Hollow fiber liquid-phase microextraction with in situ derivatization combined with gas chromatography-mass spectrometry for the determination of root exudate phenylamine compounds in hot pepper (Capsicum annuum L.)

Article abstract of DOI:10.1021/jf4003973

Hollow fiber liquid-phase microextraction (HF-LPME) with derivatization was developed for the determination of three root exudate phenylamine compounds in hot pepper (Capsicum annuum L.) by gas chromatography-mass spectrometry (GC-MS). The performance and applicability of the proposed procedure were evaluated through the extraction of 1-naphthylamine (1-NA), diphenylamine (DPA), and N-phenyl-2- naphthaleneamine (N-P-2-NA) in a recirculating hydroponic solution of hot pepper. Parameters affecting the extraction efficiency were investigated. The calibration curves showed a good linearity in the range of 0.1-10 μg mL^-1. The limits of detection (S/N = 3) for the three compounds were 0.096, 0.074, and 0.057 μg mL^-1, respectively. The enrichment factors reached 174, 196, and 230 at the concentration of 5 μg mL ^-1, and relative standard deviations (RSD) of 9.5, 8.6, and 7.8% and 8.4, 7.6, and 6.2% were obtained at concentrations of 2 and 5 μg mL ^-1 for 1-NA, DPA, and N-P-2-NA, respectively. Recoveries ranging from 90.2 to 96.1% and RSDs below 9.1% were obtained when HF-LPME with in situ derivatization was applied to determine root exudate 1-NA, DPA, and N-P-2-NA after 15 and 30 days of culture solution, respectively.

Full text of DOI:10.1021/jf4003973

Article
pubs.acs.org/JAFC
Hollow Fiber Liquid-Phase Microextraction with in Situ Derivatization
Combined with Gas Chromatography−Mass Spectrometry for the
Determination of Root Exudate Phenylamine Compounds in Hot
Pepper (Capsicum annuum L.)
Haiyan Sun†,§ and Yan Wang*,†
†Science Research Centre, Harbin Institute of Technology, Harbin 150001, China
§Test Centre, Heilongjiang Bayi Agricultural University, Daqing 163319, China
ABSTRACT: Hollow fiber liquid-phase microextraction (HF-LPME) with derivatization was developed for the determination of
three root exudate phenylamine compounds in hot pepper (Capsicum annuum L.) by gas chromatography−mass spectrometry
(GC-MS). The performance and applicability of the proposed procedure were evaluated through the extraction of 1-
naphthylamine (1-NA), diphenylamine (DPA), and N-phenyl-2- naphthaleneamine (N-P-2-NA) in a recirculating hydroponic
solution of hot pepper. Parameters affecting the extraction efficiency were investigated. The calibration curves showed a good
linearity in the range of 0.1−10 μg mL−1. The limits of detection (S/N = 3) for the three compounds were 0.096, 0.074, and
0.057 μg mL−1, respectively. The enrichment factors reached 174, 196, and 230 at the concentration of 5 μg mL−1, and relative
standard deviations (RSD) of 9.5, 8.6, and 7.8% and 8.4, 7.6, and 6.2% were obtained at concentrations of 2 and 5 μg mL−1 for 1-
NA, DPA, and N-P-2-NA, respectively. Recoveries ranging from 90.2 to 96.1% and RSDs below 9.1% were obtained when HF-
LPME with in situ derivatization was applied to determine root exudate 1-NA, DPA, and N-P-2-NA after 15 and 30 days of
culture solution, respectively.
KEYWORDS: hollow fiber liquid-phase microextraction, derivatization, phenylamine, gas chromatography−mass spectrometry
because hollow fibers are not only expendable and disposable
but can also protect the acceptor phase from the interference of
matrices and widely connected with the available apparatus.20
Therefore, HF-LPME is generally complementary to SPME
and has found wide applications.
Chromatographic separation and quantification of amine
compounds are hampered by their polarity, which can cause
tailing and incomplete adsorption. In addition, the mass
spectrometry of amines is not often ideal for their character-
ization, especially when these compounds exist at low
concentration.21 Therefore, derivatization is required to
increase extraction efficiency and improve chromatographic
performance.2,21 Derivatization can be mainly divided into
three modes. The first mode is pre-extraction derivatization.22
INTRODUCTION
■
Plants not only absorb nutrients and moisture from the
environment but continuously secrete inorganic ions and
organic compounds to the media by root during the growth
period. The composition and content of root secretions are not
only the most direct adaptive responses of plants to the
environmental stress but also an adaptive mechanism of
different ecotype plants for living environment.1 In the research
of root exudates, the methods of collection, extraction, and
identification for root exudates always are on the forefront and
represent a difficulty in the research field. 1-Naphthylamine (1-
NA), diphenylamine (DPA), and N-phenyl-2-naphthalene-
amine (N-P-2-NA) are representative root exudate phenyl-
amine substances with high polarity in hot pepper.
Some sample preparation techniques have been reported for
the pretreatment of aromatic amines in aqueous samples based
on liquid−liquid extraction (LLE)2,3 and solid-phase extraction
(SPE).4−6 Both methods are time-consuming and require large
amounts of high-purity organic solvents, which are expensive
and toxic.7 In recent years, a miniaturized technique, that is,
solid-phase microextraction (SPME), has been developed as a
solvent-free extraction method for the analysis of amine
compounds.8−13 However, this method has several disadvan-
tages. First, SPME fibers are still comparatively expensive and
have a limited lifetime. Second, they are susceptible to carry-
over from one extraction to the next.14,15 As a modification of
classic liquid−liquid extraction, an alternative miniaturized
“green” sample preparation approach, hollow fiber liquid-phase
microextraction (HF-LPME), has been considered as an
excellent invention for the sample preparation.16−19 It is mainly
The second mode is postextraction derivatization, and injection
28
port23−27 and on-column derivatization
are the two main
approaches. The third model is in situ derivatization; that is,
derivatization and extraction of analytes take place simulta-
neously in donor phase or acceptor phase. The mixture of
extraction and derivatization reagent has been held within a
hollow fiber of LPME for the simultaneous purification,
derivatization, and extraction in many research studies.29−36
This approach not only decreases solvent consumption but also
simplifies sample preparation steps. In addition, the hollow
fiber can exclude biomacromolecules and protect water-
Received: January 30, 2013
Revised: May 16, 2013
Accepted: May 24, 2013
© XXXX American Chemical Society
A
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and selected ion monitoring (SIM) mode for analytes. The column
used was an Rxi-5 ms (5% diphenyl/95% dimethylpolysiloxane) fused
silica capillary column (30 m long × 0.25 mm i.d × 0.25 μm film
thickness). The initial temperature of the column was set at 50 °C and
held for 1 min, then increased to 250 °C at a rate of 15 °C min−1 and
held for 2 min, and finally increased to 270 °C at a rate of 20 °C min−1
and held for 6 min. The injector temperature was set at 250 °C. The
MS operating conditions were as follows: Helium (99.999%) was
employed as carrier gas, and its rate was adjusted to 3 mL min−1 in
constant flow mode. The ion source and transfer line temperatures
were set at 200 and 280 °C, respectively. The electron energy (70 eV)
was operated in the EI mode. SIM mode was used to perform all
quantification experiments for three target analytes. The ions used for
the confirmation of m/z values of 1-NA, DPA, and N-P-2-NA were
143, 115, and 116; 169, 115, and 84; and 219, 218, and 115,
respectively.
HF-LPME with in Situ Derivatization and LLE Procedure. The
hollow fibers were cut into 5 cm length pieces. Each piece was
discarded after each extraction to avoid the memory effect. Hollow
fiber was sonicated for 5 min in acetone to remove any possible
contaminants prior to extraction. Then, they were removed from the
acetone, and the solvent was naturally evaporated at room temper-
ature. Ten milliliters of standard solution with 5 μg mL−1 1-NA, DPA,
and N-P-2-NA after adjustment of the pH with an FE20 pH meter
(Mettler, China) was added into a 15 mL sample vial (Supelco,
America) containing a 2 mm × 12 mm magnetic stirring bar. The
sample was stirred during the extraction with a 6798-420D (Corning,
America) magnetic heated stirrer. The mixture of extraction and
derivatization reagent was prepared by mixing toluene with AA (80:20,
v/v%) in proportion. The fiber was immersed in the organic solvent
for a few seconds to impregnate the membrane pores of the hollow
fiber wall. A 50 μL microsyringe filled with 15 μL of mixture was
inserted into the hollow fiber to fill it with the acceptor phase, and the
other microsyringe was used to seal the end and withdraw organic
solvent. Sample solution was extracted at 30 min of extraction time, 50
°C extraction temperature, and 800 rpm stirring rate. After extraction,
the solvent mixture with analyte was withdrawn into the syringe and
the hollow fiber was discarded. Then, 1.0 μL of solvent mixture was
injected into the injection port of the GC-MS.
sensitive derivatization reagents. Furthermore, extracted analy-
tes would be exhausted in the hollow fiber, which enhances
extraction velocity and quantities due to in situ derivatization.
Therefore, this technique is simple, inexpensive, time-saving,
and environmentally friendly. To the best of our knowledge,
HF-LPME with in situ derivatization combined with GC-MS
has not been reported for analyzing phenylamine compounds.
Commonly used derivatization reactions include esterification,
silylation, acetylation, and alkylation. Derivatization of amine
compounds for transformation to less polar amide compounds
by acylation is most often adopted.37,38
In this work, three derivatization methods with HF-LPME
have been attempted to improve the sample preparation of 1-
NA, DPA, and N-P-2-NA in hydroponic solution, and the
hollow fiber liquid-phase microextraction with in situ
derivatization method was proved to be the most suitable.
The derivatization reagent acetic anhydride (AA) was protected
within the hollow fiber, which allowed the derivatization and
extraction of the analytes prior to GC-MS analysis. Derivatiza-
tion and extraction conditions including the proportion of
mixed extraction/derivatization reagent, extraction time,
extraction temperature, salt addition, stirring rate, and solution
pH were discussed and optimized. The extraction performance
between HF-LPME with in situ derivatization and LLE was
compared at the same concentration level, and the proposed
method was applied to determine the concentrations of 1-NA,
DPA, and N-P-2-NA in real samples after 15 and 30 days of
culture.
MATERIALS AND METHODS
■
Reagents and Materials. 1-Naphthylamine (1-NA, 99.5%),
diphenylamine (DPA, 99.5%), and derivatization reagent acetic
anhydride (AA) were obtained from Anpel Scientific Instrument
Co., Ltd. (Shanghai, China), and N-phenyl-2-naphthalene amine (N-
P-2-NA, 99.5%) was purchased from Dr. Ehrenstorfer (Augsburg,
Germany). The individual stock solutions of 1-NA, DPA, and N-P-2-
NA (1 g L−1) were prepared by dissolving each compound in
methanol (Merck, Darmstadt, Germany). The standard solutions for
1-NA, DPA, and N-P-2-NA were prepared by diluting stock solutions
with ultrapure water to the required concentration, respectively. Other
reagents were purchased from Kermel Chemical Reagent Co., Ltd.
(Tianjin, China).
The LLE method was used for the isolation organic compounds to
compare extraction performance between LLE and the HF-LPME
with the in situ derivatization method in this paper. Four different
organic solvents (toluene, dichloromethane, ethyl acetate, and n-
hexane) were used to extract the hydroponic solution of hot pepper.
The organic phase was evaporated to dryness at 40 °C by a rotary
evaporator after dehydration by CaSO4 and brought to a volume of 1
mL by extractant. Then, 1 μL of organic solvent containing the target
analytes was used for analysis after filtration through a 0.45 μm organic
membrane.
Two 50 μL microsyringes (10F, SGE Australia) with blunt tips were
used for the HF-LPME. The Accurel Q 3/2 polypropylene hollow
fiber membrane (600 μm inner diameter, 200 μm wall thickness, 0.2
μm pore size, 75% porosity) was purchased from Membrana GmbH
(Wuppertal, Germany).
RESULTS AND DISCUSSION
■
Plant Cultivation and Root Exudates Collection. Hot pepper
seeds were obtained from the Academy of Agricultural Science,
Daqing, China. Hot pepper seeds were sterilized by 1% KMnO4
treatment for 30 min before germination, then washed several times
with distilled water, and finally placed in a Petri dish with two sheets of
moist filter paper. Seeds were cultivated for 15 days and transplanted
to a recirculating hydroponic culture system. The number of seedlings
and the solution volume were constant in every container, and carbon
dioxide and sunlight were regularly supplemented every day. The
hydroponic solution was prepared with distilled water. Real sample
solution was obtained from recirculating hydroponic solution of hot
pepper after 15 and 30 days of cultivation. The collected solutions
were maintained in brown glass bottles. Blank tests were performed
using distilled water and hydroponic solution without plants to
eliminate the effect of external factors, respectively. All of the
experiments were performed in three replicates.
Selection of Derivative Methods. The response of
phenylamine with high polarity was poor in GC-MS without
derivatization, so derivative methods were first selected. In the
first method, 15 μL of toluene was used for LPME, and 4 μL of
extractant was combined with 1 μL of AA in the injection port.
In the second method, 12 μL of toluene was held in the hollow
fiber for the extraction of the analytes. The extraction was
performed at an 800 rpm stirring rate for 30 min without NaCl.
The used toluene was retrieved into a derivative bottle, and 3
μL of AA was added into the bottle for derivatization at 60 °C
for 20 min. Then 1.0 μL of extractant was injected. In the third
method, 15 μL of a mixed solution of toluene and AA (v/v =
80:20, v/v %) was held in the hollow fiber for simultaneous
extraction and derivatization, followed by injection of 1.0 μL of
extractant.
GC-MS Analysis. The GC-MS experiments were performed using
a GC 2010 gas chromatograph coupled to a QP mass spectrometer
(Shimadzu, Japan) operating in full scan in the m/z range of 50−500
Experimental results showed that the first method might lead
to incomplete derivatization and that the derivatization reagent
B
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Figure 1. Optimization of toluene/AA: (a) optimization of toluene/AA proportion; (b) optimization of volume of toluene/AA. Conditions of HF-
LPME with in situ derivatization: extraction time, 30 min; extraction temperature, 50 °C; no NaCl; stirring rate, 800 rpm; pH, 12.0. The
concentrations of target analytes were 5 μg mL−1.
Figure 2. Optimization of the HF-LPME parameters: (a) extraction temperature; (b) extraction time; (c) salt concentration; (d) stirring rate.
Conditions of HF-LPME with in situ derivatization: toluene/AA, (v/v = 80:20, v/v %), 15 μL. The concentrations of target analytes were 5 μg mL−1.
might damage the capillary column. The second method
needed complex operation; it easily caused a high limit of
detection and low precision. The third method, in situ
derivatization, integrated derivatization into extraction, which
was simple and rapid. The comparison result suggested that the
third method was suitable for three target analytes using a
mixed solution of toluene and AA (v/v = 80:20, v/v %).
Optimization of HF-LPME with in Situ Derivatization.
Effect of Organic Solvent. The selection of organic solvent is
important for HF-LPME to gain maximum enrichment factor
(EF). The extractant should have the following properties.
First, it should be immiscible with water. Second, the polarity of
the organic solvent should match that of the derived product.
Third, it must be stable during the extraction process.39 As
analytes were derivatized at amino groups, solvents without
amino groups were chosen. For the selection of the optimum
extraction solvents, benzene, cyclohexane, isooctane, toluene,
and chloroform were evaluated. The peak areas of the derivative
product using HF-LPME with in situ derivatization were
determined by GC-MS (n = 5) in 10 mL of working solution.
Each of the extraction solvents was mixed with AA (v/v =
80:20, v/v %), and 15 μL of the mixture was held in the hollow
fiber for extraction. The largest peak area was obtained with
chloroform, but it is volatile and led to poor precision. Thus,
C
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Table 1. Analytical Performance of Developed Method for Target Analytes
a
RSD (%, n = 5)
b
c
analyte
1-NA
linearity range (μg mL−1
)
r2
LOD (μg mL−1
)
2 μg mL−1
5 μg mL−1
EF
0.1−10
0.1−10
0.9964
0.9978
0.9989
0.096
0.074
0.057
9.5
8.6
7.8
8.4
7.6
6.2
174
196
230
DPA
N-P-2-NA
0.1−10
a
b
c
Relative standard deviation. Limit of detection for S/N = 3. Target analyte concentration: 5 μg mL−1.
toluene was selected as the extraction solvent on the basis of
comprehensive consideration.
DPA, and N-P-2-NA reached equilibrium at 30 min, and no
further increase was observed after 30 min. Therefore, 30 min
of extraction time was selected in the subsequent studies.
Effect of Salt Concentration. The viscocity of sample
solution may increase with increasing salt concentration. In
addition, salt addition can decrease the solubility of analytes in
the sample solution and improve the partition coefficient into
the organic phase. Sample solutions with various concentrations
of NaCl in the range of 0−36% (w/v) were investigated (Figure
2c) to investigate the effect of salt addition. It was observed that
extraction efficiency showed a slightly decreasing trend with
increasing salt concentration. This is most likely due to the
increased viscosity of the sample solution, which limited the
movement of the analyte from the aqueous phase to the organic
phase. Therefore, no salt was added in the following
experiments.
Effect of Toluene/AA. The amount of AA required for
derivatization was investigated by mixing AA with 20, 40, 50,
60, and 80% (v/v) of toluene (Figure 1a). It was found that a
ratio of toluene/AA of 80:20 (v/v %) produced the optimal
result. Perhaps it is because the appropriate amount of organic
solvent assists the mass of analytes diffusing and transferring
from the organic solvent in the pore of the hollow fiber wall
into the mixed solution. Hence, 20% of AA was used in the
following experiments.
The effect of mixed toluene/AA solution volume was
evaluated by calculating EF according to the following
formula:28
EF = 1/(V /V + 1/K)
(1)
o
a
Effect of pH. The pH of the aqueous phase should be
adjusted to ensure that the analyte is electrically neutral so that
it can be efficiently transferred into the organic phase.32 The
pKa values of the three analytes were all <5; therefore, the
aqueous phase should be adjusted to basic condition to
maintain the neutrality and reduce the solubility of analytes
within the aqueous phase. A comparison of extraction of the
analytes was performed at pH 9.0, 10.0, 11.0, 12.0, and 13.0.
This showed that pH 12.0 was optimum to obtain the highest
extraction efficiency.
Effect of Stirring Rate. Mass transfer of target analytes
through the organic solvent in the hollow fiber can be improved
by agitation. The interface between the aqueous phase and the
organic phase will be continuously exposed with agitation.41 In
the experiment, the effect of stirring rate on the extraction
efficiency was investigated in the range of 400−1100 rpm
(Figure 2d). It was found that the extraction efficiency of 1-NA
and DPA increased with increasing stirring rate up to 800 rpm;
after that, the extraction efficiency slightly decreased with
increasing stirring rate. However, N-P-2-NA showed a slightly
increasing trend with increasing stirring rate 800 rpm. Perhaps,
their dissolution gradually increased with the increase of stirring
rate. On the basis of comprehensive consideration, the stirring
rate was set at 800 rpm in the following experiments.
Method Evaluation. Validation of HF-LPME with in Situ
Derivatization and LLE. Analytical performances for the
extraction of 1-NA, DPA, and N-P-2-NA including their
linearity, repeatability, limits of detection (LODs), and
recoveries of HF-LPME with in situ derivatization were
evaluated. The extraction results obtained by extracting
standard solutions over a concentration range of 0.1−10 μg
mL−1 are shown in Table 1. For HF-LPME with in situ
derivatization, good linearity was obtained with acceptable
correlation coefficients (r2) of 0.9964 (1-NA), 0.9978 (DPA),
and 0.9989 (N-P-2-NA); limits of detection (S/N = 3) of
0.096, 0.074, and 0.057 μg mL−1, EF up to 174, 196, and 230 at
5 μg mL−1, and relative standard deviations (RSD) below 9.5%
were obtained at concentrations of 2 and 5 μg mL−1 for 1-NA,
K is the distribution coefficient, Vo is the volume of organic
solvent, and Va is the volume of aqueous sample. K is calculated
on the basis of the two-phase equilibrium condition
K = Co,eq/Ca,aq
(2)
where Co,eq is the concentration of analyte in the organic phase
and Ca,aq is the concentration of analyte in the aqueous phase.
The experiment was performed using mixed toluene/AA (v/
v = 80:20, v/v %) solution volumes from 5 to 25 μL, and the
results were evaluated by comparison of EF (Figure 1b). It was
found that the optimum volume of mixed toluene/AA (4:1)
was 15 μL and the EF of 1-NA, DPA, and N-P-2-NA reached
174, 196, and 230, respectively.
Effect of Extraction Temperature and Extraction Time.
Temperature not only affects derivatization efficiency but also
affects the kinetics and thermodynamics of the diffusion process
of analytes during the extraction.40 In this experiment, the
extraction temperature, ranging from 30 to 70 °C, was
examined, and the amount of extracted analytes increased
with increasing temperature up to 50 °C (Figure 2a). It can be
explained that higher temperature may increase derivatization
efficiency and improve the amounts of analytes in the hollow
fiber, which results in the increase of 1-NA and DPA; however,
N-P-2-NA showed a slightly increasing trend with increasing
extraction temperature above 50 °C. It was possibly caused by
high vapor pressure of toluene that could volatilize out of the
hollow fiber and dissolve into the water solution above 50 °C.
With comprehensive consideration, 50 °C was chosen as the
extraction temperature in the following experiments.
The optimal extraction efficiency is obtained when
equilibrium time is established. Equilibrium time is another
key parameter influencing the extraction efficiency. However,
equilibrium time depends on the mass transfer of the analytes
from aqueous solution to organic solvent. Therefore, the
extraction time plays an important role in the whole process.
The extraction efficiency was evaluated by extraction time in
the range of 20−50 min in this experiment (Figure 2b). 1-NA,
D
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DPA, and N-P-2-NA, respectively. In LLE, dichloromethane
had better extraction efficiency than other solvents. Analytical
performances for the extraction of 1-NA, DPA, and N-P-2-NA
including their linearity, repeatability, LODs, and recoveries
were validated. Poor repeatability, low recoveries, and high
LODs for target analytes were obtained at 5 μg mL−1
concentration level (Table 2). The chromatograms of 5 μg
Table 3. Concentrations and Recoveries of Target Analytes
Determined in Sample Solution by HF-LPME with in Situ
Derivatization
spiked
(μg mL-
original
RSD (%,
n = 5)
recovery (%,
n = 5)
−1
(μg mL−1
)
)
analyte
1-NA
a
b
a
b
a
b
a
b
2.87
4.17
4.05
3.89
4.88
5.12
2
2
2
5
5
5
9.1
7.4
7.7
7.9
6.4
5.9
90.2
91.8
93.8
91.7
92.4
96.1
Table 2. Analytical Performance of the LLE Method for
Target Analytes
DPA
N-P-2-NA
a
a
b
LOD
(μg mL−1
n = 5)
RSD
(%,
Extraction for 15 day root culture solution. Extraction for 30 day
b
linear range
,
recovery
root culture solution.
analyte
1-NA
(μg mL−1
)
r
n = 5) (%, n = 5)
0.1−10
0.1−10
0.1−10
0.9165
0.9287
0.184
0.157
0.125
18.7
15.4
12.5
62.1
65.2
71.2
DPA
AUTHOR INFORMATION
Corresponding Author
*E-mail: shysun7908@sina.com.
■
N-P-2-NA
0.9496
a
b
Relative standard deviation. Target analyte concentration: 5 μg
mL−1.
Funding
We acknowledge financial support from the National Natural
Science Foundation of China (B21075025) and the Natural
Science Foundation of Heilongjiang Province (B200910).
mL−1 standard solution by HF-LPME with in situ derivatization
and LLE are shown in Figure 3. The result showed that higher
Notes
The authors declare no competing financial interest.
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