Determination of eucomic acid in Chinese hawthorn fruit and products by gas chromatography-mass spectrometry

Article abstract of DOI:10.14233/ajchem.2014.16312

An analytical method for determination of eucomic acid in Chinese hawthorn fruit and its products by gas chromatography-mass spectrometry (GC-MS) was developed. The sample was extracted by ethanol, esterificated with sulphuric acid-methanol solution and then extracted with dichloromethane. The final analysis was performed by GC-MS using the fragment ions m/z 107 under selected ion monitoring (SIM) for the content of eucomic acid. External standard method was used for quantification. Under optimum conditions, there was a good linearity within the range of 2-500 μg/g. The limit of detection was 0.35 μg/g, the recoveries were in the range 87-102%, the relative standard deviation (RSD) was 3.1-5.6%. This method was applied to determine the eucomic acid in hawthorn fruit and its products. The content is between 5.1-255 μg/g.

Full text of DOI:10.14233/ajchem.2014.16312

Asian Journal of Chemistry; Vol. 26, No. 16 (2014), 5053-5057
ASIAN JOURNAL OF CHEMISTRY
http://dx.doi.org/10.14233/ajchem.2014.16312
Determination of Eucomic Acid in Chinese Hawthorn Fruit and
Products by Gas Chromatography-Mass Spectrometry
*
ZHE GAO, RONG-FANG WANG, XIAO-HU KANG, AI-XIA QIN, YA-NAN JIA, TONG-SEN HE, YU-LEI PAN and TONG CUI
College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, Hebei Province, P.R. China
*Corresponding author: Fax: +86 312 7528195; Tel: +86 312 7528427; E-mail: cuitong98@aliyun.com
Received: 9 September 2013;
Accepted: 10 October 2013;
Published online: 28 July 2014;
AJC-15630
An analytical method for determination of eucomic acid in Chinese hawthorn fruit and its products by gas chromatography-mass spectrometry
(GC-MS) was developed. The sample was extracted by ethanol, esterificated with sulphuric acid-methanol solution and then extracted
with dichloromethane. The final analysis was performed by GC-MS using the fragment ions m/z 107 under selected ion monitoring (SIM)
for the content of eucomic acid. External standard method was used for quantification. Under optimum conditions, there was a good
linearity within the range of 2-500 µg/g. The limit of detection was 0.35 µg/g, the recoveries were in the range 87-102 %, the relative
standard deviation (RSD) was 3.1-5.6 %. This method was applied to determine the eucomic acid in hawthorn fruit and its products. The
content is between 5.1-255 µg/g.
Keywords: Eucomic acid, Eucomic acid 1,4-dimethyl ester, GC-MS, Crataegus pinnatifida, Flavor component.
Eucomic acid, a secondary metabolite in plants, was first
isolated from the bulbs of Eucomis punctata L' Hér. (Liliaceae)
and later identified as (2R)-2-(p-hydroxybenzyl) malic acid
by Heller and Tamm4. It was also found in Lycoris radiata5,
Cattleya trianaei6, Opuntia vulgaris Mill7, Crotalaria sessiliflora
L8, Cactus stems (Opuntia spp.)9, Opuntia dillenii10, Encyclia
michuacana11, Prichly Pear (Opuntia ficus-indica (L) Mill.)12
and Turpinia arguta Seem13. Recently, eucomic acid was iden-
tified as an endogenous metabolite in leaf movement in the
model organism, Lotus japonicus14.According to the research
on Vanda teres15, eucomic acid contributed to stimulate respi-
ratory functions in keratinocytes by increasing cytochrome c
oxidase activity and/or expression and is therefore a potential
new natural ingredient for antiaging preparations to remedy
age-related disorders. However, quantitative analysis for
eucomic acid has not been reported. Therefore, this paper aims
to develop a stable and reliable GC-MS analytical method for
the quantification of eucomic acid.
INTRODUCTION
Hawthorn have been widely used as medicinal and food
materials in China and in certain European countries1. Chinese
hawthorn is one of the most popular fruits in north China,
because of its special flavor and beneficial health effects in
treating digestive ailments, dyspnea, kidney stones and
cardiovascular disorders2. The flavor components of Chinese
hawthorn fruit were analyzed by GC-MS and a total of 90
compounds have been detected3. However, limited data on its
characteristic flavor compounds have been reported. In our
previous research of polyphenol in hawthorn fruit extracts,
we found the existence of eucomic acid (eucomic acid, Fig. 1),
which was the characteristic fragrance of hawthorn fruit. Due
to the fact that it rarely exists in other fruits, eucomic acid is a
potential index for a quality assessment of hawthorn fruit and
products.
3'
OH
4'
5'
EXPERIMENTAL
2'
1'
The Sephadex LH-20 was purchased from Sigma Chemical
Co. (Pharmacia, Sweden). Deuterium dimethyl sulfoxide
(CD3SOCD3), tetramethylsilane (TMS) and deuterated acetone
(CD3COCD3) were purchased from Beijing Boya Dabei
Technology Development Co. (Beijing, China). The methanol
and acetonitrile used for the HPLC mobile phase were HPLC
grade. The water was double distilled water. Dichloromethane,
O
1
5
6'
OH
3
HO
4
2
OH O
Fig. 1. Chemical structure of eucomic acid

5054 Gao et al.
Asian J. Chem.
4-oxo-pentanoic acid, malic acid, citric acid, isocitric acid were
analytical grade. The solution of sulphuric acid-methanol was
12:100 (v/v).
HPLC system. The elution was carried out with 45 % methanol
in 0.02 % formic acid: the flow rate was 10 mL/min at 25 °C
and the wavelength was 280 nm. The fractions at retention
time 20 min were collected, evaporated (under reduced pressure)
and lyophilized to obtain compound 2.
The hawthorn fruits (Crataegus pinnatifida Bge. var.
major N.E.Br.) were purchased from markets in Baoding,
Hebei, China and the cultivar was Dajinxing. Hawthorn juice
(I and II), Hawthorn jam, Hawthorn jelly and Hawthorn soup
were purchased from the market.
Liquid chromatography-mass spectrometry (LC-MS)
analysis of compound 1: The HPLC analysis was performed
on a Mightysil RP-18 GP Aqua column (250 mm × 4.6 mm
i.d., 5 µm). 20 % methanol in 0.3 % formic acid was employed
as mobile phase A and 30 % acetonitrile in 0.3 % formic acid
was employed as mobile phase B. The gradient procedure was
0-6 min with 0 % B, 15-25 min with 25-60 % B, 25-30 min
with 60-100 % B, 30-40 min with 100 % B and 40-42 min
with 100-0 % B. The column temperature was 40 °C and detec-
tion was carried out at 280 nm. The injection volume was 5 µL.
The mass spectrometer used nitrogen gas at a temperature of
350 °C, a flow rate of 10 mL/min, a nebulizer pressure of 50
psi and the scanning range was m/z 50-2000 in positive mode.
NMR analysis of compound 1 and 2: Each 5 mg of com-
pound 1 and 2 were dissolved in CD3SOCD3 and CD3COCD3.
TMS was the reference reagent. The 1H NMR and 13C NMR
spectra, H-H homonuclear correlation spectroscopy (COSY),
multi-key hydrocarbon relationship (HMBC) and hetero-
nuclear multiple quantum relations (HMQC) were analyzed.
Sample preparation: Each 10 g of the hawthorn fruits,
hawthorn jam, hawthorn soup and hawthorn jelly were ground
into a cream with 95 % ethanol. The cream slurry was trans-
ferred to a 50 mL volumetric flask using 95 % ethanol and
processed in an ultrasonic homogenizer for 10 min. 10 g of
the hawthorn juice was transferred to a 50 mL volumetric flask
using 95 % ethanol. 10 mL of supernatant was evaporated to
dryness with a vacuum, dissolved in 15 mL sulphuric acid-
methanol solution, sealed and then reacted and heated at 80 °C
for 1 h. After cooling, 15 mL distilled water was added to the
reaction mixture and extracted with the 8 mL of dichloromethane
3 times. Dichloromethane extracts were mixed together to a
constant volume of 25 mL after being dried by anhydrous
sodium sulfate. The solution was filtered through a 0.45 µm
membrane, a portion of the filtrate was directly used for the
determination by GC-MS.
GC-MS analysis was performed on anAgilent 7890A gas
chromatograph (Agilent Technologies, Shanghai, China),
5975C Mass Selective Detector (Agilent Technologies, Santa
Clara, CA, USA) and 7683B autosampler (Agilent Techno-
logies, Little Falls, DE, USA), fitted with a HP-5MS fused
silica capillary GC column (30 m × 0.25 mm i.d., 0.25 µm film
thickness). Analytical high-performance liquid chromato-
graphy (HPLC) systems (Agilent Technologies) fitted with a
Hypersil BDS C18 column (250 mm × 4.6 mm i.d., 5 µm).
Preparative HPLC systems (Beijing Chuangxin Tongheng
Science and Tecnology Co., Ltd, Beijing, China) fitted with a
SinoChrom ODS-BP C18 column (300 mm × 20 mm i.d, 10
µm). LC-MS analysis was performed using an Agilent 1100
Series LC/MSD Trap System (California, USA) and equipped
with an ESI source. NMR spectra were measured on a Bruker
Avnce III 600 MHz (Bruker Biospin Crop, Ettlingen, Germany).
Extraction and purification of eucomic acid: A 14 kg
(FW) fruit sample was cut into pieces and extracted with 95 %
ethanol (w/v = 1:1.5) under room temperature for 24 h. The
residue was re-extracted twice with 80 % (v/v) ethanol. Ethanol
extracts were mixed together and evaporated to 3 L in a rotatory
evaporator and extracted with 1.5 L of petroleum ether three
times to remove the lipid components and followed by 3 L of
ethyl acetate extraction for 5 times. The ethyl acetate layer
was evaporated in a rotatory evaporator at 35 °C to produce a
final extract of 150.50 g of the extract was dissolved in 100
mL water and the solution was added to a Sephadex LH-20
column (375 mm × 45 mm i.d.) at a flow rate 10 mL/min and
eluted respectively with 40, 50, 60, 70, 80 and 100 % methanol
(v/v, in 0.02 % formic acid, 2 bed volume (BV), each 1200
mL). Each 0.5 BV eluate was collected and monitored by
HPLC. The elution was carried out with 12 % acetonitrile
(v/v) in 0.02 % formic acid. The column temperature was 35 °C.
The flow rate was 0.8 mL/min and the injection volume was
5 µL. The eluates from 3rd to 4th fractions were mixed, evapo-
rated (under reduced pressure) and lyophilized and then
purified with a preparative HPLC system. The elution was
carried out with 25 % methanol (v/v) in 0.02 % formic acid at
the flow rate 10 mL/min and the wavelength for detection was
280 nm. The chromatography was repeated and the fractions
at retention time 9 min were collected, evaporated (under
reduced pressure) and lyophilized to obtain achromous solids
197 mg of compound 1.
GC-MS analysis: Operating conditions for the GC were:
the oven temperature was programmed at 60 °C for 1 min,
ramped to 160 °C at 20 °C/min, held for 1 min and then to
250 °C at 20 °C/min and held for 3 min. The injection port
temperature was 250 °C, helium was used as carrier gas at a
flow rate of 1 mL/min. The split ratio was 20:1, the injection
volume was 1 µL. The MS was operated in electron ionization
(EI) mode at 70 eV with a transfer line temperature of 250 °C,
ion source 230 °C, quadrupole temperature 150 °C. The samples
were analyzed qualitatively based on retention time of
chromatography and mass spectra of reference substance with
full scan mode (SCAN). The characterizing ions of selective
ion monitoring (SIM) mode were m/z = 107 and the external
reference method was used in the quantitative analysis.
Synthesis and purification of eucomic acid 1,4-dimethyl
ester: 30 mg compound 1 was dissolved in 6 mL methanol
and 720 µL concentrated sulphuric acid was added and then
sealed at 80 °C for a 1 h reaction.After cooling, 6 mL of distilled
water was added to the reaction mixture and extracted with
the equivalent volume of diethyl ether 3 times. Diethyl ether
extracts were mixed together, dried with anhydrous sodium
sulfate, concentrated and then purified with a preparative
RESULTS AND DISCUSSION
Structural identification of eucomic acid and its esteri-
fied esters: Compound 1 was obtained by separation and
purification from hawthorn according to the steps described

Vol. 26, No. 16 (2014)
Determination of Eucomic Acid in Chinese Hawthorn Fruit and Products by GC-MS 5055
in extraction and purification of eucomic acid. HPLC was used
to analyze the purity of the chromatographic peak of compound
1. The maximum absorption wavelength was 278 nm in the
ultraviolet spectra (UV) absorption spectrogram.As the mobile
phase acidity increased, retention time decreased. This
suggested that it might be one of organic acids. By LC-MS
analysis, compound 1 showed molecular ion peak with m/z
263 [M + Na]+ which is consistent with the eucomic acid. The
1H NMR, 13C NMR analysis were as follows: NMR δH (DMSO-
d6): 6.981 (2 H, d, J = 8 Hz, H-2', H-6'), 6.620 (2 H, d, J = 8
Hz, H-3', H-5'), 2.795 (1 H, d, J = 3.5 Hz, H-5b), 2.741 (1 H,
d, J = 3.5 Hz, H-5a), 2.692 (1 H, d, J = 16 Hz, H-3b), 2.347 (1
H, d, J = 16 Hz, H-3a). NMR δC (DMSO-d6): 175.7 (C-4),
171.7 (C-1), 156.1 (C-4'), 131.5 (C-2', C-6'), 126.3 (C-1'),
114.7 (C-3', C-5'), 75.0 (C-2), 43.8 (C-5), 42.6 (C-3). These
ion peaks m/z = 250 was also obvious. The 1H NMR, 13C NMR
analysis were observed: NMR δH (CD3COCD3): 7.035 (2 H,
d, J = 8 Hz, H-2', H-6'), 6.74 (2 H, d, J = 8 Hz, H-3', H-5'),
3.68 (3 H, d, J = 52 Hz, H-2''), 3.59 (3 H, d, J = 52 Hz, H-1''),
2.95 (1 H, d, J = 14 Hz, H-5a), 2.93 (1 H, d, J = 11 Hz, H-5b),
2.85 (1 H, d, J = 14 Hz, H-3b), 2.62 (1 H, d, J = 16 Hz, H-3a).
NMR δC (CD3COCD3): 170.51 (C-1), 156.35 (C-4'), 131.32
(C-2', C-6'), 126.15 (C-1'), 114.7 (C-3', C-5'), 75.8 (C-2), 51.67
(C-2''), 50.89 (C-1''), 44.3 (C-5), 42.6 (C-3). The chemical
structure of compound 2 was confirmed by 1H NMR, 13C NMR,
1H-1H COSY, HMQC, HMBC, NOESY spectra and mass spec-
trometry. Compound 2 was identified as eucomic acid 1,4-
dimethyl ester ((R)-2-(4'-hydroxybenzyl)-malic acid 1,4-
dimethyl ester).
Optimization of the condition of eucomic acid esterifi-
cation: In order to examine the influence of esterification in
hawthorn fruit juice, different amount of sulfuric acid were
mixed with methanol according to the method used in sample
preparation, which was followed by the quantification method
of eucomic acid methyl ester described in GC-MS analysis.
Fig. 3 showed that the esterification product was significantly
increased when the amount of sulfuric acid increased. The
esterification product was saturated at 12 mL sulfuric acid per
100 mL methanol and was stable when increasing the sulfuric
acid content. Therefore, we used the reaction solution which
had a sulfuric acid to methanol ratio equal to 12:100.
1
data and the 2D NMR analysis, including H-1H COSY,
HMQC, HMBC and NOESY spectra, were in accordance with
those reported in the literature4,5. Therefore, compound 1 was
identified as eucomic acid and was taken as the quantification
standard in this experiment.
Eucomic acid methyl ester were prepared according to
the steps described in synthesis and purification of eucomic
acid dimethyl ester and purified by preparative HPLC. Total
ion chromatogram (TIC) and EI spectra were analysed by GC-
MS. As shown in Fig. 2, retention time was 12 min, hydroxy-
benzyl ion peaks m/z 107 was the strongest and dehydrated
200000
150000
100000
50000
0
(a)
160000
120000
80000
40000
0
5
7
9
11
13
4
6
8
10
Sulfuric acid (mL)
Fig. 3. Effect of sulfuric acid on esterification
12
14
Time (min)
107.0
3
′
(b)
60000
40000
20000
0
OH
4
′
To examine the effect of temperature on esterification,
reaction temperature was respectively set at 20, 40, 60, 80 and
100 °C according to the steps described in sample preparation.
Sample analysis is described in GC-MS analysis and the results
are shown in Fig. 4. The esterification reaction was weak at
20 °C. As the temperature increased, the reaction rate accele-
rated. The highest esterification product was obtained at 80 °C,
with little increase in esterification product at 100 °C. There-
fore, this study was performed at an 80 °C water bath.
2
′
1
′
5
′
O
5
6
′
OCH3
2
3
1
H3CO
′′
4
′′
2
1
O
OH
250.1
To observe the effect of time on esterification, different reac-
tion times were used according to the steps described in sample
preparation and the analytical method in GC-MS analysis and
the results are shown in Fig. 5. We observed that the esteri-
fication product was optimized when reaction time was 1 h.
No significant improvement was observed when increasing
reaction time. Thereby, 1 h was used as the reaction time.
60
100
140
180
220
260
m/z
Fig. 2. Total ion chromatogram (a) and mass spectra (b) of eucomic acid
1,4-dimethyl ester

5056 Gao et al.
Asian J. Chem.
200000
3
1
16000
12000
8000
4000
0
2
150000
100000
50000
0
5
4
20
40
60
Temperature (°C)
80
100
Fig. 4. Effect of temperature on the esterification
5
7
9
Time (min)
11
13
300000
225000
150000
75000
0
Fig. 6. GC-MS total ion current (TIC) chromatogram of a hawthorn sample
after methyl esterification; 1 = 4-oxo-pentanoic acid; 2 = malic
acid; 3 = citric acid; 4 = isocitric acid; 5 = eucomic acid
Linearity, recovery and precision: Eucomic acid was
prepared at the concentration of 2 mg/mL in methanol. The
esterification, extraction and analysis were performed accor-
ding to sample preparation and GC-MS analysis. The deve-
loped GC-MS method showed excellent linearity (r2 = 0.9997)
in the 4-1000 µg range, with a concentration 2-500 µg/g in
sample. The detection limits (for S/N = 3) was 0.35 µg/g. The
average recovery rate was determinate by five replication at
three levels of 20, 200, 400 µg/g, respectively. The results of
average recovery and precision are shown in Table-1.
0
0.5
1.0
1.5
2.0
2.5
3.0
Time (h)
Fig. 5. Effect of reaction time on the esterification
Selection of capillary chromatographic column: KB-1
(30 m × 0.25 mm, i.d., 0.25 µm, Kromat), HP-5MS and HP-
INNOWAX (30 m × 0.25 mm, i.d., 0.25 µm, Agilent) capillary
chromatographic column were used to assess the isolation
effect. After adjusting GC conditions many times, no esterifi-
cation products of eucomic acid were monitored with KB-1.
It may be due to the fact that the retention time of the eucomic
acid peak was too short for non-polar column KB-1. Even
when the initial column temperature was at 40 °C for 10 min,
the peak also did not separate well with the solvent peak. The
esterification product of eucomic acid could be monitored by
both HP-5MS and HP-INNOWAX.Although in HP-INNOWAX,
it took a long time at a high temperature program (close to the
column temperature limit) and the peak was obviously smeared.
Therefore, we chose HP-5MS in analyzing the samples.
GC-MS analysis of eucomic acid 1,4-dimethyl ester:
Compared with the European hawthorn, one of the obvious
features of Chinese hawthorn fruit was that it contains large
amount of organic acids, such as citric, malic, oxalic, succinic
acids16,17. The methyl ester derivatives of these organic acids
can be washed out and separated in a short time under the
chromatographic condition described in GC-MS analysis.
Fig. 6 shows there is no obvious interference components near
eucomic acid 1,4-dimethyl ester. The methyl ester derivations
of other organic acids such as 4-hydroxy-pentanoic acid, malic
acid, citric acid and isocitrate can also be well separated. We
used the fragment ions m/z 107 for monitoring and quantitative
analysis under selected ion monitoring (SIM). The analysis
method had higher sensitivity, decreased other interference and
other methyl ester derivates did not give detection signals.
TABLE-1
AVERAGE RECOVERY RATE AND RELATIVE STANDARD
DEVIATION (n = 5) OF EUCOMIC ACID
Recovery (%)
RSD
Added (µg/g)
Found (µg/g)
20
17
87
90
5.6
3.1
4.5
200
400
180
410
102
Quantification of hawthorn products: The established
GC-MS method was used to determine the content of eucomic
acid in hawthorn fruit and products,which is given in Table-2.
The content of eucomic acid in hawthorn products was signi-
ficantly lower compared to hawthorn fruit. It is known that
eucomic acid is chemically stable and is not expected to be
lost in a significant amount during processing besides thermal
volatilization. The low content of eucomic acid in this study
indicated that the content of hawthorn fruit is actually not high
in these products. Research relevant to eucomic acid should
continue to identify the characteristic fragrance of hawthorn
fruit. We expect our analytical method can be applied as a
method in monitoring the quality of hawthorn products for
manufacturers and market regulators.
TABLE-2
CONTENT OF EUCOMIC ACID IN HAWTHORN
FRUIT AND PRODUCTS
Fruits
255
Juice I
15.4
Juice II
17.9
Jam
7.2
Soup
5.1
Jelly
7.8
Content
(µg/g)

Vol. 26, No. 16 (2014)
Determination of Eucomic Acid in Chinese Hawthorn Fruit and Products by GC-MS 5057
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ACKNOWLEDGEMENTS
This project was financially supported by the National
Natural Science Foundation of China (31071494), Natural
Science Foundation of Hebei Province (C2011204093) and
Project of Human Resources and Social Security of Hebei
Province 2010-195.
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