Date post: | 16-Jul-2015 |
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©2015 Waters Corporation 1
Rapid Analysis of Bisphenols (BP) A, B & E
in Baby Food and Infant Formula
using LC/MS/MS
©2015 Waters Corporation 2
Presentation Overview
Background and Potential Health Issues
Worldwide opinions on BPA
Experimental
– Waters System Solution
Results
– Method development –
– Sample preparation optimisation
– Calculation of matrix effects
Conclusion
©2015 Waters Corporation 3
BPA Background
What is BPA?
– First synthesised in 1905 by Thomas Zincke of the University of Marburg, Germany
– In 1953, Dr. Hermann Schnell of Bayer in Germany and Dr. Dan Fox of General Electric
in the United States independently developed manufacturing processes for a new plastic
material, polycarbonate, using BPA as the starting material.
– It has been an important industrial chemical not only to make polycarbonate plastic but
also epoxy resins; both of which are used in a wide variety of applications.
o Polycarbonate plastics are used in baby bottles, tableware and other food containers
• (Eyeglass lenses, medical equipment, digital media (e.g., CDs and DVDs), cell
phones, consumer electronics, computers and other electrical equipment, …)
o Epoxy resins are used in can coatings, industrial floorings, adhesives, industrial
protective coatings, powder coatings, and printed circuit boards
©2015 Waters Corporation 4
BPB ( 2,2 Bis(4-hydroxyphenyl)butane)
BPA ( 2,2-Bis(4-hydroxyphenyl)propane)
BPE (1,1-Bis(4-hydroxyphenyl)ethane)
BPA and related compounds
©2015 Waters Corporation 5
From BPA to Polycarbonate…. Synthetic pathway
©2015 Waters Corporation 6
BPA Background
What are the potential health issues?
– Can leach into food from the epoxy resin lining of cans
and from consumer products such as polycarbonate
tableware, food storage containers, water bottles, and
baby bottles.
– BPA is a weak "estrogen like" endocrine disruptor which
can mimic circulatory endogenous hormones and is
suspected to be linked to a higher incidence of certain
cancers, reproductive disorders, diabetes and heart
disease in humans
©2015 Waters Corporation 7
International Regulations
Canada - First country to take action, listed it as a toxic substance
US-FDA - Released a report in 2010 expressing some concern regarding exposure of fetuses, infants and young children to BPA. Report encourages further studies into the safety of BPA
European Union - EU directive (2011/8/EU) has banned the use of BPA in infant feeding bottles
o France Bans Bisphenol A in All Food Contact Materials: French National Assembly has been voting to ban BPA from 2014 on. Concerning baby nutrition products the ban will step into force in 2013
Japan - Between 1998-2003, canning industry voluntarily replaced its BPA-containing epoxy resin can liners with BPA-free polyethylene terephthalate (PET) in many of its products
©2015 Waters Corporation 8
Experimental Information
©2015 Waters Corporation 9
Analysis of BPA Waters Systems Solution
©2015 Waters Corporation 10
UPLC system ACQUITY UPLC System
Column: ACQUITY UPLC BEH C18 2.1 x 50 mm, 1.7 m
Column temp 40 C
Mobile phase A 0.5 % NH4OH in H2O
Mobile phase B 0.5 % NH4OH in MeOH
Elution 3 minute linear gradient from 5% (B) to 95% (B)
Flow Rate 0.5 mL/min
Injection volume
50 L
ACQUITY UPLC-Xevo TQD Conditions
MS System XEVO TQD
Ionization mode ESI Negative
Capillary voltage 3.5 kV
Cone voltage 30.0 V
Source temp 140 C
Desolvation temp 350 C
Desolvation gas 550 L/hr
Cone gas 50 L/hr
Routine targeted quantification
with information rich qualitative data
©2015 Waters Corporation 11
• Automatically sets up instrument,
• Optimises MRMs and performs system suitability
IntelliStart
• Methods generated automatically by QuanPedia,
• Clear flagging when data is out of regulatory tolerance
• Reduced risk of errors
TargetLynx
• Long term system monitoring, Lab management tool
TrendPlot
• Generates optimised methods, time scheduled MRMs
Quanpedia
• Real time QC, Intelligent ‘on the fly’ decision making
QCMonitor
Waters QUAN Workflow
©2015 Waters Corporation 12
Other Related Compounds…?
BPB ( 2,2 Bis(4-hydroxyphenyl)butane)
BPA ( 2,2-Bis(4-hydroxyphenyl)propane)
BPE (1,1-Bis(4-hydroxyphenyl)ethane)
©2015 Waters Corporation 13
Results and Discussion
©2015 Waters Corporation 14
Developing a Suitable Method
Analysis of food samples can be challenging – need to
develop a sample preparation that can:
o Reduce matrix effects (e.g. peak distortion, ion
suppression…)
o Extract relevant compounds
o Provide a robust, repeatable method
Method development to investigate different
extraction techniques can be time intensive
Useful to have tools that support this process and help
improve lab efficiency
RADAR
Matrix Calculator
LC-MS/MS Analysis
Modify analytical method
LC-MS/MS Analysis
Modify sample
preparation
©2015 Waters Corporation 15
oReduce matrix effects
oDevelop sample prep procedure
oExtract relevant compounds
oProvide a robust, repeatable method
©2015 Waters Corporation 16
Determining sample prep efficiency…
What is RADAR?
– Rapid electronics allow instrument to switch between MRM & full scan
– Added information can be gained from full scan data in routine analyses
• Intelligent decision making
• Improves method development
• Monitor matrix components
• Detect un-targeted adulterants
How do I set it up on the instrument?
- Parallel MRM with full scan data acquisition
©2015 Waters Corporation 17
The advantage of RADAR Full scan and MRM in one analysis
1. Protein precipitation 2. SPE – OASIS HLB
1. Protein precipitation
2. DisQUE (QuEChERS) 3. SPE – OASIS HLB
©2015 Waters Corporation 18
oReduce matrix effects
oDevelop sample prep procedure
oExtract relevant compounds
oProvide a robust, repeatable method
Use results from …
to Modify Sample Prep Procedure
©2015 Waters Corporation 19
Part 2 - DisQUE Part 3 – OASIS HLB
Cartridge: Oasis HLB 30 m, 60 mg/3 cc
Condition: 2 mL Methanol
Equilibrate: 2 mL Water
Load: 70 mL diluted extract
Flow rate: < 5 mL/min
Wash: 2 mL 40 % MeOH in Water
Elute: 1 mL 100 % MeOH
Sample Prep Protocol
©2015 Waters Corporation 20
Part 1 – Protein precipitation
Part 2 - DisQUE
Part 3 – OASIS HLB
Optimised Sample Prep Protocol
Precipitation (10g sample, 10mL CH3CN) Centrifuge and collect supernatant
Add contents from DisQuE tube 1. Shake.
Centrifuge & collect 10 mL supernatant
Add contents from DisQuE tube 2. Shake.
Centrifuge & collect supernatant.
Dilute supernatant with 70 ml H2O. Load on Oasis HLB (3cc)
Wash – 2 mL 40 % MeOH Elute – 1 mL 100 % MeOH
Dilute eluate with 1 mL H2O Inject 50 µL for LC-MS/MS
©2015 Waters Corporation 21
Spiked Compounds 1 pg/μL in Samples
S/N > 3
©2015 Waters Corporation 22
oReduce matrix effects
oDevelop sample prep procedure
oExtract relevant compounds
oProvide a robust, repeatable method
Calculating the Matrix effect with the
New Sample Prep Method
©2015 Waters Corporation 23
Correlation coef f icient: r = 0.998164, r^2 = 0.996332
Response type: Internal Std ( Ref 4 ), Area * ( IS Conc. / IS Area )
Curve type: Linear, Origin: Exclude, Weighting: 1/x^2,
Conc-0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0
Res
po
nse
-0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0.90
1.00
1.10Bisphenol
Infant Formula (10 pg/μL spike)
BPA 102% (3.2%)
BPB 95% (5.5%)
BPE 81% (4.6%)
Linearity and Repeatability
Bisphenol Baby food
(20 pg/μL spike)
BPA 110% (7.8%)
BPB 112% (6.7%)
BPE 99% (6.1%)
©2015 Waters Corporation 24
Robustness
TargetLynx Matrix Calculator
©2015 Waters Corporation 25
Matrix Calculator how does it work?
©2015 Waters Corporation 26
Monitoring long-term robustness?
Long term system monitoring
Essential record for auditors
Tool for effective lab management
©2015 Waters Corporation 27
Conclusions
BPA – WW very topical compound
Analysis of BPA in food and beverage samples require a robust method
Waters System Solution is rapid and robust
o Sample preparation- DisQUE and OASIS HLB
o Reliable chromatography - ACQUITY UPLC & ACQUITY BEH C18 columns
o Robust and Repeatable results - Xevo TQD
o Matrix monitoring tools - RADAR, Matrix Calculator
o Waters QUAN Workflow - TargetLynx, TrendPlot