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

0

50

100

Re

lative

Ab

un

da

nce

0

50

100

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 165.00@cid2.00 [81.00-175.00] MS 100ppbSPMSMSFS02

NL: 3.99E3Base Peak m/z= 156.50-157.50 F: + c SRM ms2 199.00@cid1.80 [100.00-210.00] MS ICIS 100ppbSPMSMSFS02

NL: 4.09E4Base Peak m/z= 134.50-135.50 F: + c SRM ms2 163.00@cid2.00 [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