Food safety in non-alcoholic beverages: Importance of testing pesticide residues
Kaushik Banerjee PhD FRSC FNAASPrincipal Scientist
ICAR-National Research Centre for GrapesPune, India
Chairman- India Section of AOAC INTERNATIONAL
Non-alcoholic beverages
vNon-carbonated •Tea, Coffee •Fruit juice (fresh, canned, blended or shake) & Veggies Juice •Mineral water •Milk beverages•Nutrition beverages v Carbonated •Soft drinks•Mock tails, Flavoured fizzy drinks (High carbonation) •Sports or isotonic beverages (low carbonation)
Stimulating (tea, coffee), Refreshing (soft drink, juices) and Nourishing drinks (milk, energy drink)
Food safety issues
• Beverage products that are marketed- Quality compliance
• Ingredients/ Components- Quality compliance
Compliance to global Regulations
Food Market
European Union
Japan
USA
Middle East
Singapore & Malaysia
EUR- Lex
http://www.ffcr.or.jp/zaidan/FFCRHOME.nsf/TrueMainE?OpenFrameset
eur-lex.europa.eu
www.fda.gov/
http://www.ava.gov.sg/
http://old.sfda.gov.sa/En/Food
MHLW
USFDA
AVA
SFDA
FBOs face mounting challenges of variable quality standards often with little clarity how to meet them
Food standards are many
http://face-cii.in/sites/default/files/final_report-version_2.pdf
Regulations expect product traceability: utmost important for sustainable implementation of
food safety
Regulation (EC) No 178/2002q Traceability means the ability
to trace and follow:Ø a food or Ø a substance intended to be, or expected to be incorporated
into a food q through all stages of:Ø production, Ø processing or Ø distribution.
What is traceability?
• Why is traceability needed?q Most of the food scares in the 90’s originated due to a lack of
traceability across the food chain:Ø BSE (‘mad cow’ disease)Ø Dioxin (Belgium)Ø Pesticides in soft drinks in India- 2003
There were 13 RASFF alerts in Europe for Indian grapes in 2003
Pesticide residues in fruit juice- Carbendazim in orange juice
http://www.fnbnews.com/Marketing/packaged-juice-market-growth-in-india--current-and-future-scenario-43861
Worldwide-Major product categories of concern for MRL non-compliances
RASSF Annual report 2017, 2016
Product Category
ClassificationsTotal 2016
2017
Fruits & Vegetables 497 504
Nuts, nut products & seeds 443 536
Fish & fish products 327 368
Cereals & bakery products 112 124
Dietetic foods, food supplements, fortified food
198 313
• Cultivation involves frequent pesticide applications• Lack of good agricultural package of practices - Recommended dose? Application time? - PHI to ensure residue dissipation before harvest
PESTICIDES IN SOFT DRINKS- 2003
-AN INCIDENCE THAT HAD SHAKEN THE ENTIRE BEVERAGE INDUSTRY
& ALSO CONTRIBUTED TO ANALYTICAL CAPACITY BUILDING IN BEVERAGE SECTOR
Pesticides in soft drinks- 2003
• An NGO reported unacceptable levels of pesticides in certain soft drinks in India
- above the MLs of BIS &- EU
• Chlorinated hydrocarbons, OPs - restricted in many of the developed countries due to their potential mammalian toxicity
Follow up actions….
• The disputed soft drinks samples were cross-examined by some of the Government official control laboratories under Ministry of Health
• These official labs also reported pesticides at various levels
A close look at the results revealed….
• Lots of variations in inter-lab test results -The detected pesticides were not the same -The residue levels were largely different
Lab-to-lab variations (Official labs)
Pesticide Sample Presence / Absence
Lab 1 Lab 2 Lab 3
MalathionSoft
drink1
Detected Detected Not detected
Ethion Not detected
Not detected
Detected
Lab-to-lab variations: unacceptably high
Sample Total pesticide residue(OC+OP in mg/L)
Lab 1 Lab 2 Lab 3
Soft drink1 0.000025 0.0187 0.00009
Soft drink2 0.001 0.011 0.00063
Soft drink3 0.002 0.0223 0.0006
Soft drink4 0.00054 0.0166 0.00033
Soft drink5 0.001628 0.0055 0.00012
VARIATIONS ≥ 100%
Science based concerns were raised based on this incidence
• Was the method fit for purpose?• Did these labs have enough capacity to analyze
such trace levels of residues?• Were the analytical methods appropriately
validated for accuracy, precision and measurement uncertainty?
• Which criteria were used to confirm the identity of these detections?
Observations
• Fitness for purpose of the sample preparation was not clearly established
• Optimization of the instrument methods was also questionable
Some more concerns….
• The instrument used was GC with ECD (for OC) and NPD (for OP)
-lack of selectivity
• Some pesticides, e.g. malathion are not supposed to be stable enough in low pH of carbonated beverage
Although MS confirmation was used, it involved single ion monitoring
• Identification based on only one mass, which was not even the molecular mass against the Regulatory criteria: SIM, 3 ions min. +/- 10% expected Relative Intensity
Criteria # of Hitsm/z 268 1144
m/z 268 (40-100% RI) 149
m/z 268 + m/z 239 (30-100% RI) 5
m/z 268 + m/z 239 + m/z 145(40-100% RI)
1
Heptachlor
Regulatory queries could not be answered
• Office of Prevention, Pesticides and Toxic Substances (OPPTS) 860.1340 - Residue Chemistry Test Guidelines:
– An enforcement method should be sufficiently specific to measure and identify the residue in the presence of residues of other pesticides which could reasonably be expected to be present on the same commodity
- Registrant must provide supporting data from interference studies, OR
- Registrant must provide an appropriate confirmatory method
Is GC or LC Specific Enough?
• OPPTS 860.1340- Although certain GC and LC detection systems possess inherent specificity, methods based on these systems should usually be supplemented by a confirmatory method which is Significantly Different from the primary enforcement method. In general confirmation by Mass Spectrometry is suitable.
Co-elution & chances of false detection in a multiresidue situation
Unless we use unique MS criteria, detections could be ambiguous
Pentachloroaniline
Aldrin
Cyperazine
Desmetryn
Dimethochlor
Pretilachlor
2,4-DDD
Oxadiazon
Co-elution- Chances of false detection
• Cyfluthrin, cypermethrin, flucythrinate and ethofenprox could not be separated in TIC because of co-elution.
• RT Separation of cypermethrin and cyfluthrin isomers was possible by MS/MS with MRM 165 > 127.
• Flucythrinate I & II could be separated by MS/MS with MRM transition from m/z 199 > 157.
• Ethofenprox (163>135)
RT: 15.28 - 17.00 SM: 7G
15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8Time (min)
0
50
100
0
50
100
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100
Re
lative
Ab
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RT: 16.25SN: 134RMS
RT: 16.57SN: 90
RT: 15.72SN: 88
RT: 15.71SN: 419 RT: 16.25
SN: 387
RT: 16.27SN: 176RMS
RT: 16.57SN: 163RMS
RT: 16.58SN: 3121RMS
NL: 1.27E4Base Peak m/z= 180.50-181.50+205.50-206.50 F: + c Full ms [50.00-520.00] MS 100ppbSPMSMSFS02
NL: 1.68E3Base Peak m/z= 126.50-127.50 F: + c SRM ms2 [email protected] [81.00-175.00] MS 100ppbSPMSMSFS02
NL: 3.99E3Base Peak m/z= 156.50-157.50 F: + c SRM ms2 [email protected] [100.00-210.00] MS ICIS 100ppbSPMSMSFS02
NL: 4.09E4Base Peak m/z= 134.50-135.50 F: + c SRM ms2 [email protected] [100.00-173.00] MS ICIS 100ppbSPMSMSFS02
Isobaric interferences- Chances of false detection
Acceptable GC-MS Criteria:
• Full scan EI MS• SIM, 3 ions min.- +/- 10% expected RI
• But these criteria were not referred by the laboratories while reporting results of soft drinks analysis
Need of an standard method• The stakeholders felt the requirements of a standard
method
• Official methods (e.g. AOAC) do not exist for pesticide residues in soft drinks
AOAC INTERNATIONAL Presidential Task Force on Pesticides in Soft Drinks (PSD)
• At the request of the soft drink industry, AOAC INTERNATIONAL formed a Task Force on Pesticides in Soft Drinks (PSD)
- to collaborate in the method development- evaluation and validation of consensus-based
analytical method(s) for the analysis of pesticide residues in soft drinks
Why? Because AOAC Official MethodsSM are considered the “gold standard”
• AOAC methods are accepted and recognized by regulatory agencies and international organizations, and can be used worldwide
• The task force aimed to bring stakeholders to come to consensus on the fitness for purpose of an analytical method/s that can be used and adopted by the national governments and others for the analysis of pesticide residues in soft drinks
The PSD Task Force constituted stakeholders who were– Scientific experts in pesticides
– Representatives from the Indian government and agencies charged with protecting the public
– Other stakeholders from industry and other governments
– AOAC Methods Committee on Residues & Related Topics
Expert Review Panel:
– Members were selected based on scientific expertise– Review analytical methods requirements– Review available analytical methods– Recommend methods and/or additional studies that may
be necessary
ERP- list of pesticides & call for methods
• 15 Candidate methods• Each method was reviewed by 2 reviewers• Strengths & Weaknesses considered• Consensus on preferred method
Action plan• Method validation- SLV• Multi-lab validation
Scope and Fitness for Purpose• Concentration range: LOQs in bottled water was
referred.- performance limit was 0.1 ppb - Total 0.5 ppb if multiple residues are present- GC-MS and LC-MS based methods, qualitative
and quantitative• Method classification: Intended use- screening,
quantification, confirmation
Practicality: the Task Force guided to establish overall "fitness for purpose" requirements
• Standard operating procedure defined• Single laboratory validation• Collaborative study• Data processing, statistical evaluation
Current scenario & challenges
• Testing for food contaminants is very limited• Methods are still not defined for many of the
matrices
• MRLs are not available for beverages - FSSAI finds difficulty to set MRL for juice - Processing factors are not available
2018-2020 EU coordinated multiannual control programme (171 compounds)
BitertanolBoscalidCaptanCarbarylCarbendazim Carbofuran ChlorantraniliproleChlorfenapyrChlormequat Chlorothalonil ChlorprophamChlorpyrifosChlorpyrifos-methyCyfluthrin CymoxanilCypermethrin CyproconazoleCyprodinilCyromazineDeltamethrin DiazinonDichlorvosDicloranDicofol DiethofencarbDifenoconazoleDiflubenzuronDimethoateDimethomorphEthephonEthionl
Etofenprox FamoxadoneFenamidoneFenamiphosFenarimolFenazaquinFenbuconazoleFenbutatin oxideFenhexamidFenitrothionFenoxycarbFenpropathrinFenpropidinFenpropimorphFenpyroximateFenthion Fenvalerate FipronilFlonicamidFludioxonilFlufenoxuronFluazifop-P-butylFlubendiamideFluopyramFluquinconazoleFlusilazoleFlutriafolFolpetFormetanate
Compounds frequently found in
Indian samples
(EU) 2017/660 of 6 April 2017
HexaconazoleHexythiazoxImazalilImidaclopridIndoxacarb LufenuronMalathion MandipropamidMepanipyrim MepiquatMetalaxyl MethamidophosMethidathion Methiocarb Methomyl and thiodicarbMethoxyfenozideMonocrotophosThiaclopridParathion methyl PenconazolePencycuronPendimethalinPermethrin
Phosmet Pirimicarb Pirimiphos-methylProcymidoneProfenofosPropamocarb PropargiteThiamethoxam Thiophanate-methylTolclofos-MeTolylfluanid TriazophosTrifloxystrobinTriflumuronVinclozolin
Concerns of food safety in Indian fruits/vegetables
• Many of the detected pesticides are:- banned / restricted in most countries- have potential endocrine disrupting
properties- toxicity to non-target organisms, e.g.
neonicotinoids on honey bees
Threats of emerging contaminants
• PGRs• Antibiotics• Pharmaceuticals• Personal care products• Disinfectants, etc.
Many of these are polar/ionic in natureLevels are regulated at default levelAnalytical challenges
Regulatory limitation
• Majority of the commonly detected pesticides remain on the fruit surface and may not appear in juice
• But, in absence of MRL, default MRL (0.01 mg/kg) is applied
Food safety & traceability in
horticultural fresh commodities
Farm registration to consumer level
Mango.NetCitrus.Net
Plot
MH-01 -01 -01 -0001 -01
State codeDistrict code
Sub-district codeProduct code
Farm code
A complete geographical traceability system
Traceability- Salient FeaturesHortinet
• An Internet based traceability software system for fresh grapes exported from India to the European Union.
Unique coding system of traceability
• Geographical traceability of produce from farm to consumer
Internal alert system
• If a farm’s produce fails the EU MRL requirements, an internal alert is issued for this farm’s produce, restricting it from export to European Union.
Integration of all stake holders
• Farmers, State Government Horticulture Departments, The testing laboratories, Agmark Certification Department, The Phyto-sanitary department, Pack houses, Exporters, NRL and APEDA.
- “Though India's share in the global market is still nearly 1% only, there is increasing acceptance of horticulture produce from the country.
This has occurred due to concurrent developments in the areas of quality assurance measures.”
http://apeda.gov.in/apedawebsite/six_head_product/FFV.htm
Residue testing is the backboneIt costs money but adds value to a traceable product
To meet the quality standards, residue testing ensures- Domestic food safety
- Export compliance
- Risk assessment for fixation of MRL
- Facilitates trade and enhances income
NRL has methods for juice & fresh commodities
• Extraction of 10 mL sample with 250 µL chloroform• The organic layer was pipetted out, evaporated to near
dryness, reconstituted in ethyl acetate + cyclohexane (1:9) mixture resulting in an enrichment factor of 400.
• Method detection limit (MDL) ranged from 1 to 250 ng/L
• The MDLs were 400 times lower than the instrument LoQ.
• The method was validated in in a range of beverages (e.g. apple, lemon, pineapple, orange, grape and pomegranate juice)
Analysis of PPCPs
Sample PreparationTwo methods employed
Extraction with Acetonitrile
Cleanup
Centrifuge & dilute
Dilute
and
Shoot
10 mL juice
Dilute with water
Filter and Inject
Way forward
• Standardization & Method Verification in various beverage matrices
• Why a method for fresh commodities require verification in beverage matrices
- Composition of beverages varies - Chances of false positives/negatives - Matrix effects might significantly vary
Applicability (matrix and concentration) LOD/LOQ Accuracy Precision (repeatability / reproducibility) Recovery Selectivity Sensitivity Linearity
Measurement uncertainty
Method Validation
Standard Method
Non standard and Lab-developed Method
Slide from Barbara deSantis
What AOAC-IBA collaboration can offer
• Facilitate establishment of processing factors and help FSSAI to decide risk based MRLs for beverages
• Contribute to FSSAI methods manual
• Help FSSAI and other national/ international regulatory bodies for dispute resolutions
• Identify candidate methods• Evaluate existing methods• Establish fitness for purpose
for intended use• Coordinate SLV• Collaborative study• Recommendation to FSSAI • Coordinate with AOAC
International to organize PT rounds
AOAC-IBA collaboration- a long way to go
• Proactive action to prevent future disaster• Food safety is a shared responsibility.- FSSAI-AOAC official collaboration is already 1 year old• IBA and AOAC can join hand to make
beverages more safe by - Implementation of fit for purpose methods - Facilitate capacity building on beverage testing
THANKS FOR THE ATTENTION