Quality Analysis of Virgin Olive Oils –

Part 4
Nutritive benefits - Quantification of tocopherols and
tocotrienols in virgin olive oil using the Agilent 1260
Infinity Bio-inert Quaternary LC system

Application Note
Food Analysis & Agriculture

Author LU
a-Tocopherol

5
Sonja Schneider
Agilent Technologies, Inc. 4

Waldbronn, Germany 3
g-Tocopherol
b-Tocopherol

b-Tocotrienol

0
0 5 10 15 20 min25

Abstract
This Application Note shows the analysis of tocopherols and tocotrienols in virgin
olive oil samples using normal phase chromatography on the Agilent 1260 Infinity
Bio-inert Quaternary LC system with fluorescence detection. Using the bio-inert
system, huge improvements of retention time and area precision were observed
in comparison to the standard stainless steel system. Very good linearity was
obtained for all eight vitamin E isomers together with limits of detection (LOD) and
limits of quantitation (LOQ) in the low µg/mL range. The analysis of nine olive oil
samples revealed an a-tocopherol content between 100 to 390 mg/kg olive oil. No
difference in the vitamin E content was detected between partly refined and virgin
Verified for Agilent
lent olive oils.
1260 Infinity II LC
Bio-inert System
Introduction Due to the diversity of biological activ- Tocopherols are known for being
ity, which can be related to most of the sensitive to degradation processes
Virgin olive oil is obtained from the single tocopherols and tocotrienols in into products such as, for example,
fruits of the olive tree (Olea europea L.) cancer or infarction prevention, it is tocored, tocopurple, para-tocopherol,
only by mechanical procedures, and essential to gain a complete vitamin E quinone, and dimerization reactions7.
without the use of any thermal or profile6. Suitable chromatographic Degradation processes occur espe-
chemical treatment. To ensure the techniques that separate, identify, cially by photooxidation but also by
authenticity of virgin olive oil regarding and quantify all vitamin E isoforms oxidation in the presence of Fe3+ or
adulteration, mislabeling, characteriza- are important elements for complete Cu2+ ions7. Due to the latter, liquid
tion, or misleading origin, numerous vitamin E profiling. Both, reversed (RP) chromatographic analysis in stain-
methods have been developed for the and normal phase (NP) high pressure less steel HPLC/UHPLC systems, is
evaluation of vegetable oils in the last liquid chromatography (HPLC) can be prone to result in very poor retention
two decades1. The analysis of thermally applied for the analysis of tocopherols time and area precision. To compare
treated olive oils has been shown in and tocotrienols. This Application Note reproducibility regarding retention time
previous application notes2,3,4. used normal phase ultra high pres- (RT) and area, both a stainless steel
sure chromatography (UHPLC) due to (Agilent 1260 Infinity Quaternary LC
Regarding nutritive benefits, virgin the good separation of all vitamin E system) and a bio-inert system (Agilent
olive oil is a good source of several isomers6. The fluorescing properties of 1260 Infinity Bio-inert Quaternary
bioactive components related to the vitamin E isomers permit detec- LC system) are compared in this
highly chemoprotective effects on tion through sensitive fluorescence Application Note.
human health. In addition to the high detection.
amount of mono-unsaturated and
polyunsaturated fatty acids, it contains
highly valuable antioxidants such as
phytosterols, carotenoids, phenols, R1 R2
and vitamin E5,6. The levels of these a-Tocopherol/trienol CH3 CH3
bioactive components are dependent b-Tocopherol/trienol CH3 H
on genetic, agronomic, and environ-
g-Tocopherol/trienol H CH3
mental factors. The name vitamin E Tocopherol
comprises four tocopherol (a-,b-,g-, d-Tocopherol/trienol H H
and d-tocopherol) and four tocotrienol
(a-,b-,g-, and d-tocotrienol) isomers.
All eight forms contain a chromanol
ring and an isoprenyl with three double
bonds in tocotrienols, see Figure 1.
In virgin olive oil, 95% of the total
Tocotrienol
tocopherol/tocotrienol content is
a-tocopherol6.
Figure 1
Tocopherol and tocotrienol structure.

2
Experimental To perform normal phase chromatogra- Solvents
phy on the 1260 Infinity Quaternary LC N-heptane and methyl tert-butyl ether
The Agilent 1260 Infinity Bio-inert systems (stainless steel and bio-inert), (tBME) were LC grade and purchased
Quaternary LC system consisted of the the wash and piston seals have to from Sigma-Aldrich, St. Louis, MO,
following modules: be exchanged from reversed phase USA.
(standard) to normal phase:
• Agilent 1260 Infinity Bio-inert
• Piston seals - normal phase
Columns
Quaternary Pump (G5611A)
(p/n 0905-1420) Agilent LiChrospher DIOL,
• Agilent 1260 Infinity Bio-inert 4 × 250 mm, 5 µm (p/n 79925DI-584)
High Performance Autosampler • Wash seals (PE) (p/n 0905-1718)
(G5667A) Software
Sample
• Agilent 1290 Infinity Thermostat The Tocotrienol and Tocopherole Agilent OpenLAB CDS ChemStation
(G1330B) for sample cooling Mixed Solution Standard (in Hexane) Edition for LC & LC MS Systems, Rev.
was purchased from LGC Standards, C.01.04 [35]
• Agilent 1290 Infinity Thermostatted
Column Compartment (G1316C) with Teddington, UK. Several olive oils
bio-inert solvent heat exchanger (virgin and partly refined olive oil) were
purchased in local stores. For normal
• Agilent 1260 Infinity Fluorescence
phase analysis, the olive oil samples
Detector (G1321B), equipped with
were diluted with heptane to a concen-
bio-inert standard FLD flow cell
tration of 100 mg/mL before injection
The Agilent 1260 Infinity Quaternary into the UHPLC system.
LC system consisted of the following
modules:

• Agilent 1260 Infinity Quaternary Chromatographic conditions

Pump (G1311B)
Long run
• Agilent 1260 Infinity High Mobile phase: tBME:Heptane (5:95)
Performance Autosampler
Flow rate: 1 mL/min
(G1367E)
Isocratic run: Stop time – 45 minutes
• Agilent 1290 Infinity Thermostat Injection volume: 10 µL
(G1330B) for sample cooling
Temperature thermostat: 4 °C
• Agilent 1290 Thermostatted Column Temperature TCC: 35 °C
Compartment (G1316C)
DAD: Ex: 298 nm, Em: 330 nm
• Agilent 1260 Infinity Fluorescence Peak width: > 0.05 minutes (1 second resp. time) (9.26 Hz)
Detector (G1321B), equipped with Table 1
standard FLD flow cell Chromatographic conditions.

3
Results and Discussion LU

a-Tocotrienol
a-Tocopherol

d-Tocotrienol
g-Tocotrienol
30
Figure 2 shows the successful sepa-

g-Tocopherol

d-Tocopherol
ration of all vitamin E isomers of the
25
tocopherol and tocotrienol standard

b-Tocopherol
from LGC Standards using NP chroma- 20
tography. The precision of retention
time and area was determined on two

b-Tocotrienol
15
systems, the 1260 Infinity Quaternary
LC system and the 1260 Infinity Bio- 10
inert Quaternary LC system. Figure 2
shows an overlay of six runs on the 5
bio-inert system with excellent relative
standard deviations, regarding both 0
retention time and area. 0 5 10 15 20 25 30 35 40 min

Table 2 summarizes the results for Figure 2

vitamin E isomer analysis on both Overlay of six runs of all vitamin E isomers on the Agilent Infinity Bio-inert Quaternary LC system.

systems. On the bio-inert system,

the results were much better for RT
Agilent 1260 Infinity Bio-inert Agilent 1260 Infinity
precision and area due to oxidation
Quaternary LC system Quaternary LC system
processes of the isomers in presence
of Fe3+ ions7. Regarding RT precision, RSD RT (%) RSD area (%) RSD RT (%) RSD area (%)
the bio-inert system showed up to ten a-Tocopherol 0.069 0.63 0.677 2.64
times better performance resulting in a-Tocotrienol 0.062 0.94 0.658 2.88
very good RT precision values under b-Tocopherol 0.053 0.64 0.65 2.78
0.07% for example for a-tocopherol in
g-Tocopherol 0.103 0.61 0.691 2.73
contrary to 0.68% RSD on the stain-
less steel system. Area precision also b-Tocotrienol 0.103 0.79 0.708 3.32
improved between three and ten (four g-Tocotrienol 0.097 0.84 0.67 3.78
times for a-tocopherol) times better on d-Tocopherol 0.062 0.29 0 3.16
the bio-inert system. d-Tocotrienol 0 0.34 0 2.74
Table 2
Precision of retention time and area on the Agilent 1260 Infinity SST Bio-inert Quaternary LC system and the Agilent
1260 Infinity Bio-inert Quaternary LC system.

4
To determine linearity, LOD and LOQ, relationship was determined between Very good linearity was determined for
the tocopherol and tocotrienol standard the peak area and the corresponding all vitamin E isomers with correlation
solution was diluted in n-heptane in 1:3 concentrations. LOD and LOQ were coefficients >0.9998. LOD and LOQ
dilution steps, see Table 3. defined as the signal to noise ratio of were found in the low µg/mL range
3:1 respectively 10:1. Table 4 shows the where, for example in olive oil, small
Eight different concentration results for linearity and LOD and LOQ amounts of b-tocopherol and b-tocot-
levels were prepared and the linear for all vitamin E isomers. rienol are found.

Level 1 Level 2 Level 3 Level 4 Level 5 Level 6 Level 7 Level 8

Analyte (µg/mL) (µg/mL) (µg/mL)] (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL)
a-Tocopherol 463.00 154.33 51.44 17.15 5.72 1.91 0.64 0.21
a-Tocotrienol 447.00 149.00 49.67 16.56 5.52 1.84 0.61 0.20
b-Tocopherol 203.00 67.67 22.56 7.52 2.51 0.84 0.28 0.09
g-Tocopherol 430.00 143.33 47.78 15.93 5.31 1.77 0.59 0.20
b-Tocotrienol 135.00 45.00 15.00 5.00 1.67 0.56 0.19 0.06
g-Tocotrienol 467.00 155.67 51.89 17.30 5.77 1.92 0.64 0.21
d-Tocopherol 423.00 141.00 47.00 15.67 5.22 1.74 0.58 0.19
d-Tocotrienol 414.00 138.00 46.00 15.33 5.11 1.70 0.57 0.19
Table 3
Dilution series of the tocopherol and tocotrienol standards.

Analyte Correlation LOD (µg/mL) LOQ (µg/mL)

a-Tocopherol 0.99984 3.34 11.14
a-Tocotrienol 0.99995 2.97 9.89
b-Tocopherol 0.99998 1.70 5.66
g-Tocopherol 0.99992 1.45 4.82
b-Tocotrienol 0.99998 1.75 5.82
g-Tocotrienol 0.99997 1.73 5.76
d-Tocopherol 0.99992 1.03 3.44
d-Tocotrienol 0.99990 0.99 3.29
Table 4
Linearity of tocopherols and tocotrienols standard dilution series analyzed
on the Agilent 1260 Infinity Bio-inert Quaternary LC system.

5
Figure 3 shows the analysis of virgin
LU
olive oil for its comprising vitamin E iso-

a-Tocopherol
mers. Like reported in literature6, 95%
of all vitamin E isomers is a-tocopherol 5

in virgin olive oil. In addition, small

amounts of g-tocopherol, b-tocopherol 4

and b-tocotrienol were found.

3
A total of nine olive oil samples

g-Tocopherol
were analyzed for their vitamin E 2

b-Tocotrienol
b-Tocopherol
content, see Table 5. The amounts
of determined vitamin E isomers: 1
a-,b-,g-tocopherol and b-tocotrienol
was correspondent to the amounts 0
described in literature, for example, for 0 5 10 15 20 25 30 35 40 min
a-tocopherol from approximately 100
to 390 mg per kg olive oil6,8. There was Figure 3
Analysis of virgin olive oil for vitamin E isomers.
no difference found between partly
refined (sample #3) and virgin olive oils
(#1, 2 and #4 - #8) regarding vitamin E
content.

#1 #2 #3 #4 #5 #6 #7 #8 #9
Olive oil mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg
a-Tocopherol 389.7 315.8 179.8 152.1 109.8 168.2 98.9 114.2 337.3
b-Tocopherol 5.4 3.5 2.5 2.0 3.8 3.0 2.7 2.6 4.3
g-Tocopherol 9.1 11.8 12.1 6.6 5.8 9.2 12.0 1.8 4.1
b-Tocotrienol 12.3 5.8 5.3 4.8 3.6 4.9 4.2 5.8 13.3
d-Tocopherol - - - - - - 0.4 - -
Table 5
Vitamin E content of nine analyzed olive oil samples.

6
Conclusion References 5.
Owen et al., “Phenolic compounds and
Tocopherols are prone to degradation 1. squalene in olive oils: the concentra-
processes by oxidation in the presence Aparicio and Ruiz, “Authentication tion and antioxidant potential of total
of Fe3+ or Cu2+ ions7. Therefore, it is of vegetable oils by chromato- phenols, simple phenols, secoiridoids,
highly recommended to use the Agilent graphic techniques,” Journal of lignans and squalene,” Food and
1260 Infinity Bio-inert Quaternary LC Chromatography A, 881, 93–104, 2000 Chemical Toxicology, 38, 647-659, 2000
system for the analysis of tocophe-
nols and tocotrienols for the highest 2. 6.
precision of retention time and area. Schneider, “Quality Analysis of Virgin Cunha et al., “Quantification of
With the bio-inert system, an up to 10x Olive Oils – Part 1,” Agilent Application Tocopherols and Tocotrienols in
improvement of retention time preci- Note, Publication Number 5991- Portuguese Olive Oils Using HPLC with
sion was observed. In addition, area 1894EN, 2013 Three Different Detection Systems,”
precision improved approximately 10x Journal of Agricultural and Food
3. Chemistry, 54, 3351-3356, 2006
on the bio-inert system.
Schneider, “Quality Analysis of Virgin
Using the bio-inert system together Olive Oils – Part 2,” Agilent Application 7.
with normal phase chromatography Note, Publication Number 5991- Cort et al., “Stability of Alpha- and
using heptane, very good linearity 1985EN, 2013 Gamma-Tocopherol: Fe3+ and Cu2+
was obtained for all eight vitamin Interactions,” Journal of Food Science,
4. 43, 797-798, 1978
E isomers together with LODs and
Schneider, “Quality Analysis of Virgin
LOQs in the low µg/mL range. The 8.
Olive Oils – Part 3,” Agilent Application
tocopherol/toctrienol amount of nine Psomiadou et al., “a-Tocopherol
Note, Publication Number 5991-
olive oil samples was found in a range Content of Greek Virgin Olive Oils,”
1896EN, 2013
between 100 to 390 mg/kg olive oil for Journal of Agricultural and Food
example for a-tocopherol. No differ- Chemistry, 48, 1770-1775, 2000
ence in the vitamin E content was
detected between partly refined and
virgin olive oils.

7
www.agilent.com/
chem/1200BioLC
© Agilent Technologies, Inc., 2013-2016
Published in the USA, May 1, 2016
5991-2180EN