©2012 Waters Corporation 1

Senior Manager Global Program Management

Engineering & Technology Abbot Nutrition

Tom Seipelt

Food Safety and Ingredient Quality in the Post-Melamine Era

Tom Seipelt Jim Denison

Abbott Nutrition Abbott Nutrition Global Program Management Global Research Services Supply Chain R&D Columbus, Ohio Columbus, Ohio

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Overview

Introduction / Background

Food Safety & Ingredient Quality • Defining the Post-Melamine Era • Defining the Analytical Problem

Analytical strategies

• Approaches for Targeted and Non-Targeted Analysis • Masslynx Application Managers • New workflows in UNIFI

Future Direction & Challenges

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

For over 100 years, Abbott Nutrition (formerly know as Ross Laboratories) has been well recognized as one of the world's most respected names for nutritional products

About Abbott Nutrition…

Products include:

Infant Nutrition • Similac Advance • Similac Isomil Advance • Similac Alimentum

Child Nutrition • Pediasure, Pedialyte

Adult Nutrition • Ensure, ZonePerfect, EAS

Diabetes • Glucerna

Medical • Nepro, Jevity, Oxepa

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Pre-Melamine Food Safety Strategy

Primary control of contaminants in finished products through quality systems applied to ingredient supply • Ingredient Qualification

– Evaluate supplier’s ability to provide acceptable ingredient

• Ingredient Specifications – Develop specifications and supplier test certification program

• Ingredient Acceptance – Rapid screening tests – Vendor Certificate of Analysis

• Ingredient Quality Assurance / Surveillance – Ingredient verification and monitoring

Quality systems based primarily on identity, purity, and targeted screening for contaminants

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Melamine Crisis – Summary

Sporadic outbreaks of renal failure in pets attributed to melamine first emerged in 2004 and escalated in 2007.

A major incident was spawned in China in 2008 when melamine was intentionally added to diluted raw milk to artificially inflate the apparent protein value. Kidney maladies affected at least 300,000 Chinese infants and six fatalities were reported.

This incident represented one of the largest deliberate adulterations of food and sent shockwaves throughout the Food System.

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Economics of Melamine

Adding 1g of melamine (0.0018 cents) per Kg of milk is enough to raise the apparent protein content from 2.7% (cheapest grade of milk in China @ 17.6 cents/Kg) to 3.1% (most expensive grade @ 26.5 cents/Kg). If milk is diluted the profits can be even higher.

Source: Washington Post Nov. 10, 2008

Melamine

N

N

N

NH2

NH2 NH2

Cyanuric Acid

N

N

N

OH

OH OH

N

N

N

O

O O

H

H

H

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Food Adulteration is Not New

China Makes Arrests Over Food Safety Push Follows a Spate of Contamination Incidents

Officials Close Thousands of Businesses

FDA: Melamine found in baby formula made in China

Historical Examples • Wine dilution (ancient Rome) • Flour (Alum adulteration - 19th century)

Modern Examples Moore, et.al. (2012) J Food Sci, 77(4): R118-126

• Olive Oil • Milk • Honey • Spices (Saffron) • Juices (Orange / Apple) • Apple juice

Post-Melamine Era - What has changed? • News Reporting / Internet • Consumer Expectations • Regulations • Analytical Capabilities

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Analytical Challenge

GOAL: Assure quality and safety of food supply

Strategies to screen for unexpected contamination / adulteration

Targeted / non-targeted screening

• Universal detection with good sensitivity and qualitative capability • Proposed screening methods

– GC-MS – DESI-MS – FTIR – Amino Acid Analysis

Chemometrics using Principle Component Analysis (PCA) to differentiate ‘good’ vs. ‘bad’ ingredients

Question: How to enable chemometrics by defining ‘good’

– LC-MS – NMR – NIR – Fatty Acid Analysis

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Ingredient Qualification

Chemometric Library Generation

Contaminant Screening

Target Compound Analysis

Pesticides

Quantitative Target Compound

Analysis (as needed)

Ingredient Acceptance Driven by Qualification

Heavy Metals Melamine

Etc….

GC-MS Analysis

Target Compound

Screen

Non-Target Compound

Screen

LC-MS Analysis

Target Compound

Screen

Non-Target Compound

Screen

Testing requirements beyond standard identity & purity tests

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

After the Crisis – Routine Melamine Testing

Waters ACQUITY TQD • Sample Preparation

– Screening Precipitate protein with 2% formic acid Dilute 1:10 with mobile phase B

– Confirmation SPE cleanup (Oasis MCX or MAX)

• UPLC Conditions – BEH HILIC column (2.1 x 150 mm) – Mobile Phase (0.7 mL/min flow): A: 10 mM NH4OAc in H2O B: 10 mM NH4OAc in 97/3 CH3CN/H2O

• MS Conditions – ESI(-) : 0.3 – 1.1 min m/z 128 42; 128 85

– ESI(+) : 1.1 – 6.0 min m/z 127 68; 127 85 Time

0.40 0.50 0.60 0.70 0.80 0.90 1.00

%

0

100

0.40 0.50 0.60 0.70 0.80 0.90 1.00

%

0

100

0.40 0.50 0.60 0.70 0.80 0.90 1.00

%

0

100121108-s-035 Sm (Mn, 3x4) 1: MRM of 4 Channels ES-

128 > 85986

0.81

0.64

121108-s-024 Sm (Mn, 3x4) 1: MRM of 4 Channels ES- 128 > 85

1520.39

0.300.46 0.990.860.67

121108-s-026 Sm (Mn, 3x4) 1: MRM of 4 Channels ES- 128 > 85

3.27e30.78

Time1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50

%

0

100

1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50%

0

100

1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50

%

0

100121108-s-033 Sm (Mn, 3x4) 2: MRM of 4 Channels ES+

127 > 859.60e3

2.76

2.011.511.21 3.373.16

121108-s-024 Sm (Mn, 3x4) 2: MRM of 4 Channels ES+ 127 > 85

1.09e43.68

3.573.453.22

2.95

2.701.621.40 2.071.89 2.45

121108-s-026 Sm (Mn, 3x4) 2: MRM of 4 Channels ES+ 127 > 85

5.52e42.80

Melamine Cyanuric Acid

Standard (0.25 ng/mL)

Unfortified Infant Formula

Fortified Infant Formula (0.1 µg/g)

Standard (2.0ng/mL)

Unfortified Infant Formula

Fortified Infant Formula (0.6 µg/g)

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Analytical Workflow – Target / Non-Target Screening

“Ideal” Workflow

Xevo G2 QTof Workflow

Overnight Operation

Sampling / Shipping

Sample Prep

Instrument Analysis

Data File

Target / Non-Target

Compound Analysis

Preliminary Report

Data Review & Approval

Database Storage

Overnight Operation

Sampling / Shipping

Sample Prep

Database Storage

LC-MS/MSESI+

Data File

Target / Non-target

Compound Analysis

Preliminary Report

Data Review & Approval

LC-MS/MSESI-

Data File

Target / Non-target

Compound Analysis

Preliminary Report

Data Review & Approval

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

60 min - ?? ~4 hrs

Overnight Operation

Sampling / Shipping

Sample Prep

Database Storage

RPLC-MS/MSESI+

Data File

Target Compound

Analysis

Preliminary Report

Data Review & Approval

Non-Target Compound

Analysis

Preliminary Report

Data Review & Approval

RPLC-MS/MSESI-

Data File

Target Compound

Analysis

Preliminary Report

Non-Target Compound

Analysis

Preliminary Report

Data Review & Approval

Data Review & Approval

HILIC-MS/MSESI+

Data File

Target Compound

Analysis

Preliminary Report

Non-Target Compound

Analysis

Preliminary Report

Data Review & Approval

Data Review & Approval

HILIC-MS/MSESI-

Data File

Data Review & Approval

Target Compound

Analysis

Preliminary Report

Non-Target Compound

Analysis

Preliminary Report

Data Review & Approval

Xevo G2 QTof Acquity UPLC with Column Manager

POSIT VE Software 20-30 min per worklist

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Example – Milk Protein Qualification RP-UPLC-MSE using ESI+

Sodium Cas R2

Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100

1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00 16.00

%

0

100032912-8-025 1: TOF MS ES+

TIC1.23e7

11.28

10.92

10.771.01

0.66

0.48

9.55

8.71

8.01

4.28

7.106.05 7.47

8.59

9.449.09

10.5010.24

10.159.94

11.75

12.38

12.30

12.71

12.97

13.8713.03

13.7714.38 16.09

032912-8-024 1: TOF MS ES+ TIC

1.60e712.5110.86

10.73

0.97

0.66

0.48

10.239.548.70

7.99

4.286.03

8.599.449.09

10.15

9.76

10.49

11.2411.73 12.41

12.34

11.86

12.72

12.79

12.88

13.08

13.8713.34

14.04

14.38 14.7116.0915.33

Unspiked Casein QuEChERS Extract

Spiked Casein QuEChERS Extract 45 Antibiotics – 5 ppb each

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Manual Data Processing – EIP Chromatograms of 12 Antibiotics (5 ppb spike)

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

POSIT VE Template

Target Compound

Identification Criteria

Semi-Quantitation Reference Standard

Internal Standard

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

ChromaLynx Output from POSIT VE Software

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

TargetLynx: Quantitation of Identified Target Compounds

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

ChromaLynx: Non-Targeted Analysis

Using modified parameters, Chromalynx detected 65 unknowns in the casein QuEChERS extract (reduced from >1500 using standard parameters)

• Based on standard software threshold filters • Need semi-quantitative threshold to limit the scope of the data review

Typical mass accuracy of 5 ppm used to generate elemental composition and isotope ratios using i-FIT

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

ChromaLynx: Incorporation of MSE data

MSE data are incorporated into structural analysis

Integration into ChemSpider search to determine suggested structures

MassFragment assists interpretation of MSE data for identification

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Unknown Identification

ChemSpider correctly identified ‘unknown’ as Clenbuterol-D9

Elimination of known or

target compounds from ChromaLynx peak list is needed to eliminate redundancy

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

UNIFI workflow

Moving toward ideal workflow - limitations are in chemistry and not software

60 min - ?? ~4 hrs

Xevo G2 QTof Acquity UPLC with Column Manager

Unify Software 20-30 min per data file

Overnight Operation

Sampling / Shipping

Sample Prep

Database Storage

RPLC-MS/MSESI+

Data File

Target / Non-target Compound

Analysis

Preliminary Report

Data Review & Approval

RPLC-MS/MSESI-

Data File

Target / Non-target Compound

Analysis

Preliminary Report

Data Review & Approval

HILIC-MS/MSESI+

Data File

Target / Non-target Compound

Analysis

Preliminary Report

Data Review & Approval

HILIC-MS/MSESI-

Data File

Target / Non-target Compound

Analysis

Preliminary Report

Data Review & Approval

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Future Direction and Challenges

Current Status

• Targeted Analysis – Posit ve simplifies targeted / semi-quant screening – 10-15 samples/day throughput

• Non-targeted Analysis – ChromaLynx identifies peaks for investigation – Peaks identified in Posit ve workflow are not flagged – Need semi-quantitative threshold to limit number of peak identifications – 10-15 samples/week (dependent on the number of peaks)

Future UNIFI Software

• Software development ongoing to simplify workflow

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Acknowlegements

Advanced Testing Laboratories

• Bindiya Sameem

Waters Corporation

• Jennifer Burgess • Gareth Cleland • Paul Young

Ingredient Quality in Post-Melamine Era Date: May 19, 2012

Waters Users’ Meeting – ASMS 2012 © 2012 Abbott

Thank You!

Questions?