Food Sci. Biotechnol.

21(1): 107-111 (2012)

DOI 10.1007/s10068-012-0013-5

RESEARCH ARTICLE

Validation of HPLC Method for Determination of E- and Z-Ajoene in

Oil-macerated Garlic Juice
Miyoung Yoo, Sanghee Lee, Sunyoung Kim, and Dongbin Shin

Received: 13 August 2011 / Revised: 4 October 2011 / Accepted: 15 October 2011 / Published Online: 29 February 2012
KoSFoST and Springer 2012

Abstract HPLC method for determination of ajoene

isomers in garlic oil products was optimized and validated.
E- and Z-Ajoene were extracted with ethyl acetate and
followed by the sensitive and selective determination of 2
isomers in a single run using normal phase HPLC
equipped with silica gel column. The mobile phase was nhexane and 2-propanol (85/15, v/v) with an isocratic
condition as a flow rate of 1.0 mL/min and 240 nm of
HPLC UV detector. All calibration curves of E- and Zajoene in oil-macerated garlic showed good linearity
(r=0.998). Overall, intra- and inter-day were in the range of
0.12-2.30 and 2.84-5.26%, respectively. Recovery was in
range of 87.17-98.53% for E-ajoene and 85.16-99.23% for
Z-ajoene. The validated method was applied to determine
contents of ajoene in macerate garlic juices prepared with
various vegetable oil. There were no any matrix effects in
all chromatograms. The proposed method may be useful
for quality control and evaluation of garlic oil products.
Keywords: E- and Z-ajoene, oil macerate garlic juice,
validation, analytical method, HPLC

Introduction
Garlic is a bulbous plant belonging to the genus Allium
and a very popular foodstuff as well as medical
vegetable to improve human health in many areas of the
world. Numerous studies have been previously reported
that garlic has many health benefits including cholesterol
reduction, cancer prevention, inhibition of carcinogenesis,
and anti-inflammatory (1-3). The major causal substances
Miyoung Yoo, Sanghee Lee, Sunyoung Kim, Dongbin Shin ( )
Korea Food Research Institute, Seongnam, Gyeonggi 463-746, Korea
Tel: +82-31-780-9126; Fax: +82-31-780-9280
E-mail: shindb@kfri.re.kr

providing these beneficial effects are S-allyl-L-cysteine,

thiosulfinates (allicin), ajoene, and other volatile sulfurcontaining compounds (diallyl sulfide, diallyl disulfide,
diallyl trisulfide, etc.).
Ajoene [(E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide]
was first reported as a key compound in oil macerated
garlic products by Block and Ahmad (4). Block et al. (5)
suggested that S-thioallylation of allicin followed by Copetype elimination and re-addition of allylsulfenic acid
should give ajoene while unimolecular decomposition of
allicin.
Ajoene is well known as a strong inhibitor of platelet
aggregation. It impairs platelet aggregation by inhibiting
the functional exposure of platelet integrins GPIIa/IIIb (6).
Ajoene has also been reported to exhibit antimutagenic and
antidiabetic effects (7,8). Nishikawa et al. (9) proposed that
ajoene inhibits the skin-tumor promotion in mice. Hattori
et al. (10) also found protective effect of ajoene on hepatic
injury.
Ajoene, which can be subdivided into the trans isomer
E- and the cis isomer Z-form (Fig. 1), is not found in garlic
bulbs, and only formed on incubation of garlic pulp as
garlic homogenate in organic solvents (methanol, ethanol,
or oils). It has been known that Z-isomer has a strong
bioactivity as compared with E-ajoene, while E-isomer is
more stable than Z-isomer during storage. The E/Z ratio
and yield depend on the polarity of the solvent system,
reaction condition during processing and cultivar. In oil
macerated products prepared with fresh garlic, the Z-ajoene
has always been found to be dominant over the E-isomer,
usually about 2-4-fold. But garlic homogenates using
processed garlic, like frozen garlic paste and fried garlic,
contained a higher amount of E-ajoene than Z-ajoene (1113). Aforementioned factors regarding to the E/Z ratio,
yield, and biological effects of ajoene might give different
effects although it appears the same in garlic oil products.

108

Therefore, the accurate and sensitive method for

determination of ajoene isomers is absolutely necessary for
the development and quality control of garlic oil products
containing ajoene.
Until now, it has been reported that the determination of
ajoene isomers in garlic products was only used the HPLC
procedure due to the instability of the ajoene in a heated
GC column (14-16). However, these methods were not
sufficiently validated with respect to the analytical
performance characteristics despite quantitative analysis. In
this study, we established and validated HPLC method for
determination of ajoene isomers derived from oil
macerated garlic juice. The established method was applied
to determine contents of Z- and E-ajoene in 8 different oil
macerated juices.

Materials and Methods

Chemicals E- and Z-Ajoene were purchased from
Medigen (Daejeon, Korea). 2-Propanol, n-hexane, and
ethyl acetate were obtained from J. T. Baker (Paris, KY,
USA). Water (DW) was purified through a Milli-Q system
(Millipore, Bedford, MA, USA) for all sample preparation
and mobile phase. All other chemicals and solvent were
used in HPLC grade.
Preparation of stock and working solutions Stock
solutions of E- and Z-ajoene were prepared by transferring
a weighted amount of the pure substances in a brown
volumetric flask and subsequently dissolved by 50% ethyl
acetate in n-hexane to obtain an ultimate concentration of
0.57 and 0.53 mg/g, respectively. The stock solution was
stored at 80oC. The working standard solutions, used to
spike the garlic matrixes or construct the calibration curves,
were prepared by diluting with 50% ethyl acetate in nhexane on the same day of the experiment. Calibration
curves were obtained with 7 concentrations for E-ajoene
(2.4, 23.6, 47.2, 70.8, 141.5, 235.9, and 283.0 g/g) and for
Z-ajoene (2.2, 21.9, 43.9, 65.8, 131.7, 219.4, and 263.3
g/g).
Analytical conditions for E- and Z-ajoene To determine
the amounts of ajoene isomers formed in oil macerated
garlic juice, homogenate garlic was carried out centrifuge
(Brinkman Instruments, Westbury, NY, USA) at 4,000g
for 10 min. Five mL of supernatant, oil fraction, was
transferred to a 20-mL tube containing 5 mL ethyl acetate
and mixed for 1 min using a vortex mixer (Thermo Scientific,
Dubuque, IA, USA). Ethyl acetate layer was filtered with
syringe filter (0.2-m, Sartorious Stedim Biotech,
Geottingen, Germany) and injected on HPLC system.
HPLC system equipped with UV-2075 plus detector,

Yoo et al.

2089 plus pump, and cool auto sampler (Jasco, Kyoto,

Japan) was used. The separation was carried out using a
silica gel column (Si 60 column, 4.6150 mm, 5 m,
Agilent Technologies, Santa Clara, CA, USA) at 25oC and
UV/Visible detector was set at 240 nm. The mobile solvent
was n-hexane and 2-propanol (85:15, v/v) at a flow rate of
1.0 mL/min. Concentration of ajoene isomers was determined
based on the chromatographic data of standards. The
calibration curves (peak area vs. concentration) for individual
compound were obtained for a wide concentration range.
Preparation of different oil-macerated garlic juices
Garlic bulbs harvested in 2010 was purchased from a
cultivator at Shinan in Korea. Soybean oil, corn oil, olive
oil, grape seed oil, rice bran oil, perilla oil, flaxseed oil, and
sesame oil were purchased from local market (Seongnam,
Korea). Oil macerated garlic samples were prepared as
follows; Garlic juice was prepared using a DH 850
laboratory blender (Oscar, Kimhae, Korea). The garlic
juice obtained from the peeled garlic cloves (10 kg) was
packed by vacuum film and then stored at 80oC before
the day for preparation of oil macerated garlic samples.
Edible oil (20 g) was added to the garlic juice (5 g), and
mixed using a vortex mixer (Thermo Scientific) for 1 min
and sufficiently mixed in ultrasonic cleaner (Bronsonic,
Danbury, CT, USA) at 40oC for 20 min. Then the mixture
was incubated at 80oC for 4 h to form completely ajoene
isomers.
Method validation The method was performed according
to the FDA guidelines for validation of analytical procedures
(17).
Statistical analysis All experiments were performed in
triplicate. Statistical significant tests were performed using
SAS software (version 8.2; SAS Institute, Inc, Cary, NC,
USA). Significant difference was verified by Duncans
multiple range tests at 95% confidence level.

Results and Discussion

Optimization of HPLC analysis method Based on the
previous studies (12,16) regarding to the analytical method
of ajoenes and preliminary tests, the extraction and
separation conditions for accurate determination of ajoene
isomers presented in oil macerated garlic juice were
selected as mentioned above. For the extraction of E- and
Z-ajoene, 5 mL of supernatant, which oil macerated garlic
juice was centrifuged, mixed thoroughly with 5 mL ethyl
acetate followed by filtration, and ethyl acetate layer was
injected on HPLC system. As shown in Fig. 2 and Table 3,
there were no any matrix effects, and we could get a good

109

HPLC Analysis of Ajoenes in Garlic Products

Fig. 1. Chemical structures of ajoene isomers.

recovery (>85%). The best HPLC column and mobile

phase to resolve ajoene isomers and other compounds were
a silica gel normal phase column and n-hexane/2-propanol
(85:15, v/v). Retention time of Z- and E-ajoene was 14.39
and 16.40 min, respectively. (R=14.2 between Z-ajoene
and other garlic compound, and R=4.4 between E-ajoene
and Z-ajoene; R is resolution). The only analytical method
which has been reported for the determination of ajoenes is
the HPLC procedure due to the instability of the ajoenes in
a heated GC column (14-16). Voigt and Wolf (14) developed
a reversed phase HPLC method for ajoene and other sulfur
compounds without separation of the isomeric or homologous
ajoene components. Iberl et al. (15) and Lawson et al. (16)
achieved the separation of E- and Z-ajoene with normal

phase (Si) HPLC column using hexane/isopropanol (92/8,

95/5, v/v) as the mobile phase. However, these methods
were not sufficiently validated with respect to the
analytical performance despite quantitative analysis.
Previous analytical methods (14-16) for assay of ajoene
were often modified to suit laboratory systems and
performance. Validation of analytical method from an
international guideline (17) is very useful for developing
and implementing an official method to determine ajoene
isomers in garlic sample. The proposed HPLC method for
determination of ajoene isomers in oil macerated garlic
juice samples was validated.
Method validation The proposed HPLC method for
ajoene isomers in oil macerated garlic juice was validated
with regard to selectivity, linearity, limit of detection, limit
of quantification, precision, and accuracy. In order to
investigate the presence or absence of any interference in
the extraction procedures of oil macerated garlic sample
and blank sample that was not contained garlic juice, it was
analyzed using the proposed HPLC method. Z- and EAjoene were very well separated and eluted in 14.39 and
16.40 min, respectively. However, no peaks for ajoenes
were found in the chromatogram of blank sample, as
shown in Fig. 2C.
Linearity was examined using a calibration curves

Fig. 2. HPLC chromatograms of 2 ajoene isomer standards (A), garlic macerated in oil (B), and blank (C).

110

Yoo et al.

Table 1. Linearity and sensitivity data of E- and Z-ajoene in oil-macerated garlic

Regression equation1)

Compound

Linearity range
(g/g)

Slope

Intercept

r2)

LOD (g/g)

LOQ (g/g)

E-Ajoene
Z-Ajoene

2.4283.0
2.2263.3

12,357301
13,214231

71,7385338
71,1167095

0.998
0.998

1.43
1.77

4.32
5.37

1)

Regression equation is y=ax+b, where y is the peak area, x refers to the concentration of compounds (g/g), a is the slope, and b is the
intercept.
r is the correlation coefficient.

2)

Table 2. Intra-day and inter-day precision data of E- and Zajoene in oil-macerated garlic

Table 3. Recovery data of E- and Z-ajoene spiked to garlic juice

Compound

1)

RSD (%)
Compound

E-Ajoene

Z-Ajoene

Concentration
(g/g)

Intra-day

Inter-day

(n=3)

(n=9)

23.6
70.8
235.9

2.3
1.12
0.12

4.78
3.56
4.76

21.9
65.8
219.4

1.96
1.33
2.97

5.26
2.84
4.45

1)

RSD stands for relative SD.

obtained by standard working solutions with 7 different

concentrations for 2 ajoene isomers. Calibration curves of
E- and Z-ajoene were obtained by linearity relationship
between concentration of standard compounds and
corresponding peak areas. Each solution was injected with
3 times. Linearity data, as well as limit of detection (LOD)
and limit of quantitation (LOQ) for ajoene isomers were
shown in Table 1. The calibration curves were generated to
be linear over the 2.4-283.0, and 2.2-263.3 g/g range for
E- and Z- ajoene, respectively. The correlation coefficients
for each analyte are greater than 0.998. The LODs and
LOQs of the proposed methods were calculated on the
basis of 3.3 /S and as 10 /S, respectively (; the standard
deviation of y-intercepts of regression analysis, and S; the
slope of the calibration curve) (18). Under analytical
condition, LODs of E- and Z-ajoene were 1.43 and 1.77
g/g, while LOQs of these compounds were from 4.32 and
5.37 g/g, respectively.
The precision based on intra-day repeatability was
evaluated by replicate (n=3) measurements from standard
working solutions at 3 different concentration levels of low,
medium, and high as shown Table 2. The inter-day
precision was established using samples at the same
concentration range as mentioned above. A triplicate
determination of each concentration over a period of 3
consecutive days was followed by same experimental
procedures. The method exhibited excellent precision, as
shown in Table 2. The intra-day data (repeatability)
resulted in low relative standard deviations (RSDs <3.0%)

Spiked conc. Detected conc.1)

(g/g)
(g/g)

Recovery
(%)

E-Ajoene

94.1
188.2

92.724.42
163.712.06

98.534.70
87.171.10

Z-Ajoene

223.2
446.3

190.037.52
443.052.74

85.163.37
99.230.61

1)

Values are shown as meanSD of triplicate.

for 2 isomers. The inter-day data conducted on 3 different

days also displayed good values (RSDs <5.3%), confirming
the excellent reproducibility of the method.
Recovery (%) of E- and Z-ajoene was calculated when
2 different concentrations of each ajoene was spiked to oil
macerated garlic sample using garlic juice extracted from
blenched garlic clove (blank sample). Recovery was
determined by comparing the found concentration with the
spiked concentration. Data of these experiments were
shown in Table 3. Satisfactory recovery values were
obtained, with recoveries ranging from 85.16 and 99.23%.
These values demonstrated the proposed method was quite
adequate for the detection and quantification of E- and Zajoene in oil macerated garlic juice.
Analysis of ajoenes in different oil macerated garlic
products The validated method was applied to determine
amount of ajoene isomers in different oil macerated garlic
juices prepared using aforementioned method. Homogenate
of oil and garlic juice were incubated at 80oC for 4 h. The
yield and ratio of E- and Z-ajoene in 8 oil macerated garlic
samples were shown in Table 4. The total amount of ajoene
was significantly different in oil macerated samples. The
highest total amount of ajoene (792.975.2 g/g garlic
juice) was obtained from rice oil. Oil macerated samples
with olive, flaxseed, and sesame oil contained larger
amount of 2 isomers than samples with corn and perilla oil.
The samples using grape seed and soybean oil showed the
lowest total amount of ajoenes, 544.434.4 and 543.7
46.8, respectively. All oil macerated garlic samples
contained higher amount of Z-ajoene than E-ajoene. The Z/
E ratio of ajoene in eight samples was found to be 1.7 and
2.4 by the proposed HPLC method. The amount of Z- and

111

HPLC Analysis of Ajoenes in Garlic Products

Table 4. Contents of E- and Z-ajoene in oil-macerated garlic from different oils
Refined
edible oil
Corn
Perilla
Olive
Flaxseed
Rice bran
Grape seed
Sesame
Soybean

Concentration (g/g, garlic juice) 1)

Z-Ajoene
a,b,c 2)

467.074.4
399.321.7b,c
555.856.1a
487.156.4a,b
526.048.1a
374.247.7c
517.350.5a
367.945.6c

E-Ajoene

Z/E
Total ajoenes

b,c

206.436.7
237.832.0a,b
229.328.5a,b
247.825.6a,b
266.930.7a
155.627.0c
235.437.5a,b
175.82.6c

a,b,c

673.4110.1
641.422.2b,c
785.172.4a,b
734.882.0a,b
792.975.2a
544.434.4c
752.767.1a,b
543.746.8c

ratio
2.3
1.7
2.4
2.0
2.0
2.2
2.2
2.1

1)

Values are shown as meanSD of triplicate.

There are significant differences (p<0.05) among 8 different edible oils using Duncans multiple comparison test between the samples having
the different letter in a column.

2)

E-ajoene in different oil macerated garlic juices was ranged

from 367.9-555.8 and 155.6-266.9 g/g garlic juice,
respectively. These results agreed with the previously
reported study (12, 13, 15). Naznin et al. (12) reported that
maximum yield of Z-(476.3 g/g of garlic) and E-(172.0
g/g of garlic) ajoene was obtained in Japanese garlic oil
macerate with rice oil incubated at 80oC for 4 h. Iberl et al.
(15) suggested that the Z/E ratio and yield depend on the
polarity of the solvent system and the reaction conditions
during processing. Hibi (13) reported that the fatty
composition of oil and fat was one of key factors for
increasing of ajoene formation. He proposed that oil with
a medium-chain fatty acid triglyceride provided a relatively
large amount of ajoenes with oil macerated garlic. Among
edible vegetable oils used in this study, olive oil and rice
bran oil were rich source of oleic acid. On the other hand,
grape seed oil and soybean oil had higher amount of
linoleic acid (19). As a consequence, we carefully suggested
that vegetable oils with high content of oleic acid used for
efficient preparation to increase the formation of ajoene
isomers in oil macerated garlic.

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