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Real World-Application Methods From a Food Testing Laboratory
Sonja RiedikerNestlé Quality Assurance Center
Singapore
21 March 2006
44
Overview
Nestlé Quality
Nestlé Laboratory Network
LC-MS/MS in Routine Analysis
LC-MS/MS: Advantages and Challenges
Conclusions
45
Nestlé Products ... A Diversity as the Consumers
Beveragese.g. Nescafé, Milo, Nestlé AquarelMilk productse.g. Nespray, Nido, Molico, LC1
Nutrition Productse.g. Lactogen, Cerelac, PowerBarCulinary productse.g. Maggi, Buitoni, ThomyChocolate, Confectionarye.g. Crunch, CaillerPetCaree.g. Purina Friskies, FelixAlconPharmaceutical companyetc.
More than 130 different brands:
46
Nestlé Quality: General Approach
Distribution
Trade
Consumer
Transport
DownstreamUpstream
Transport
Primary & SecondaryTransformation
AgriculturalProduction
Production
People are involved all along the Supply Chain
47
Nestlé Quality: Three Dimensions
SocialPrice/Performance
ConvenienceCultural, Conviviality,
Pleasure
SensoryAspect, ColourAroma, FlavourTexture, Sound
HealthNutritional Value, Functionality
Food SafetyRegulatory Compliance
48
Nestlé Quality: Health
Our approach:Usage of high-quality and safe ingredients
Release parameters for production (physical, microbiological, and chemical)Monitoring parameters for due diligence
On- and in-line quality control during production
Quality control of finished productsRelease parameters (physical, microbiological, and chemical)Monitoring parameters for due diligence
49
Release and Monitoring Parameters
Examples:
Hygiene indicators and pathogens
Contaminantse.g. pesticides, mycotoxins, veterinary drugs, processing contaminants, toxic metals, …
Authenticity (adulteration), GMO
Compositional parameterse.g. vitamins, minerals, carbohydrates, fat content
Claimse.g. low sodium, sugar-free, MSG-free
50
Nestlé Laboratory Network
Factory Laboratoriesrapid tests or alternative methods, high throughput (on-, in-line, on-site)
Regional Laboratoriesaccredited by local authorities, internal proficiency testing (P-test)highly specialized or expensive analyses (e.g. HPLC-UVD, LC-MS/MS, ICP-MS, GC-ECD, etc)
External Laboratoriesaccredited, participation in international or internal proficiency testing
51
Nestlé Research Center (NRC)
Quality and Safety DepartmentMethod development, modifications(ca. 250 Laboratory Instructions)Groups with focus on
ContaminantsIngredients and AuthenticityPackaging, Off-FlavourMicrobiological SafetyChemical Food Safety
Coordination of Laboratory Network
Performance of P-Tests
52
LC-MS/MS in Routine Analysis
Laboratories equipped with LC-MS/MS (Triple Quadrupole)
Weiding (Germany): Quattro LC (Waters), API3000 (Applied Biosystems)
Cergy (France): API4000
Dublin (USA): API3000
Singapore: Quattro Micro (Waters), API3000, API2000
NRC: different suppliers (Waters, Applied Biosystems, Agilent, ...)
53
LC-MS/MS Methods for Routine
Applications forBanned compoundsCompounds, for which other detection techniques are not suitable
Methods available or in preparationChlormequat, Acrylamide Chloramphenicol, Nitrofuran antibiotics, Spinosadresidues, Beta-lactam antibiotics, 5-NitroimidazolesIllegal colorantsMulti-residue methods for pesticides and veterinary drugs (in preparation)
54
LC-MS/MS Methods for Routine
Main purpose
Confirmation of analyte absence/presenceCriteria in accordance to EU Commission Decision 2002/657/EC
Quantitation of analyte
Tampering cases: structural informationisotopes, molecular weight, functional groups, etcin combination with other techniques (e.g. NMR)
55
MS Acquisition Mode
SRM Mode (Selected Reaction Monitoring):
Q1: mass filter for molecular ion (precursor)Q2: collision cell to generate product ionsQ3: mass filter for one product ion per time unit
Q1 Q2 Q3
56
Confirmation: EU 2002/257/EC
DefinitionConfirmatory methods for organic residues or contaminants shall provide information on the chemical structure of the analyte.
Identification points:
Banned compounds: ≥ 4
⇒ at least 1 precursor and 2 product ions
⇒ at least 2 SRM traces
57
Confirmation: EU 2002/257/EC
Criteria
Positive response in at least two SRM traces (signal-to-noise: ≥ 3)
Ion ratios (response ratios)respect of max. permitted tolerances or even more stringentmake use of natural isotopic distribution (e.g. 35Cl/37Cl)
Retention time ratio (analyte/internal standard)
58
Nitrofuran Antibiotics Added to Honey
0.5 µg/kgAMOZ
1.4 µg/kg AH
0.35 µg/kgAOZ
1.1 µg/kgSC
m/z 335 >291
m/z 236 >134
m/z249 >134
m/z209 >166
Matrices coveredHoney, poultry, seafood,milk powder
ReferencesSeu-Ping Khong et al., J. Agric. Food Chem. 2004, 52, 5309-5315
P. Mottier et al. J. Chromatogr. A, 1067 (2005) 85-91
59
AMOZ Added to Honey
m/z 335 >128
m/z 335 >262
m/z 335 >291
m/z 340 >265
m/z 340 >296
0.5 µg/kgAMOZ
AMOZ-d512.5 µg/kg
Quantifier
Qualifier
Qualifier
Quantifier
Qualifier
60
Chloramphenicol Added to Milk Powder
0.43 µg/kg CAP
5 µg/kg CAP-d5
Time7.00 8.00 9.00 10.00 11.00 12.00
%
0
100
7.00 8.00 9.00 10.00 11.00 12.00
%
0
100
7.00 8.00 9.00 10.00 11.00 12.00
%
0
100
7.00 8.00 9.00 10.00 11.00 12.00
%
0
100
7.00 8.00 9.00 10.00 11.00 12.00
%
0
100 8.13
8.13
8.19
6.80 7.56 10.119.4310.34 10.85
8.16
7.747.15 8.938.69 9.22 9.69
8.16
7.89
m/z 326 > 262
m/z 328 > 262
m/z 323 > 152
m/z 321 > 257
m/z 321 > 152
37Cl-isotope
35Cl-isotope
37Cl-isotope
35Cl-isotope
35Cl-isotopeP. Mottier et al., Food Additives and Contaminants, 2003, 20 (4),335-342
61
Illegal Colorants Added to Chilli Powder
25 µg/kgSudan Red 7B
100 µg/kgSudan IV
50 µg/kgSudan III
50 µg/kgSudan II
m/z353 > 156
m/z 381 > 276
m/z 380 > 169
m/z 277 > 156Time
10.00 12.50 15.00 17.50 20.00
%
0
100
10.00 12.50 15.00 17.50 20.00
%
0
100
10.00 12.50 15.00 17.50 20.00
%
0
100
10.00 12.50 15.00 17.50 20.00
%
0
100 19.35
19.48
14.74
9.75
Other matricesSpice powderse.g. turmeric, curry, paprikaOleoresinse.g. capsicum, paprikaPalm oilSynthetic food colours e.g. Sunset Yellow
Other coloursSudan I, Para Red, Butter Yellow, Toluidine Red
62
Quality Control: Method Performance
Method verificationas a part of method implementation (accredited by local authorities)
Common controlsreagent blanks (contamination)FAPAS samples if available, otherwise spiked sample blanks (recovery and detection limit)solvent blanks (carry-over)instrumental controls (sensitivity)
Participation in interlaboratory studiesFAPAS, Nestlé P-Test
63
LC-MS/MS: Advantages
High selectivity in SRM modelow detection limits even in complex food matricesbaseline separation of analytes not mandatory
Confirmatory analysisone analysis sufficient to prove analyte presence/absence
Feasible to analyse ‘difficult’ moleculese.g. Acrylamide, Chlormequat without derivatisation
64
LC-MS/MS: Challenges
Broad variety of food matricesraw materials e.g. honey, milk, cereals, vegetables, fruitssemi-processed ingredients e.g. chicken meat, milk powderflavouringsfinished products
Methods have to be applicable for varying matrices
Challenges due to sample matrixvisible and invisible interferences (preferable 3 SRM traces)impact on detection limit (ion suppression)more frequent MS cleaning necessary (e.g. honey, oleoresin)
65
LC-MS/MS: Challenges
Varying LC-MS/MS instrumentsdifferent fragmentation patterne.g. Acrylamide: m/z 27 as product ion with Quattro Micro
m/z 44 as product ion with API3000varying LODs
Quantitation internal standards needed for reliable quantitation(otherwise: standard addition, which is time-consuming)
Data evaluation tools able toquantify the analyte levelscalculate ion ratiosidentify ion ratios out-of-tolerance range
66
LC-MS/MS: Challenges
Banned compoundsNestlé policy: If an ingredient is detected positive for abanned compound, the ingredient will be rejected.
with Minimum Required Performance Level (MRPL)LOD at most MRPL set by European Unionif LOD lower than MRPL and sample found positive⇒ rejection of ingredient
without MRPLLOD at least at the same level as commercial and/or food authority laboratories (e.g. Sudan colorants)
67
Conclusions
LC-MS/MStoday a routine technique in Nestlé laboratories
a powerful technique to confirm analytes in complex food matrices at low concentration level
reliable quantitation possible in complex food matrices by using isotopic-labelled internal standards
implementation of applications for multi-residue screening in near future
68
Acknowledgment
NQAC SingaporeWee Siew-MoiDaniel TanChan Wai-Chinn
NRCPhilippe GuyPascal MottierEric GremaudSeu-Ping Khong….
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