rapid analysis of bisphenols (bp) a, b & e in baby food and infant formula - waters corporation...

©2015 Waters Corporation 1

Rapid Analysis of Bisphenols (BP) A, B & E

in Baby Food and Infant Formula

using LC/MS/MS


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


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


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


From BPA to Polycarbonate…. Synthetic pathway


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


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


Experimental Information


Analysis of BPA Waters Systems Solution


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


• 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


Other Related Compounds…?

BPB ( 2,2 Bis(4-hydroxyphenyl)butane)

BPA ( 2,2-Bis(4-hydroxyphenyl)propane)

BPE (1,1-Bis(4-hydroxyphenyl)ethane)


Results and Discussion


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


oReduce matrix effects

oDevelop sample prep procedure

oExtract relevant compounds

oProvide a robust, repeatable method


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


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






Use results from …

to Modify Sample Prep Procedure


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


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


Spiked Compounds 1 pg/μL in Samples

S/N > 3






Calculating the Matrix effect with the

New Sample Prep Method


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%)


Robustness

TargetLynx Matrix Calculator


Matrix Calculator how does it work?


Monitoring long-term robustness?

Long term system monitoring

Essential record for auditors

Tool for effective lab management


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

rapid analysis of bisphenols (bp) a, b & e in baby food and infant formula - waters corporation...

Science

waters corporation

bpa background

use of bpa

baby food

baby bottles

polycarbonate tableware

o polycarbonate plastics

o epoxy resins