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Journal of Chromatography A
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Short communication

Analysis of volatile compounds responsible for kiwifruit aroma by

desiccated headspace gas chromatographymass spectrometry
Chun-Yun Zhang a,b , Qiong Zhang a , Cai-Hong Zhong a , Ming-Quan Guo a,b,
a
Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074,
China
b
Sino-African Joint Research Center, Chinese Academy of Sciences, Wuhan 430074, China

a r t i c l e

i n f o

Article history:
Received 14 January 2016
Received in revised form 17 February 2016
Accepted 18 February 2016
Available online xxx
Keywords:
Volatile compounds
Organoleptic quality
Desiccated headspace sampling
Kiwifruit
GCMS

a b s t r a c t
A new method for desiccated headspace (DHS) sampling of aqueous sample to GCMS for the analysis
of volatile compounds responsible for kiwifruit aroma in different kiwifruit cultivars has been developed based on the complete hydrate formation between the sample solvent (water) with anhydrous
salt (calcium chloride) at an elevated temperature (above the boiling point of the aqueous sample) in a
non-contact format, which overcame the water-effect challenge to directly introduce aqueous sample
into GCMS analysis. By means of DHS, the volatile compounds in three different kiwifruit cultivars were
analyzed and compared under the optimized operating conditions, mainly time and temperature for
headspace equilibration, column temperature program for GCMS measurement. As a result, 20 peaks
of volatile compounds responsible for kiwifruit aroma were detected and remarkable differences were
found in the relative contents of three major volatile compounds among the three different kiwifruit cultivars, i.e., acetaldehyde, ethanol and furfural. The DHS sampling technique used in the present method
can make the GCMS analysis of volatile compounds in the aqueous sample within complex matrix possible without contaminating the GCMS instrument. In terms of the analysis of volatile compounds in
kiwifruit, the present method enabled a direct measurement on the ltrate of the aqueous kiwifruit pulp,
without intermediate trap phase for the extraction of analytes, which will be more reliable and simpler
as compared with any other headspace method in use. Thus, DHS coupled with GCMS will be a new
valuable tool available for the kiwifruit related research and organoleptic quality control.
2016 Elsevier B.V. All rights reserved.

1. Introduction
Kiwifruit (Actinidia chinensis) is one of the most valuable fruit
crops that is native to China and now gaining international commercial importance [1]. It is widely appreciated by consumers for
its avor and nutritional qualities, e.g., high levels of vitamin C and
health-promoting effects [2,3]. Together with sweetness and acidity, kiwifruit aroma is one of the crucial factors that contribute to
the kiwifruit avor, which is the result of a subtle mixture of volatile
compounds [4]. Therefore, techniques that can efciently and effectively analyze these volatile compounds of kiwifruit will be of great
importance for the explanation of the fruit aroma and the breeding
of new kiwifruit cultivars with better organoleptic quality.

Corresponding author at: Key Laboratory of Plant Germplasm Enhancement

and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences,
Wuhan 430074, China. Fax: +86 27 87518018.
E-mail address: guomq@wbgcas.cn (M.-Q. Guo).

Recently, more and more consumers have paid more attention

to organoleptic quality of kiwifruit, even a growing percentage of
consumers are willing to pay a premium for kiwifruit cultivars with
higher organoleptic quality. Nowadays, with an increasing demand
for kiwifruit around the world, the determination of the organoleptic quality of kiwifruit is becoming highly important. Since the
organoleptic quality of kiwifruit is often ascribed to its volatile
aroma components, many methods have been developed for the
analysis of the aroma compounds in kiwifruit to date. Because of
the ability of simultaneously separating and identifying multiple
volatile components, GCMS was commonly used for the analysis of aroma compounds in kiwifruit [5]. However, prior to GCMS
measurement, it is mandatory to isolate the volatile species from
the complex sample matrix. Those techniques commonly used
for the isolation of volatile compounds, including solvent extraction [6], distillation [7,8], static or dynamic headspace (SH or DH)
[9,10] and solid phase micro-extraction (SPME) [11], were typically based on the volatility or solubility of the volatile compounds.
In more details, solvent extraction and distillation were aiming at

http://dx.doi.org/10.1016/j.chroma.2016.02.056
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analyzing the whole set of volatiles in the matrix because of their

high accessibility of volatile compounds. However, they are timeconsuming and easy to introduce other chemical impurities, such
as some co-existing non-volatile components and some remaining
solvents, which also cause contamination problems in the GCMS
analysis. Headspace based extraction techniques, e.g., SH, DH and
SPME, have been proposed as the simpler techniques for extracting volatile components from fruits and vegetables [1214]. Since
SH is based on an in-situ releasing and sampling of the volatiles,
different from that of DH and SPME where an intermediate trap
phase is involved, it is promising to be developed as an in-situ and
simple method for the analysis of volatiles in fruits and vegetables [15]. Generally, SH is employed at an elevated temperature to
obtain higher amount and more components in gas phase partition
in order to increase sensitivity and adaptability of detection [15].
However, for the aqueous kiwifruit pulp sample, this elevated temperature, especially higher than the boiling point of water, would
result in high stream pressure of water vapor introduced to GC
and then to MS with high risk of damaging the ion source of MS
detector [16], which is not allowed in methodology. Thus, measures that can remove this high stream pressure of water should be
taken in the high-temperature SHGCMS measurement of aqueous samples. Recently, an application of acetone acetals as water
scavengers was reported to remove water and increase the detection sensitivity in SHGC analysis [17]. However, the by-products
of the reaction between the scavenger and water, such as methanol
and acetone, could cause impurities when the whole set of volatiles
in sample were tackled. Previous works on SHGC analysis have
also shown that adding anhydrous salt can effectively remove the
water pressure by the formation of hydrate, and increase the detection sensitivity of volatiles [18,19]. Thus, we believe this technique
can also be used in the high-temperature SHGCMS measurement
of volatile compounds in the aqueous kiwifruit sample to achieve a
desiccated headspace (DHS) sampling and make the non-damaging
GCMS measurement possible.
In this work, a desiccated headspace GCMS (DHSGCMS)
method has been developed for the analysis of volatile compounds
in kiwifruit. The main focuses were on the feasibility and applicability of this new methodology. After the optimization of the
headspace equilibration conditions and the column temperature
program of GC, the volatile compounds of three different kiwifruit
cultivars were identied and compared as cases of studies. Meanwhile, the precision and adaptability of the present method were
also evaluated.

2. Experimental section
2.1. Chemicals and materials
Anhydrous calcium chloride used in the experiment is of analytical grade and purchased from commercial sources without further
purication. Samples of three different kiwifruit cultivars, i.e., Jintao, Maohua and Jinkui, were collected from Wuhan botanical
garden in December, 2015. The samples were stored in room temperature for 57 days, and then prepared for the analysis.
2.2. Apparatus and operations conditions
An automated headspace sampler (Agilent 7697A, US) equipped
with a sample loop volume of 1 mL, a GC system (Agilent 7890A,
US) equipped with HP-5 capillary column, and MS system (Agilent 5975C, US), were used for the analysis of volatile components
in different kiwifruit cultivars. The headspace operating conditions
were as follows: Equilibration time = 60 min; Equilibration temperature = 160 C; Pressing time = 0.5 min; Extracting time = 0.2 min;
Injecting time = 0.5 min. GC operating conditions were as follows:
The carrier gas was helium at a ow rate of 1 mL/min; the column temperature program of GC was initially set at 30 C for 5 min,
and gradually increased to 120 C at 6 C/min, then kept there for
10 min before gradually increased to 250 C at 12 C/min, and then
kept there for 10 min. For GCMS detection, electron ionization (EI)
system was used with ionization energy at 70 eV.
2.3. Procedures for sample preparation
50 g of pulverized fruit tissue was centrifuged and ltered
through a membrane lter (0.45 m). 1 mL of ltrate was measured into a half-open glass tube (2 mL), and then the whole tube
was embedded into a headspace vial containing 5 g of anhydrous
calcium chloride. After that, the sample vial was sealed with a
PTFE/silicone septum and an aluminum cap and equilibrated at
150 C for a proper time (about 3 h) in the oven to completely
remove the water in sample by the formation of hydrate with anhydrous calcium chloride in a non-contact format (shown in Fig. 1).
Headspace extraction, followed by GCMS measurement, was performed on the sample vial after further equilibrating in the oven of
headspace sampler at 160 C for another one hour.
3. Results and discussion
3.1. Principle of the method
When the liquid sample was placed into a closed headspace vial
without anhydrous salt, there is a tendency for the volatile species

Fig. 1. The schematic of the principle of the method.

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to release from the liquid sample phase to the headspace phase due
to the concentration difference between the two phases. The mass
balance of the analyte can be expressed as
C0 Vl0 = Cg Vg + Cl Vl

(1)

where C0 is the initial concentration of analyte in the liquid sample.

Vl0 is the initial liquid sample volume. Cg and Cl are the concentrations of analyte in the gas and liquid phase, respectively. Vg and Vl
are the volumes of gas and liquid phase, respectively.
When the vaporliquid equilibrium of analyte was achieved at
an elevated temperature, it can be described by the Henrys law,
i.e.,
K=

Cl
Cg

(2)

where K is the vaporliquid equilibrium partition coefcient.

Substituting Eq. (2) into Eq. (1) and making an arrangement, we
have
C0 =

Vg + KVl
Vl0

Cg

C0 =

fVl0

at 150 C is about 476 kPa) with high risk damaging the GCMS
system. Therefore, to remove water evaporated from the aqueous sample, anhydrous salt was added into the headspace vial to
form hydrate with water vapor in a non-contact format (shown in
Fig. 1a).
When the equilibrating time and the amount of hydrous salt
are enough to remove all water in the aqueous sample (shown in
Fig. 1b), and the liquid volume tends to reach zero, i.e.,Vl 0, Eq.
(4) can be revised as
C0 =

Vg
fVl0

(5)

According to Eqs. (4) and (5), the disappearance of liquid phase

can increase the detection sensitivity for the same sample. In this
case, the vapor pressure of water tends to reach zero and no water
will be introduced into the GCMS system, which makes the desiccated headspace (DHS) coupled to GCMS analysis successful.

(3)

Since the GCMS signal count is linearly proportional to the vapor

solute concentration in the headspace; i.e., A = fCg , Eq. (3) can be
rewritten as
Vg + KVl

(4)

where A is the peak area of the GCMS signal, and f is the response
factor of peak area to analyte concentration in the headspace upon
HSGCMS measurement.
Eq. (4) is the quantitative basis of the conventional headspace
analysis. The vapor pressure of water in the headspace for an aqueous sample is the saturated vapor pressure of water under the
given temperature. Clearly, increasing equilibration temperature
and decreasing the volume of liquid phase (through reducing the
value of K and Vl ) can improve the detection sensitivity of the
analytes. In this paper, to obtain better detection sensitivity and
more composition information of the volatiles from kiwifruit, it is
better to employ the sample equilibration at a temperature (e.g.,
150 C) much higher than the boiling point of the aqueous sample
(e.g., 100 C). However, this high temperature will not only produce
boiling liquid contaminating headspace sampling system, but also
cause high water pressure (the saturated vapor pressure of water

3.2. Time and temperature for headspace equilibration

According to the principle of the present method, it is required to
remove all amount of water evaporated from the aqueous sample.
Thus, the equilibration time should be long enough for the complete hydrate formation. In this work, 3 h was chosen as the proper
time for the equilibration of headspace vial in the oven based on
the observation of the liquid remaining in tube (shown in Fig. 1b).
Another one hour for equilibration in the headspace sampler was
needed for the evaporating of volatiles before headspace sampling
to GCMS.
Generally, higher temperature is preferred to obtain more
amounts of volatile compounds partitioned in the headspace phase.
However, the temperature is mandatory to be below the maximum
tolerable temperature of the containing and sealing equipments,
e.g., rubber septum that seals the headspace sample vial. Otherwise, leakage will occur from the vial as a consequence of the high
pressure generated at the high temperature. Thus, in this work,
we chose moderate high temperatures, i.e., 150 C for equilibration
in the oven and 160 C for headspace sampler, for the headspace
equilibrating and sampling process, according to the tolerable temperature of the equipments.

Table 1
Identication and comparison of volatile compounds from three kiwifruit cultivars.
Peak no.

Retention time, min

Volatile compounds

Relative contents, %
Jintao

Maohua

Jinkui

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20

1.197
1.292
1.378
1.457
1.466
1.641
1.837
1.921
2.032
2.519
2.654
3.247
3.408
4.445
6.894
7.91
10.773
11.589
12.391
18.86

Acetaldehyde
Ethanol
Acetone
Ethyl formate
Acetic acid
2-Methyl-propanal
2,3-Butanedione
2-Methyl-furan
Ethyl acetate
3-Methyl-butanal
2-Methyl-butanal
2,3-Pentanedione
3-Hexyne-2,5-diol
Dimethyl disulde
Dihydro-2-methyl-3(2H)-furanone
Furfural
1-(2-Furanyl)-ethanone
Carbonodithiotic acid, S,S-dimethyl ester
5-Methyl-2-furancarboxaldehyde

13.4
28.5
4.81
0.340

2.09
2.06
0.854
0.178
7.22
2.50
3.93
0.107
0.440
1.97
23.6
0.579

7.44

45.8
1.14
12.48
2.83

2.81
1.42
1.65
0.0677
2.13
1.55
2.70
0.0964
0.608
2.10
14.7
0.706
0.499
6.79

17.8
1.40
5.52
0.266
0.129
0.900
4.06
1.53
0.565
4.53
2.13
6.50
0.0836
0.0227
1.92
37.1
2.25

8.37
2.17

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Fig. 2. Chromatogram in the GCMS analysis of volatile compounds from three kiwifruit cultivars.

3.3. Chromatogram in the GCMS analysis

Under the above headspace conditions, the column temperature
program of GC has been optimized for the DHSGCMS analysis
of volatile compounds from kiwifruit. In this work, the volatile
compounds from three different kiwifruit cultivars, i.e., Jintao,
Maohua and Jinkui, were all analyzed as the case studies of the proposed method. Fig. 2 shows the chromatogram in the DHSGCMS
analysis of volatile compounds from the three kiwifruits. It was
observed that up to 20 peaks of volatile compounds from the three
kiwifruits can be well separated and measured by GCMS under
the optimized operating conditions, indicating that the optimized
operating conditions can be used to analyze the volatile compounds
from kiwifruit.
3.4. Identication and comparison of the volatile compounds
from three kiwifruit cultivars
The 20 peaks in Fig. 2 as volatile compounds from the three
kiwifruit cultivars were identied by comparing their mass fragmentation pattern with those stored in the NIST database using
NIST11. The results are listed in Table 1, in which the volatile
compounds were quantitated by relative peak areas of the total
ion chromatography (TIC) from the MS signals. It was found that
the volatile compounds from the three kiwifruit measured by the
present method were mainly aldehydes, together with alcohols and
esters, which were also reported on the volatile compounds from
other kiwifruit cultivars or with other analytical methods [2022].
Table 1 also shows that almost similar chemical compositions
of volatile compounds were found in these three kiwifruit cultivars. However, differences were found in relative contents of the
chemicals, which are shown in Fig. 3. It can be seen that most of
the compounds with relatively small contents have corresponding
RC (1:1) among the three kiwifruit cultivars. Three components
(circled in red in Fig. 3) showed remarkable differences among the

Fig. 3. Differences of relative contents (RC) of volatile compounds from three

kiwifruit cultivars. (For interpretation of the references to color in the text, the reader
is referred to the web version of this article.)

three cultivars, which are acetaldehyde, ethanol and furfural. In

detail, acetaldehyde is the one of the major volatile compounds in
kiwifruit, accounting for 45.8% in Maohua, while 13.4% and 17.8%
in Jintao and Jinkui, respectively. Ethanol is a major volatile compound in Jintao with RC of 28.5%, different from that of Maohua
and Jinkui with RC of only 1.14% and 1.40%, respectively. Furfural
is a major component in all the three types of kiwifruit, but their
contents are quite different, which are 23.6%, 14.7% and 37.1% in
Jintao, Maohua and Jinkui, respectively.

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3.5. Evaluation of the method

References

3.5.1. Method precision

The precision of the present method was investigated by triplicate measurements of the same sample (Jintao) under the same
operating conditions. The peak areas of three major components,
i.e., acetaldehyde, ethanol and furfural, were used to calculate the
uncertainty (expressed as the relative standard deviation, RSD)
of the present method. The results showed that the uncertainties
of the three components were in the range from 1.64% to 4.81%,
indicating that the present method based on the DHSGCMS measurement has a good precision in analysis of volatile compounds in
kiwifruit.

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3.5.2. Method adaptability

The present method is based on a direct measurement on the
ltrate of the aqueous kiwifruit pulp using DHSGCMS. Thus, the
following points on the method adaptability were highlighted: (a)
hydrophobic volatile compounds, mainly esters in kiwifruit, are not
sensitive to the present method because of the limited solubility in
the ltrate; (b) the full evaporation and absorption of the sample
solvent (water) results in the disappearance of the sample matrix,
which makes the simple external standard (inuenced by sample
matrix) available for further quantitative study of these volatile
compounds; (c) in-situ measurement on the ltrate of the aqueous kiwifruit pulp, without intermediate trap phase for extraction,
can make the method more reliable and simpler.
4. Conclusion
A simple method for the analysis of volatile compounds from
kiwifruit has been developed. Based on the complete hydrate
formation between sample solvent (water) and anhydrous salt
(calcium chloride), the desiccated headspace sampling to GCMS
measurement on the aqueous kiwifruit pulp was achieved. The
results of the analysis of three different kiwifruit cultivars showed
that 20 peaks of volatile compounds can be detected by the present
method and differences were found on the relative contents of
volatile compounds among the three different kiwifruit cultivars.
Since the method was based on a direct measurement on the ltrate
of the aqueous kiwifruit pulp, without intermediate trap phase for
analytes extraction, it will be more reliable and simpler in the analysis of volatile compounds in kiwifruit and will be a new valuable
tool available for the kiwifruit related research and its organoleptic
quality control.
Acknowledgements
This work was jointly supported by the Hundred Talents Program from Chinese Academy of Sciences (Grant No.
29Y429291a0129 to M. Guo), and the Sino-Africa joint research
project (Grant No. SAJC201328 to M. Guo). Both funders played no
roles in the study design, data collection and analysis, and decision
to publish.

Please cite this article in press as: C.-Y. Zhang, et al., Analysis of volatile compounds responsible for kiwifruit aroma by desiccated
headspace gas chromatographymass spectrometry, J. Chromatogr. A (2016), http://dx.doi.org/10.1016/j.chroma.2016.02.056