Natural Compounds and Additives – Food Compendium

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Analysis of sweeteners in food and
beverages with the Agilent 1120 Compact
LC system
Application Note
Rongjie Fu,
Zhixiu Xu
Abstract
An analysis method for three of the most popular artificial sweeteners
was developed in this application, and the sweeteners were analyzed in
Agilent Equipment a food and a beverage. The system suitability results showed that the
• Agilent 1120 Compact LC
Agilent 1120 Compact LC is the system of choice for conventional, ana-
(cid:129) EZChrom Elite Compact software
lytical scale liquid chromatography. This integrated HPLC system was
Application Area
designed for ease of use, performance, and reliability. The quantitative
(cid:129) Food and beverage
analysis of typical samples is demonstrated in this Application Note.
Introduction For this application, the most (cid:129) Water was obtained from a
widely used sweeteners were ana- Millipore water purifier.
lyzed in samples of yogurt and a (cid:129) Acetonitrile (Fisher Scientific)
Artificial sweeteners are widely
beverage. The analysis was per- was HPLC purity. All other
used all over the world, and some
formed with the Agilent 1120 reagents were analytical purity.
of them have a long history. For
Compact LC, which is the system
example, saccharin was invented
of choice for conventional, analyti- Sample preparation
nearly 100 years ago. Artificial
cal scale liquid chromatography. Yogurt: 5 mL was diluted with
sweeteners taste similar to cane
It is an integrated HPLC system 5 mL methanol, and then the mix-
sugar, but are low-calorie. They
designed for ease of use, perfor- ture was stirred and centrifuged.
benefit overweight people and
mance, and reliability. It is ideally The sample was filtered with a
those who have problems with
suited for routine analyses in 0.45 µm filter prior to injection.
sugar metabolism. Artificial
the food industry because of its
sweeteners are also cheaper than
capability to achieve very precise Diet cola: The sample was treated
natural sugar and can reduce the
retention times and peak areas, as with an ultrasonic for 10 minutes,
cost for some foods and bever-
well as low detection limits for the and then was filtered with a 0.45
ages. However, scientific research
analyzed compounds. µm filter prior to injection.
has shown that some of them can
cause tumors in certain animals,
Chromatographic conditions
so to prevent potential danger to
humans, it is necessary to control Experimental (cid:129) Column: Agilent TC-C18(2),
4.6 x 250 mm, 5 µm
the amount of sweeteners in foods
(cid:129) Mobile phase: A = 20 mM
and beverages. Equipment
KH PO buffer, pH 3.0;
• Agilent 1120 Compact LC system 2 4
B = acetonitrile
with gradient pump (degasser
(cid:129) Gradient:0 min 15 %B
Regulations set an upper limit on inside), autosampler, column
5 min 35 %B
the concentration of artificial compartment, and variable
10 min 80 %B
sweeteners in foods and bever- wavelength detector (VWD)
(cid:129) Flow rate: 1 mL/min
ages. The labels of foods and bev- (cid:129) EZChrom Elite Compact
(cid:129) Wavelength: 214 nm
erages should list what kinds of software
(cid:129) Injection volume: 5 µL
sweeteners are used. Quality con-
(cid:129) Temperature: 30 °C
trol or spot-checking can use a Chemicals and reagents
conventional HPLC method to (cid:129) Reference standards were
determine the amount of the purchased from Sinopharm
sweeteners in the samples. Chemical Reagent Co. Ltd.,
Shanghai, China.
2
Results and discussion
1000
2 Saccharin
The separation of standards of
three sweeteners (acesulfame,
800
saccharin, and aspartame) was 3 Aspartame
1 Acesulfame Standard
done in eight minutes. To make
sure the other components of the
600
real sample were eluted from the
column, the final method needed AU
m Yogurt
11 minutes runtime.
400
By overlaying the chromatograms
of the standards and the real sam-
200 Diet cola
ples, one can easily find out which
kind of sweeteners are used in
specific samples. As shown in fig-
0
ure 1, the samples of yogurt and
0 1 2 3 4 5 6 7 8 9 10 11 12
diet cola that were used for this Time [min]
test contained both acesulfame Figure 1
and aspartame, but no saccharin. Overlaid chromatograms of sweetener standards and real samples.
The linearity of the compounds Peak Compound Range (ng) r2
was tested within the amount 1 Acesulfame 18.75 - 1500 0.99999
range from 18.75 to 1500 ng, 2 Saccharin 18.75 - 1500 0.99997
which covers the most likely 3 Aspartame 18.75 - 1500 0.99998
amounts injected on to the col- Table 1
umn in real sample analysis. The Linearity of the sweetener standards.
results are shown in table 1. The
data shows that very good regres- Peak Compound % RSD retention times % RSD areas
sion factors (values of r2) were 1 Acesulfame 0.075 0.09
achieved for each compound. 2 Saccharin 0.070 0.24
3 Aspartame 0.033 0.23
The system reproducibility was Table 2
Reproducibility of the 10 injections of sweetener standards.
also tested with the three com-
pounds. The high precision of the
Acesulfame Aspartame
retention times gives high confi-
Yogurt 0.09 mg/mL 0.027 mg/mL
dence when comparing the stan-
Diet cola 0.205 mg/mL 0.146 mg/mL
dards and real samples.
Table 3
The amount of sweeteners in the real samples.
The quantitative results from the
two samples are shown in table 3.
and system suitability experiments
showed the robustness and high
Conclusion
precision for this kind of applica-
The Agilent 1120 Compact LC is tion. The high precision of the
ideal for the routine analysis of retention times and peak areas
sweeteners in foods and bever- ensures reliable results when
ages. Excellent resolution and quantitation is needed for quality
good separation were achieved, control.
3
References
1.
X. Ping, “Analysis of sweeteners in
food and beverage using Agilent
TC C18”, Agilent Technologies
Application Note, publication
number 5989-3890CHCN,2005.
2.
W. Liu, “Determination of
Antiseptic and Sweetener in wine
by HPLC”, Liaoning Chemical
Industry, 35(4), 238,2006.
3.
X. Sheng, “Solid phase extraction-
liquid chromatography/mass spec-
trometry for simultaneous deter-
mination of artificial synthetic
sulfa sweeteners in food”, Chinese
Journal of Analysis Laboratory,
25(7), 75,2006.
Rongjie Fu and Zhixiu Xu are
Application Chemists at Agilent
Technologies in Shanghai, China.
www.agilent.com/chem/1120
© 2008 Agilent Technologies Inc.
Published October 1, 2008
Publication Number 5989-8961EN
Ultrafast analysis of synthetic
antioxidants in vegetable oils using
the Agilent 1290 Infinity LC system
Application Note
Food
Authors
Standard solution
Gerd Vanhoenacker, Frank David, mAU
10 BP
Pat Sandra 8 TH
6
RKeensenaerdcyhp aInrks t2it6ute for Chromatography 024 PG TBHQ NDGA BHA Ionox-100 OG DG BHT AP
B-8500 Kortrijk 0.2 0.4 0.6 0.8 1 1.2 1.4 min
Belgium
Abstract
The addition of synthetic antioxidants in edible vegetable oils is regulated in Europe
and the US. The official method was translated into an ultrafast LC method using the
Agilent 1290 Infinity LC equipped with an Agilent ZORBAX Rapid Resolution High
Definition (RRHD) column. High throughput is obtained in 2 min with a backpressure
of 1120 bar, which is below the 1200 bar upper limit of the column. Optimization of the
mobile phase composition and the temperature are discussed. The figures of merit are
illustrated using standard solutions and spiked vegetable oil (sunflower, rapeseed, and
olive) extracts. Limits of detection are 1 mg/kg or less in the oil samples. Using a sim-
ple methanol extraction, good recovery was obtained for all antioxidants in the oil
samples.
Introduction
O O O O O O O C3H7
Lipid oxidation causes rancidity and C3H7 C8H17 C12H25
OH
odor problems and decreases the nutri-
tional value of food products. Synthetic
HO OH HO OH HO OH HO
ascorbyl palmitate and phenolic antioxi-
OH OH OH OH
dants are often added to foods to pre-
Propyl gallate Octyl gallate Dodecyl gallate 2,4,5-trihydroxybutyrophenone
vent oxidation of unsaturated fatty
(PG) (OG) (DG) (THBP)
acids in oils and fats. Combinations of
antioxidants are commonly used to
enhance the antioxidative effect. The OH HO O O
structures and abbreviations of the C(CH3)3 OH C(CH3)3
HO
investigated antioxidants are shown in
Figure 1. OH C(CH3)3
OH OH OH
Regulatory agencies in Europe1and the
tert-butyl-hydroquinone Nordihydroguaiaretic acid 2- and 3-tert-butyl-4-hydroxyanisole
US2have imposed maximum levels for (TBHQ) (NDGA) (BHA)
some antioxidants while the use of oth-
ers has been forbidden. The determina-
tion of antioxidants in foods and food OH OH OH
components is therefore an important (H3C)3C C(CH3)3 (H3C)3C C(CH3)3 O O O
C H
15 31
analysis. The limits are given in Table 1.
HOOH OH O
HO
3,5-di-tert-butyl-4- 2,6-di-tert-butyl-4-hydroxymethylphenol Ascorbyl palmitate
hydroxytoluene (BHT) (Ionox-100) (AP)
Figure 1
Structures and codes of the investigated antioxidants.
Antioxidant Europe1 US2
AP Quantum satis No restriction
PG ~200 mg/kg, individual or combined ~200 mg/kg, individual or combined
OG
DG
BHA
BHT ~100 mg/kg
TBHQ Not allowed
THBP Not allowed Not allowed
NDGA Not allowed Not allowed
Ionox-100 Not allowed Not allowed
Table 1
Limits for antioxidants in edible oils in Europe and US.
2
In the official method for the determina- result is desirable for economical and Solutions and samples
tion of the antioxidants in edible oils, practical reasons.
Sample and standard solutions were
columns of 15 to 25 cm in length with
an internal diameter of 4.6 mm, and This Application Note describes the prepared according to Perrin and
packed with 5-µm octadecyl silica parti- analysis of 10 antioxidants in vegetable Meyer.4The solvent for the standards
cles are used.3The mobile phase is oils using the Agilent 1290 Infinity LC. and extraction is a solution of citric acid
composed of diluted acetic or phos- The original method was translated into (1 mg/mL) and isoascorbic acid (1
phoric acid (eluent A) and methanol/ a high throughput method by optimizing mg/mL) in methanol. For the spiked
acetonitrile 50/50 volume to volume the mobile phase composition and the samples, a stock solution of the antioxi-
(eluent B). Analysis times are between temperature. The figures of merit are dants in the solvent was added prior to
15 to 25 min. presented for vegetable oil and spiked extraction. The extraction was carried
oil extracts. out by weighing 1 g of oil and adding 10
There are two reasons for increasing mL of the solvent. This mixture was
Experimental
the speed of analysis for this applica- vortexed for 30 s, allowed to stand for 2
tion. First, instability of some of the tar- min, and vortexed once more for 30 s.
Instrumentation and method
gets (for example, AP) have been The sample was then centrifuged at
reported and long residence times of An Agilent 1290 Infinity LC system with 5000 x g for 5 min and the supernatant
samples in an autosampler can already the configuration in Table 2 was used: was transferred into an autosampler
lead to significant degradation of the vial for injection.
compounds. Perrin and Meyer could
enhance the stability of sample and Part number Description
standard solutions by using citric and G4220A Agilent 1290 Infinity Binary Pump with integrated vacuum degasser
isoascorbic acid.4They were able to
G4226A Agilent 1290 Infinity Autosampler
stabilize AP at room temperature for
G1316C Agilent 1290 Infinity Thermostatted Column Compartment
about 7 h. However, QC laboratories in
G4212A Agilent 1290 Infinity Diode Array Detector
edible oil and fat processing industries
have a need for increased analysis
speed. The presence or absence, and
Method parameters:
assay of antioxidants have to be carried
out prior to loading or unloading oils Column ZORBAX RRHD Eclipse Plus C18, 50 mm L × 2.1 mm id, 1.8 µm dp
and fats. A fast, accurate, and precise Mobile phase A = 0.02% phosphoric acid in water
B = Acetonitrile/methanol 50/50 or 75/25 v/v
Flow rate Variable
Gradient Variable
Temperature Variable
Injection 2 µL
Detection DAD, 40 or 80 Hz
Phenolic antioxidants Signal 280/10 nm, Reference 400/50 nm
Ascorbyl palmitate Signal 255/10 nm, Reference 400/50 nm
Table 2
Conditions
3
Results and Discussion
A B C
Methanol/Acetonitrile 50/50 75/25 75/25
The analysis was first carried out with
ratio (v/v)
the mobile phase used in the official
Flow rate 0.4 mL/min 0.4 mL/min 1.9 mL/min
method. The flow rate was set at a
Gradient 0–7.5 min: 35–100% B 0–7.5 min: 30–100% B 0–1.6 min: 30–100% B
moderate 0.4 mL/min. The analysis
time was 8 min (see Figure 2A). The Temperature 30 °C 45 °C 45 °C
synthetic phenolic antioxidants are all Detector speed 40 Hz 40 Hz 80 Hz
detected at 280 nm while for ascorbyl Maximum pressure 375 bar 270 bar 1120 bar
palmitate (AP) 255 nmwas used. The
eluent B composition was then modi-
fied from methanol/acetonitrile 50/50
to 75/25 volume to volume to lower the A.
viscosity and enable a faster separa- mAU
tion. The selectivity changed consider- 120
P
ably with this mobile phase adaptation 100 THB
and the temperature was optimized to
obtain sufficient separation between all 80
target antioxidants. Note that at 45 °C, 60
ta2ahAnt ed4 a 5eBt l °u3HC0tTi oc° inCos .l ourAermdvlleen crr sot oeemfmd pd pcoooeudmrneapcdtuyasrlr eweg.da e lrtloea tFseit ga(uDbrlGee) 42000 PG TBHQ NDGA BHA Ionox-100 OG BHT DG AP
1 2 3 4 5 6 7 min
An additional advantage of the
increased temperature is the decrease B.
of the backpressure. When the flow mAU
rate was increased to 1.9 mL/min the 120 BP
last peak eluted under 1.5 min and the TH
100
pressure on the column was 1120 bar
(Figure 2C). 80
60
42000 PG TBHQ NDGA BHA Ionox-100 OG DG BHT AP
1 2 3 4 5 6 7 min
C.
mAU
P
B
H
80 T

60
42000 PG TBHQ NDGA BHA Ionox-100 OG DG BHT AP
0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 min
Figure 2
Analysis of 10 µg/mL standard solution under the various conditions.
4