1 Tocopherol Content in Vegetable Oils Using a Rapid HPLC Fluorescence Detection

2 Method

4 Constantin BELE, Cristian T. MATEA*, Camelia RADUCU, Vioara MIRESAN,

5 Octavian NEGREA

7 University of Agricultural Sciences and Veterinary, 3-5 Manastur Street, RO-400372 Cluj-

8 Napoca, Romania (* corresponding author: [Link]@[Link])

10 Abstract. A quick and direct method based on reversed phase high performance liquid

11 chromatography with fluorescence detector for measuring tocopherols (α , β + γ and δ) has

12 been developed. Oils are diluted in methanol : hexane: tetrahydrofuran (neither previous

13 extraction of tocopherols nor saponification procedure are required) and after being vortexed

14 and centrifuged, an aliquot of the overlay was injected directly into an Alltima C 18 column.

15 Acetonitrile and methanol (50: 50) mixture was used as a mobile phase with a flow rate of 1

16 mL min-1. Quantification of tocopherols was performed by fluorescence detector at 290 nm

17 excitation wavelength and 325 nm emission wavelength. Tocopherols were separated at 250C

18 in less than 10 min after injection. The method has good limit of detection (9 ng g-1 for α-

19 tocopherol and 8 ng g-1 for β-, γ- and δ- tocopherols) and reproducibility (CV< 2.9 %). This

20 method can be used to assess the influence of genetic modification of oil seeds on the

21 distribution of tocopherols or the effect of tocopherols on the oxidative stability of edible

22 plant oils.

23

24 Keywords : Food analysis , Tocopherols, Oils, RP-HPLC

25
26 Introduction

27

28 Vegetable oils are probably the major dietary source of vitamin E (Hammond, 2003),

29 with a variable isomer profile according to the oil identity (Amaral et al., 2005). Tocopherols

30 protect food from oxidation by protecting the stability of oils and fats (Tangolar et al., 2011).

31 Tocopherols are a group of fat soluble antioxidants with a chromanol ring and a hydrophobic

32 side chain and exist in four different congeners (vitamers) called alpha, beta, gamma and

33 delta, which differ in the methylation pattern of the benzopyran ring (Boschin and Arnoldi

34 2011; Schwarz et al., 2008). Alpha tocopherol has three methyl groups, beta and gamma

35 forms have two methyl groups and the delta has one methyl group. The biological activities of

36 these compounds are mainly attributed to their radical-chain-breaking antioxidant in

37 membranes and lipoproteins, as well as in foods (Kamal-Eldin and Appelqvist 1996). The

38 most active form of vitamin E is α-tocopherol which is believed to protect the body against

39 degenerative malfunction, particularly cancer and cardiovascular disease (Hasani et al., 2008;

40 Zingg 2007). γ-Tocopherol has been reported to be more potent than α-tocopherol in

41 decreasing platelet aggregation, delaying intra-arterial thrombus aggregation and LDL

42 oxidation (Li et al., 1999; Saldeen et al., 1999).

43 Several methods have been described for the analysis of tocopherols by gas

44 chromatography (GC) and high performance liquid chromatography (HPLC) (Habib et al.,

45 2013; Lee et al., 2012; Slavin and Yu 2012). Analysis of tocopherols in vegetable oils by

46 HPLC can employ either normal- or reversed phase columns, as well in isocratic as in

47 gradient elution, with fluorescent, electrochemical and UV detection (Jedlicka and Klimes

48 2005). The normal phase columns provide separation of all tocopherols, while reversed phase

49 columns (usually C18) are unable to separate the β- and γ- tocopherols (Andres et al., 2011).

50 RP-HPLC is preferred over normal-phase systems due to the reproducibility of retention

52 phases (Cert et al., 2000). When the separation of β- and γ-tocopherols is not the point of

53 analysis, reversed phase columns are preferred (Gliszczynska-Swiglo and Sikorska 2004).

54 Fluorescence detection permits to get lower detection limits (Gimeno et al., 2000).

55 In this work we optimized a quick and simple method for routine analysis of

56 tocopherols in vegetable oils by RP-HPLC with fluorescence detection. The oil was diluted in

57 methanol / hexane/ tetrahydrofuran and after being vortexed and centrifuged, an aliquot of the

58 overlay was injected directly into an Alltima C18 column.

59

60 Materials and methods

61

62 Reagents and standard solutions

63

64 Acetonitrile, methanol, tetrahydrofuran and hexane were HPLC grade and were

65 purchased from Merck (Darmstadt, Germany). α-, β-, γ- and δ-Tocopherols standards were

66 obtained from Calbiochem (Merck Biosciences, Darmstadt, Germany). Standard stock

67 solutions of tocopherols were prepared in ethanol and stored at -200C in dark bottles for up

68 to a month. The exact concentrations were determined spectrophotometrically (Ryynanen et

69 al., 2004). Working standard solutions of analytes were prepared from these solutions directly

70 before analysis.

71

72 High –performance liquid chromatography (HPLC)

73

74 Separation by HPLC was carried out using a Shimadzu liquid chromatograph system

75 equipped with two delivery pumps (LC-10 AD), a FL detector (FR-10 AXL), a degasser and a
 76 model 7725i manual injector valve with a 20µl sample loop. The column was an Alltima RP

77 C-18 (250x4,6 mm, 5 µm, Alltech Associates Inc.).

78 The mobile phase was a mixture of acetonitrile and methanol (50: 50, v/v) and eluted at a

79 flow rate of 1.0 mL min-1. The analytical column was kept at 250C. The fluorescence detector

80 was set at 290 nm excitation wavelength and 325nm emission wavelength. The total

81 separation time was 10 min. The injection volume was 20µL. The tocopherols were identified

82 by comparison of the retention times with standards of the α-, β-, γ- and δ-tocopherols.

83

84 Tocopherols quantification

85

86 An external calibration was performed prior to analyses of edible plant oils, by

87 injecting different volumes (10 and 20 µL) of tocopherol working solutions ( 0.2- 5 mg L-1)

88 on column. Standard curves (concentration versus peak area) were calculated by linear

89 regression analysis. Injections in triplicate were made at each concentration for both standards

90 and samples. The calibration curves were constructed using standard solutions of α-, β-, γ-

91 and δ-tocopherol and used for quantification. The total tocopherol content is expressed as

92 milligrams per gram oil.

93

94 Sample preparation

95

96 Corn, walnut, grape seed, rice, virgin olive, sesame, peanut, sunflower oils were

97 purchased in a local market. The samples were stored in the dark at (21±2)0C until the

98 measurements were performed. Pure oil samples were weighted (about 50 mg) and diluted 10

99 times in hexane. Thereafter, 50 µL of above solution was taken into a screw-capped tube and

100 diluted with 1 mL of a mixture of methanol: hexane: tetrahydrofuran ([Link], v/v/v). The
101 sample was vortexed for 5 min and centrifuged for 10 min. at 5000 rpm. After that, the

102 sample was filtered through a 0.45 µm pore size filter and an aliquot of the clear liquid was

103 directly injected into HPLC column.

104

105 Results and discussion

106

107 Method assessment and validation

108

109 Typical chromatograms obtained for the oils tested are presented below (Fig .1). A

110 good resolution and acceptable retention times were obtained. Tocopherols were further

111 identified by comparing retention times with those of authentic standards. The retention times

112 of δ-, (β+γ)- and α-tocopherols were about 7.85min, 8.65min and 9.72 min., respectively and

113 were determined from their authentic standards which were injected both individually and as

114 a mixture .The analysis time is lower than found in the literature (Breithaupt and Kraut 2006;

115 Delgado-Zanarreno et al., 2006; Kuhn et al., 2008).Similar results were found by other

116 authors with different protocols for sample preparation and HPLC separation of tocopherols

117 (Chen et al., 2011; Gliszczynska-Swiglo and Sikorska 2004).

118

119 Fig. 1 Typical chromatograms of corn and peanut oils

121 Validation results are summarized in Table 1. Calibration curves (n = 8 points) were

122 linear between 0.05-10 µg g-1. Linear correlation coefficient (r) for all standard curves were

123 not lower than 0.995. The reproducibility of the analysis (sample dilution, injection and

124 chromatographic run) was measured by CV% of α- and (β+γ)-tocopherol (Chen et al., 2011)

125 in three replicates of olive oil and of δ-tocopherol in three replicates of walnut oil. Accuracy

126 was tested by the standard addition procedure (% of recovery). The standard mixture was

127 added to aliquots of grape seed oil at three concentration levels (n=3 replicates). The results

128 demonstrate good recovery for the tocopherols (ranging from 97.5 % to 101.0 %). The limits

129 of detection (LOD) calculated as the concentration corresponding to three times the standard

130 deviation of the baseline noise were not higher than 9 ng mL-1. The limits of quantification

131 (LOQ) were investigated by sample dilution and were not higher than 28 ng mL-1.

132

133

134

135

136

137

138

139

140

141

142

143

144
145 Tab. 1. Method validation parameters for determination of tocopherols in edible plant oils

Parameter α-Tocopherol β-Tocopherol γ-Tocopherol δ-Tocopherol

tR= 9.72± 0.1 tR= 8.65± 0.1 tR= 8.65 ± 0.1 tR=7.85±0.1
min min min min
Standard linearity

Range (µg g-1) 0.05-10 0.05-10 0.05-10 0.05-10

R2 0.9956 0.9967 0.9974 0.9979

Precision (CV%) 1.6% 2.9% 2.7% 2.3%

Sensitivity

LOD ( ng g-1)a 9 8 8 8

LOQ (ng g-1) b 28 23 23 23

Accuracy

Mean recovery (%) 98.2 97.5 98.7 101.0

146 a
Calculated based on a S/N ratio of three.

147 b
Calculated as 3x LOD.

148

149 Quantification of tocopherols in edible plant oils

150

151 The distribution of individual tocopherols and their total content in assessed oils are

152 reported in Table 2. All values are arithmetic mean of at least three separate determinations.

153 α-Tocopherol dominates in olive, grape seed, peanut and sunflower oils. In corn,

154 walnut and sesame oils (β+ γ)- tocopherols dominate. The concentration of δ-tocopherol in all

155 oils is not higher than 0.032 mg g-1. α-Tocopherol is absent in sesame oil and δ-tocopherol in

156 olive oil. The results obtained are generally in agreement with the literature data (Berasategi

157 et al., 2012; Rammell and Hoogenboom, 1985; Sanchez-Perez et al., 2000). Lack of

158 separation of β- and γ- tocopherols in the case of edible oils introduce rather small error in
159 quantification of these isomers (Gliszczynska-Swiglo and Sikorska, 2004). This is because,

160 according to literature (Crawley, 1993; Gregory, 1996) plant oils contain small quantities of

161 β-tocopherol as compared with γ- tocopherol. When necessary, all tocopherols can be fully

162 separated using normal phase chromatography.

163

164 Tab. 2. Distribution of α-, (β+ γ)-,δ- tocopherols and total tocopherol content in assessed oils

Oil sample α-Tocopherol (β+γ)-Tocopherols δ-Tocopherol Total Tocopherols

(mg per 100g) (mg per 100g) (mg per 100g) (mg per 100g)

Olive 19.53±0.1 0.91±0.01 - 20.44

Grape seed 12.45±0.01 2.14±0.01 0.71±0.04 15.3

Corn 5.60±0.02 41.12±0.4 1.82±0.04 48.54

Walnut 1.47±0.01 19.13±0.07 3.23±0.04 23.83

Sesame - 29.37±0.4 0.57±0.07 29.84

Peanut 19.22±0.2 9.32±0.2 0.91±0.03 29.45

Rice 3.31±0.05 3.56±0.03 0.62±0.05 7.49

Sunflower 73.02±0.3 1.98±0.06 0.82±0.04 75.82

165 Data are calculated from three replicated analysis of each sample ± SD.

166

167 Conclusion

168

169 This study presents a simple, fast and precise method for determination of tocopherols

170 in edible oils. The method proposed a new fast sample preparation which avoids

171 quantification errors (dilution of the oil in methanol/hexane/tetrahydrofuran mixture, no

172 saponification procedures, then the sample was vortexed-mixed and centrifuged), direct

173 injection of the overlay in a low-cost RP- HPLC column and is based on a high sensitivity and

174 selectivity of the fluorimetric detector. Tocopherols can preserve their stability by using the
175 protocol proposed for sample preparation. Lack of separation of β- and γ- tocopherols by RP-

176 HPLC did not introduce major error in the determination of latter one because vegetable oils

177 contain small quantities of β- tocopherol as compared to γ- tocopherol. Therefore, the method

178 proposed can be useful for the routine analysis of α-, (β+γ)– and δ- tocopherols in edible

179 vegetable oils and is comparable only to a few published methods (Chen et al., 2011;

180 Gliszczynska-Swiglo and Sikorska, 2004). This method can be used to assess the influence of

181 genetic modification of oil seeds on the distribution of tocopherols or the effect of tocopherols

182 on the oxidative stability of edible plant oils.

183

184 Acknowledgements

185

186 This work was partially financed by grant USAMV-CJ RU no. 1283/2013. The

187 authors acknowledge the support of the Society for Interdisciplinary Applicative Research.

188

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