Analysis of pesticide residues in food using ethyl acetate extraction and detection with GCMS/MS and LC-MS/MS (SweEt)

Tuija Pihlström

National Food Agency LAPRW 2015 Santiago de Chile Sweden

Outlines of presentation

 SweEt an alternative method for analysis

of pesticides in food  Validation according to EU SNACO 12571/2013 ”Analytical Quality Control Procedures for Pesticide Residue Analysis i Food and Feed”  Example of reinforced import control (EC 669/2009) using the SweEt method

LAPRW 2015 Santiago de Chile

Official control of pesticides at the National Food Agency (NFA) in Sweden How is it organized? Laboratories National Reference Laboratory (NRL) and one official laboratory (Eurofins), both accredited

Method development • Most of the method development at the NFA. Transfer of the methods

to the official laboratory for validation and for monitoring of pesticides

Analytical methods  Mainly multi residue methods based on ethyl acetate extraction and

determination using LC-MS/MS and GC-MS/MS

Analysis • 1800 of 2000 of the samples analysed by OfL (Eurofins) •

history of the SweEt method • Multi residue methods are a self-evident choice for analysis of

• • • •

pesticide residues in food Since 1989 multi residue method based on extraction with ethyl acetate method has been used At that time GC with various detectors was the main detection technique The method has since then continuously been improved including simplified sample preparation and faster analysis Introduction of animal product 2009 has further enlarged the scope of the method

The simplified NFA´s MRM for FV (448 analytes; 145 GC, 303 LC) Extraction 10 g sample + 3 g NaHCO3 20 ml ethyl acetate and 10 g Na2SO4 Falcon tube SpexSample Prep 3 minutes Centrifugation in 3 min. (3800 g) Filtration Filtrate the crude extract 0.20 µm PTFE filter

Inject to GC-MS/MS and UPLC-MS/MS Sample conc. 0.5g/ml

Fruit and vegetables, MRM Steps Extraction Centrifugation Filtration

MIn 3

Concentration/redissolve Clean up

15 45

Concentration/redissolve Filtrate Solvent change

10 2 15 95

5

Min 3 3 2

Ethyl acetate method

The simplified SweEt

Extraction 75 g sample+200 ml EtoAc Waring blendor 3 min

Extraction 10 g sample+20 ml EtoAc Falcon tube SpexShaker prep 3 min

↓

↓

Decant and filtrate through Na2 SO4

Centrifugation

↓ Concentration and redissolve

↓

↓ 2

GPC clean up

↓

Concentration and redissolve

↓

10

↓ Filtration

Filtration↓ ↓

Solvent switch for LC ↓

↓

↓

Inject to GCMS/MS

Inject to LCMS/MS

1.5 g/ml

2.5 g/ml

Inject to GCMS/MS

Inject to UPLC-MS/MS

0.5 g/ml

0.5 g/ml

FV +honey

Cereals 315

AO10% fat 76↓

96

Fat determination Extraction 10 g sample 20 ml EtAc+NaHCO3 3 min Spex shaker Na2SO4

Extraction

Extraction

5 g sample 10 ml water 10 ml EtAc (1 % Hac) 3 min Spex shaker Na2SO4

5 g sample PSA + C18 10 ml EtAc 3 min Spex shaker Na2SO4

↓ Extraction 0.5 g fat 10 ml EtAc:cyhex (1+1) 3 min Spex shaker Na2SO4 ↓

Centrifugation ↓ Filtration

Centrifugation

Centrifugation

Centrifugation

Filtration ↓

Filtration

GPC SX-3 (1+1 EtOAc/cyklohexane)

↓ GC-MS/MS

UPLCMS/MS

GCMS/MS ↓

Filtration ↓

UPLCMS/MS ↓

GC-MS/MS

↓

UPLCMS/MS

Evaporation ↓ Reconstitution in EtOAc ↓

↓

GC-MS/MS

UPLC-MS/MS

Advantages of the SweEt method • Fast and non-laborious • Negligible sample work up costs • No evaporative concentration steps • The extract is injected directly into the GC and LC • The need of clean up has been eliminated (almost)

• Low amounts of matrix components as proteins and sugar which often interfere with the chromatographic are co extracted. National Food Agency, Sweden

LAPRW 2015 Santiago de Chile

The study of different extraction solvents and the amount of co extracted matrix components J. Klein and L. Alder: Development of a Multi-Analyte / Multi-Matrix Method Based on LC-MS/MS; EPRW 2002. Rome 28.- 31.05.2002

Type of solvent Matrix

Matrix type

Amount of extracted sample

Acetone /water 2+1

Tomato

High water content

1.0g

38mg

36mg

Citrus

High acid and high water content

1.0g

79mg

Raisins

High sugar an low water content

1.0g

Avocado

High oil content

1.0g

National Food Agency, Sweden

Acetonitrile Methanol /water 2+1 /water 2+1

Water

Ethyl acetate

34mg

33mg

8mg

69mg

81mg

-

3mg

370mg

148mg

333mg

302mg

4mg

33mg

40mg

30mg

43mg

332mg

LAPRW 2015 Santiago de Chile

Even the sun has spots……….

Limitations Distorted or split peak shapes Solvent composition ”solvent effect” •

The injection solvent is stronger than the mobile phase (MP)

•

EtOAc not diluted immediately with MP, some of the analyte molecules are diluted and migrated with the MP resulting in split peaks

•

Early Peaks (low k) most affected (2-5 min)

•

It is important to keep the injection volume low 2 µl is recommended to avoid the peak split.

National Agency, Sweden

LAPRW 2015 Santiago de Chile

Oxydemetonmethyl in MeOH

MP: Buffert/MeOH (90/10)

Oxydemetonmethyl in EtOAc

National Food Agency,

National Food Agency Sweden Sweden

LAPRW 2015 Santiago de Chile

Validation in multi residue methods

Requirements for official laboratories 1. Implementation of Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food an feed SANCO12571/2013

1. Accredited according to the ISO/IEC 17 025 standard 1. Participation in EU Proficiency tests

Guidance document on analytical quality control and validation procedures for pesticide residues analysis in food an feed SANCO12571/2013

Why do we need the guidelines?  To reach a complete harmonization of quality assurance measures for pesticide residue analysis within Europe  To help monitoring laboratories achieve an acceptable standard  The reported results are reliable and consistent with other similar results

The key objectives of the guidelines

 To harmonize cost effective AQC in the EU (to find an optimum between cost and output (efficiency/quality) To ensure false positives or false negatives are not reported  To support compliance with ISO/IEC 17025 accreditation standard

Guidance in  The whole analytical chain (from sampling to reporting results)  To define minimum criteria for validation and analysis (allows free choice of method)

Table of contents • • • • • • • • • • • •

Accreditation Sampling, transport, processing and storage of samples Pesticide standards, calibration solutions, etc. Extraction and concentration Contamination and interference Analytical calibration, representative analytes Analytical methods and analytical performance Method validation Routine recovery determination Proficiency testing and analysis of reference materials Confirmation of results Reporting of results

Example of representative commodities fruit vegetables Commodity Categories

Commodities included in this category

Typical representative commodities

1.High water content

Pome fruit

Apples, pears

Stone fruit

Apricots, carrots

2.High acid content Citrus fruit and high water content

Lemon, mandarin, orange

Honey, dried fruit 3.High sugar and low water content 4a. High oil content Tree nuts and very low water content 4b. High oil content Oily fruits and products and intermediate water content

Honey, raisins, dried apricots, dried plums, fruit jams Walnuts, hazelnuts, chestnuts

Olives, avocados and pastes thereof

Example of validation according to the SANCO 12571/2013 Full validation in three different commodity groups high water and high acid content, e.g. orange high water content, e.g. cucumber or carrot high sugar and low water content, e.g. raisins Recovery study: 5 spiked samples at target LOQ (0,01 mg/kg) 5 spiked samples at 2-10 * target LOQ (0,05 mg/kg)

Table 1. Validation parameters criteria. Parameter

What/how

Criterion

Linearity

Through calibration curve

Residuals < ±20%

Matrix effect

Comparison of response from solvent standards and matrixmatched standards By definition: lowest level for which it has been demonstrated that criteria for trueness and precision have been met Response in reagent blank and control samples

20% small effect

Precision (RSDr)

Determine repeatability RSDr, determine for both spike levels

≤ 20%

Precision* (RSDwR)

Determine within-laboratory reproducibility* Can be derived from on-going method validation / verification through establishing average recovery and RSDwR? Determine the average recovery for spike levels

≤ 20%

LOQ

Specificity

Robustness

Trueness (bias)

≤ MRL

< 30% of LOQ

See above

70-120%

Validation results of fruit and vegetables GC analytes 145, LC ES+/ES- analytes 303 Spiking level Mean recovery (mg/kg) (%)

Mean RSD (%)

Number of results

Matrix group

Detection

High acid content and high water content

GC-MS/MS

0,01

97

8,95

204

High acid content and high water content

GC-MS/MS

0,05

100

7,25

142

High acid content and high water content

LC-MS/MS ES-

0,01

87

9,59

25

High acid content and high water content

LC-MS/MS ES-

0,05

89

6,32

26

High acid content and high water content

LC-MS/MS ES+

0,01

89

7,51

262

High acid content and high water content

LC-MS/MS ES+

0,05

89

4,87

272

High sugar and low water content

GC-MS/MS

0,01

100

8,72

148

High sugar and low water content

GC-MS/MS

0,05

104

6,91

149

High sugar and low water content

LC-MS/MS ES-

0,01

94

4,4

11

High sugar and low water content

LC-MS/MS ES-

0,05

98

3,91

13

High sugar and low water content

LC-MS/MS ES+

0,01

94

4,52

274

High sugar and low water content

LC-MS/MS ES+

0,05

97

3,18

271

High Water Content

GC-MS/MS

0,01

101

9,24

207

High Water Content

GC-MS/MS

0,05

103

6,35

140

High Water Content

LC-MS/MS ES-

0,01

93

8,13

23

High Water Content

LC-MS/MS ES-

0,05

88

5,64

23

High Water Content

LC-MS/MS ES+

0,01

95

4,92

259

High Water Content

LC-MS/MS ES+

0,05

94

4,34

262

Matrix effects – Orange, LC-MS/MS Matrix effect (%)

60,0 40,0 20,0 0,0 -20,0

-40,0 -60,0

Pesticide

Only 24 of 219 pesticides have >20 % matrix effect. – in samples were those are found, true matrix-matching is used. Standard in blank carrot extract. Tested at 0.05 mg/kg. Agilent 6460.

Matrix effects – Orange, GC-MS/MS Matrix effect (%)

30,0

20,0 10,0 0,0 -10,0 -20,0 -30,0 Pesticide

Only 1 of 129 analyte has >20 % matrix effect (Tetrachloroaniline-2,3,5,6, +27 %). – in samples were found, true matrix-matching is used. Standard in blank cucumber extract. Tested at 0.05 mg/kg. Thermo TSQ Quantum XLS.

Matrix effects – Raisins, LC-MS/MS

Matrix effect (%)

60,0 40,0 20,0

0,0 -20,0 -40,0 -60,0

Pesticide •

Only 17 of 219 pesticides have >20 % matrix effect. – in samples were those are found, matrix-matching is used for quantification. Standard in blank carrot extract. Tested at 0.05 mg/kg. Agilent 6460.

Matrix effects – Raisins, GC-MS/MS 45

Matrix effect (%)

35 25 15 5 -5 -15 -25 -35 Pesticide

26 of 129 pesticides have a matrix effects >20 %) - in samples were those are found, matrix-matching is used. Standard in blank cucumber extract. Tested at 0.05 mg/kg. Thermo TSQ Quantum XLS.

SweEt in EU RL Proficiency tests:

Proficiency Test 1-4 for cereals

Fat content in selected food Ref. The Food Database. NFA

Product

*Total fat %

Triglycerides %

Milk 3 %

3.1

2.3

Egg

9.7

8.5

Liver

3.0

2.8

Butter

82

79.3

*Phospholipid. Triglycerides, stereols National Food Agency, Sweden

LAPRW 2015 Santiago de Chile

Analysis of pesticides in products with animal products Extraction 5 g sample + 0.2 g PSA and 0.2 g C18 10 ml ethyl acetate Spex SamplePrep 3 min 0.5 g Na2SO4

Centrifugation and filtration In 3 min. (3800 g). filtrate the crude extract with 0.20 µm PTFE filter

Inject to GC-MS/MS and UPLC-MS/MS Sample conc. 0.5g/ml National Food Agency, Sweden

LAPRW 2015 Santiago de Chile

Results EU PT AO 9 Test material: Liquid Whole Egg Pesticide Dieldrin Endosulfansulfat Endrin gamma-HCH (Lindane) Trans- Heptachlorepoxid Diazinon Parathion Phoxim Deltamethrin Permethrin

National Food Administration. Sweden

Conc mg/kg Conc mg/kg Conc mg/kg NFA Eurofins Spiked Assigned Found Z score Z score 0,03 0.0268 0.031 0,6 0,8 0,045 0.0289 0.036 0,9 1 0,015 0.0141 0.011 -0,9 2 0,033 0.0256 0.033 1,2 0,9 0,051 0.0438 nd -3,5 0,9 0,065 0.0588 0.062 0,2 0,6 0,105 0.0862 0.097 0,5 1 0,15 0.122 0.17 1,6 0,4 0,075 0.0562 0.078 1,6 1,1 0,3 0.251 0.34 1,4 0,8 LAPRW 2015 Santiago de Chile AOAC2015

Multi method for analysis of pesticides in products with high fat content Fat determination Gravimetrical determination after extraction with EtOAc:Cyhe (1+1)

Extraction Amount of fresh sample that corresponds to 0.5 g of fat (2.5 g cream) 10 ml ethyl acetate + cyclohexane (1 + 1) 2.5g Na2SO4

Centrifugation & Filtration 3 min. 3800g Filtrate crude extract 0.2 µm PFTE filter

Clean up 5.0 ml to GPC SX-3 Ethyl acetate + cyclohexane (1 + 1)

Evaporation To dryness with N2 Re-dissolve in 0.5 ml ethyl acetate

Inject into GC-MS/MS and LC-MS/MS LAPRW 2015 Santiago Matrix conc. 0.5 g/mlde Chile

76 analytes

Butter – our challenge (slide from 2010. Heidi Pekar)

GPC Fat Profile 0.5 g butter 385 Residue (mg)

285

Pesticide Fraction Start

185

Pesticide Fraction End

85

4

National Food Agency, Sweden

8

12

16

20

24

28

LAPRW 2015 Santiago de Chile

32 Time (min)

EU PT AO7 results 2012 - liquid cream NFA. Sweden (GPC) Fat Content: 29% Pesticide Z-scores Bifentrin -0.2 cis-Chlordane -0.6 trans-Chlordane -0.2 DDE -1.0 Diazinon -0.5 Deltamethrin -0.1 Endosulfansulfate -0.5 Hexachlorbenzene -0.8 alfa-Endosulfan -0.1 alfa-HCH -0.6 beta-HCH -0.4 Lindane -0.9 Parathion-ethyl -0.1 Boscalid -0.5 Tetraconazole -0.4 Thiachloprid -1.3 National Food Agency, Sweden

Eurofins, Sweden Mandatory pesticides (GPC) Fat Content: 29% Pesticide Z-scores Bifentrin -0.8 cis-Chlordane -0.4 trans-Chlordane -0.6 DDE -1.6 Diazinon -1.3 Deltamethrin -0.2 Endosulfansulfate -1.0 Hexachlorbenzene -1.4 alfa-Endosulfan -0.7 alfa-HCH -0.9 beta-HCH -0.9 Lindane -1.1 Paration-ethyl -1.0

LAPRW 2015 Santiago de Chile

The presented method available: NMKL 195. 2013 ”Pesticide residues. Analysis in foods with ethyl acetate extraction using gas and liquid chromatography with tandem mass spectrometric determination“

EU RL website (fruit and vegetables only) Proposal at CEN level (animal products) LAPRW 2015 Santiago de Chile National Food Agency,

National Food Sweden Administration. Sweden

LAPRW 2015 Santiago de Chile

EU reinforced import control Case: Vine leaves Specific framework for the official feed and food controls

Regulation (EC) No 882/2004 includes requirements for the official control of vegetables being imported from third countries. Article 15(5) provides a list of certain products on the basis of known or emerging risks and be subject to increased controls at points of entry into the EU. The consignments may not be placed on the market before the analysis is complete. Regulation 669/2009 implementing Regulation 882/2004 sets out rules for the increased level of official controls.

National Food Agency, Sweden

LAPRW 2015 Santiago de Chile

Sample- vine leaves Dolma is a family of stuffed vegetable dishes originating from Ottoman Empire. The most-well known type is stuffed vine leaves. A lot of 8 300 kg vine leaves from Turkey was subjected to the increased control.

The results originates from a 4.5 kg composite (bulk) sample containing 10 bags of wine leaves á 450 g National Food Agency, Sweden

LAPRW 2015 Santiago de Chile

Table 1 Results in vine leaves Analyte Acetamiprid Azoxystrobin Bifenthrin Boscalid

Found residue mg/kg 0.014 2,00 0.052 1.4

Carbendazim

0.1

MRL

Analyte

0.01* 0.05* 0.05* 0.05*

Imidacloprid Indoxacarb Iprodion Kresoxim-methyl

40 analytes Found residue mg/kg 0.063 0.065 0.86 0.15

MRL 2,00 0.02*

Lambda-cyhalothrin

0.046

0.02*

0.005 0.017 0.5 0.82 0.03 0.08 0.016 0.011

0.01*

0.05*

Chlorantraniliprole Chlorpyrifos Cyflufenamid Cymoxanil Cypermethrin Deltamethrin Difenoconazole Dimethomorph

0.019 0.3 0.019 0.013 0.016 0.035 0.036 0.13

20,0 0.05* 0.02* 0.05* 0.5 0.05* 0.01*

Mandipropamid Metalaxyl Methoxyfenozide Metrafenone Myclobutanil Penconazole Propargite Proquinazid

Dithiocarbamates Emamectin Ethirimol Famoxadone

10.4 0.014 0.018 0.037

0.05* 0.01* 0.05* 0.02*

Pyrimethanil Quinalphos Tebuconazole Tebufenpyrad

0.92 0.013 0.42 0.066

0.05* 0.02* 0.05*

Fenbutatin Oxide

10.9

0.05*

Tetraconazole

0.052

0.02*

Fluopyram Hexythiazox

0.084 0.061

0.01* 0.5

Triadimefon (sum) Trifloxystrobin

0.15 0.082

0.1 0.02*

LAPRW 2015 Santiago de Chile

0.02* 0.02* 0.05* 0.02*

Results In total 40 residues were found thereof one pesticide using the single residue method. In 26 cases the MRL was exceeded. Taking into account the 50% measurement uncertainty, 17 pesticides (analytes) fulfilled the criteria for enforcement. However, none of the detected quantities of residues exceeded the acute reference dose (ArfD). Preliminary results from further analysis of the same lot (bulk sample) carried out by EURL FV, Almeria University (ES) NVWA (NL) Fera Science Ltd (U.K.) EURL SRM; CVUA (DE) EU RL CER, DTU (DK) (pending) gave similar, consistent results (taking into account the inhomogeneity in the bulk sample) LAPRW 2015 Santiago de Chile

Analysis of vine leaves Pesticide

Homogenate of same sample NFA / EURL-FV, Eurofins, Spain Sweden mg/kg mg/kg

Homogenate of same sample

Other individual sample

NVWA, NL mg/kg

Fera Science Ltd U.K. mg/kg

CVUA DE mg/kg Pesticide

NFA / EURL-FV, Eurofins, Spain Sweden mg/kg mg/kg

Other individual sample

NVWA, NL mg/kg

Fera Science Ltd U.K. mg/kg

CVUA DE mg/kg

Acetamiprid

0,014

0,019

n.d.

n.d

0,001

Hexythiazox

0,061

0,095

0,010

n.d

0,006

Azoxystrobin

2,0

3,24

1,32

0,35

0,83

Imidacloprid

0,063

0,065

0,060

0,019

0,060

0,052

0,045

n.d.

n.d

0,024

Indoxacarb

0,065

0,092

0,010

0,059

0,12

1,4

1,90

0,92

1,2

5.6

Iprodion

0,86

1,12

n.d.

0,22

0,015

0,10

0,14

0,025

0,014

0,15

0,25

0,13

0,15

0,41

Chlorantraniliprole

0,019

0,045

0,015

0,010

n.d

Mandipropamid

0,005

0,018

n.d.

n.d