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©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