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Food contaminants Dhaka University August 2014
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Programme Partners Funded by
Trends in Food Contaminantsand Residue Analysis
Dr. Margarita CorralesInternational ConsultantFAO-Bangladesh
What are Food Contaminants?
• Contaminants are substances that can unintentionally enter food during its production or marketing.
• Residues are also food contaminants but occur in foodstuffs as traces or derivatives thereof from the use of agrochemicals or food processing
Food Contaminants
• Naturally occurring toxicants – Aflatoxins – Ochratoxins..
• Improper use of Agrochemicals– Pesticides, veterinary drug residues
• Industrial and environmental pollutants– Heavy metals, Dioxins, Polychlorinated biphenyls (PCBs)..
• Food Additives over permitted levels– Dyes, sweeteners, antioxidants..
• Emerging chemical hazards– Process-related: Acrylamide, furans, polycilic Aromatic Hydrocarbons (PaHs)– Adulterants:Melamine
Maximum Residue Levels (MRLs)
• MAXIMUM RESIDUE LEVELS (MRLs) are the upper legal levels of a concentration for residues in or on food or feed based on good agricultural practices and to ensure the lowest level of exposure
• AIMS:– Protect consumers– Ensure fair trade practices in the food trade– Promote coordination of all food standards work
undertaken by international governmental and non governmental organisations
Codex Alimentarius MRLs
Pesticide Food Commodity *MRL [mg/kg]
Endosulfan Poultry meatPotatoTomato
0.030.050.5
Methyl Parathion ApplePotatoCabbage
0.20.050.05
* Codex Alimentarius Commission
Regular upgrading of national standards and MRLs in line
with international standards
Food Contaminant Control
Ideal Situation • Rigorous controls• Identify the main sources of contamination (raw materials,
supply chain)• New emerging pathogens and contaminants
Needs• Improve prevention, detection and control strategies along
the food production chain• Use powerful and sophisticated technologies• Anticipate and control emerging pathogens, new
contaminants• Develop methods for the unequivocal identification/detection
of potential chemical contaminants
Methods of Analysis, Detection and Control
Aspect1 : Screening Procedures • Fast, cheap, reliable• Portable, field, in/on-line• High sample throughput • Selective <-> Multi- Analyte/Residue/ Pathogen• Stable & robust• Low LoD & LoQ• Test-Kits & sensors
Aspect 2 : Confirmatory Procedures • Sophisticated & reliable• Selective <-> Multi- Analyte/Residue• Stable• Low LoD & LoQ• Low solvent extraction• Selective clean-up step• Selective end-point• e.g.LC-MS-MS,GC-MS-MS, • Matching screening procedure(s)
Aspect 3 : Reliability
• Intra-lab quality control / assurance• Inter-lab quality control / assurance• Acceptance criteria • Low suspected false negative & positive results• Benchmarking• Accreditation• Standardisation (guide)• Communication (results)
Aspect 1: Screening ProceduresField rapid, reliable sensitive methods
Dipstick methodColorimetric testsBionanosensorsTime Resolved Fluorometres (veterinary drugs)
Interlaboratory Method Validation
Detection of sulfonamides, tylosin, chloramphenicol and fluoroquinolones in honey
Aspect 2: Confirmatory Methods
• Sample Extraction• Sample Clean-up• Analysis by high throughput technologies
– High Performance Liquid Chromatography• UV-Detector• Fluorescence detector• Mass Detector/ tandem mass spectrometry (MS/MS)
– Gas Chromatography• Electron Capture Detector• Flame Ionisation Detector• Mass Detector/tandem mass spectrometry (MS/MS)
– Atomic Absorption Spectrometry
Multi-Residue/Analyte Methods
Analysis of 300 pesticides in 30min by LC-MS/MS
GOAL
Mission of the National Food SafetyLaboratory (NFSL)
• Act as Reference Laboratory related to Food Safety Examination and Procedure
• Analysis of food samples collected by sanitary inspectors as per standard
• Reporting of results after sample analysis• Conduct research and training programmes related to
food safety• Coordinate and networking with all laboratories related
to food safety
Selection of Methods (ISO 17025)
• Published in international or national standards• Published by reputable technical organisations• Published in scientific journals• Specified by the manufactures of the equipment• In-house developed laboratory methods
Methods have to be validated !!
Aspect 3: Reliability
• Method Validation is to:– Identify an analyte in a test sample– Quantify accurately an analyte in a test sample
• Method Validation is to provide evidence that a method is fit for purpose. It does what it’s intended to do with high level of precision and accuracy
• A properly validated method should provide confidence in the results
Method Validation
Method Validation Parameters
• Specificity/Selectivity• Linearity• Range• Limit of Detection• Limit of Quantification• Accuracy/Trueness• Precision/Repeteability• Reproducibility• Ruggedness/Robustness
Method Selectivity
• The identification of a target analyte is correct and not hindered by the presence of one or more interferences
• The presence of interferences is not leading to a false positive
• The quantification is correct and not influenced notably by the matrix
• Selectivity can be confirmed using a matrix-added calibration curve
Linearity / Range
• The regression analysis of test results vs analyte concentration must be linear.
• The linearity must cover the range of the expected concentration
0
500
1000
1500
2000
2500
3000
3500
4000
4500
0 50 100 150 200
Concentration
Peak
Are
a
LOD and LOQ
Limit of Detection (LOD): It is the point at which an analysis is feasible
Limit of Quantification (LOQ): Concentration at which quantitative results can be reported with a high degree of confidence.
Limit of QuantificationSignal / Noise Ratio 10:1
Limit of DetectionSignal / Noise Ratio 3:1
Signal-to-noise examples
Accuracy/Recovery
• Accuracy is a qualitative term referring to whether there is agreement between a measurement made on an object and its true (target or reference) value.
• Accuracy can be assessed on samples spiked with known amounts of the analyte
• At least 3 repetitions at 3 concentration levels (low, medium and high)
• Accuracy can be expressed as percentage recovery from the added amount with confidence intervals
Acceptance Criteria (AOAC). Recoveries ≥ 80%
Precision/Repeteability
• Calculation of the (relative) standard deviation of a sufficient number of sample aliquots. This can be of three levels, three repetitions or 6 determinations at 100%.
Acceptance Criteria (AOAC) ≤ 15%
Reproducibility
• Multiple chemists in multiple labs run samples. Results should be reproducible and comparable for method precision.
• Example: Samples run by 3 Chemist, 3 labs and 3 different instruments, 3 different days
20
Robustness
• Change some method parameters and compare the differences with the core method
• Shows the reliability of the analytical method with respect to variations in the method
• Some parameters that can be changed:
- pH - temperature- % Organic
- buffer Concentration- column Lot
21
Example 1: Organochlorine Pesticides by GC-ECD
GC-ECD Chromatogram of 20 organochlorine pesticides spiked at a concentration of 200ng/ml in dry fish (matrix-matched). α-BHC, Rt: 3.26; 2) δ-BHC, Rt: 3.69; 3) β-BHC, Rt:3.82; 4) γ-BHC, Rt:4.10; 5) Heptachlor, Rt: 4.46; 6) Aldrin, Rt:5.01; 7) Heptachlor Epoxide, Rt: 6.26; 8) γ-Chlordane, Rt:6.55; 9) α-Chlordane, Rt: 6.87; 10) α-Endosulfan, Rt: 7.14; 11) 4 ,4’ DDE, Rt: 7.15; 12) Dieldrin, Rt: 7.77; 13) Endrin, Rt: 8.35; 14) 4,4’ DDD, Rt: 8.70; 15) β-Endosulfan, Rt: 8.99; 16) 4, 4’DDT, Rt: 9.59; 17) Endrin Aldehyde, Rt: 10.42; 18) Endosulfan sulphate, Rt:11.78; 19) Methoxychlor, Rt: 12.24; 20) Endrin Ketone, Rt:13.60.
Multi-Residue Method for determination of organochlorine pesticides in fish
Validation ResultsMatrix free Matrix Match (Loiytta Fish)
Pesticide R2 LOD [ppb]
LOQ [ppb]
Range [ppb]
R2 LOD [ppb]
LOQ [ppb]
Range [ppb]
α-BHC 0.99 22.33 74.44 25-500 0.99 61.40 204.67 50-500δ-BHC 0.99 23.09 76.98 25-500 0.99 55.90 186.36 50-500β-BHC 0.99 48.91 163.05 50-500 0.99 37.19 123.98 50-500 γ-BHC 0.99 50.87 169.59 50-500 0.99 69.56 231.87 50-500Heptachlor 0.99 23.81 79.38 25-500 0.99 37.38 124.61 50-500Aldrin 0.99 8.50 28.33 10-500 0.99 63.33 211.11 50-500
Pesticide (Spiked Level 80[ng /g])
Recovery [%] RSD [%]Repeatability
RSD [%]Precision
α-BHC 89.61 11.68 3.76δ-BHC 85.79 6.99 3.19β-BHC 107.12 6.50 9.24Heptachlor 99.96 5.04 13.01Heptachlor Epoxide 100.31 10.12 16.86γ-Chlordane 83.67 16.57 8.92
α-Chlordane 86.82 16.46 7.67
Example 2: Formalin Detection by HPLC
1 2 3 4 5 6 7 8 9 10 11
10 6
20 6
30 6
40 6
Time [min]
Pea
k A
rea
[u
V]
1 2 3 4 5 6 7 8 9 10 11
Pea
k A
rea
[u
V]
10 6
20 6
30 6
40 6
Time [min]
A)
1 2 3 4 5 6 7 8 9 10 11
10 6
20 6
30 6
40 6
Pea
k A
rea
[u
V]
Time [min]
B)
1 2 3 4 5 6 7 8 9 10 11Time [min]
Pea
k A
rea
[u
V]
10 6
20 6
30 6
40 6C) D)
1 1
2
1 1
2
Selectivity HPLC Chromatogram: DNPH (A); 2mg/L derivatized standard (HCHO-DNPH (B); derivatised mango sample (spiked with 5mg/L formaldehyde). 1: DNPH, 2:CH2O
METHOD VALIDATION PARAMETERS: Formalin in FoodsParameter Matrix-free Matrix-matched
Mango Milk FishLinear Equation y= 488092x +
4440.9y=209862x +
189675y=202380 + 52100 y=27507.17 +
125966.7
R2 0.99 0.99 0.99 0.99LOD [mg/L] 0.39 0.32 0.67 1.75LOQ [mg/L] 1.30 1.08 2.23 5.83Range [mg/L] 1.30-250 1.08-150 1.0-150 5.0-150
Moving Forward
• Bangladesh Food Safety Laboratory Network– Harmonisation and upgrading of analytical methods
in the country– Improve the level of confidence of analytical results– Interlaboratory comparison– Provide scientific-based data to support legislation– Support authorities in the control and monitor of
improper business practices.
Programme Partners Funded by
Thank you for your attention