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

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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

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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)

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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

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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

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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

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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

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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

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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

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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)

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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)

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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….