a

1. Journal of Cereal Science 93 (2020) 102975
Available online 3 April 2020
0733-5210/© 2020 Elsevier Ltd. All rights reserved.
Identification and quantitative determination of 2-acetyl-1-pyrroline using
GC-TOF MS combined with HS and HS-SPME pretreatment
Zhenling Guo a
, Siqi Huang b
, Mingxue Chen b
, Yanxia Ni b
, Xianqiao Hu b,**
, Nan Sun a,*
a
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou, China
b
Rice Product Quality Supervision and Inspection Center, Ministry of Agriculture and Rural Affairs, China National Rice Research Institute, Hangzhou, China
A R T I C L E I N F O
Keywords:
Headspace (HS)
Headspace-solid phase micro-extraction (HS-
SPME)
2-Acetyl-1-pyrroline (2-AP)
Gas chromatography-time-of-flight mass spec­
trometry (GC-TOF MS)
A B S T R A C T
2-Acetyl-1-pyrroline (2-AP) was recognized as the key characteristic volatile in aromatic rice. In this paper, two
precise and rapid methods for quantitative determination of 2-AP by headspace-gas chromatography-time-of-
flight mass spectrometry (HS-GC-TOF MS) and headspace-solid phase micro-extraction-gas chromatography-
time-of-flight mass spectrometry (HS-SPME-GC-TOF MS) have been presented and compared. Chromatograms of
2-AP and internal standard (2-methyl-3-heptanone) in samples were extracted by accurate masses, and the
response ratios of 2-AP to internal standard were used for constructing matrix-matched standard curve with the
blanks deducted. Pretreatment conditions such as temperature and extractant amount were optimized. Linear
ranges of two methods were 1–150 ng g 1
with linear correlation coefficients (r) of 0.9998 and 0.9997,
respectively. The limit of detection (LOD) and limit of quantitation (LOQ) of HS-GC-TOF MS method were 0.68
ng g 1
and 2.27 ng g 1
. LOD and LOQ of HS-SPME-GC-TOF MS method were 0.46 ng g 1
and 1.50 ng g 1
, and
could be reduced to 0.02 ng g 1
and 0.06 ng g 1
, respectively, in the case of splitless mode. At spiking level of
100 ng g 1
, recoveries were 94.19–116.00% and 95.57–107.49% for HS-GC-TOF MS method and HS-SPME-GC-
TOF MS method, respectively.
1. Introduction
Rice is a complex system whose aroma quality is determined by the
comprehensive result of many different volatile compounds instead of
by one or two volatiles. Until now, more than 300 volatile compounds
have been reported in rice (Champagne, 2008; Wakte et al., 2017).
Among these numerous volatile compounds, 2-acetyl-1-pyrroline (2-AP)
was recognized as the key characteristic volatile in aromatic rice. It
possesses odor thresholds of 0.1 ng g 1
in water and 0.02–0.04 ng L 1
in
air (Buttery et al., 1988) and contributes a popcorn-like aroma.
Many evaluation methods have been reported for rice aroma anal­
ysis, such as sensory evaluation, electronic nose, near-infrared spec­
troscopy (NIRS), gas chromatography (GC) method and gas
chromatography-mass spectrometry (GC-MS) method. Sensory evalua­
tion using human nose as the detector, provided final human sense and
was considered as a direct, effective, unique and intuitive method
(Wilkie et al., 2004; Lu et al., 2019). It needed no complex instruments
and equipment and could carry out anywhere. Although widely used for
rice evaluation, lack of unified sensory evaluation criteria and effective
training were the main problems for objective and repetitive sensory
evaluation. Electronic nose based on sensors array and an appropriate
pattern recognition method can mimic human smell to evaluate food
quality (Lu et al., 2015a). It provided whole scene of volatiles instead of
quantitative analysis of volatiles (Ito et al., 2013; Lu et al., 2015b), and
was used mostly for rice classification, predicting the degree of differ­
ence between samples and investigating changes of aroma compounds
during storages (Lin et al., 2018). NIRS was also used to evaluate rice
aroma quality due to its non-destructive nature, rapidity, low cost, and
environmental friendliness (Lapchareonsuk and Sirisomboon, 2015;
Maneenuam et al., 2015). NIRS evaluated the content of volatile by
predicting using an established model instead of determining directly.
There was no significant difference between NIR-predicted values and
reference values obtained by headspace-gas chromatography method
(Maneenuam et al., 2015). However, the model for prediction needed
great deal of work and data.
GC method includes extraction of aroma volatiles using a proper
extraction method, separation of them using a proper GC column and
following determination by a proper detector. Extraction method
* Corresponding author.
** Corresponding author.
E-mail address: sunnan@zjut.edu.cn (N. Sun).
Contents lists available at ScienceDirect
Journal of Cereal Science
journal homepage: http://www.elsevier.com/locate/jcs
https://doi.org/10.1016/j.jcs.2020.102975
Received 19 September 2019; Received in revised form 11 March 2020; Accepted 25 March 2020

2. Journal of Cereal Science 93 (2020) 102975
2
included steam distillation, indirect steam distillation, solvent extrac­
tion, supercritical fluid extraction (Lin et al., 1990; Mahatheeranont
et al., 2001) and headspace (HS) method, such as static headspace,
dynamic headspace, headspace sorptive extraction and headspace-solid
phase micro-extraction (HS-SPME) (Sansenya et al., 2018; Lee et al.,
2019). Among them, HS method was more favored by researchers now
because the volatiles extracted from headspace were closer to that
perceived during consuming (Lin et al., 2018). HS-SPME was thought to
be a simple, rapid, and sensitive method for collecting headspace vola­
tiles, and was widely used for quantitative determination of 2-AP in rice
(Grimm et al., 2001; Maraval et al., 2010; Mathure et al., 2011; Ying
et al., 2011; Hopfer et al., 2016). Flame ionization detector (FID) has
been the most widely used gas chromatography detector due to the
advantages of high sensitivity, fast response, and accurate quantification
(Sriseadka et al., 2006; Mathure et al., 2011; Lee et al., 2019). However,
it has limited ability in qualitative analysis. Mass spectrometry (MS) was
also a commonly used detector with the advantages of high sensitivity
and excellent qualitative ability (Yoshihashi, 2002; Hien et al., 2006;
Hopfer et al., 2016). MS can detect all compounds that be ionized,
obtaining mass spectrum at each time point which will provide infor­
mation of molecular structure of compounds. Combined with the high
separation efficiency of GC and quantitative ability of MS, qualitative
and quantitative work can be performed simultaneously by GC-MS.
Single ion monitoring (SIM) mode and MS/MS mode were used for
quantitative determination of 2-AP in rice (Yoshihashi, 2002; Hien et al.,
2006). Time-of-flight mass spectrometry (TOF MS) was superior to
general mass spectrometry in terms of resolution, mass accuracy,
sensitivity, scan speed, detection limit and qualitative capability (Gar­
cía-Reyes et al., 2006). The mass-to-charge ratio (m/z) for target frag­
ment obtained by TOF MS was consistent with calculated theoretical
value with mass accuracy error lower than 2 ppm (García-Reyes et al.,
2006). It eliminated the interference of fragments with similar m/z.
Besides, it was suitable for compound analysis without standard mate­
rials. Hence, gas chromatography-time-of-flight mass spectrometry
(GC-TOF MS) has played an important role in compound analysis
especially in screening analysis.
However, no work on quantitative analysis of 2-AP in rice using GC-
TOF MS had been reported. In this paper, GC-TOF MS combined with HS
or HS-SPME extraction method was applied for determination of 2-AP in
rice. The conditions for headspace-gas chromatography-time-of-flight
mass spectrometry (HS-GC-TOF MS) and headspace-solid phase micro-
extraction-gas chromatography-time-of-flight mass spectrometry (HS-
SPME-GC-TOF MS) were optimized and the performance were
demonstrated.
2. Material and methods
2.1. Chemicals and instrument
2-Acetyl-1-pyrroline (25 mg, 10% w/w in Toluene) was from Tor­
onto Research Chemicals (Toronto, Ontario, Canada). And the internal
standard, 2-methyl-3-heptanone (purity 99.9%), was from Sigma-
Aldrich (St. Louis, Missouri, USA). Solvents, toluene (purity 99.9%)
and ethanol (purity 99.5%), were all from TEDIA Company Inc. (Ohio,
USA).
2-AP and 2-methyl-3-heptanone were extracted and concentrated by
using SPME fiber (1 cm, 50/30 μm Divinylbenzene/Carboxen/Poly­
dimethylsiloxane (DVB/CAR/PDMS)) (SAAB-57329U) attached to
SPME manual holder (SAAB-57330U) (Supelco, Bellefonte, PA, USA) or
using the headspace system (7697 A Headspace Sampler, Agilent, Cali­
fornia, USA). Separation of collected volatiles was performed by using
GC system (7890 B GC, Agilent) with DB-WAX column (30 m � 0.25 mm
� 0.25 μm, Agilent). Identification of them was done by using GC/MS
system (7200 Q-TOF GC/MS, Agilent, California, USA).
2.2. Rice samples and pretreatment
Eight types of rice samples (Matrix sample: Zhongzao 39; Samples 1
to 3: Yuexiuyou 376, Nanjingxiangzhan, and Ruanhuayoujinsizhan;
Samples 4 to 7: different batches of Yumizhan) were provided by Rice
Product Quality Supervision and Inspection Center, Ministry of Agri­
culture and Rural Affairs. Thai aromatic rice purchased from a local
store was used as a sample for optimization experiments. Before testing,
rice samples were husked and milled until most of bran and part of
embryo had been removed. Then, part of milled rice samples was ground
into flour by using a Cyclotec 1093 sample mill (Foss Tecator, Sweden).
The milled rice and rice flour samples were ready for use.
2.3. Determination of 2-AP using HS-GC-TOF MS method
1 g matrix flour sample, 50 μL ethanol and 10 μL 0.102 μg mL 1
internal standard solution was added to 20 mL headspace vial in order
under ice bath conditions. Next, 10 μL 0, 0.1, 0.5, 1, 5, 10 and 15 μg
mL 1
2-AP standard solutions, respectively, were added to vial to pre­
pare matrix-matched standard samples. After sealed, the vials were
transposed into the headspace system, and each sample was repeated
twice.
After stabilized in the headspace system at 90 �
C for 30 min, the
upper gas was injected into GC-TOF MS for testing. The split ratio was
set at 5:1 and the solvent delay time was set to 5 min. The oven tem­
perature was kept at 40 �
C for 5 min, then programmed to 240 �
C at a
rate of 30 �
C∙min 1
and kept for 5 min. The injector, ion source,
quadrupole, and transfer line temperatures were set at 250 �
C, 230 �
C,
150 �
C, and 280 �
C, respectively. Ionization was performed under
electronic impact (EI) mode and the electron ionization source was 70
eV. High purity helium (purity > 99.999%) was used as carrier gas with
a flow rate of 1 mL min 1
. Full scan mode with a scan range of m/z
40–500 was adopted.
After data acquisition was completed, the chromatograms of 2-AP
and internal standard were extracted with m/z of 83.0728 and
128.1191, respectively. Among them, the sample without adding 2-AP
standard solution was set as the blank group. Calculated the response
ratios of 2-AP to internal standard in the other six matrix-matched
samples with the blanks deducted. Matrix-matched standard curve
was constructed based on the response ratios and the spiking content of
2-AP.
When measuring samples, 1 g rice flour sample, 50 μL ethanol and
10 μL 0.102 μg mL 1
internal standard solution was added to 20 mL
headspace vial in order under ice bath conditions. After sealed, the vials
were transposed into the headspace system, and each sample was
repeated twice. Other conditions were consistent with those of matrix-
matched standard samples. After data acquisition was completed, the
response ratios of 2-AP to internal standard were first calculated, and
then 2-AP content in the samples were calculated according to matrix-
matched standard curve.
2.4. Determination of 2-AP using HS-SPME-GC-TOF MS method
Except for the use of 200 μL extractant, the preparation processes for
matrix-matched standard samples and test samples were the same as
those in section 2.3. After the sample preparation was completed, the
SPME fiber was exposed to the headspace of the vial, and the extraction
device was fixed in a water bath at 80 �
C. After 10 min of stabilization
and 40 min of extraction, the SPME fiber was inserted to the GC injector
for desorption at 250 �
C for 5 min.
In HS-SPME-GC-TOF MS method, the parameters of GC-TOF MS were
exactly the same as those in section 2.3 except for the split ratio and the
solvent delay time. The split ratio was set at 20:1 and the solvent delay
time was set to 7 min. Each sample was also repeated twice. The pro­
cesses of establishing matrix-matched standard curve and calculating 2-
AP content of samples were the same as those in section 2.3.
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3. Journal of Cereal Science 93 (2020) 102975
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2.5. Sensory evaluation
Sensory evaluation of rice aroma was performed according to the
agricultural industry standard of China NY/T 596–2002 “Aromatic rice”
with some modification. Firstly, five cooked rice water samples and one
cooked rice sample were prepared for each sample. 2 g milled sample
was placed into porcelain bowl. After adding 50 mL distilled water, the
porcelain bowl was covered with the lid and stewed for 15 min. After
slightly cooled, the prepared cooked rice water sample was ready for
sensory evaluation with respect to flavor intensity, flavor types and
flavor descriptions. At the same time, cooked rice sample was also
prepared for identifying aroma retention intensity, aroma retention
duration and comprehensive impression. 40 g milled rice was washed
for twice and soaked with 1.2 times mass of water for 30 min before
cooked for 40 min, and finally simmer for 20 min.
Then, five skilled panelists scored the cooked rice water sample and
cooked rice sample by using their sense. Based on the sensory evaluation
of one panelist, overall sensory score was obtained as: overall sensory
score ¼ flavor intensity score þ flavor type score þ aroma retention
intensity score þ aroma retention duration score þ comprehensive
impression score. Finally, ultimate score of test sample was determined
as the average value of five sensory scores.
Fig. 1. Mass spectrum of 2-AP (A) and 2-methyl-3-heptanone (D) obtained by using HS-GC-TOF MS method. (A) 10 μL 10 μg mL 1
2-AP was added for analysis. (D)
10 μL 0.102 μg mL 1
2-methyl-3-heptanone was added for analysis. (B) EIC chromatogram for 2-AP, m/z of 83.0728 was used for extraction. (C) EIC chromatogram
for 2-methyl-3-heptanone, m/z of 128.1191 was used for extraction. a, b and c represent three repeats under the same experimental conditions, respectively.
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4. Journal of Cereal Science 93 (2020) 102975
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3. Results and discussion
3.1. Qualitative and quantitative analysis of 2-AP and 2-methyl-3-
heptanone
Searching NIST library combined with comparing retention index
(RI) was widely used for qualitative analysis of volatiles. However, it
was more suitable for qualitative analysis of pure compounds than
complex samples. GC-TOF MS plays an important role in compound
analysis, especially in screening analysis due to its advantage of high
resolution, high mass accuracy and high sensitivity. In this paper, exact
mass obtained from GC-TOF MS as well as the retention time was used
for qualitative analysis of target compounds. The fragment of target
compound was extracted from scan mode data using exact mass with
mass accuracy error of 10 ppm. Due to the high-resolution of data, the
target compound could be separated from background interference,
eliminating the interference of other compounds in sample and conse­
quently reducing the detection limit. The m/z of 111, 83, and 68 were
often mentioned as characteristic fragments of 2-AP in the literature
(Ying et al., 2011; Liu et al., 2015; Peddamma et al., 2018). Fig. 1(A)
showed a mass spectrum of 2-AP standard solution obtained by using
HS-GC-TOF MS method and the m/z of fragments were 111.0672,
83.0728, 82.0646, 69.0563, 68.0491, 55.0424, 45.0336, 43.0181, etc.
Since the information of mass spectra obtained by using HS-GC-TOF MS
method and HS-SPME-GC-TOF MS method was more detailed and ac­
curate, they could both be stored as high-resolution mass spectra of 2-AP
standard. Among them, the fragment ion with m/z of 83.0728 was a
base peak generated by α-cleavage reaction of a carbonyl group, and its
fragment ion was þ
C5H9N. By using m/z of 83.0728, the extracted ion
chromatogram (EIC) of 2-AP was extracted from the total ion chro­
matogram (Fig. 1(B)). The retention time of 2-AP obtained by using
HS-GC-TOF MS method was 8.885 � 0.003 min, the average peak area
was 5.53eþ06 (relative standard deviation (RSD) was 5.56%). The
retention time of 2-AP obtained by using HS-SPME-GC-TOF MS method
was 8.556 � 0.001 min.
Internal standards were often used to overcome the effects of 2-AP
instability, which was caused by volatility and oxidation of 2-AP. The
physicochemical properties of internal standard should be similar to
those of 2-AP (e.g., relative molecular mass, melting point, density, etc.),
while it could be completely separated from 2-AP during detection.
Besides, it should not be present in the sample or react chemically with
the sample. From the perspective of internal standard selection, isotope
was the most ideal internal standard (Yoshihashi, 2002; Hopfer et al.,
2016). The stable isotope dilution method and isotope internal standard
method were the future research trends. However, 2-methyl-3-hepta­
none was selected as internal standard in this paper due to the limited
sources and expensive cost of stable isotopes. In addition, the process of
sample preparation was completed under ice bath conditions to mini­
mize errors caused by the instability of 2-AP.
Exact mass and retention time were also used for qualitative analysis
of internal standard solution. The internal standard was diluted to 0.102
μg mL 1
. Then 10 μL internal standard solution was extracted and
analyzed (n ¼ 3). Fig. 1(D) showed a mass spectrum of 2-methyl-3-hep­
tanone obtained by using HS-GC-TOF MS method and the m/z of frag­
ments were 128.1191, 86.0720, 85.0646, 71.0488, 57.0700. 43.0545,
41.0388, etc. Among them, the fragment ion with m/z of 57.0700 had
the highest intensity, which was one of the main fragment ion peaks
generated by the α-cleavage reactions, and its fragment ion was þ
C4H9.
However, the fragment ion þ
C4H9 was not only detected in 2-methyl-3-
heptanone but also in other compounds, besides, it was a common
source of pollution in the equipment. Therefore, this fragment ion
couldn’t be used to extract a clean EIC from the total ion chromatogram.
In this paper, EIC of internal standard was extracted by using the mo­
lecular ion peak with m/z of 128.1191. It could be seen from Fig. 1(C)
that the retention time of 2-methyl-3-heptanone obtained by using HS-
GC-TOF MS method was 7.358 � 0.001 min, the average peak area
was 2.74eþ06 (RSD ¼ 3.85%). The retention time of 2-methyl-3-hepta­
none obtained by using HS-SPME-GC-TOF MS method was 7.223 �
0.005 min.
Furthermore, in this paper, the quantitative analysis of 2-AP in
sample was carried out by using standard curve method combined with
internal standard method. The standard curve method was particularly
suitable for the analysis of a large number of samples, and the internal
standard method to a certain extent eliminated the effects of the pre­
treatment conditions (e.g., operating conditions, sample volume, etc.)
on the quantitative results (Amirahmadi et al., 2013; Shoeibi et al.,
2014).
3.2. Optimization of extraction conditions
Thai aromatic rice with high aroma and high 2-AP content was used
as the test sample in this paper for optimization of extraction conditions
such as extractant, extractant amount and extraction temperature.
3.2.1. Extractant
Hopfer et al. (Hopfer et al., 2016) used ethyl acetate as extractant in
combination with isotope internal standard method to obtain the lowest
detection limit to date. In addition, ethanol (Hu et al., 2014; Wakte et al.,
2017) has been one of the most commonly used extractants. Compared
to other organic solvents, ethanol and ethyl acetate were relatively safe
and green. Therefore, the extractant was selected among these two
organic solvents in this paper.
During HS extraction, 1 g Thai aromatic rice flour sample was
extracted at a steady temperature of 70 �
C for 30 min both without
extractant and with 100 μL ethanol or ethyl acetate. As shown in Fig. 2
(A), the extraction efficiency of 2-AP could be increased by 3–5 times
after adding extractant. It was preferred to add an extractant to the
sample in this paper. The extraction efficiency of adding ethanol was
about 1.5 times that of ethyl acetate. Therefore, ethanol was used as the
extractant in HS-GC-TOF MS method. During HS-SPME extraction, 1 g
Thai aromatic rice flour sample was extracted at water bath temperature
of 60 �
C for 50 min both without extractant and with 100 μL ethanol or
ethyl acetate. It could be seen from Fig. 2(B) that the extraction effi­
ciency of adding ethanol was about 2.5 times that of ethyl acetate.
Therefore, ethanol was also used as the extractant in HS-SPME-GC-TOF
MS method.
3.2.2. Amount of extractant and extraction temperature
The extraction conditions for the combination of five extractant
amounts (0, 50, 100, 200, and 400 μL) and four extraction temperatures
(60, 70, 80, and 90 �
C) were tested under both two extraction methods.
When using HS extraction method, the extraction efficiency
increased first and then decreased with the increase of extractant
amount at the same extraction temperature, as shown in Fig. 2(C).
Among them, the extraction efficiency of 50 μL and 100 μL extractant
was better than that of 0, 200 and 400 μL in most cases. Excessive
extractant competed with the target during extraction process, reducing
the extraction efficiency of the target. Besides, it showed that the
extraction efficiency was increased with the increase of extraction
temperature in the case of the same amount of extractant in Fig. 2(C).
Increased temperature led to intense molecular thermal motion which
improved extraction efficiency of the target. However, too high pre­
treatment temperature would also lead to the synthesis of new 2-AP in
sample, resulting in higher results (Yoshihashi, 2002; Maraval et al.,
2010; Liu et al., 2015; Hopfer et al., 2016). There is no consensus on
whether high temperature will lead to synthesis of new 2-AP in rice
sample. The extraction temperatures were set around 50 �
C (Hopfer
et al., 2016; Peddamma et al., 2018) in some literature while some were
set at 80 �
C (Grimm et al., 2001; Hu et al., 2014). Reliable results had
also been obtained at 120 �
C (Sriseadka et al., 2006; Sansenya et al.,
2018). In order to ensure the reliability of results, the extraction tem­
perature was controlled below 100 �
C in this paper. Based on the
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5. Journal of Cereal Science 93 (2020) 102975
5
comprehensive analysis of extraction results, extraction temperature of
90 �
C and extractant amount of 50 μL was selected as the final extraction
condition for HS extraction method.
Similarly, when using HS-SPME extraction method, the extraction
efficiency increased first and then decreased with the increase of
extractant amount at the same extraction temperature (Fig. 2(D)). The
extraction efficiency of 100 μL and 200 μL extractant was better than
that of 0, 50 and 400 μL. The extraction efficiency was not remarkable
with low amount extractant. When the amount of extractant was
excessive, extractant would cause adsorption competition in the SPME
fiber head, resulting in a decrease in the adsorption effect of the target. It
could also be seen from Fig. 2(D) that the extraction efficiency also
increased first and then decreased with the increase of extraction tem­
perature with the same amount of extractant. Increased temperature
improved extraction efficiency of 2-AP, but too high temperature would
reduce it. By observing the experimental phenomena, we speculated that
too high temperature caused expansion of the septum and consequently
broken of the seal of vial, causing loss of target and reducing the
Fig. 2. Effect of extractant (A, B), extractant amount and extraction temperature (C, D) on extraction efficiency. (A) Effect of different extractants on extraction
efficiency by using HS extraction method. Extraction temperature: 70 �
C, stabilization time: 30 min, amount of extractant: 100 μL; (B) Effect of different extractants
on extraction efficiency by using HS-SPME extraction method. Extraction temperature: 60 �
C, extraction time: 50 min, amount of extractant: 100 μL; (C) Effect of
extraction temperature and extractant amount on extraction efficiency by using HS extraction method; (D) Effect of extraction temperature and extractant amount on
extraction efficiency by using HS-SPME extraction method.
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6. Journal of Cereal Science 93 (2020) 102975
6
extraction stability. Base on the comprehensive analysis of extraction
results, extraction temperature of 80 �
C and extractant amount of 200 μL
was selected as the final extraction condition for HS-SPME extraction
method.
Under the selected extraction conditions, HS extraction method was
more convenient than HS-SPME extraction method due to its high
automation, while the response signal of HS-SPME extraction method
was higher than that of HS extraction method due to the strong
enrichment ability of SPME fiber.
3.3. Performance
2-AP had a very low sensory threshold (Buttery et al., 1988), and its
content in most rice samples was low. The extraction efficiency of 2-AP
in rice flour sample was quite different from that in standard solution
because of a large matrix effect. Even adding an extractant in rice flour
sample, the extraction efficiency was still somewhat different from that
in standard solution. At spiking level of 100 ng g 1
, recoveries of 2-AP in
HS-GC-TOF MS method were less than 10%. Although the SPME fiber
had a strong enrichment ability, recoveries of 2-AP in HS-SPME-GC-TOF
MS method were also only 69.60%. Therefore, the matrix effect of the
rice flour sample couldn’t be ignored. Matrix-matched standard curve
method was preferred to avoid matrix effects (Jung et al., 2019; Lee
et al., 2019). In this paper, the matrix effect caused by starch adsorption
was reduced by using matrix-matched standard curve established by
adding 2-AP standard solution to rice matrix (Zhongzao 39). At the same
spiking level of 100 ng g 1
, recoveries of 2-AP increased to
94.19–116.00% (n ¼ 3) and 95.57–107.49% (n ¼ 3) for HS-GC-TOF MS
method and HS-SPME-GC-TOF MS method, respectively.
Matrix-matched standard curves of these two methods were obtained
by testing seven types of matrix-matched samples at different spiking
levels of 2-AP (0, 1, 5, 10, 50, 100 and 150 ng g 1
, respectively). Matrix-
matched standard curve equation in HS-GC-TOF MS method was y ¼
0.0018x-0.0001 (r ¼ 0.9998), the limit of detection (LOD, signal-to-nose
ratios (S/N) ¼ 3) was 0.68 ng g 1
, and the limit of quantitation (LOQ, S/
N ¼ 10) was 2.27 ng g 1
. When using HS-SPME extraction method, a
large amount of collected volatiles was desorbed into the instrument due
to the strong enrichment ability of SPME fiber. Excessive target caused
signal saturation in the detector of the mass spectrometer, leading to
signal errors. Therefore, a larger split ratio mode was preferred to use in
HS-SPME-GC-TOF MS method. In the case of a large split ratio, low
limits of detection could still be obtained despite a significant reduction
of the content of targets entering the instrument. When the split ratio
was 20:1, the matrix-matched standard curve equation was y ¼ 0.0097x-
0.0007 (r ¼ 0.9997), LOD was 0.46 ng g 1
, and LOQ was 1.50 ng g 1
. In
addition, LOD and LOQ in splitless mode could be reduced to 0.02 ng g 1
and 0.06 ng g 1
, respectively, which were below the sensory threshold.
The performances of methods established in this paper and some
literature were listed in Table 1. The detection limits of the two methods
established in this paper were lower than that of methods in the litera­
ture (Ying et al., 2011; Liu et al., 2015; Peddamma et al., 2018). In the
splitless mode, LOD of HS-SPME-GC-TOF MS method was close to that of
SPME-GC-MS/MS method established by Hoper et al. (2016). As far as
these performances concerned listed in Table 1, both two methods in
this paper met the requirements of daily testing and research.
3.4. Determination of 2-AP in rice flour samples
The sensory scores and 2-AP contents of seven samples were
measured in this paper. As shown in Fig. 3, 2-AP contents measured by
two methods ranged from 81.84 ng g 1
to 1.17 ng g 1
and from 72.00
ng g 1
to 1.00 ng g 1
, respectively. Using paired t-test, there was no
significant difference in 2-AP content measured by two methods (P >
0.05), indicating the reliability of results obtained by two methods. A
weak positive correlation (r ¼ 0.45) was obtained between sensory score
and 2-AP content. According to the agricultural industry standard of
aromatic rice (NY/T 596–2002), only five types of samples were aro­
matic rice (sensory score � 60 points). During the sensory evaluation,
samples 3 and 5 were judged as non-aromatic rice because strong rubber
flavor affected the overall flavor. However, 2-AP contents measured in
these two types of samples were 55.87 and 17.33 ng g 1
, respectively,
which was not lowest 2-AP content among the seven types of samples. In
addition, sample 7 with the content of only 1 ng g 1
was also aromatic
rice in the sense of sensory. These phenomena indicated that other
compounds besides 2-AP also affected the aroma intensity of rice. The
most likely reason was that the key compounds for aroma in rice was not
a single 2-AP (Hien et al., 2006). 2-AP has always been the most obvious
feature to distinguish aromatic rice from non-aromatic rice due to its low
sensory threshold. However, the aroma flavor of rice may be produced
integrated expression of more than one volatile compound.
4. Conclusion
In this paper, HS and HS-SPME extraction methods combined with
GC-TOF MS were establish for quantitative analysis of 2-AP. LOD and
LOQ of 0.68 ng g 1
and 2.27 ng g 1
, recoveries of 94.19–116.00%
(spiking level: 100 ng g 1
) were obtained for HS-GC-TOF MS method.
Meanwhile, LOD and LOQ of 0.46 ng g 1
and 1.50 ng g 1
, recoveries of
95.57–107.49% (spiking level: 100 ng g 1
) were obtained for HS-SPME-
GC-TOF MS method. Both two methods had good linearity (1–150 ng
g 1
, r > 0.9995), low detection limits, and high recoveries. And there
was no significant difference in results obtained by these two methods
(p > 0.05), indicating the reliability of the established methods. HS-GC-
TOF MS method was suitable for daily batch detection of samples due to
Table 1
Performances of 2-AP determination methods established in this paper and in some literature.
Sample
volume
Extraction method Split
mode
LOD LOQ RSD Linear range Recovery Ref.
1 g SPME 20:1 0.46 ng g 1
1.50 ng
g 1
7.02–7.77% 1–150 ng g 1
95.57–107.49% Experimental data
No Split 0.02 ng g 1
0.06 ng
g 1
/ / /
HS 5:1 0.68 ng g 1
2.27 ng
g 1
2.34–13.98% 1–150 ng g 1
94.19–116.00%
0.5 g HS No Split 1 ng g 1
5 ng g 1
<5% 0.1–250 ng g 1
/ Peddamma et al.
(2018)
1 g SPME No Split 0.039 ng
g 1
0.103 ng
g 1
5–33% 0.053–5.38 ng
g 1
107–109% Hopfer et al. (2016)
1 g SPME No Split 45.5 ng g 1
152 ng g 1
8.54% 500–4000 ng
g 1
96.3–103.5% Liu et al. (2015)
3 g (NAFION/PDDAC)Homemade SPME
fiber head
/ 0.10 ng
mL 1
/ 5.79% 0.5–8.00 ng
mL 1
105.1–103.9% Hu et al. (2014)
1 g SPME No Split 10 ng g 1
/ 5.09% / 82.57% Ying et al. (2011)
Z. Guo et al.

7. Journal of Cereal Science 93 (2020) 102975
7
its advantages of high automation, simple operation and short prepa­
ration time, while HS-SPME-GC-TOF MS method was more suitable for
identifying and analyzing complex samples with low content target due
to the strong enrichment ability of SPME fiber. In addition, sensory re­
sults of seven samples had weak positive correlation (r ¼ 0.45) with 2-
AP content. This phenomenon indicated that the aroma flavor of rice
was produced by the comprehensive expression of multiple volatile
compounds, although 2-AP was one of the key components of the aroma.
Other key volatile compounds were needed for further study.
Declaration of competing interest
The authors declare that there is no conflict of interest.
CRediT authorship contribution statement
Zhenling Guo: Investigation, Data curation, Writing - original draft,
Writing - review & editing. Siqi Huang: Investigation, Data curation.
Mingxue Chen: Resources, Funding acquisition. Yanxia Ni: Investiga­
tion. Xianqiao Hu: Resources, Writing - review & editing, Supervision,
Funding acquisition. Nan Sun: Resources, Writing - review & editing.
Acknowledgement
This work was supported by Zhejiang Provincial Natural Science
Foundation of China (grant no. LQ15C200007); Agricultural Science
and Technology Innovation Program of CAAS (grant no. CAAS-
XTCX2019024); and the earmarked fund for China Agriculture Research
System (grant no. CARS-01-47).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jcs.2020.102975.
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