0.00 0.02 0.04 0.06
6.0
×
10
4
8.0
×
10
4
1.0
×
10
5
1.2
×
10
5
1.4
×
10
5
Amount of 200 μL HCl (mol/L)
Peak area
(a)
0 100
200
300
6.0
×
104
8.0
×
104
1.0
×
105
1.2
×
105
1.4
×
105
Amount of saturated sodium nitrite (μL)
Peak area
(b)
0 20406080
6.0
×
104
8.0
×
104
1.0
×
105
1.2
×
105
1.4
×
105
Temperature (ºC )
Peak area
(c)
0 102030
6.0 × 10
4
8.0 × 10
4
1.0 × 10
5
1.2 × 10
5
1.4 × 10
5
Time (min)
Peak area
(d)
Figure 3: e effects of hydrochloric acid concentration and quantity (a), the amount of saturation solution of sodium nitrite (b), derivative
reaction temperature (c), and time (d).
4Journal of Analytical Methods in Chemistry
acetate were evaluated as shown in Figure 4. e study
revealed that dichloromethane and chloroform afforded
optimum extraction of the derivative. Finally, dichloro-
methane was selected as the extraction reagent. 2.0 mL of
dichloromethane was used to extract the derivative, and
1.5 mL organic layer was transferred to a tipped glass tube
and dried with a slow stream of nitrogen at room temperature.
e dried substances were dissolved in 100 µL ethyl acetate
before GC analysis. However, the experiment resulted in poor
precision as indefinite derivative was blown away in the ni-
trogen blowing process. To improve the extraction efficiency
and stabilization, 0.5 mL dichloromethane was added and
vortex mixed for 1 min followed by standing for 10 min to
extract the derivative.
An attempt to detect morpholine (400 μg·L
1
) by GC-MS
without any derivatization process failed, as the method
could not detect any signal of the compound (Figure 5(a)).
In another attempt, morpholine produced similar signal
abundance in two different samples (20 mg·L
1
in
dichloromethane without derivatization and 400 μg·L
1
in
pure water after derivatization) (Figures 5(b) and 5(c)). e
proposed derivatization method was about 65 times more
sensitive than the direct detection. Kataoka [16] had com-
pared the effects of commonly used derivatization reagents,
such as acylation, silylation, dinitrophenylation, per-
methylation, carbamate formation, sulfonamide formation,
and phosphoamide formation for analysis of secondary
amines by GC. However, sodium nitrite and hydrochloric
acid are preferred as derivatization agents, since they are
cheaper and easily available compared to other reagents.
Sacher et al. [18] established a method based on de-
rivatization of the amines with benzenesulfonyl chloride.
However, usage of many reagents and long derivatization
process (1 hour) and operation time (morpholine peak
appeared at 18 min) made this method practically in-
convenient. In comparison, the proposed method involves
Figure 4: Extraction effects of different extraction reagents.
Time (min)
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
0
500
1000
1500
2000
2500
3000
3500
4000
4500
Abundance
A
B
C
Figure 5: e total ion current chromatograms of morpholine resulting from different methods: direct detection of 400 μg·L
1
(A) and
20 mg·L
1
(B) morpholine prepared in dichloromethane and detection of 400 μg·L
1
morpholine prepared in pure water after the proposed
derivatization (C).
Journal of Analytical Methods in Chemistry 5
less derivatization process (5 min) and operation time
(morpholine peak appeared at 7.72 min).
e proposed method has many advantages compared to
previously published methods [14, 15, 22] (Table 1). Use of MS
detector in this study produced similar sensitivity as HILIC-
ESI-MS/MS [22], and both the methods yielded better results
than flame ionization detection (FID) [15]. Dawei Chen et al.
[14] established a reliable method to determine morpholine
residues by UHPLC-HRMS combined with dispersive micro-
solid-phase extraction (DMSPE). e method is more sen-
sitive but involves complicated sample pretreatment process
and costly instrumentation.
3.3. Selection of Chromatographic Column. In the pre-
liminary experiment, HP-5 nonpolar chromatographic
column (dimensions: 30 m ×0.32 mm ×0.25 µm; stationary
phase: 5% phenyl-95% methylpolysiloxane) and TM-1701
medium polarity chromatographic column (dimensions:
30 m ×0.32 mm ×0.5 μm; stationary phase: 14% cyanopropyl
phenyl-86% dimethyl polysiloxane) were employed. HP-5
column resulted in peak tailing of peak and high baseline.
TM-1701 column provided better peak shape under the
optimized conditions and therefore suited the experiment.
3.4. Method Validation
3.4.1. Linearity, Detection Limit, and Quantitative
Limit. Calibration curve was constructed by plotting the
peak area against the concentration range from 10 to
500 µg·L
1
of morpholine. e linear regression equation
was A471.2c2263.8, in which ccorresponds the con-
centrations and Acorresponds the peak areas. e results
obtained a good linearity of the analytical range which was
10–500 µg·L
1
with the coefficient of determination (R
2
) of
the calibration curve for morpholine higher than 0.999. In
this method, the limit of detection (LOD) and the limit of
quantification (LOQ) were calculated as 3 and 10 times the
S/Nratio, which indicated 7.3 µg·L
1
and 24.4 µg·L
1
,
respectively.
3.4.2. Accuracy and Precision. rough adding standard
solution of morpholine with high concentration to the apple
juice and ibuprofen blank samples, three different spiked
samples with final concentration levels of 50, 200, and
400 µg·L
1
were obtained. e samples with each concen-
tration level were determined on six times a day over three
consecutive days. e spiked recovery rate, intraday re-
peatability, and interday reproducibility were 94.3%–109%,
2.3%–4.4%, and 4.8%–5.2% for apple juice spiked samples,
and 96%–107.9%, 2%–4.4%, 3.3%–7% for ibuprofen spiked
samples, respectively (Table 2). e results indicated that the
method was suitable for determining morpholine with
favourable accuracy and precision.
3.5. Application to Real Samples. Samples of apple juice and
ibuprofen granules were analysed by this method under
Table 1: Comparison of the proposed method with previously published methods.
Sample e test process of sample LOQ Reference
Sample pretreatment Derivatization reaction Determination
Apple juice and
ibuprofen Centrifugation and filtration Sodium nitrite under
acidic condition
Gas chromatography-mass
spectrometry (GC-MS) 24.4 μg·L
1
is work
Steam
condensate — —
Chromatography with multimode
inlet and flame ionization
detection (GC-MI-FID)
100 μg·L
1
[15]
Citrus and
apples
15mL 1% acetic acid in
methanol
Hydrophilic interaction liquid
chromatography with
electrospray ionization and
tandem mass spectrometry
(HILIC-ESI-MS/MS)
10 μg·kg
1
[22]
Citrus and
apples
Dispersive micro-solid-phase
extraction (DMSPE)
Ultrahigh performance liquid
chromatography-high resolution
mass spectrometry
(UHPLC-HRMS)
5μg·kg
1
[14]
Table 2: Recovery and precision of three spiked levels.
Sample Spiked concentration (μg·L
1
) Recovery (%) Intraday repeatability (%) Interday reproducibility (%)
Apple juice
50 109.0 4.4 5.2
200 94.3 2.3 4.8
400 98.4 3.3 5.0
Ibuprofen
50 96.0 4.4 3.3
200 100.9 2.5 7.0
400 107.9 2.0 5.5
6Journal of Analytical Methods in Chemistry
optimal conditions using standard addition method.
However, morpholine was not detected in any real samples.
us, using the standard addition method, morpholine was
detected in apple juice and ibuprofen granules samples. e
total ion current chromatograms of the real samples of apple
juice and ibuprofen granules and their spiked samples
(400 μg·L
1
) are shown in Figure 6.
4. Conclusions
According to that morpholine could react with sodium
nitrite to generate the stable and volatile N-nitrosomorpholine
under acidic conditions, we established a rapid, sensitive,
simple, low-cost, and reliable method to detect morpholine in
apple juices and drugs. is method had been successfully
analysed of spiked samples with low detection limit and
favourable accuracy and precision. It can provide technical
support to establish the national standards of morpholine in
fruit juices and pharmaceuticals and monitor the residue of
morpholine in the future.
Conflicts of Interest
e authors declare no conflicts of interest.
Authors’ Contributions
Kaoqi Lian and Weijun Kang conceived and designed the
experiments. Mengsi Cao, Pingping Zhang, Yanru Feng,
Huayin Zhang, and Huaijiao Zhu performed the experi-
ments. Kaoqi Lian and Mengsi Cao analysed the data and
wrote the paper. All authors read and approved the final
manuscript.
Acknowledgments
is work was supported by the National Natural Science
Foundation of China (no. 81302471) and the Natural Science
Foundation of Hebei Province (no. H2014206345).
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Abundance
5.00 5.50 6.00 6.50 7.00 7.50 8.00
1000
1600
2200
2800
3400
4000
4600
5200
5800
Time (min)
(a)
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4500
5000
5500
Time (
min)
(b)
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Journal of Analytical Methods in Chemistry 7
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8Journal of Analytical Methods in Chemistry
... The method used whole fruits rather than separately analyzing the peel and pulp of fruit samples [17,18]. A previous study developed a gas chromatography-mass spectrometry (GC-MS) method for morpholine analysis in apple juice and whole apples [28] but was not used to examine the fruit peel itself. Because of the high lipid contents in fruit peel, lipids may lower extraction efficiency. ...
... A previously described sample preparation method for morpholine was modified by adding a lipid removal step and changing the pH during the derivatization step [28]. Sequential extraction was performed. ...
... Sequential extraction was performed. The first step employed the lipid removal method and the second step involved a derivatization step to N-nitroso-morpholine. As described in the Introduction, Cao et al., prepared samples of apple juice rather than of fruits [28]. When the method of Cao et al., was used for fruit samples, particularly fruit peels, the final extract solution was unclear, possibly because of the presence of lipids. ...
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Morpholine salts of fatty acids have been used in wax coatings on the surfaces of fruit and vegetable commodities in China and the United States, etc. However, morpholine usage was prohibited in many other countries because of safety concerns. We optimized analytical methods to determine morpholine in the peel and pulp of fruits and vegetables by gas chromatography-mass spectrometry (GC-MS). This morpholine analysis method was applied to real samples of apples, citrus fruits, and vegetables from Korea, China, and the U.S. The method was validated using apple and citrus fruit peels and pulp. The method detection limit (MDL) was 1.3–3.3 µg/kg. The recovery rates of morpholine were 88.6–107.2% over a fortified level of 10–400 µg/kg. Intra-day and inter-day precisions were 1.4–9.4% and 1.5–2.8%, respectively. The morpholine concentrations were n.d. (not detected)–11.19 and n.d. (not detected)–12.82 µg/kg in apple and citrus peels, respectively. Morpholine was not detected in citrus or apple pulp samples or in vegetable samples.
... (tetrahydro-2H-1,4-oxazine) is a heterocyclic secondary amine, a colourless and clear liquid, which has been widely used as an accelerant in rubber manufacturing, a sulfuration agent, a cleanser, a descaling agent, a surfactant and an agent in textile printing (Van Stee et al. 1981;IARC 1989;Cao et al. 2018). In addition, morpholine fatty acid salts are an important emulsifying agent, which added to some waxes is used in the preparation of a wax coating for some fruits and vegetables, and the wax coating can protect fruits and vegetables against insects and fungal contamination (IPCS 1995;Mcguire and Dimitroglou 1999;US FDA 2018). ...
... Second, food consumption data used in this study were from the China National Nutrition and Health Survey of 2002, and consumption levels of fruit and juice may have changed during the 16-year interval since the study. Third, only exposure through food was calculated, which does not account for additional exposure pathways, such as drugs, cosmetics, snuff and chewing tobacco (Brunnemann et al. 1982;CIR,1989;Cao et al. 2018). Fourth, in the ADI (480 µg/kg bw/day) estimated by Health Canada, which was derived from animal experiments, there were some uncertainties in the extrapolation from animal experimental data to humans. ...
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Morpholine is a common chemical used as emulsifier in the preparation of wax coatings for some fruit to help them remain fresh and protect against insects and fungal contamination. It has been reported that morpholine has acute toxic effects on rodents. In the present study, morpholine concentrations were analysed in fruits (citrus fruits, apples, strawberries and grapes) and juices (apple juice and orange juice) in order to determine dietary exposure among the Chinese population. A total of 732 fruit and juice samples were collected during 2015–2016, which covered major foods in China. Fruit and juice consumption data were taken from China National Nutrient and Health Survey (2002) and include data from 16,407 fruit or juice consumers. It was found that mean dietary exposure to morpholine residues from fruits and/or juices for general Chinese consumers and children 2–6 years old were 0.42 and 1.24 µg/kg bw/day, respectively. The 97.5% intake in general Chinese consumers and children 2–6 years old were 2.25 and 6.90 µg/kg bw/day, respectively. The primary food sources of the morpholine dietary intake of general Chinese consumers were citrus fruits (57.4%) and apples (40.8%). These findings suggested that dietary exposure to morpholine in the Chinese population was lower than the acceptable daily intake of morpholine, and there are no health concerns.
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A new analytical method involving gas chromatography-tandem mass spectrometry (GC−MS/MS) was developed for the determination of morpholine residues in fruit and fruit juice samples. The samples were extracted with acidic acetonitrile-water mixed solution, purified with a mixed cation exchange column, and subjected to gas chromatography-tandem mass spectrometry (GC–MS/MS). The isotope internal standard (morpholine-d8) method was used for quantification. The average recoveries ranged from 85.4 to 108.9%. The limit of quantification (LOQ) for all samples was 10.0 μg/kg. The positive samples were confirmed by gas chromatography-quadrupole-orbitrap high-resolution mass spectrometry (GC−Orbitrap HRMS). The developed method has good precision and accuracy.
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A reaction of bis[(2-chlorocarbonyl)phenyl] diselenide with various mono and bisnucleophiles such as aminophenols, phenols, and amines have been studied as a convenient general route to a series of new antimicrobial and antiviral diphenyl diselenides. The compounds, particularly bis[2-(hydroxyphenylcarbamoyl)]phenyl diselenides and reference benzisoselenazol-3(2H)-ones, exhibited high antimicrobial activity against Gram-positive bacterial species (Enterococcus spp., Staphylococcus spp.), and some compounds were also active against Gram-negative E. coli and fungi (Candida spp., A. niger). The majority of compounds demonstrated high activity against human herpes virus type 1 (HHV-1) and moderate activity against encephalomyocarditis virus (EMCV), while they were generally inactive against vesicular stomatitis virus (VSV).
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Wax application plays an important role in prolonging fruit quality, and the addition of imazalil (IMZ) furthermore protects fruit against green mould caused by Penicillium digitatum. The objectives of this study were to evaluate green mould control and quality preservation effects of carnauba or polyethyl-ene citrus coatings supplemented with IMZ, as well as the effect of synthetic or horsehair brush types used on sweet orange fruit. Single applications of IMZ at 3000 g mL −1 at rates of 0.6, 1.2 and 1.8 L t −1 resulted in residues that increased with increasing coating loads on navel oranges (1.31 to 3.32 g g −1) and Valencia oranges (3.22 to 6.00 g g −1). Coating with IMZ generally provided poorer curative control (≈14%) than protective control (≈58%), with less sporulation in treatments using horsehair (≈59%) than synthetic brushes (≈64%). More fruit weight and firmness losses were found in fruit treated with the poly-ethylene coating (≈1.18 and ≈0.93 ratios of treated vs. untreated, respectively) and lower in carnauba treated fruit (≈0.76 and ≈0.74 ratios, respectively). However, polyethylene coatings resulted in shinier fruit before (≈10.85 shine ratio) and after storage (11.60), whereas carnauba coatings resulted in lower shine ratios (≈7.45 and 10.15, respectively). Gas (CO 2) exchange ratios remained similar for both waxes (≈0.67). Higher polyethylene coating loads (1.8 L t −1) resulted in off-tastes similar to uncoated control fruit (≈2.21 rating on a 5-point scale) and higher than the rating for carnauba coated fruit (≈1.82) at this rate. Scanning electron micrographs showed an amorphous crystallised natural wax layer with uncov-ered stomatal pores on the surface of uncoated fruit. The thickness of the applied wax layer increased with increasing coating load. A single application of IMZ in wax provided good protective green mould control and sporulation inhibition, with differing effects on some fruit quality parameters due to coating and brush types.
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Vibrational spectral analysis and quantum chemical computations based on density functional theory have been performed on the anti-neuronal drug 4-(2-aminoethyl) morpholine. The geometry, intermolecular hydrogen bond, and harmonic vibrational frequencies of the title molecule have been investigated with the help of B3LYP method. The calculated molecular geometry has been compared with the experimental data. The various intramolecular interactions have been exposed by natural bond orbital analysis. Analysis of SERS bands in comparison to the normal Raman spectrum indicates the chemisorption of the drug on the silver surface. The analysis of the electron density of HOMO and LUMO gives an idea of the delocalization and low value of energy gap indicates electron transport in the molecule and thereby bioactivity. Effective docking of the drug molecule with 2C6C protein also enhances its bioactive nature.
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This paper presents a new analytical method for the determination of morpholine residue in citrus and apples using a novel dispersive micro solid phase extraction (DMSPE) followed ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS). Samples were extracted with 1% formic acid in acetonitrile/water (1:1 v/v) and then cleaned up using DMSPE procedure. Morpholine from the extract was adsorbed to a polymer cation exchange (PCX) sorbent and eluted with ammonium hydroxide/acetonitrile (3:97 v/v) through the 1 mL syringe with a 0.22 μm nylon syringe filter. All of the samples were analyzed by UHPLC-HRMS/MS on a Waters Acquity BEH HILIC column using 0.1% formic acid and 4 mM ammonium formate in water/acetonitrile as the mobile phase with gradient elution. The method showed a good linearity (R2> 0.999) in the range of 1-100 μg/L for the analyte. The LOD and LOQ values of morpholine were 2 μg/kg and 5 μg/kg respectively. The average recoveries of morpholine from the citrus and apple samples spiked at three different concentrations (5, 20 and 100 μg/kg) were in a range from 78.4 to 102.7%.
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An analytical method was developed for the determination of morpholine on apples and citrus. The method utilized acidified methanol extraction, centrifugation and determination by hydrophilic interaction liquid chromatography with electrospray ionization and tandem mass spectrometry (HILIC-ESI-MS/MS). Validation of the method occurred at the Pacific Agricultural Laboratory (PAL, Portland, OR) and the Trace Analytical Laboratory (TAL, UC Davis, CA). Method validation recoveries from control apple, orange, lemon and grapefruit samples ranged from 84-120% over three levels of fortification (0.01, 0.04 and 0.2 µg/g). The limit of quantitation (LOQ) for all commodities was 0.01 µg/g and the calculated method detection limit (MDL) ranged from 0.0010-0.0040 µg/g.