Routine Quantification of Pesticides in Food Matrices using UPLC APGC-MSMS - Waters Corporation Food Safety

©2016 Waters Corporation 1

Routine Quantification of Multi-Residue

Pesticides in Food Matrices using

UPLC and APGC coupled to MS/MS


Overview

Challenges and method requirements for multi-residue analysis

Single instrument platform for LC and GC amenable pesticides

Sample preparation and analysis workflow

Acquisition modes – MS/MS, RADAR, PICS

Quanpedia database - Pesticides, metabolites, toxins and other contaminants

Collaboration (Nofa Lab) - Validation study for analysis of 460 pesticides in 10 different food commodities using APGC-MS/MS

Summary and Conclusions


Challenges in Multi Residue Pesticide Analysis of Food Products

Changing Legislation

Demanding Performance

Multitude of Food Types

Variety of Pesticides


High throughput: Generic QuEChERS clean up

Quantification & confirmation: gold standard MS/MS

Sensitivity: 0.01 mg/kg

Method robustness: System up time, instrument

maintenance

Multi Residue Pesticides in Food Method Requirements

Food product

Representative extract

GC with MS/MS detection

LC with MS/MS detection


Xevo TQD

Xevo TQ-S

XEVO Tandem Quadrupole Systems

Xevo TQ-S micro

Routine UPLC-MS/MS

RADAR acquisition mode

Universal ion source

Compact with high sensitivity



Fast data acquisition

Xtended Dynamic Range

Ultimate sensitivity



ScanWave Enhanced

product ion scanning

Ultimate sensitivity and reliability

Sensitive, reliable and compact

Robust, reliable and proven


Extended Dynamic Range

Provides up to 6 orders of linear dynamic range

Easier to transfer methods from other instruments with higher or lower

sensitivities

Accessible sensitivity

Time0.80 0.90 1.00 1.10 1.20 1.30 1.40 1.50 1.60 1.70 1.80 1.90 2.00 2.10 2.20

%

0

1x, 1 ng

1x, 100 pg

10x, 10 pg

82x, 1pg

876x, 100 fg

10,000x, 10 fg

10,000x magnification, 1 fg


Increased Capability LC and GC Workflow on one MS Platform

UPLC-MS/MS APGC-MS/MS

Quantitative & Confirmatory Analysis LC and GC with XEVO TQ-S micro


Quantitative & Confirmatory Analysis LC and GC with XEVO TQ-S micro

10 µg/kg

Compound name: Pyracarbolid

Correlation coefficient: r = 0.999741, r^2 = 0.999482

Calibration curve: 2921.65 * x + -539.66

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Standard Addition Concentration : -0.184711

ppb-0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

-0

100000

200000

ppb

Re

sid

ua

l

-2.00

0.00

2.00

Compound name: Pyracarbolid

Correlation coefficient: r = 0.999741, r^2 = 0.999482

Calibration curve: 2921.65 * x + -539.66

Response type: External Std, Area

Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

ppb-0 10 20 30 40 50 60 70 80 90 100

Re

sp

on

se

-0

100000

200000

ppb

Re

sid

ua

l

-2.00

0.00

2.00


Generic Cleanup with QuEChERS

Homogenisation 15 g sample

DisQuE extraction 15 mL 1 % acetic acid in ACN

Centrifuge 3500 RPM 5 min

DisQuE Tube 2 186004832

Dilute 1 in 10 in water

Solvent Exchange 500 µL ethyl acetate

APGC UPLC


UPLC-MS/MS Diluted QuEChERS extract, spiked with LC pesticide mix LC multi-residue pesticide mix ~204 compounds Solvent curve - 1 to 500 ppb Matrix match spiked curve - 1 to 500 ppb

LC - 204 pesticides mix

Experiments APGC-MS/MS After solvent exchange of QuEChERS extract, spiked with GC pesticide mix GC multi-residue pesticide mix ~203 compounds Solvent curve – 0.1 to 100 ppb Matrix match spiked curve – 0.1 to 100 ppb

Accurately detect and quantify multi-residue pesticides in a wide range of fruits, vegetables, and other commodities by UPLC-MS/MS

Full kit contains 204 pesticides, formulated and

grouped for maximum long-term stability Optimized multi-residue pesticide method with

UPLC conditions and MS/MS transitions available


Determine Sample Prep Efficiency

What is RADAR?

– Rapid electronics allow instrument to switch between MRM & full scan

– Added information can be gained from full scan data in routine analyses

• Intelligent decision making

• Improves method development

• Monitor matrix components

• Detect un-targeted adulterants

How do I set it up on the instrument?

- Parallel MRM with full scan data acquisition


Grape

Avocado

Marjoram

Ginger

Ultra Trace Analysis: Sample Complexity

Xevo TQ-S


Ultra trace analysis: Matrix effects

DisQuE Ginger QC extract

pesticides @ 0.01 mg/kg (10 ppb)

Cyromazine

Simetryn

Atrazine

Azinphos-ethyl

Poor peak shape

Low calculated recoveries

High Matrix background

Xevo TQ-S


Ultra trace analysis: Reduced matrix effects (10x Dilution)

Cyromazine

Simetryn

Atrazine

Azinphos-ethyl

10 x dilution of Ginger extract

All peaks detected, recoveries good

Reduced Matrix background by 10X dilution

Improved peak shape

-54%

-58%

-47%

Reduced ion suppression


Multi-Residue Pesticide Analysis in Green Grapes

Using UPLC-MS/MS


UPLC-MS Conditions

LC System: Waters ACQUITY H-Class Column: ACQUITY UPLC BEH C18 2.1 x 100 mm, 1.7 µm Column Temp: 45 ˚C Flow Rate: 0.450 mL/min Injection volume: 10 µL Mobile Phase A: 10mL of 1M ammonium acetate (pH5.0) + 990mL Water Mobile Phase B: 10mL of 1M ammonium acetate (pH 5.0) + 990mL MeOH Gradient: Min %A %B Curve Initial 98.0 2.0 0.25 98.0 2.0 6 12.25 1.0 99.0 6 13.00 1.0 99.0 6 13.01 98.0 2.0 6 17.00 98.0 2.0 6

MS System: Xevo TQ-S micro

Capillary (kV): 1.0

Source Temp.: 150°C

Desolvation Temp.: 500°C

Desolvation Gas Flow: 1000 L/Hr

MRM Transitions: TQ-S micro Quanpedia


• Is my system ready, does it satisfy lab quality requirements?

Set-up and check analytical system

Automated Quan Workflow: From System Setup to Data Processing

• The QuanPedia™ database allows for efficient method management

Select or create LC-MS/MS method

• TargetLynx XS streamlines automated quantitative data review and reporting

Comprehensive data processing and review

• Trending inter- and intra-batch QC and sample results over time is easy with TrendPlot

Compare data day to day, week to week

• Automated real-time QC checking with QCMonitor prevents wasting valuable samples

Critical QC Information Delivered Real Time


IntelliStart: Ready to Use System

IntelliStart

In-built Fluidics

Automated delivery

of reference

solutions

Automated flow

switching


Quanpedia for XEVO TQ-S micro

Compound database includes

– LC-MS/MS method files (UPLC and MRM conditions)

– TargetLynx quantification

– PICS

– Options for modifying and optimizing methods

Analyses include:

Alternaria toxins Diquat and Paraquat Patulin Polyphenols

Aminoglycosides Endocrine disrupting compounds Perfluorinated Progestogens

Amphetamines Glyphosate and AMPA Pesticide polarity switching Quinolones

Androgens and progestogens

Heterocyclic amines Pesticide screening Sudan dyes 1

Anthelminitics Hormones Pesticides (APEDA- India) Sulfonamides

Antibiotics Japanese positive list Pesticides 375 Tetracyclines

Avermectins Marine biotoxins Pesticides in baby food Thyreostats

Beta agonists Melamine Pharmaceutical and personal

care products Triarylmethane dyes

Bisphenol Mycotoxins Pharmaceuticals Triarylmethane dyes and

metabolites

Chloramphenicol Nitrofuran metabolites Phenolics Veterinary drug residues

Cyanotoxins Nitroimidazoles Phenoxy acid herbicides Water soluble vitamins

Dexamethasone and Betamethasone

Nitrosamines Phthalates Waters LC 204 multi residue

pesticides standards kit


Sample Analysis in QuanPedia

Step 1 : Run samples

Step 2 : Select an analysis

MS method

LC method

TargetLynx method

Step 3: Select methods to be generated

Step 4: Input your sample details


Automated Quan Workflow: QuanPedia

QuanPedia


Expanding the Scope of QuanPedia Import New Compounds

Excel spreadsheet

Notepad or Word

Optimised Compounds

PICS


PIC Scan

Acquisition

MRM

Acquisition

Product Ion Confirmation (PIC) Scan: Rapid Switching Provides Increased Confidence

Post Apex

PIC SCAN


Overlay UPLC-MS/MS Chromatogram Grapes Post-Spiked at 10 ppb


Selected MRM Chromatograms at 10ppb (MRL)


Automated Quan Workflow: TargetLynx XS Data Processing and Review


Linearity by UPLC-MS/MS in Green Grape Matrix

Compound Range R2

Acetamiprid 1 ppb to 100 ppb 0.998

Aldicarb 1 ppb to 500 ppb 0.996

Aldicarb sulfone 1 ppb to 500 ppb 0.997

Ametryn 1 ppb to 500 ppb 0.994

Aminocarb 1 ppb to 500 ppb 0.999

Azoxystrobin 1 ppb to 500 ppb 0.993

Benalaxyl 1 ppb to 500 ppb 0.998

Bendiocarb 1 ppb to 500 ppb 0.999

Benzoximate 1 ppb to 500 ppb 0.998

Bitertanol 1 ppb to 500 ppb 0.998

Buprofezine 1 ppb to 500 ppb 0.998

Butoxycarboxim 1 ppb to 500 ppb 0.997

Carbendazim 1 ppb to 500 ppb 0.995

Carbetamide 1 ppb to 500 ppb 0.997

Carbofuran 1 ppb to 500 ppb 0.999

Carbofuran 3-hydroxy 1 ppb to 500 ppb 0.998

Chlortoluron 1 ppb to 500 ppb 0.999

Clofentezine 1 ppb to 500 ppb 0.998

Desmedipham 1 ppb to 500 ppb 0.999

Dimoxystrobin 1 ppb to 500 ppb 0.997

Diuron 1 ppb to 500 ppb 0.994

Ethiofencarb 1 ppb to 500 ppb 0.999

Ethofumesate 1 ppb to 500 ppb 0.998

Fenamidone 1 ppb to 500 ppb 0.998

Fenbuconazole 1 ppb to 500 ppb 0.997

Fenobucarb 1 ppb to 500 ppb 0.996

Compound Range R2

Fenoxycarb 1 ppb to 500 ppb 0.996

Fenpyroximate 1 ppb to 500 ppb 0.997

Fenuron 1 ppb to 500 ppb 0.999

Hexaconazole 1 ppb to 500 ppb 0.998

Imidacloprid 1 ppb to 500 ppb 0.996

Isoprocarb 1 ppb to 500 ppb 0.995

Isoproturon 1 ppb to 500 ppb 0.999

Linuron 1 ppb to 500 ppb 0.997

Mandipropamid 1 ppb to 500 ppb 0.998

Mefenacet 1 ppb to 500 ppb 0.999

Mepronil 1 ppb to 500 ppb 0.994

Metalaxyl 1 ppb to 500 ppb 0.997

Oxamyl 1 ppb to 500 ppb 0.997

Phenmedipham 1 ppb to 500 ppb 0.999

Picoxystrobin 1 ppb to 500 ppb 0.994

Piperonyl butoxide 1 ppb to 500 ppb 0.998

Pirimicarb 1 ppb to 500 ppb 0.997

Promecarb 1 ppb to 500 ppb 0.995

Prometryn 1 ppb to 500 ppb 0.998

Propargite 1 ppb to 500 ppb 0.996

Propoxur 1 ppb to 500 ppb 0.997

Pyracarbolid 1 ppb to 500 ppb 0.999

Simetryn 1 ppb to 500 ppb 0.999

Spirotetramat 1 ppb to 500 ppb 0.997

Tebuconazole 1 ppb to 500 ppb 0.997

Tebuthiuron 1 ppb to 500 ppb 0.998


Recovery by UPLC-MS/MS in Green Grape Matrix

Name Average (n=3) %RSD

Acetamiprid 99.3 4.8

Aldicarb 98.9 6.8

Aldicarb sulfone 99.2 5.8

Ametryn 100.8 5.3

Aminocarb 90.8 5.2

Azoxystrobin 101.0 6.0

Benalaxyl 103.9 4.8

Bendiocarb 103.3 5.8

Benzoximate 99.4 6.9

Bitertanol 97.6 9.1

Bupirimate 107.7 9.6

Buprofezin 102.0 7.1

Butafenacil 107.8 8.1

Butocarboxim 98.0 6.5

Butoxycarboxim 98.2 6.0

Carbaryl 96.8 4.4

Carbendazim 97.8 6.0

Carbetamide 97.7 5.6

Carbofuran 102.4 5.4

Carbofuran-3-hydroxy 103.7 6.4

Carboxin 99.5 5.3

Carfentrazone-ethyl 109.8 16.6

Chlorantraniliprole 89.2 14.0


Incurred Residues by UPLC-MS/MS Green Grapes

Name Retention

Time Match

Ion Ratio Match

Incurred Residue (mg/kg)

EU MRLs (mg/kg)

Boscalid 0.006 5

Buprofezin 0.001 1

Fenhexamid 0.036 15

Imidacloprid 0.012 1

Methoxyfenozide 0.018 1

Pyraclostrobin 0.001 1

Trifloxystrobin 0.001 3

Incurred residues detected are well below the EU MRLs


Automated Quan Workflow: QC Monitor

Run samples

Decision time Reinject Troubleshoot

Criteria defined by the requirements of your

lab and method


TrendPlot - Monitoring Long-Term Robustness

Long term system

monitoring

Essential record

for auditors

Tool for effective

lab management


Multi-Residue Pesticides Analysis in Green Grapes

Using APGC-MS/MS


Ion Source Versatility

– Universal source architecture: Enables the widest range of ionisation

techniques to be utilised

– Simple and quick: Allowing you to change between ion source options in

minutes

Universal Source Options: UPLC to APGC


APGC is an inlet option of the XEVO universal source, readily

interchangeable with UPLC, etc.

Operates

– At atmospheric pressure, thus allowing higher gas flows to be applied

– By APCI like ionisation using a corona discharge pin, thus allowing ionisation

by proton and charge transfer

Atmospheric Pressure Gas Chromatography


APGC Video


Ionisation Comparison: APGC vs EI

Standrad D 2PPM Split 10:1 DRE H2O

m/z25 50 75 100 125 150 175 200 225 250 275

%

0

100

jv250809test005 533 (9.336) Cm (531:534-518:526) TOF MS AP+ 3.50e4230.0066198.9643

170.9724

124.9845

93.0114

232.0065

APGC - Proton Transfer

C5H12NO3PS2 M+· 229

Classic EI

Mix C 1 ppm

m/z60 80 100 120 140 160 180 200 220 240

%

0

100

API033 533 (10.107) Cm (527:533-465:517) TOF MS EI+ 5.46e487.0137

58.0303

63.0009

93.0101124.9828

93.0561

104.0112

201.0790

186.0549173.0479

158.0325142.9955 203.0784229.0006

Dimethoate


GC-MS Conditions

GC System: Agilent 7890 Column: DB-5 30m x 0.25 mm x 0.25 µm Injection Mode: Splitless Inlet Liner: Single gooseneck SKY deactivated Injector Temp: 280 °C Flow Rate: 2.0 mL/min Injection volume: 1.0 µL Transfer Line Temp: 320 °C Carrier Gas: Helium Oven Program: Ramp (°C/min) Temp (°C) Hold (min) 35 1.2 25 320 2

MS system: Xevo TQ-S micro

Corona: 5.0 uA

Source Temp: 150°C

Auxiliary Flow: 250 L/hr

Cone Gas Flow: 50 L/hr


Overlay APGC-MS/MS Chromatogram Grapes post-spiked at 10 ppb


Example MRM chromatograms at 10ppb (MRL)


Linearity by APGC-MS/MS in Green Grape Matrix

Compound Range R2

Malathion 0.25 ppb to 50 ppb 0.998

Chlozolinate 0.5 ppb to 50 ppb 0.998

Pirimiphos-ethyl 0.25 ppb to 50 ppb 0.999

Prochloraz 0.25 ppb to 100 ppb 0.999

Carfentrazone ethyl 0.25 ppb to 100 ppb 0.999

Biphenyl 0.1 ppb to 100 ppb 0.999

Terbacil 1.0 ppb to 100 ppb 0.999

Diclobenil 0.25 ppb to 100 ppb 0.999

Chlorpropham 1.0 ppb to 50 ppb 0.999

Tefluthrin 0.5 ppb to 50 ppb 0.999

Anthraquinone 0.5 ppb to 100 ppb 0.999

Propachlor 0.5 ppb to 100 ppb 0.999

Terbutylazine 0.5 ppb to 50 ppb 0.999

Clomazone 0.5 ppb to 50 ppb 0.999

Etridazole 0.5 ppb to 100 ppb 0.998

Linuron 0.5 ppb to 50 ppb 0.999

Hexazinone 0.25 ppb to 50 ppb 0.999

Phorate 1.0 ppb to 50 ppb 0.998

Fenson 0.25 ppb to 100 ppb 0.999

Diallate 0.25 ppb to 50 ppb 0.999

Fenitrothion 0.5 ppb to 50 ppb 0.998

Fenthion 0.5 ppb to 100 ppb 0.999

4,4'-Methoxychlor Olefin 0.5 ppb to 50 ppb 0.996

Sulfotep 0.5 ppb to 100 ppb 0.996

Fenchlorphos 0.5 ppb to 50 ppb 0.995

Sulprofos 0.25 ppb to 50 ppb 0.996

Flutolanil 0.5 ppb to 50 ppb 0.995

Compound Range R2

Pyridaphenthion 0.5 ppb to 100 ppb 0.997

Phenothrin 0.5 ppb to 50 ppb 0.995

Phosalone 0.1 ppb to 50 ppb 0.999

Transfluthrin 0.25 ppb to 100 ppb 0.997

Fluazifop-p-butyl 0.1 ppb to 50 ppb 0.999

Ethion 0.25 ppb to 50 ppb 0.998

Flucythrinate 1 0.5 ppb to 100 ppb 0.999

Flucythrinate 2 0.5 ppb to 50 ppb 0.996

Deltamethrin 0.25 ppb to 50 ppb 0.998

Tetrahydrophthalimide 0.5 ppb to 100 ppb 0.998

2-Phenylphenol 1.0 ppb to 100 ppb 0.999

Bifenthrin 0.5 ppb to 50 ppb 0.997

Mevinphos 0.25 ppb to 100 ppb 0.993

Pebulate 0.25 ppb to 50 ppb 0.991

Cycloate 1.0 ppb to 50 ppb 0.998

Atrazine 0.5 ppb to 100 ppb 0.994

Propanil 0.5 ppb to 50 ppb 0.996

2,3,5,6-Tetrachloroaniline 1.0 ppb to 100 ppb 0.997

o,p'-DDD 0.25 ppb to 50 ppb 0.996

Diphenamid 0.25 ppb to 50 ppb 0.996

Propyzamide 0.5 ppb to 50 ppb 0.996

Dimethachlor 0.25 ppb to 100 ppb 0.992

Methyl parathion 0.1 ppb to 50 ppb 0.999

Paclobutrazol 1.0 ppb to 100 ppb 0.997

Bioallethrin 1.0 ppb to 100 ppb 0.996


Incurred Residues by APGC-MS/MS Green Grapes

Name Retention

Time Match

Ion Ratio Match

Incurred Residue (mg/kg)

EU MRLs (mg/kg)

Pyriproxyfen 0.0009 0.05

Tetrahydrophthalimide 0.0003 0.02

2-Phenylphenol 0.0007 0.05

Pyrimethanil 0.008 5

Anthraquinone 0.0004 0.01

Incurred residues detected are well below the EU MRLs


Analysis of 460 Pesticides in 10 different Food Commodities by APGC-MS/MS

Collaboration with NOFA Lab, Netherlands


Commodity independent

– Semi quantitative screening

– 10 commodities screened against solvent curve

460 pesticides (92.4 %) passed validation criteria

% Recovery of between 70 and 120 %

% rRSD < 20 %

LoD < 5 µg/kg

32 pesticides (7.6%) failed due to poor analyte recovery

(<70%)

– Including nitro- and chloro- phenols, captan and folpet

courtesy of NofaLab, the Netherlands, 2015

NOFA Lab – Analysis of 460 Pesticides in 10 different Food Commodities


Method results

courtesy of NofaLab, the Netherlands, 2015


APGC vs EI: Benefits

APGC EI Customer benefit

Injection of sample in mg

0.1- 0.5 mg 5- 50 mg (LOD still higher) Inject less, chromatography

longer stable

Retention times Neglect able shifts (small

scan windows) Large shifts (large scan

windows) More confidence, less reprocess, save time

Response Stable Large variation and sudden

drop Less maintenance

Cleaning the source Once or twice a year

(preventive) Once a week (when

needed)

No need to vent the system, clean the source, change the

filament

Replacing the front column/liner (inject

less)

Every two weeks (preventive)

Once or twice a week (when needed)

Less downtime

Replacing the analytical column (inject less)

3 to 6 months (when needed)

Once a month (when needed)

Less downtime, save consumable

Downtime A week per year (most

preventive)

One or two months per year (most trouble

shooting) Positive ROI

Inject Less

Clean Less

Replace Less

Down Less

courtesy of NofaLab, the Netherlands


Improved efficiencies for LC and GC amenable multi-residue pesticides

analysis on a single instrument platform

Innovative workflow strategies for MS quantification and to maximize laboratory efficiency

APGC-MS/MS

UPLC-MS/MS

Summary

− IntelliStart

− QuanPedia

− TargetLynx XS

− TrendPlot

− RADAR

− PICs


Multi-Residue Pesticide Analysis in Food Matrices – Other Applications

Resources:

• LC- and GC- MS/MS pesticides in tea: 720004819en

• LC- MS/MS pesticides in chilli powder: 720005285en

• LC-MS/MS pesticides in okra: 720004789en

• LC- MS/MS pesticides in chilli powder: 720005559en

• GC- MS/MS pesticides in fruits and vegetables: 720004952en

• LC- and GC- MS/MS pesticides in botanicals: 720005659en

•LC- and GC- MS/MS pesticides in ginseng: 720005006en

• Dilute and shoot pesticides in fruit juices: 720004403en

• Food Testing Application Notebook: 720002565en


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