validation of hplc method for determination of e- and z-ajoene in

8/17/2019 Validation of HPLC Method for Determination of E- And Z-Ajoene In

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Food Sci. Biotechnol. 21(1): 107-111 (2012)

DOI 10.1007/s10068-012-0013-5

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 n-

hexane 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 Z -

ajoene 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% forZ -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

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

thiosulfinates (allicin), ajoene, and other volatile sulfur-

containing 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 Cope-

type 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 inhibitingthe 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 ismore 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 (11-

13). 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.

Miyoung Yoo, Sanghee Lee, Sunyoung Kim, Dongbin Shin ( )Korea Food Research Institute, Seongnam, Gyeonggi 463-746, Korea Tel: +82-31-780-9126; Fax: +82-31-780-9280E-mail: [email protected]

RESEARCH ARTICLE


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108 Yoo et al.

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 heatedGC 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 brownvolumetric 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 n-

hexane 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 µg/g) and for

Z -ajoene (2.2, 21.9, 43.9, 65.8, 131.7, 219.4, and 263.3

µg/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 4,000× g

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-µm, Sartorious Stedim Biotech,

Geottingen, Germany) and injected on HPLC system.

HPLC system equipped with UV-2075 plus detector,

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, 4.6×150 mm, 5 µm,

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 determinedbased 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 accordingto 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 Duncan’s

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


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HPLC Analysis of Ajoenes in Garlic Products 109

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 aninternational 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 E -

Ajoene 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. 1. Chemical structures of ajoene isomers.

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


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110 Yoo et al.

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 µg/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 σ/S and as 10 σ/S, respectively (σ; 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

µg/g, while LOQs of these compounds were from 4.32 and

5.37 µg/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


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HPLC Analysis of Ajoenes in Garlic Products 111

E -ajoene in different oil macerated garlic juices was ranged

from 367.9-555.8 and 155.6-266.9 µg/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 µg/g of garlic) and E -(172.0

µg/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 ricebran 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.

References

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organosulfur compounds: Human and animal studies. J. Nutr. Suppl.131: 989S-993S (2001)2. Yang CS, Chhabra SK, Hong JY, Smith TJ. Mechanisms of

inhibition of chemical toxicity and carcinogenesis by diallyl sulfide(DAS) and related compounds from garlic. J. Nutr. Suppl. 131:1041S-1045S (2001)

3. Lamm DL, Riggs DR. Enhanced immunocompetence by garlic:Role in bladder cancer and other malignancies. J. Nutr. Suppl. 131:1067S-1070S (2001)

4. Block E, Ahmad S. ( E ,Z )-Ajoene: A potent antithrombotic agentfrom garlic. J. Am. Chem. Soc. 106: 8295-8296 (1984)

5. Block E. Ahmad S, Catalfamo JL, Jain MK, Apitz-Castro R.Antithrombotic organosulfur compounds from garlic: Structural,

mechanistic, and synthetic studies. J. Am. Chem. Soc. 108: 7045-7055 (1986)

6. Romano EL, Montano RF, Brito B, Apitz R, Alonso J, Romano M,Gebran S, Soyano A. Effects of ajoene on lymphocyte andmacrophage membrane – dependent functions. Immunopharm.Immuno. 19: 15-36 (1997)

7. Ishikawa K, Naganawa R, Yoshida H, Iwata N, Fukuda H, Fujino T,Suzuki A. Antimutagenic effects of ajoene, an organosulfurcompound derived from garlic. Biosci. Biotech. Bioch. 60: 2086-2088 (1996)

8. Hattori A, Yamada N, Nishikawa T, Fukuda H, Fujino T.Antidiabetic effects of ajoene in genetically diabetic KK-A(y) mice.J. Nutr. Sci. Vitaminol. 51: 382-384 (2005)

9. Nishikawa T, Yamada N, Hattori A, Fukuda H, Fujino T. Inhibitionby ajoene of skin-tumor promotion in mice. Biosci. Biotech. Bioch.66: 2221-2223 (2002)

10. Hattori A, Norihiko Y, Nishikawa T, Fukuda H, Fujino T. Protectiveeffect of ajoene on acetaminophen-induced hepatic injury in mice.Biosci. Biotech. Bioch. 65: 2555-2557 (2001)

11. Koch HP, Lawson LD. Garlic: The Science and TherapeuticApplication of Allium sativum L. and Related Species. 2th ed.Williams & Wilkens, Baltimore, MD, USA. pp. 37-137 (1996)

12. Naznin MT, Akagawa M, Okukawa K, Maeda T, Morita M.Characterization of E - and Z -ajoene obtained from different varietiesof garlics. Food Chem. 106: 1113-1119 (2008)

13. Hibi T. Method of processing garlic and preparing ajoene-containingedible oil products. U.S. Patent 5,612,077 (1997)

14. Voigt M, Wolf E. HPLC-bestimmung von knoblauchwirkstoffen inextrakten, pulver und fertigarzneimitteln (HPLC determination of active compounds in garlic extracts). Dtsch. Apoth. Ztg. 126: 591-593 (1986)

15. Iberl B, Winker G, Knobloch K. Products of allicin transformation:Ajoenes and dithiins, characterization and their determination byHPLC. Planta Med. 56: 202-211 (1990)

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quantification of sulfides and dialk(en)yl thiosulfinates incommercial garlic products. Planta Med. 57: 363-370 (1991)

17. DHHS. Center for Veterinary Medicine (CVM): Guidance forIndustry, Bioanalytical Method. Center for Drug Evaluation andResearch (CDER), Food and Drug Administration, US Departmentof Health and Human Services, Washington, DC, USA (2001)

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Table 4. Contents of E - and Z -ajoene in oil-macerated garlic from different oils

Refinededible oil

Concentration (µg/g, garlic juice) 1) Z / E

Z -Ajoene E -Ajoene Total ajoenes ratio

Corn 467.0±74.4a,b,c 2) 206.4±36.7b,c 673.4±110.1a,b,c 2.3

Perilla 399.3±21.7b,c 237.8±32.0a,b 641.4±22.2b,c 1.7

Olive 555.8±56.1a 229.3±28.5a,b 785.1±72.4a,b 2.4

Flaxseed 487.1±56.4a,b 247.8±25.6a,b 734.8±82.0a,b 2.0

Rice bran 526.0±48.1a 266.9±30.7a 792.9±75.2a 2.0

Grape seed 374.2±47.7c 155.6±27.0c 544.4±34.4c 2.2

Sesame 517.3±50.5a 235.4±37.5a,b 752.7±67.1a,b 2.2

Soybean 367.9±45.6c 175.8±2.6c 543.7±46.8c 2.1

1)Values are shown as mean±SD of triplicate.2)There are significant differences ( p

validation of hplc method for determination of e- and z-ajoene in

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