Analysis of Phenolic Antioxidants in Edible Oil/Shortening Using the PerkinElmer Altus UPLC System with PDA Detection

A P P L I C AT I O N N O T E
Liquid Chromatography
Author:
Wilhad M. Reuter
PerkinElmer, Inc.
Shelton, CT
Analysis of Phenolic
Antioxidants in Edible
Oil/Shortening Using
the PerkinElmer Altus
UPLC System with
PDA Detection
Introduction
Phenolic antioxidants are commonly used in
food to prevent the oxidation of oils. Oxidized
oil and fats cause foul odor and rancidity in food
products, which is a major cause for concern to
the food industry. Globally, regulations vary, but
current maximum allowable levels are as low as
100 µg/g (100 ppm).
This application note presents a UHPLC method
for the analysis of the ten most common phenolic
antioxidants that may be found in such products. The application was carried out with
minor modifications to the AOAC Official Method 983.15 (1). This method applies to
the analysis of finished food products. A 2.7-μm SPP (superficially porous particle) C18
column was used, allowing one to achieve very high throughput at a back-pressure
considerably lower than that for UHPLC columns.
This method was then applied to a commercial vegetable shortening product, which
per label claim, was reported to contain at least one of the antioxidants being analyzed.
Method conditions and performance data, including linearity and repeatability,
are presented.
Solvents, Standards and Samples All solvents and diluents
used were HPLC grade and filtered via 0.45-µm filters.
Experimental
Hardware/Software
For all chromatographic separations, a PerkinElmer® Altus™
UPLC® System was used, including the Altus A-30 Solvent
delivery Module, Sampling Module, A-30h Column Module and
PDA (photodiode array) Detector with a 10-mm path-length
flow cell. All instrument control, analysis and data processing
was performed using the Waters® Empower® 3 Chromatography
Data Software (CDS) platform.
Method parameters
Using a 100-mL volumetric flask, a 100-ppm stock standard
was made up by dissolving 10 mg of each of the ten antioxidant
standards in methanol and then bringing the flask up to the
mark with methanol. Individual calibrant standards were
prepared using the 100-ppm stock solution.
The HPLC method parameters are shown in Table 1.
Table 1. UHPLC Method Parameters
HPLC Conditions
Column:
PerkinElmer Brownlee™ 2.7 µm 2.1 x 100 mm C18
(Part# N9308404)
Mobile Phase:
Solvent A: Water; Solvent B: Acetonitrile
Solvent program:
Flow Rate
0%A
(mL/min)
Time
(min)
The phenolic antioxidant standard kit #2 (catalog# 40048U) was obtained from Supelco® (Irvine, CA). This included
nordihydroguaiaretic acid (NDGA), propyl gallate (PG), octyl
gallate (OG), lauryl gallate (dodecyl gallate (DG)), 2-tert-butyl4-hydroxyanisole (BHA), 2,6-di-t-butyl-4-hydroxymethylphenol
(Ionox 100), tert-butylhydroquinone (TBHQ), 3,5-di-t-butyl4-hydroxytoluene (BHT) and ethoxyquin. In addition, a
2,4,5-trihydroxybutyrophenone standard (THBP; catalog# 26201-X9) was obtained from SynQuest® (Alachua, FL).
%B
%C
%D
Curve
1
Initial
0.600
60.0
40.0
0.0
0.0
Initial
2
4.50
0.600
45.0
55.0
0.0
0.0
6
3
7.00
0.600
18.0
82.0
0.0
0.0
6
4
10.00
0.600
18.0
82.0
0.0
0.0
6
5
10.10
0.600
60.0
40.0
0.0
0.0
11
The sample (“Sample X”) was a commercially available vegetable
shortening purchased at a local food market. The sample was
prepared by dissolving 3 grams of Sample X in 15 mL of hexane
in a 50-mL centrifuge tube and vortexing for 5 minutes. The
resulting solution was then extracted with three 30-mL portions
of acetonitrile, combining the three extracts into a 250-mL
evaporation dish. The combined extract was evaporated down to
1-2 mL and reconstituted to 6 mL with methanol.
ChromatogramOnly Report
Prior to injection, all calibrants and samples were filtered through
0.22-µm filters to remove small particles.
Equil. Time (“Next inj. Delay Time”): 3 minutes
Analysis Time:
10 min.
Flow Rate:
0.6 mL/min. (maximum pressure during run: 6600 psi)
Oven Temp.:
35 ºC
Detection:
Altus A-30 PDA; wavelength channels: 280 and 220 nm
Injection Volume:
1 µL
Results and Discussion
Figure 1 shows the chromatographic separation of the 10
phenolic antioxidants in under nine minutes. Figure 2 shows
the overlay of 10 replicate 50-ppm standard injections,
THB P
0.30
PG
0.35
0.25
AU
0.20
DG
B HT
Ionox 100
B HA
0.05
E thoxyquin
TB HQ
0.10
NDGA
OG
0.15
0.00
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
Figure 1. Chromatogram of 50-ppm phenolic antioxidant standard; wavelength = 280 nm.
2
6.00
7.00
8.00
9.00
10.00
demonstrating exceptional reproducibility. Retention time %
RSDs ranged from 0.10 (early eluters) to 0.03 (later eluters).
In a previous application note (2), it has been noted that
ethoxyquin may not be well detected at 280 nm. However, we
did not observe this, and we could easily detect the analyte
at 5-ppm levels. The same injection was also captured on a
separate channel, set to 220 nm, as shown in Figure 3. At this
wavelength, it is evident that the ethoxyquin has approximately
two times the signal intensity. However, this additional signal
intensity was not really required here, as current maximum
allowable concentrations for phenolic antioxidants only go down
to 100 ppm, which was easily handled at 280 nm.
0.30
0.25
0.20
AU
0.15
0.10
0.05
ChromatogramOnly Report
0.00
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Minutes
Figure 2. Overlay of 10 replicates of 50-ppm check standard; wavelength = 280 nm.
0.80
PG
0.90
0.70
P roject Name:
E thoxyquin
Reported by User: S ystem
Report Method: Overlay Report
0.60
Report Method ID: 1803
Page: 1 of 1
0.50
B HT
DG
Ionox 100
B HA
0.10
NDGA
0.20
THB P
TB HQ
0.30
OG
AU
0.40
P henolic Antioxidants
Date P rinted:
1/22/2015
11:28:36 AM US /E astern
0.00
-0.10
-0.20
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
Minutes
Figure 3. Chromatogram of 50-ppm phenolic antioxidant standard; wavelength = 220 nm.
3
P rocessing Method: P henolicAntoxidant S ample280nm P roject Name:
P henolic Antioxidants
P rocessing Method ID: 1827
S ystem:
Wilhads First S ystem
Channel:
P DA Ch1 [email protected]
Calibration ID:
1839
P roc. Chnl. Descr.:
****
Figure 4Date
shows
three representative
calibration
resultsAM
overE Sa Tconcentration range of 5 to 100 ppm. All ten components had linearity
Calibrated:
1/22/
2015 10:21:24
coefficients > 0.999 (n = 3 at each level).
C a libra tion P lot
250000
TBHQ X Value
Level
Name
1
E thoxyquin
Level 1
3
E thoxyquin
Level 1
2200000
E thoxyquin
4
E thoxyquin
5150000
E thoxyquin
6
Area
7
E thoxyquin
E thoxyquin
Level 1
Level 2
Level 2
Level 2
Level 3
5.000000
5.000000
5.000000
10.000000
10.000000
10.000000
25.000000
Peak: Ethoxyquin
R esponse
Calc. Value % Deviation Manual Ignore
4180.550000
4414.930001
4278.369980
10363.410017
10201.120056
10215.014999
28429.939914
4.318036
-13.639
Yes
No
4.404122
-11.918
Yes
No
9.616364
-3.836
4.524300
9.759185
9.628592
25.658410
-9.514
Yes
-2.408
Yes
Yes
-3.714
Yes
2.634
Yes
8100000
E thoxyquin Level 3
25.000000
28483.275113
25.705347
2.821 Yes
P rocessing Method:
P henolicAntoxidant
S ample280nm
P roject
Name:
9
thoxyquin Level 3
25.000000
28456.070181
25.681406
2.726 Yes
P rocessing Method EID:
1827
S ystem:
10 E thoxyquin Level 4
50.000000
56468.419900
50.333325
0.667 Yes
Channel:
P DA Ch1 [email protected]
Calibration
ID:
11 50000
E thoxyquin Level 4
50.000000
56591.655240
50.441777
0.884 Yes
P roc. Chnl. Descr.:
**** Level 4 50.000000 57545.764468
12 E thoxyquin
51.281429
2.563 Yes
Date Calibrated: 13 E thoxyquin
1/22/
2015
10:21:24
AM E S T 99.936509
Level
5 100.000000
112833.269883
-0.063 Yes
14
15
1
0
E thoxyquin
E thoxyquin
Level 5
Level 5
100.000000
100.000000
X Value
Ionox 100 Level 1
5.000000
Ionox 100 Level 1
4
Ionox 100 Level 2
3 Ionox 100 Level
Ethoxyquin
1
5.000000
120000.0
Reported by User: S100000.0
5ystem
Ionox 100 Level 2
6 Ionox
Report Method: WMR
Cal100
Report
Level 2
80000.0
7 Ionox
100 Level 3
Report Method ID: 1860
1860
8 Ionox 100 Level 3
Page: 1 of 1
60000.0
Area
99.586066
-0.875
Yes
-0.414
Yes
No
No
No
No
No
No
No
WMR Cal Report
P henolic Antioxidants
Wilhads First S ystem
1839
No
No
No
No
R2=0.999834
4364.180067
C a libra
tion P lot-11.676
4.416189
Yes
No
-7.682 Yes
No
100.00
Name 5.000000
TB HQ; E4539.660024
quation Y =
2.20e+003-8.457
X -1.05e+003;
R^2 0.999834
4.577142
Yes
No
2
9
112435.055810
99.125131
No
100
20.00 Peak: Ionox40.00
60.00
80.00
CONC.
R esponse Calc. Value
% (PPM)
Deviation Manual Ignore
0.00
Level
Name
111911.288762
No
Ionox 100 Level 3
10 Ionox 100 Level 4
40000.0
11
Ionox 100 Level 4
12 Ionox 100 Level 4
20000.0
10.000000
10.000000
10.000000
25.000000
25.000000
25.000000
50.000000
50.000000
50.000000
4581.929966
10318.269980
10297.460106
10161.870040
27359.725085
27233.360184
27073.160146
54778.930195
54755.159915
54701.550188
13 Ionox 100 Level 5 100.000000 108693.381248
14 Ionox 100 Level 5 100.000000 107803.295527
0.0
15 Ionox 100 Level 5 100.000000 108025.861196
4.615913
9.877366
9.858279
-1.226 Yes
No
P rojectNoName:
-1.417 Yes
9.733914
-2.661 Yes
No
25.392130
1.569 Yes
No
25.508034
25.245193
50.657324
50.635521
50.586350
100.108435
99.292035
99.496176
2.032 Yes
0.981 Yes
1.315 Yes
1.271 Yes
1.173 Yes
0.108 Yes
-0.708 Yes
-0.504 Yes
No
P henolic Antioxidants
Date P rinted:
2/9/2015
3:01:31 P M US /E astern
No
No
No
No
No
No
No
R2=0.999686
-20000.0
0.00
20.00
40.00
60.00
CONC. (PPM)
80.00
100.00
Name E thoxyquin; E quation
Y =tion
1.14e+003
C a libra
P lot X -7.26e+002; R^2 0.999686
120000.0
Ionox 100
Area
Reported by User: S ystem
Report Method: WMR
Cal Report
100000.0
1880
Report Method ID: 1880
Page: 1 of 1
80000.0
P roject Name:
P henolic Antioxidants
Date P rinted:
2/9/2015
3:03:16 P M US /E astern
60000.0
40000.0
20000.0
0.0
R2=0.999824
-20000.0
0.00
20.00
40.00
60.00
80.00
CONC. (PPM)
100.00
Name Ionox 100; E quation Y = 1.09e+003 X -4.51e+002; R^2 0.999824
Figure 4. Three representative results of 5-level calibration sets for the phenolic antoxidants; wavelength = 280 nm.
4
Reported by User: S ystem
Report Method: WMR Cal Report
P roject Name:
P henolic Antioxidants
Date P rinted:
0.18
0.16
AU
Figure 5 shows the chromatographic results of Sample X overlaid
with the 50-ppm standard. A peak eluting at exactly the time
0.14
of TBHQ (tert-butylhydroquinone) was observed. This was
consistent with the product label claim. By back-calculating the
0.12
concentration
in the original sample, it was determined that
Sample X contained approximately 12-ppm of TBHQ. The actual
concentration could not be verified as it was not provided in the
0.10
product’s label claim.
Per Figure 6, upon closer examination of the chromatogram
of Sample X, a small peak at about 8.23 minutes was also
observed. This matched the elution time for DG (dodecyl gallate)
in the standard mix. If this was indeed DG, its concentration was
Overlay R eport
below the calibration curve, estimated
to be <0.5 ppm. Further
verification of the identity of this peak was not pursued.
0.080.26
0.24
0.060.22
0.20
0.04
TBHQ
0.18
0.02
0.16
0.14
AU
0.000.12
0.10
-0.020.08
0.06
0.00
1.00
0.04
2.00
3.00
4.00
5.00
Minutes
0.02
6.00
0.00
-0.02
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
6.00
7.00
8.00
9.00
10.00
ChromatogramOnly R eport
Figure 5. Chromatogram of Sample X (blue) overlaid with 50-ppm standard (black); wavelength = 280 nm.
R eported by User: S ystem
0.035
R
eport Method: Overla y R eport
R eported by User: S ystem
R eport Method:
Overla y R epor
R epor
t Method
ID:t 1803
R eport Method ID: 1803
P age:
1
P age: 1 of
1 of 1
0.030
P roject Na me:
P henolic Antioxidants
Da te P rinted:
1/22/2015
ChromatogramOnly R eport
TB HQ
5:08:17 P M US /E astern
0.025
0.020
AU
8.10
8.15
8.20
8.25
8.30
8.35
Minutes
8.40
8.45
8.55
8.50
8.60
8.65
0.015
0.010
R e ported by User: S ystem
R e port Me thod: Chroma togra mOnly R eport
R e port Me thod ID:
1851
1851
P age: 1 of 1
P roject Na me:
P henolic Antioxida nts
Da te P rinted:
1/22/
2015
10:55:55 AM US /E a stern
DG
0.005
0.000
0.00
1.00
2.00
3.00
4.00
5.00
Minutes
6.00
Figure 6. Chromatogram of Sample X with zoomed in area just after 8 minutes; wavelength = 280 nm.
7.00
8.00
9.00
10.00
5
Conclusion
References
This work has demonstrated the effective chromatographic
separation of ten phenolic antioxidants using a PerkinElmer Altus
UPLC® with a PDA detector and the Empower® 3 CDS system. The
results exhibited excellent retention time repeatability as well as
exceptional linearity over the tested concentration ranges. At an
analytical wavelength of 280 nm, the sensitivity for all 10 phenolic
antioxidants was found to be more than adequate to accommodate
the current maximum allowable concentration limit of 100 ppm.
1. Official Methods of Analysis, Method 983.15, Association of
Official Analytical Chemists (AOAC), Arlington, VA USA
We were able to identify and quantitate the phenolic antioxidant
content in a commercial vegetable shortening product and the
results matched the label claim of the manufacturer.
2. Monitor Antioxidant Additives in Foods, Using HPLC
(AOAC Method 983.15) or Capillary GC and a Supelco
Reference Standards Kit, Application Note 78, Sigma-Aldrich/
Supelco, 2004
3. Analysis of Common Antioxidants in Edible Oil with the
PerkinElmer Flexar™ FX-15 System Equipped with a PDA,
Application Note, PerkinElmer, Inc.
From a food quality perspective, considering the ever growing emphasis
on food monitoring, this application is intended to serve as a valuable
guide for the monitoring of edible oils/shortening. It should be noted
that in the U.S., per label claims, only some of the vegetable shortenings
reported any amount of phenolic antioxidant. None of the edible oils
that were found in stores reported any phenolic antioxidants. However,
although only edible vegetable shortening was tested for this study,
the provided sample preparation procedure and chromatographic
application easily lend themselves to the analysis of edible oils as well.
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