Application Note
Food Testing &Agriculture

Analysis of Parabens in Soy Sauce
and Vinegar by HPLC Using
Agilent Chem Elut S Supported Liquid
Extraction (SLE) Cartridges
AuthorXia Yang
Agilent Technologies, Inc.

Abstract
This Application Note demonstrates the use of Agilent Chem Elut S 12 cc supported
liquid extraction (SLE) cartridges for the analysis of seven parabens from soy sauce
and vinegar by LC/DAD detection. Both traditional liquid/liquid extraction (LLE)
and four commercially available SLE products were tested for comparison. As
a technique, SLE provides a simplified workflow compared to traditional LLE,
with significant savings in time and effort. Chem Elut S uses a novel synthetic
media demonstrating improved performance with the consideration of recovery,
reproducibility, capacity, and cleanliness.

Introduction
LLE is a traditional sample preparation
technique that can be applied to manyaqueous sample extractions by altering
the ion strength of the aqueous phaseand the extraction solvent polarity. It
has demonstrated the ability to extract
analytes from various aqueous matrices
with good recovery and reproducibility.
However, LLE can be time-consuming
and labor-intensive in sample mixing foranalyte partition, clear phase separation
for removing the organic phase, andemulsion formation. LLE can also bemuch more challenging for multiple
extractions, which requires significantly
more time and effort, and can be difficultfor high-throughput laboratories seeking
automated workflows.
SLE is an improved technique that canreplace LLE with the advantages oftime and effort savings, no emulsion
formation, and easy automation.
Conventional SLE products use natural
diatomaceous earth (DE) material, which
is difficult to control for sorbent quality
and particle consistency, which cancause product performance variability.
The novel synthetic Chem Elut S material
is manufactured in a controlled processwith strict monitoring of sorbent quality
and consistency. Chem Elut S has greatly
improved product performance andreproducibility.
Parabens are para-hydroxybenzoic acid
esters (Figure 1), which are widely added
as preservatives in foods such as baked
goods, sauces, juices, and jellies. The useof parabens can prolong the shelf life offood products by preventing mold, yeast,and bacteria growth. In some countries
such as the US and China, parabens arerecognized as safe at certain permissiblelevels, while they are banned in someother countries such as Malaysia.

2

Experimental

There are some suggestions for foodsample preparation using LLE in Chinese
regulation method GB5009.31‑2016.1
However, the method is verytime‑consuming, with multiple steps
of extraction, and is environmentally
unfriendly due to large amounts ofdiethyl ether. This Application Notedemonstrates the use of Chem Elut S
12 cc cartridges for the analysis of sevenparabens in soy sauce and vinegar using
ethyl acetate as the extraction solvent.
Compared with the regulation methodof LLE, the Chem Elut S method useda low volume of a less toxic solvent.
This method also provided a simplified
workflow, saving time and effort whileachieving excellent performance andcleanliness.
O

Reagent and chemicals
All regents and solvents were HPLCor analytical grade. Methanol (MeOH)
was from Honeywell (Muskegon, MI,
USA), ethyl acetate (EtOAc) was fromDikma (Lake forest, CA, USA), andsodium chloride was from SinopharmChemical Reagent (Shanghai, China).
The paraben standards methyl
paraben (MP), ethylparaben (EP),propylparaben (PP), isopropylparaben(iPP), butylparaben (BP), heptylparaben(HP), and benzylparaben (BnP) were from
AccuStandard (New Haven, CT, USA).
Soy sauce and vinegar were purchasedfrom a local supermarket.

O

O

O

O

O

O
O

OH
Methyl paraben (MP)LogP: 1.8

O

OH

OH

Ethyl paraben (EP)
LogP: 2.5

OH

Propyl paraben (PP)
LogP: 3.0

OO

isoPropyl paraben (iPP)
LogP: 2.8

O
HO
O

OH
Butyl paraben (BP)LogP: 3.6

OH
Benzyl paraben (BnP)
LogP: 3.6

O
Heptyl paraben (HP)
LogP: 4.8

Figure 1. The structures and logP values of seven parabens investigated in soy sauce and vinegar.

Standards and solutions

Sample preparation

A combined standard spiking solution
was prepared at 1 mg/mL in MeOH,
diluted with an appropriate volume ofindividual stock solutions, and stored in–20 °C until use. A 1% NaCl solution was
prepared by dissolving 1 g of NaCl in
100 mL of Milli-Q water.

The combined standard spiking solution
was used to spike the soy sauce andvinegar to determine the recoveries.
The method used was revised froma published LLE and SLE method
developed for measuring parabens inbody wash.2 Figure 2 describes the
sample preparation. The soy sauce
and vinegar samples were preparedaccordingly before the extraction.
Six replicates were performed for both
SLE and LLE, respectively, at 20 ng/mL
spiking level. Sodium chloride was

Instrument and equipment
The analysis was performed on an
Agilent 1290 Infinity II LC with an
Agilent 1290 Infinity II high speed pump,an Agilent1290 Infinity II multisampler,
inline degasser, and an Agilent 1290
Infinity II diode array detector. Productcleanliness was evaluated using GC/MS
full scan by an Agilent Intuvo 9000 GC
combined with an Agilent 7000 triple
quadrupole GC/MS. Agilent OpenLab andAgilent MassHunter software were used
for data acquisition and analysis.
Other equipment and materials used forsample preparation included:•

Eppendorf pipettes and repeater•

Eppendorf centrifuge 5810R(Hamburg, Germany)•

Agilent Chem Elut S 12 cc, 3 mL
(p/n 5610-2008)•

Four commercially available SLE
12 cc products••

SPEX SamplePrep 2010
Geno/Grinder (Metuchen, NJ, USA)
Agilent Vac Elut 20 Manifold(part number 12234101)

added to increase the ion strengthof the vinegar sample. Chem Elut S(12 cc) and four commercially available
SLE cartridge products (12 cc) were
investigated and compared. LLE wasconducted in a centrifuge tube. To
improve the extractive efficiency, a Geno
Grinder was used for mixing the samples
thoroughly. After centrifugation, theEtOAc layer was transferred to a clean
glass tube with extra caution to avoid
transferring part of the water layer, anddried down with nitrogen. Finally, theresidue was reconstituted with 1 mL of
MeOH for injection.

Weigh 1.0 g of the sample.

Spike the mixed standard solution, and mix well.

Add 2 mL 1% NaCl solution (add water instead for soy sauce).

Vortex for 30 seconds, and the sample is ready for extraction.

SLE: 50 minutes(n = 6)

LLE: 80 minutes(n = 6)

Load prepared sample into SLE cartridge with
2~3 psi pressure (2 minutes).

Add 10 mL of EtOAc into the prepared sample(2 minutes).

Wait for absorption(5 minutes).

Mix with a GenoGrinder(5 minutes).

Extract with 10 mL of EtOAc by gravity, andapply ~5 psi to dry the cartridge at the end(10 minutes).

Centrifuge at 4,000 rpm(10 minutes).

Transfer the EtOAc layer to a 15 mL centrifuge tube
with caution (30 minutes).
Dry the eluent with N2 at 40 °C(30 minutes).
Dry the eluent with N2 at 40 °C(30 minutes).
Reconstitute with 1 mL of MeOH
(3 minutes).
Reconstitute with 1 mL of MeOH
(3 minutes).

Figure 2. Protocol used to extract parabens from soy sauce and vinegar using SLE and LLE.

3

HPLC conditions
HPLC columns were investigated forthe separation of seven parabens,
especially the isomers of PP and iPP.
The Agilent InfinityLab Poroshell 120
PFP column demonstrated the best
separation compared to C18 columns.
Figure 3 shows the overlappedchromatograms of neat standard
solution of seven parabens (20 ppb),
and the matrix blanks of soy sauce andvinegar after LLE. The main interferences
eluted before three minutes and did
not interfere with the target parabenschromatographically.

Configuration
Component

Agilent 1290 Infinity II high speed pump (G7120A)
Agilent 1290 Infinity II multisampler (G7167B)
Agilent 1290 Infinity II multicolumn thermostat (G7116B)
Agilent 1290 Infinity II diode array detector (G7117B)

ColumnAgilent InfinityLab Poroshell 120 PFP, 3.0 × 100 mm, 2.7 µm (p/n 695975-308）

Column Temperature
DAD Parameter

35 ℃

Injection Volume

2 µL

Mobile PhaseA) waterB) MeOH

Gradient

Time (min)
0
1.0
4.0
6.0
8.0

Stop Time

10 minutes

260 nm

%B
30
45
65
95
95

Flow rate (mL/min)
0.5
0.5
0.5
0.5
0.5

Results and discussion

20 ppb Standard solution
Vinegar blank
Soy sauce blank

mAU

SLE versus LLE
Figure 2 details the extraction protocols
and processing time for SLE and LLE.
The total time required to prepare
six replicates was 80 minutes for LLE
and 50 minutes for SLE. LLE was moretime-consuming and labor-intensive.
The separation of the aqueous phasefrom the organic phase required extra
attention, and longer drying time may
be needed for LLE if a water layer wastransferred accidently. As a result, it waschallenging to prepare multiple samples
simultaneously. However, SLE showeda much easier walk-away extractions
workflow without special precautions,which improved the sample extraction
productivity significantly.
Figure 4 shows the results of recovery
and reproducibility at 20 ng/g in both soy
sauce and vinegar with six replicates.
Both LLE and synthetic SLE(Chem Elut S) methods generated goodresults in terms of analyte recoveries(88 to 110%) and reproducibility(RSD <10%).

4

300
iPP

250
MP
200

BP BnP

EP

PP

5

6

HP

150
100
50
0

0

1

2

3

4

7

8

Time (min)Figure 3. Typical chromatograms of parabens standard solution (red) for MP, EP, PP, iPP, BP, HP, and BnP,
vinegar matrix blank after LLE (green), and soy sauce matrix blank after LLE (blue).

Chem Elut S versus competitors' SLE
We compared the performance ofChem Elut S and four commercial SLE
products on the seven parabens in soy
sauce and vinegar at 20 ng/g spiking
level. Competitors 1, 3, and 4 wereDE‑based sorbent, and competitor 2 wasa synthetic SLE sorbent. There was nota big difference between products forsoy sauce, but Chem Elut S showed an
obvious advantage for vinegar, especiallyfor the most hydrophobic paraben: HP.
Chem Elut S and competitor 2’s productshowed a slight advantage (RSD <5%)overall. Generally, Chem Elut S isequivalent to or slightly better than theother four SLE products when analyzing
HP (Figure 5).

Recovery% of paraben in soy sauce (20 ng/g, n = 6)

Agilent Chem Elut S
LLE

Recovery% of paraben in vinegar (20 ng/g, n = 6)

120

120

100

100

80

80

60

60

40

40

20

20

0

MP

EP

iPP

PP

BP

BnP

HP

0

Agilent Chem Elut S
LLE

RSD% of paraben in soy sauce (20 ng/g, n = 6)

10

8

8

6

6

4

4

2

2
0
MP

EP

iPP

PP

BP

BnP

EP

iPP

PP

BP

MP

HP

EP

iPP

PP

BnP

HP

Agilent Chem Elut S
LLE

RSD% of paraben in vinegar (20 ng/g, n = 6)

10

0

MP

Agilent Chem Elut S
LLE

BP

BnP

HP

Figure 4. Recovery and RSD results from Agilent Chem Elut S and LLE methods at 20 ng/g spiking level with six replicates.

Agilent Chem Elut S
Competitor 1

Competitor 2Competitor 3

Competitor 4

Recovery% of paraben in soy sauce (20 ng/g, n = 6)

Agilent Chem Elut S
Competitor 1

120

120

100

100

80

80

60

60

40

40

20

20

0

0
MP

EP

iPP

PP

Agilent Chem Elut S
Competitor 1

BP

BnP

Competitor 2Competitor 3

HP

MP

EP

iPP

PP

Agilent Chem Elut S
Competitor 1

BP
Competitor 2Competitor 3

BnP

HP
Competitor 4

RSD% of paraben in vinegar (20 ng/g, n = 6)

10

10

8

8

6

6

4

4
2

2
0

Competitor 4

Recovery% of paraben in vinegar (20 ng/g, n = 6)

Competitor 4

RSD% of paraben in soy sauce (20 ng/g, n = 6)

Competitor 2Competitor 3

0
MP

EP

iPP

PP

BP

BnP

HP

MP

EP

iPP

PP

BP

BnP

HP

Figure 5. Recovery and reproducibility results from Agilent Chem Elut S and four competitors’ SLE at 20 ng/g spiking level with six replicates.

5Chem Elut S sorbent provides higher
water-holding capacity. As a result, therecommended sample loading volume is
3 mL, which is 1.5 times more than therecommended loading volume (2 mL)from other competitors' equivalent
format cartridges. This feature certainly
improved the method sensitivity, given
similar analyte recoveries due to thelarger sample size. Figure 6 shows thechromatograms for the same sample
concentration (5 ng/g in vinegar)
analysis using different SLE products.
Chem Elut S (light green) demonstrates
the highest peak response and sensitivity
without obvious interferences, while
co‑extracts were observed from theresults of competitors 2 (red) and 4(blue).

6

mAU

Agilent Chem Elut S
Competitor 1
Competitor 2Competitor 3

200

Competitor 4
BnP
BP

MP

EP

150

PP

HP

iPP

100

50

0
4

5

6

7

Time (min)Figure 6. Chromatographic comparison of the same format (12 cc) cartridge with a sample of
5 ng/g parabens in vinegar.

8Conclusion
This Application Note conducted acomprehensive comparison of LLE
and SLE using multiple SLE products
in soy sauce and vinegar. SLE uses
a simple extraction without sample
emulsion formation, which reduces
extraction time by 40% compared to
LLE. Compared to LLE, Chem Elut S
also demonstrated superior recovery
and precision for parabens analysis infood, excellent water-holding ability,and qualified sorbent cleanliness.
When compared to other commerciallyavailable SLE products, Chem Elut S
demonstrated improved or equivalentprecision and analyte recovery, especiallywith more hydrophobic analytes. Lastly,
the Chem Elut S method delivered aneasier workflow, higher sensitivity, better
reproducibility, and eliminated sample
contamination.

A
Counts×107
5Competitor 1

0Counts×107
5Competitor 2
0Counts×107
5Competitor 3

0Counts×107
5Competitor 40×107
CountsThere were unexpected contamination
peaks found with some of the SLE
products. To verify the source of thecontamination, we tested the cleanlinessof the SLE products with water insteadof the matrix with a similar protocol.
Figure 7 shows that the cleanliness ofthe products was investigated using both
UV detection at 260 nm and GC/MS fullscan detection. Competitor 2 (synthetic
SLE) showed many more interferencesfrom the cartridge with both UV detection
and GC/MS full scan. Competitor 4 SLE
cartridges also showed interference
peaks. Chem Elut S and competitor 1
and competitor 3 products have fewer
interferences. The results of UV detection
are consistent with the interference
peaks in the paraben analysis of soy
sauce and vinegar in terms of retention
time and peak height. This proves thatthe interferences were sourced from SLE
products rather than food matrix.

5

Agilent Chem Elut S

0
4

6

8

10

12

14

16 18 20 22 24
Acquisition time (min)

26

28

30

32

34

36

B
mAU
Agilent Chem Elut S

800

Competitor 2

700

Competitor 1
Competitor 3

600

Competitor 4

500
400
300
200
100
0
2

4

6
Time (min)810Figure 7. Cleanliness testing with neat solvent for 12 cc SLE cartridges by GC/MS full scan (A) and 260 nm
UV detection (B).

7

References
1.

Chinese National Food Safety
Standard - Determination of
p-Hydroxybenzoic Acid Esters inFoodstuffs: GB5009.31-2016.

2.

Zimmerman, D. C.; et al.
Determination of Parabens inBody Wash using Solid‑Supported
Liquid-Liquid Extraction,
Agilent Technologies Application
Note, publication number
5991‑2735EN, 2013.

www.agilent.com/chem
This information is subject to change without notice.
© Agilent Technologies, Inc. 2019
Printed in the USA, May 24, 20195994-1018EN