Simultaneous Analysis of Malondialdehyde, 4-Hydroxy-2-hexenal

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Simultaneous Analysis of Malondialdehyde, 4-Hydroxy-2hexenal, and 4-Hydroxy-2-nonenal in Vegetable Oil by Reversed-Phase High-Performance Liquid Chromatography Lukai Ma, and Guoqin Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04566 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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Journal of Agricultural and Food Chemistry

Simultaneous Analysis of Malondialdehyde, 4-Hydroxy-2-hexenal, and 4-Hydroxy-2-nonenal in Vegetable Oil by Reversed-Phase High-Performance Liquid Chromatography

Lukai Ma †, Guoqin Liu*, †, ‡

†

School of Food Science and Engineering, South China University of Technology, Guangzhou

510640, China ‡

Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety,

South China University of Technology, Guangzhou 510640, China

Corresponding author *G. Liu, Mailing address: No.381 Wushan Road, Tianhe District, Guangzhou 510640, China. Tel: 8620-8711-4262. Fax: 8620-8711-3875. E-mail: [email protected].

ACS Paragon Plus Environment


A

group

of

toxic

aldehydes

such

as,

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malondialdehyde

(MDA),

1

ABSTRACT:

2

4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) have been found in various

3

vegetable oils and oil-based foods. Then simultaneous determination of them holds a great

4

need in both the oil chemistry field and food field. In the present study, a simple and efficient

5

analytical method was successfully developed for the simultaneous separation and

6

detection of MDA, HHE and HNE in vegetable oils by reversed-phase high-performance

7

liquid chromatography (RP-HPLC) coupled with photodiode array detector (PAD) at

8

dual-channel detection mode. The effect of various experimental factors on the extraction

9

performance, such as coextraction solvent system, butylated hydroxytoluene addition, and

10

trichloroacetic acid addition were systematically investigated. Results showed that the

11

linear ranges were 0.02 ‒ 10.00 µg/mL for MDA, 0.02 ‒ 4.00 µg/mL for HHE, and 0.03 ‒

12

4.00 µg/mL for HNE with the satisfactory correlation coefficient of > 0.999 for all detected

13

aldehydes. The limit of detection (LOD) and limit of quantification (LOQ) of MDA, HHE, and

14

HNE were ~0.021and 0.020 µg/mL, ~0.009 and 0.020 µg/mL, and ~0.014 and 0.030 µg/mL,

15

respectively. Their recoveries were 99.64‒102.18%, 102.34‒104.61%, and 98.87‒103.04%

16

for rapeseed oil and 96.38–98.05%, 96.19‒101.34%, and 96.86‒99.04% for French fries,

17

separately. Under the selected conditions, the developed methods was successfully

18

applied to the simultaneous determination of MDA, HHE and HNE in different tested

19

vegetable oils. The results indicated that this method could be employed for the quality

20

assessment of vegetable oils.

21 22

KEYWORDS: malondialdehyde, 4-hydroxy-2-hexenal, 4-hydroxy-2-nonenal, vegetable oil,

23

oxidation, reversed-phase high-performance liquid chromatography


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24 25

INTRODUCTION

26

Free radical-induced lipid oxidation is one of the major cause of deterioration of

27

vegetable oil and oil-based food in the presence of oxygen, especially those containing

28

polyunsaturated fatty acid (PUFA).1 Oxidation of PUFA leads to the formation of primary

29

lipid oxidation products, peroxide or hydroperoxide, while aldehydes are the main

30

secondary lipid oxidation products.2 Malondialdehyde (MDA) and α, β-unsaturated

31

aldehyde such as 4-hydroxy-2-hexenal (HHE) and 4-hydroxy-2-nonenal (HNE) are formed

32

among other saturated or unsaturated aldehydes. MDA, a three-carbon dialdehyde, is

33

known to be formed from both ω-3 and ω-6 PUFAs and has been claimed to be the most

34

important end-product of lipid autoxidation.3-5 While HHE and HNE, possessing double

35

bond and hydroxyl group in structure, are related to the oxidation of ω-3 and ω-6 PUFA,

36

respectively.6, 7 MDA, HHE, and HNE have been identified as the toxic substances due to

37

their high chemical reactivity with proteins and DNA, leading to the structural damage and

38

alteration of their functionality.8,

39

several diseases such as adult respiratory distress syndrome, atherogenesis, diabetes, and

40

even cancer.10,

41

vegetable oils, especially those which are rich in PUFA and processed at high

42

temperature.12, 13 Meanwhile, these three reactive aldehydes can be incorporated into fried

43

foods along with oxidized vegetable oil and be a part of our diet, and then be absorbed from

44

the gut into the blood system.14-17 Thus, it can be easily understood that PUFA-rich

45

vegetable oil and oil-based food can be a major intake source of MDA, HNE and HHE,

46

which threatens human health due to high consumption of PUFA products around the world.

11

9

Moreover, they have also been considered to cause

Unfortunately, MDA, HHE, and HNE have been found in various



47

Recently, the Belgian Superior Health Council has considered MDA and HNE as a major

48

concern for human health.18 What’s more, a threshold of toxicological concern (TTC) level

49

of exposure for MDA was 30 µg/kg bw/day, and 1.5 µg/kg bw/day for HHE and HNE in the

50

suggestion of European Food Safety Authority (EFSA) scientific committee.19 Consequently,

51

increasing attention has been gained to the formation and inhibition of MDA, HHE, and

52

HNE in vegetable oil, which are of great urgency. However, the determination of these three

53

aldehydes is an important foundation and necessity.

54

Due to the great difference in functional group (apart from the aldehyde group, see

55

Figure 1) between MDA and HHE/HNE, their chemical polarities differ largely, and they lack

56

suitable visible chromophore, which increases the difficulty of simultaneous determination

57

of these three aldehydes. Thus, the previous study mainly focused on the separated

58

determination of MDA and HHE/HNE in vegetable oil and oil-based food.16, 20-23 Moreover,

59

some of these methods, such as the thiobarbituric acid (TBA) method, lack of specificity

60

and require a high derivatization temperature (90 °C) which may lead to an overestimation

61

or underestimation of the actual content present in the samples.16, 21 Besides, some other

62

of the methods published previously either involve relatively complicated synthesis or

63

pretreatment steps for the use of isotopically labeled chemical regents.22, 23 Furthermore,

64

most of these methods focus scarcely determination of MDA, HHE and HNE in the same

65

run, and cannot meet the need of simultaneous routine determination.

66

To date, just one published paper reports a method for the simultaneous determination of

67

MDA, HHE, and HNE in vegetable oil by liquid chromatography coupled to tandem mass

68

spectrometry (LC-MS/MS).24 However, the measurement method has some drawbacks

69

such as the requirement of expensive apparatuses, high testing cost, and insufficient


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70

precision and reproducibility for HHE and HNE (79‒101% of recoveries with 13‒23%

71

coefficient of variation), which seems to suggest that it is not adequate for the routine

72

analysis of MDA, HHE, and HNE in vegetable oil. Therefore, based on the deficiency and

73

need as described above, the present study aims to develop a rapid and expedient

74

reserved-phase high-performance liquid chromatography (RP-HPLC) method for the

75

simultaneous

76

4-dinitrophenylhydrazine (DNPH) as derivatization reagent (The derivatization reactions

77

are shown in Figure 1) in vegetable oil. The proposed method is used to assess the

78

formation of toxic aldehydes MDA, HHE, and HNE in vegetable oil. Furthermore, MDA,

79

HHE, and HNE are analyzed in five types of commercial vegetable oils, that is, palm oil

80

(PO), corn oil (CO), rapeseed oil (RO), camellia oil (CLO), and linseed oil (LO), which are

81

treated by accelerated oxidation test at 60 and 180 °C.

82

MATERIALS AND METHODS

routine

determination

of

MDA,

HHE,

and

HNE

using

2,

83

Materials and Chemicals. PO was donated by Yihai Kerry (Tianjin) Investment Co., Ltd.

84

(Tianjin, China), and it was from Malaysia. CO was purchased from Yihai (Guangzhou) Oils

85

and Grains Industrial Co., Ltd. (Guangzhou, China). RO was obtained from Dongguan

86

Fuzhiyuan Feedstuff Protein Development Co., Ltd. (Dongguan, China). CLO was

87

purchased from Yongxing Taiyu Camellia Oil Co., Ltd. (Chenzhou, China). LO was friendly

88

supplied by Gansu A Best Bio-Technology Co., Ltd. (Jinchang, China). All vegetable oils

89

used in this study had been fully refined and were freshly prepared. They contained no

90

exogenous antioxidants.

91

1, 1, 3, 3-Tetraethoxypropane (TEP), trichloroacetic acid (TCA), butylated hydroxytoluene

92

(BHT), and DNPH (97%) were purchased from Sigma-Aldrich (St. Louis, MO). Stock



93

solutions of HHE and HNE in ethanol (10 mg/mL) were obtained from Cayman Chemicals

94

(Ann Arbor, MI), which both were stored at ‒80 °C. HPLC grade methanol (CH3OH) and

95

acetonitrile (CH3CN) were purchased from Merck (Darmstadt, Germany). Dichloromethane

96

and ethanol were purchased from Aladdin (Shanghai, China). Ultrapure water (>18 MΩ/cm)

97

was obtained from reverse osmosis purification system (Chongqing, China).

98

Preparation of MDA, HHE, and HNE Standard Solution. As mentioned above,

99

standard stock solutions of HHE and HNE were directly obtained from the reagent company.

100

For MDA, 10 mg/mL of stock solution was obtained by hydrolysis of TEP in 5% (v/v) of TCA

101

solution. All stock solutions were stored for maximum eight weeks at 0 °C. Before use,

102

standard working solutions of various concentrations (0.02, 0.05, 0.1, 0.5, 1, 2, 5, 10 µg

103

mL-1 for MDA, 0.03, 0.05, 0.1, 0.5, 1, 2, 3, 4 µg mL-1 for HNE, and 0.02, 0.05, 0.1, 0.5, 1, 2,

104

3, 4 µg mL-1 for HNE) were prepared by diluting appropriate amounts of stock solutions in

105

ethanol/water 50:50 (v/v).

106

Preparation of French Fries Sample. Fresh oils were used for frying French fries. After

107

the tested vegetable oils were heated at 180 °C for 5 h in an experimental fryer (Lecon co.

108

Foshan, China), about twenty grams of raw potato sticks (about 5.0 × 1.0 × 1.0 cm )

109

purchased from the local fast food restaurant were added into the oils and fried for 10 min.

110

Subsequently, the French fries were taken out, cooled, and then placed on the filter paper

111

to remove the oil on the surface. The French fries were minced and stored at ‒18 °C until

112

further use. Vegetable oils after frying French fries were transferred into glass bottles, then

113

sealed and stored at -18 °C until further use, too.

114

Sample Preparation (See Figure 2). The oil in French fries sample was extracted

115

according to the previous report.25 Subsequently, two grams of oil was weighted and mixed


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116

with 2.5% (w/v) of TCA, 0.1% (w/v) of BHT, and DNPH reagent (0.05 M in ethanol/HCl 12 M

117

9:1 (v/v)) prepared by slightly adapting the methods from Fenaille et al.26 The tube was

118

vortexed for 1 min and then stood overnight at room temperature. The DNPH derivatives

119

were extracted from the oil by ethanol/water mixture and centrifuged at 5000 g for 5 min on

120

a ST 16R centrifuge from Thermo Fisher Scientific (Waltham, MA). The supernatant was

121

transferred into another tube. The same procedure was performed in duplicate, and

122

supernatants from both extracts were combined. Subsequently, the DNPH derivatives were

123

re-extracted by dichloromethane, and this extraction procedure was also repeated two

124

times. The combined extracts were removed by blowing N2 gas to remove dichloromethane.

125

Then the concentrated solution was reconstituted by acetonitrile to 10 mL, and vortexed for

126

2 min for the homogeneous mixing. 100 µL aliquots were filtered by 0.45 µm Nylon66

127

disposable needle filter purchased from Troody (Shanghai, China). Three parallel tests

128

were carried out. The derivatization process of MDA, HHE and HNE standard solutions was

129

the same as that of oil sample above.

130

Analysis of MDA, HHE, and HNE. Separation and detection of MDA-, HHE-, and

131

HNE-DNPH derivatives were performed using a Dionex P680 HPLC system (Dionex,

132

Sunnyvale), which equipped with ASI-100 Automated Sample injector (Dionex, Sunnyvale),

133

Thermostatted column compartment TCC-100 (Dionex, Sunnyvale), and PDA-100

134

Photodiode Array Detector (PDA) (Dionex, Sunnyvale). Separation was achieved on a

135

ZORBAX Eclipse XDB-C18 column (4.6×250 mm, 5 µm) from Agilent Technologies (Santa

136

Clara, California). The detector model was dual-channel detection mode. The wavelength

137

of UV-VIS-1 was set at 310 nm for MDA-DNPH, and the wavelength of UV-VIS-2 was set at

138

378 nm for HHE-DNPH and HNE-DNPH. The flow rate was 1.0 mL/min, and the column



139

temperature was set at 30 °C. The injection volume was 10 µL. The mobile phase was

140

acetonitrile (solvent A) and water (solvent B). The gradient elution conditions were 45%

141

solvent A for the first 18 min, linear gradient from 45% solvent A to 70% in 5 min, and then

142

isocratic at 70% solvent A for 15 min. Three parallel samples were set up for each injection.

143

MDA, HHE, and HNE was finally quantified with the respective external standard curve.

144

The peak area was calculated using Chromeleon 7 software (Thermo Fisher Scientific, MA).

145

The concentration of MDA, HHE and HNE was expressed as µg/g. Data were presented as

146

mean value of triplicates with standard deviation (SD) in the result report.

147

Linearity, Limit of Detection (LOD), and Limit of quantification (LOQ). Calibration

148

curve (peak area versus concentration) of each tested aldehyde was built by plotting the

149

known concentrations of MDA (0.02‒10 µg/mL), HHE (0.02‒4.00 µg/mL), and HNE (0.03‒

150

4.00 µg/mL) against the corresponding peak area and fitting the data with linear equation.

151

LOD and LOQ under the chromatographic conditions were calculated at signal-to-noise

152

ratios of 3 and 10, respectively.

153

Recovery. Recovery of the method was respectively evaluated by the RO samples (n=6)

154

and French fries samples spiked with known amounts of MDA (2.0, 3.0, and 4.0 µg/g oil),

155

HHE (0.4, 0.5, and 0.6 µg/g oil) and HNE (1.4, 1.7, and 2.0 µg/g oil) standards. The

156

amounts of MDA, HHE and HNE was determined in triplicate in each case.

157

Matrix Effects. Matrix effects were evaluated by comparing the slopes of calibration

158

curves (ksolvent) by external standard method as shown in Linearity, Limit of Detection (LOD),

159

and Limit of quantitation (LOQ) section and standard addition calibration curves (kmatrix) by

160

spiking the blank oil matrices with the known amounts of MDA (1.0‒10.0 µg/g), HHE (0.1‒

161

1.0 µg/g), and HNE (0.2‒2.0 µg/g). The matrix factor (Mf) was calculated by the following


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162


equation: Mf= kmatrix / ksolvent.

163

Accelerated Oxidation Test. Accelerated oxidation tests of PO, CO, RO, CLO, and LO

164

were performed based on schaal oven test and simulated frying process. Briefly, about 5

165

mL of fresh oil was added into 10 mL glass bottles, kept open and heated at 60 °C for up to

166

30 days or 180 °C for 5 h in a ventilated oven (Shanghai Keelrein instruments Co., Ltd.,

167

Shanghai, China) at normal atmospheric pressure. The oil during schaal oven test was

168

sampled periodically and cooled rapid to room temperature in ice bath, and then stored at –

169

18 °C until analysis. Three parallel samples were set up for all the experimental oils.

170

Statistical Analysis. For each standard/sample, all MDA, HHE and HNE determinations

171

were done in triplicate, and data were expressed as mean ± standard deviation (SD).

172

Statistical analyses were performed with Origin 2016 (OriginLab Corporation, Northampton,

173

MA, USA).

174

RESULTS AND DISCUSSION

175

Optimization of the HPLC Conditions. The derivatization reaction of MDA, HHE, and

176

HNE with DNPH occurs at room temperature, which can therefore inhibit the possible

177

formation of aldehydes during sample pretreatment and avoid the overestimation of

178

analysis results. Conversely, the traditional derivative, TBA assay occurs at about 100 °C

179

and is not specific for aldehydes,16 leading to the wide result of aldehyde determination in

180

various experimental conditions. To our knowledge, the second oxidative products resulting

181

from vegetable oil oxidation consist of many aldehydes, such as alkanals, alkenals,

182

oxygenated saturated aldehydes, and oxygenated α, β-unsaturated aldehydes, etc.,27

183

which all exists in heat-oxidized vegetable oil and can also react with DNPH. Thus, to

184

obtain the baseline separation of MDA-, HHE-, and HNE-DNPH derivatives with the



185

separation factors of over 1.5, acetonitrile and water were used as the mobile phase for the

186

ZORBAX Eclipse XDB-C18 column. According to the peak shapes of standard solutions

187

(Figure 3B), the present study did not add the acetic acid, phosphoric acid, or

188

tetrahydrofuran into the mobile phase, which was different to the method reported

189

previously about the separated determination the determination of MDA or HHE/ HNE.25, 28,

190

29

191

respectively, for the best separation and highest resolution for the three aldehydes. Due to

192

the difference of functional group between MDA and HHE/HNE, the characteristic

193

absorption wavelengths were 310 nm for MDA-DNPH derivative and 378 nm for

194

HHE/HNE-DNPH derivative. Under these HPLC conditions, MDA-, HHE-, and HNE-DNPH

195

derivatives were well separated (Figure 3C). After the optimization of gradient eluted mobile

196

phase, a single run of these three aldehydes could finish in 30 min and the retention time

197

was 9.88, 16.31, and 29.16 min, respectively (Figure 3B).

The column temperature and flow rate were optimal at 30 °C and 1.0 mL/min,

198

Optimization of the Sample Pretreatment. Effect of Extraction solvent system. Due to

199

the great difference in functional group of MDA and HHE/HNE, extraction solvent system is

200

an important factor influencing the simultaneous analysis of these three aldehydes.

201

Therefore, to achieve the coextraction of MDA-, HHE-, and HNE-DNPH derivatives from oil

202

sample, different proportions of ethanol and water from 40:60 to 60:40 (v/v) were evaluated.

203

The results were listed in Table 1. Obviously, the coextraction of these three

204

aldehyde-DNPH derivatives was obtained with the solvent system of 50:50 ethanol/water,

205

which was used in the subsequent experiment.

206

Effect of BHT Addition. Based on the previous report that MDA, HHE, and HHE are

207

formed during oil peroxidation due to the decomposition of unsaturated fatty acid,


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208

especially long-chain unsaturated fatty acid.4,

30

209

pretreatment process or chromatographic analysis, that will lead to the extra formation of

210

target aldehydes and thus the overestimation of the analysis results. Therefore, to enhance

211

the accuracy of the developed method, the addition of BHT in different types of vegetable

212

oil samples for the inhibition of oil oxidation was evaluated by using no BHT addition as

213

control during sample pretreatment process.

If oil oxidation occurs in sample

214

Figure 4 showed the comparison of peak area of MDA-, HHE-, and HNE-DNPH

215

derivatives in PO, CO, RO, CLO, and LO with BHT addition and no BHT addition in sample

216

pretreatment. From Figure 4A, BHT addition or not in sample pretreatment did not affect the

217

determination of target aldehydes in different types of vegetable oil except LO. LO consists

218

of relatively higher content of polyunsaturated fatty acid (>70%) and thus is readily oxidized

219

to generate MDA.31 Similarly, when BHT was added into LO in sample pretreatment, the

220

peak area of HHE-DNPH derivative decreased significantly compared to the sample with

221

no BHT addition, suggesting that the precursor of HHE formation might exist in LO but not

222

in PO, CO, and CLO for no detectable HHE (Figure 4B). However, for HNE, only CO with

223

BHT addition in sample pretreatment presented significant difference with control in peak

224

area (Figure 4B), which was possibly due to the high content of linoleic acid in CO. It has

225

been identified that HNE and HHE are formed from the oxidation of ω-6 and ω-3

226

polyunsaturated fatty acid, respectively.32 Consequently, the addition of BHT is necessary

227

for the simultaneous determination of MDA, HHE, and HNE in vegetable oil with high

228

amount of polyunsaturated fatty acid, such as CO, LO, etc.

229

Effect of TCA Addition. It has been well known that MDA, HHE, and HNE are highly

230

reactive and can react with amino groups in proteins, nucleic acid, and phospholipids to



231

form the corresponding bound aldehydes, which is not able to be determined directly. Thus,

232

if the determined sample possesses certain amounts of compounds with amino groups, the

233

free aldehyde release treatment that converts bound aldehydes into free form is needful for

234

the accurate determination of total target aldehydes. As far as we know, TCA has been well

235

used to precipitate proteins and release bound aldehydes in food and biological samples.21,

236

33

237

TCA addition in sample pretreatment on MDA, HHE, and HNE analysis in heated RO, frying

238

RO, and RO extracted from French fries (Figure 5). Figure 5A and 5B indicated that TCA

239

addition did not affect the analysis results of the target aldehydes in heating RO and frying

240

RO with only few proteins. Generally, there are very few amino compounds in vegetable oil,

241

suggesting the absence of bound aldehydes during sample pretreatment process, which

242

accounts for this phenomenon in Figure 5A and 5B. However, TCA addition in sample

243

pretreatment improved the MDA determination in RO extracted from French fries, which

244

was possibly due to a certain amount of proteins in potato leading to the presence of bound

245

aldehydes in extracted oil. However, compared with MDA level, the HHE and HNE levels

246

were far lower, which are thus not enough to react with small amount of proteins.

Therefore, to verify this above hypothesis, the present study investigated the effect of

247

Method Validation. Matrix Effects. Matrix effects of the developed method above were

248

evaluated by comparing the slopes of external standard (ksolvent) and standard addition

249

calibration curve (kmatrix), which was constructed by the pure aldehydes added in

250

ethanol/water mixtures and different blank oil matrices (five kinds of vegetable oils and the

251

French fries), respectively. kmatrix= ksolvent, that is, the matrix factor (kmatrix/ksolvent) = 1

252

represents that there is no matrix effect. The signal enhancement occurs if the matrix factor

253

is > 1, and conversely, the signal suppression occurs if the matrix factor is < 1. As shown in


Page 12 of 35

Page 13 of 35


254

Table 2, the matrix factors of MDA, HHE, and HNE in PO and RO matrix samples were

255

found to be close to 1, which indicated that matrix effect did not existed in PO and RO. In

256

CLO and French fries matrix samples, the signals of these three aldehydes were

257

suppressed as their matrix factor ranged from 0.86‒0.91 and 0.75‒0.86, respectively, which

258

indicated that CLO and French fries samples presented a statistically significant matrix

259

effect for MDA, HHE, and HNE (P < 0.05). Additionally, HHE was enhanced remarkably in

260

CO and LO matrix samples as the matrix factors were 1.13 and 1.21, respectively, but MDA

261

and HNE were not influenced by matrix effects. Consequently, to reduce the matrix effects

262

for some matrix samples, such as CO, CLO, LO, etc., matrix-matched calibration curves

263

were built for the accurate determination of the target aldehydes.

264

Linearity, LOD, and LOQ. The linearity in the present study was evaluated without an oil

265

matrix for the varied matrix effects in different oil types. The linearity of the proposed

266

method under the optimal conditions was tested from 0 to 10 µg/mL for MDA and from 0 to

267

4 µg/mL for HHE and HNE with respective eight levels. As shown in Table 3, good linearity

268

of these three aldehydes was obtained in the concentration range of 0.02 ‒ 10.00 µg/mL for

269

MDA, 0.02 ‒ 4.00 µg/mL for HHE, and 0.02 ‒ 4.00 µg/mL for HNE, and the correlation

270

coefficients (R2) were all better than 0.999, which was higher than that of the previous

271

report that is the first time to simultaneously determine these three aldehydes by

272

LC-MS/MS.23 The LOD and LOQ, calculated at the signal-to-noise of 3 and 10, respectively,

273

were below 0.012 and 0.020 µg/mL for MDA, below 0.009 and 0.020 µg/mL for HHE, and

274

below 0.014 and 0.030 µg/mL for HNE, which were lower than those of Douny et al.24

275

Recovery and Precision. The recoveries of MDA, HHE, and HNE determination were

276

measured by spiking unoxidized blank RO and French fries samples with these three



277

aldehyde standards at three different levels (2.0, 3.0, and 4.0 µg/g for MDA, 0.4, 0.5, and

278

0.6 µg/g, and 1.4, 1.7, and 2.0 µg/g for HNE). The recovery values and RSDs were

279

recorded in Table 4. In RO, the average recovery values for MDA, HHE, and HNE were in

280

the range of 99.64‒102.18%, 102.34‒104.61%, and 98.87‒103.04%, with the RSD less

281

than 0.53, 1.53, and 0.66%, respectively. In French fries, the average recovery values for

282

MDA, HHE, and HNE were in the range of 96.38–98.05%, 96.1‒101.34%, and 96.86‒

283

99.04%, with the RSD less than 0.61, 0.55, and 0.87%, respectively. The results indicated

284

that the proposed RP-HPLC method could be applied to the simultaneous determination of

285

MDA, HHE, and HNE in thermally oxidized vegetable oil.

286

Analysis of MDA, HHE, and HNE Contents in Vegetable Oil during Accelerated

287

Oxidation Test at 60 °C. According to the proposed RP-HPLC method above, the contents

288

of MDA, HHE, and HNE in PO, CO, RO, CLO, and LO during accelerated oxidation test at

289

60 °C were determined. The results were shown in Figure 6. From Figure 6A, MDA

290

contents in five types of vegetable oils used in this study increased with the increasing

291

treatment time at 60 °C. Moreover, the increasing rates in RO and LO were higher than that

292

in other vegetable oils, which might be due to the relative higher degree of unsaturation in

293

RO and LO. It has been well known that MDA is formed from the decomposition of

294

unsaturated fatty acids, particularly long-chain polyunsaturated fatty acid.30, 34 MDA level

295

was up to 2.92 and 4.23 µg/g in RO and LO, respectively, stored at 60 °C for 30 days. As

296

well, with the increase of treatment time, HHE contents also increased in RO and LO, but

297

were not detected in PO, CO, and CLO (Figure 6B), which was attributed to the fact that

298

HHE was formed from the oxidation of ω-3 polyunsaturated fatty acid, especially linolenic

299

acid. Only very small levels of linolenic acids exist in PO, CO, and CLO, which accounts for


Page 14 of 35

Page 15 of 35


300

the absence of HHE in them. Also, it was well understood that LO with high levels of

301

linolenic acids exhibited a sharp increase of HHE contents during accelerated oxidation test,

302

which was consistent with the earlier report.24 For HNE, with the increase of treatment time

303

in the first 12 days, HNE levels increased gradually in five types of vegetable oils. However,

304

after 12 days of oxidation treatment, HNE levels presented a sudden increase in LO.

305

Similar phenomenon also occurred in CO and RO after day 17, and HNE levels were up to

306

3.70 and 1.70 µg/g at day 30, respectively.

307

Although nowadays no exposure limiting regulation about MDA, HHE, and HNE contents

308

in food is available, their potential toxicity to humans in biological systems has been

309

identified.35, 36 Therefore, to reduce the uptake of MDA, HHE, and HNE from vegetable oil, it

310

is essential to protect the oils and fats from oxidation or to keep a short storage time before

311

further use of oils and fats. In our following work, a mathematical model will be developed

312

for the further description of the relationship between MDA, HHE, and HNE formation and

313

oil oxidation to predict their contents in vegetable oil, and then more fully assess the quality

314

of vegetable oil.

315

Analysis of MDA, HHE, and HNE Contents in Vegetable Oil at Frying Temperature.

316

To confirm the accuracy of the proposed method above, five types of vegetable oils were

317

heated at 180 °C for 5 h. It was observed that MDA, HHE, and HNE in all original oils were

318

not detected, which might be attributed to the nonoccurrence of oil oxidation and thus the

319

non-formation of these three aldehydes. However, when these commercial vegetable oils

320

were heated at 180 °C for 5 h, MDA and HNE were detected in all tested vegetable oils,

321

while HHE was detected in RO and LO (Table 5). As mentioned above, small levels of

322

precursors of HHE, mainly linolenic acid, were not detected in PO, CO, and CLO, which



323

thus accounted for the absence of these three aldehydes in spite of high temperature

324

exposure. Additionally, compared with MDA, HHE, and HNE contents in vegetable oil

325

treated at 60 °C (Figure 6), the contents of the tested aldehydes were significantly higher in

326

corresponding vegetable oil treated at frying temperature (Table 5), which suggested that

327

MDA, HHE, and HNE levels might be used as the new evaluation indexes for the quality of

328

frying oil. Thus, in future the further research should focus on the inhibition and elimination

329

of MDA, HHE and HNE in frying oil.

330

In conclusion, the present study developed for the first time the simultaneous

331

determination method of MDA, HHE, and HNE in vegetable oil by RP-HPLC. Due to the

332

great oxidative sensitivity of unsaturated fatty acid, the sample pretreatment process could

333

be as simplified as possible. The extraction conditions, such as coextraction solvent system,

334

BHT addition, and TCA addition, were optimized in order to obtain satisfactory extraction

335

performance. Compared with the LC-MS/MS method reported previously, the RP-HPLC

336

method can reduce the testing cost and analyze rapidly and accurately the contents of

337

MDA, HHE, and HNE in vegetable oil, including heat-oxidized oil and frying oil. The method

338

possessed minimal sample preparation, low LOD and LOQ, and good linearity and

339

reproducibility, which can be used for the simultaneous routine analysis of MDA, HHE and

340

HNE in vegetable oil.

341

AUTHOR INFORMATION

342

Corresponding Author

343

*Tel: 8620-8711-4262. Fax: 8620-8711-3875. E-mail: [email protected].

344

Funding

345

The work was financed by the National Key Research and Development Program of


Page 16 of 35

Page 17 of 35


346

China (Project No. 2016YFD0400401‒5), the National Natural Science Fund of China

347

(Project Nos. 31771895, 31471677, 31271885), the National Hi-tech Research and

348

Development Project of China (Project No. 2013AA102103), the Public Welfare (Agriculture)

349

Research Project (Project No. 201303072), the Fundamental Research Funds for the

350

Central Universities of China, and Guangdong Province Laboratory for Green Processing

351

of Natural Products and Product Safety.

352 353

Notes

354

The authors declare no competing ﬁnancial interest.

355

ABBREVIATIONS USED

356

MDA, malondialdehyde; HHE, 4-hydroxy-2-hexenal; HNE, 4-hydroxy-2-nonenal; DNPH, 2,

357

4-dinitrophenylhydrazine, TCA, trichloroacetic acid; BHT, butylated hydroxytoluene; PO,

358

palm oil; CO, corn oil; RO, rapeseed oil; CLO, camellia oil; LO, linseed oil; PUFA,

359

polyunsaturated

360

chromatography; LOD, limit of detection; LOQ, limit of quantification; RSD, relative

361

standard deviation.

fatty

acid;

RP-HPLC,

reversed-phase


high-performance

liquid


362 363 364 365 366 367 368

Page 18 of 35

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the in

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Determination

Food

Matrices

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Malondialdehyde and Three Other Aldehydes in Vegetable Oil Using Liquid Chromatography Coupled to

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Tandem Mass Spectrometry (LC-MS/MS) and Application to Linseed Oil. Food Anal. Method. 2015, 8,

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1425-1435.

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Frying Oils and Their Distribution in French Fries Revealed by LC-MS-Based Chemometrics. J. Agric. Food

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Chem. 2016, 64, 3881-3889.


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prolonged heating at frying temperature: Presence of toxic oxygenated α,β unsaturated aldehydes. Food

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4-Hydroxy-2-Nonenal in Foods by Pre-Column Fluorigenic Labeling with 1,3-Cyclohexanedione and

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Reversed-Phase High-Performance Liquid Chromatography with Fluorescence Detection. J. Liq. Chromatogr.

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Salmon. J. Agric. Food Chem. 2016, 64, 487-96.

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Larondelle, Y., Linseed oil presents different patterns of oxidation in real-time and accelerated aging assays.

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459


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Page 23 of 35


460

Figure Captions

461

Figure 1. Scheme of derivatization reaction of MDA, HHE, and HNE with 2, 4-DNPH.

462

Figure 2. Analytical scheme for the simultaneous determination of MDA, HHE, and HNE in

463

vegetable oil

464

Figure 3. HPLC chromatograms of DNPH solution (A), standard solution spiked with MDA,

465

HHE, and HNE at the concentrations of 5, 2, and 2 µg/g, respectively (B), and heat-oxidized

466

linseed oil sample (C). Analysis conditions: the mobile phase was acetonitrile (solvent A)

467

and water (solvent B); the gradient elution conditions were 45% solvent A for the first 18

468

min, linear gradient from 45% solvent A to 70% in 5 min, and then isocratic at 70% solvent A

469

for 15 min; the flow rate was 1.0 mL/min; the column was ZORBAX Eclipse XDB-C18

470

column (4.6 × 250 mm, 5 µm, Agilent); the column temperature was set at 30 °C; the

471

injection volume was 10 µL; the dual detection wavelengths were 310 nm for MDA and 378

472

nm for HHE and HNE.

473

Figure 4. Influence of BHT addition in sample pretreatment on MDA (A), HHE (B), and HNE

474

(C) analysis in palm oil (PO), corn oil (CO), rapeseed oil (RO), camellia oil (CLO), and

475

linseed oil (LO). Different letters in the same vegetable oil denote significant difference

476

between data (P < 0.05).

477

Figure 5. Influence of TCA addition in sample pretreatment on MDA, HHE, and HNE

478

analysis in heated RO (A), frying RO (B), and RO extracted from French fries (C). The

479

heating and frying temperature were set at 180 °C in this research. Values labeled by

480

different letters for each aldehyde were significantly different (P < 0.05).

481

Figure 6. Evolution of MDA (A), HHE (B), and HNE (C) Contents in Five Types of Vegetable

482

Oils during Accelerated Oxidation Test for 30 Days.



Page 24 of 35

Table 1. Solvent systems used for the extraction of aldehyde-DNPH derivativesa ethanol/water 40:60 MDA HHE HNE

*

ethanol/water 45:55

ethanol/water 50:50

ethanol/water 55:45

ethanol/water 60:40

* *

* *

*

* * *

a

The presence of each aldehyde-DNPH derivative under different solvent systems was indicated by an asterisk (*).


Page 25 of 35


Table 2. Matrix Effects (kmatrix / ksolvent) of MDA, HHE, and HNE in Five Types of vegetable oils and French fries

MDA HHE HNE

PO

R2

CO

R2

RO

R2

CLO

R2

LO

R2

French friesa

R2

0.96 0.95 0.97

0.997 0.993 0.992

0.98 1.13* 1.03

0.992 0.994 0.989

0.96 1.03 0.98

0.992 0.993 0.995

0.86* 0.87* 0.91*

0.995 0.992 0.987

1.12 1.21* 0.97

0.995 0.992 0.988

0.86* 0.75* 0.79*

0.997 0.992 0.991

a

The potato sticks were fried in RO. *Statistically significant difference between matrix slope (kmatrix) and solvent slope (ksolvent) (P < 0.05). PO, palm oil; CO, corn oil; RO, rapeseed oil; CLO, camellia oil; LO, linseed oil.



Page 26 of 35

Table 3. Linear Ranges, Regression Equations, Coefficients, and Detection Limits (LOD and LOQ) of the Proposed Method Standard curve

Analyte

Linearity (µg/mL)

LOD (µg/mL)

Linear equation

R

LOQ (µg/mL)

MDA HHE

0.02 ‒ 10.00 0.02 ‒ 4.00

y = 0.5265x ‒ 0.0391 y = 0.3269x + 0.0130

0.9991 0.9996

0.012 0.009

0.020 0.020

HNE

0.03 ‒ 4.00

y = 0.0926x + 0.0038

0.9992

0.014

0.030


2

Page 27 of 35


Table 4. Recoveries and Precision of the Spiked MDA, HHE, or HNE from RO and French fries Determined by RP-HPLCa MDA Sample

RO

French fries

HHE

added (µg/g)

recovery (%)

RSD (%)

added (µg/g)

2.0 3.0 4.0 2.0 3.0 4.0

102.18±0.47b 99.64±0.53 101.16±0.36 97.64±0.39 96.38±0.59 98.05±0.34

0.46 0.53 0.36 0.40 0.61 0.35

0.4 0.5 0.6 0.4 0.5 0.6

HNE

recovery (%)

RSD (%)

added (µg/g)

102.34±0.43 104.61±0.51 103.58±0.37 98.25±0.54 101.34±0.49 96.19±1.16

0.88 1.53 1.14 0.55 0.48 0.12

1.4 1.7 2.0 1.4 1.7 2.0

a

recovery (%)

RSD (%)

101.34±0.52 103.04±0.38 98.87±0.62 96.86±1.03 98.51±0.86 99.04±0.61

0.44 0.38 0.66 0.10 0.87 0.62

Analysis conditions: the mobile phase was acetonitrile (solvent A) and water (solvent B); the gradient elution conditions were 45% solvent A for the first 18 min, linear gradient from 45% solvent A to 70% in 5 min, and then isocratic at 70% solvent A for 15 min; the flow rate was 1.0 mL/min; the column was ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm, 5 µm, Agilent); the column temperature was set at 30 °C; the injection volume was 10 µL; the dual detection wavelengths were 310 nm for MDA and 378 nm for HHE and HNE. bMean ± SD.



Page 28 of 35

Table 5. Contents of MDA, HHE, and HNE in Five Types of Vegetable Oils Heated at Frying Temperature (180 °C) for 5 h. Oil sample PO CO RO CLO LO a

MDA (µg/g oil) 2.04 ± 0.12a 2.24 ± 0.08 3.24 ± 0.14 1.83 ± 0.15 5.83 ± 0.24

HHE (µg/g oil)

HNE (µg/g oil)

NDb ND 2.23 ± 0.15 ND 5.96 ± 0.27

1.51 ± 0.15 4.78 ± 0.18 2.71 ± 0.13 1.40 ± 0.11 2.80 ± 0.23

Mean value of triplicate determination ± SD; bnot detected.


Page 29 of 35


Figure 1



Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6 ACS Paragon Plus Environment

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TOC graphic


Simultaneous Analysis of Malondialdehyde, 4-Hydroxy-2-hexenal

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