Analysis of Bisphenol A in Toys by HPLC with Fluorescence Detection

Introduction
Bisphenol A (BPA), shown in Figure 1,
has made headlines over the past few
years due to its potential effects on
human health. The headlines began with
concerns over BPA in baby bottles and spread to include other types of contact materials
such as re-useable plastic Tupperware, can linings, thermal paper, cosmetics, and plastic toys.
BPA is a chemical byproduct used in the production of plastics for its optical clarity and heat
resistance properties. It has been shown to exhibit hormone-like properties, causing concerns
over possible health risks. Since 2008, several governments have supported on-going studies
to investigate its safety and to establish safe levels of exposure. In the U.S., the Food and
Drug Administration (FDA) set the safe level to 5 mg/kg (by body weight) per day, while, in
Europe, the European Food Safety Authority (EFSA) recently lowered its safe level to 4 µg/kg
(by body weight) per day.1, 2
Analysis of Bisphenol A
in Toys by HPLC with
Fluorescence Detection
A P P L I C A T I O N N O T E
Authors:
Chi Man Ng
Wilhad M. Reuter
PerkinElmer, Inc.
USA
Liquid Chromatography

2
As a result of the health concerns over human exposure to BPA,
this compound is now closely monitored in specific products,
including children’s goods. This application focuses on the
extraction and HPLC analysis of BPA in three store-bought
children’s toys. Method conditions and performance data,
including linearity and repeatability, are presented.
Table1.HPLCMethodParameters.
HPLC Conditions
Column:
PerkinElmer Brownlee™
HRes Biphenyl 1.9 µm,
50 x 2.1 mm (Part No. N9303912)
Mobile Phase:
Solvent A: water
Solvent B:Acetonitrile
Solvent program (isocratic):
Analysis Time: 4.0 min.
Flow Rate: 0.5 mL/min. (5500 psi; 367 bar)
Oven Temp.: 40 ºC
Detection:
Altus A-30 FL detector
Excitation: 275 nm; Emission: 313 nm
Injection Volume: 3 µL
Sampling (Data) Rate: 5 pts./sec
Time
(min)
Flow Rate
(mL/min)
%A %B %C %D Curve
1 Initial 0.500 60.0 40.0 0.0 0.0 Initial
Figure1.StructureofBisphenolA(BPA).
Experimental
Hardware/Software
For all chromatographic separations, a PerkinElmer Altus™
HPLC System was used, including the Altus A-10 solvent/sample
module, integrated vacuum degasser, A-10 column module
and Altus A-30 fluorescence (FL) detector. All instrument control,
analysis and data processing was performed using the Waters®
Empower®
3 CDS platform.
Method Parameters
The HPLC method parameters are shown in Table 1.
Solvents, Standards and Samples
All solvents and diluents used were HPLC grade and filtered
via 0.45 µm filters.
The BPA standard was obtained from Sigma-Aldrich, Inc®
(Allentown, PA). A stock 100-ppm BPA standard was made
using purified water as diluent. The lower level standards were
then prepared from this stock solution.
The children’s toy samples were purchased at a local store.
These included a toy car, pony and keychain (Figure 2). The
extraction procedure used was intended to simulate the contact
route through which children are likely to encounter BPA. The toy
samples were immersed in 800 mL of purified water, at 40 ºC for
24 hours. Following the extraction, all water extracts were filtered
and then chromatographed using the conditions listed in Table 1.
Prior to injection, all calibrants and samples were filtered through
0.45-µm filters to remove any small particles.
Results and Discussion
Figure 3 shows the chromatogram of the 1-ppm BPA Standard,
using the optimized conditions described above. BPA eluted at
0.95 min. A second peak, eluting at 2.57 min, was also detected in
the standard. As this second peak was also observed in subsequent
blanks (see Figure 6), this peak was not further pursued.
Figure2.Children’stoysamplesanalyzedforBPAinthisapplication:A)car,B)pony,
C)keychain.

3
Figure 4 shows the overlay of ten replicate injections of the 1-ppm BPA Standard, demonstrating exceptional reproducibility.
The retention time %RSD was 0.04%.
Figure3.Chromatogram of the 1-ppm BPA Standard.
EU
0.00
50.00
100.00
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0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
Figure4.Overlay of 10 replicates of the 1-ppm BPA Standard.
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Figure 5 shows the calibration result for the BPA Standard over a concentration range of 0.1 to 1.0 ppm. BPA followed a linear
(1st
order) fit and had an R2
coefficient of 0.99821 (n = 3 at each level).
Figure5.Results of 5-level calibration set for BPA.
Area
-1x106
0
1x106
2x106
3x106
4x106
5x106
6x106
Amount
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00
R² = 0.99821

4
Figure6.Chromatographic overlay of the water blank (red) and 0.1-ppm BPA Standard (black).
Figure7.Chromatographic overlay of water-extracts from the car sample (blue), keychain sample (green) and 1-ppm BPA Standard (black). The peaks at 0.5 and 2.6 minutes
were not identified.
EU
-4.00
-2.00
0.00
2.00
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6.00
8.00
10.00
12.00
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00
EU
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Figure 6 shows the overlay of a 0.1-ppm standard and the following water blank, confirming that the system was free of any BPA
contamination or carry over.
Using the same chromatographic conditions, the three toy
sample water-extracts were then analyzed. As shown in Figure 7,
the chromatograms for both the car and keychain water-extracts
showed no quantitatable level of BPA. However, as shown in
Figure 8, the chromatogram of the water extract from the pony
showed a significant level of BPA. Based on the calibration curve,
the amount of water-extractable BPA in the pony sample was
calculated to be 0.274 mg. As an example, if one considers the
average weight of a 4-year old child to be 15 kg, the amount
of BPA extractable from the pony sample was within the FDA’s
safe maximum level of 5 mg/kg (by body weight) per day.
However, using the above example, this amount exceeded the
EFSA’s safe maximum level of 4 µg/kg (by body weight) per day.

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Figure8.Chromatographic overlay of water-extracts from the pony sample (red) and 1-ppm BPA Standard (black).
EU
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Conclusion
This work has demonstrated the effective chromatographic
separation of BPA using a PerkinElmer Altus HPLC System with FL
detection. The results show very good retention time repeatability
as well as excellent linearity over the tested concentration range.
Considering the lower calibration levels that were run, the
sensitivity levels were well below the maximum safe levels
stipulated by both the FDA and EFSA regulatory agencies.
This work also focused on the BPA extraction and analysis of three
store bought toys, comparing them both chromatographically
and quantitatively. It effectively identified one of the toys as
containing a significant amount of water-extractable BPA. This
is especially relevant in today’s environmentally-sensitive climate,
as the uncertainties surrounding potential health effects of BPA
exposure, especially in plastics, have raised widespread concern.
References
1. http://www.fda.gov/NewsEvents/PublicHealthFocus/
ucm064437.htm [Online]. [Accessed 15 February 2015].
2. http://www.efsa.europa.eu/en/press/news/150121.htm
[Online]. [Accessed 15 Febrary 2015].

Analysis of Bisphenol A in Toys by HPLC with Fluorescence Detection

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