Modification

and

re-validation

of

the

ethyl

acetate-based

multi-residue

method

for

pesticides

in

produce

The

ethyl

acetate-based

multi-residue

method

for

determination

of

pesticide

residues

in

produce

has

been

modified

for

gas

chromatographic

-LRB-

GC

-RRB-

analysis

by

implementation

of

dispersive

solid-phase

extraction

-LRB-

using

primary

secondary

amine

and

graphitized

carbon

black

-RRB-

and

large-volume

-LRB-

μL

-RRB-

injection

The

same

extract

before

clean-up

and

after

a

change

of

solvent

was

also

analyzed

by

liquid

chromatography

with

tandem

mass

spectrometry

-LRB-

LC

MS

MS

-RRB-

All

aspects

related

to

sample

preparation

were

re-assessed

with

regard

to

ease

and

speed

of

the

analysis

The

principle

of

the

extraction

procedure

-LRB-

solvent

salt

-RRB-

was

not

changed

to

avoid

the

possibility

invalidating

data

acquired

over

past

decades

The

modifications

were

made

with

techniques

currently

commonly

applied

in

routine

laboratories

GC

MS

and

LC

MS

MS

in

mind

The

modified

method

enables

processing

-LRB-

from

homogenization

until

final

extracts

for

both

GC

and

LC

-RRB-

of

samples

per

eight

hours

per

person

Limits

of

quantification

-LRB-

LOQs

-RRB-

of

mg

kg

were

achieved

with

both

GC

MS

-LRB-

full-scan

acquisition

mg

matrix

equivalent

injected

-RRB-

and

LC

MS

MS

-LRB-

mg

injected

-RRB-

for

most

of

the

pesticides

Validation

data

for

pesticides

and

degradation

products

are

presented

A

compilation

of

analytical

quality-control

data

for

pesticides

routinely

analyzed

by

GC

MS

-LRB-

compounds

-RRB-

and

LC

MS

MS

-LRB-

compounds

-RRB-

in

over

different

matrices

obtained

over

a

period

of

months

are

also

presented

and

discussed

At

the

mg

kg

level

acceptable

recoveries

were

obtained

for

-LRB-

GC

MS

-RRB-

and

-LRB-

LC

MS

MS

-RRB-

of

pesticide

matrix

combinations

Introduction

For

monitoring

and

control

of

pesticide

residues

multi-residue

methods

are

very

cost-effective

and

are

used

in

many

laboratories

The

pesticides

are

usually

first

extracted

with

an

organic

solvent

of

high

or

medium

polarity

Typical

solvents

used

for

this

purpose

are

acetone

-LSB-

-RSB-

ethyl

acetate

-LSB-

-RSB-

-LRB-

Table

-RRB-

and

acetonitrile

-LSB-

-RSB-

With

all

three

options

pesticides

are

partitioned

between

an

aqueous

phase

and

an

organic

phase

With

acetone

and

acetonitrile

this

is

done

in

two

successive

steps

with

ethyl

acetate

in

one

step

With

regard

to

extraction

efficiency

ethyl

acetate

has

been

shown

to

be

equivalent

to

the

water-miscible

solvents

for

both

polar

and

non-polar

pesticides

in

vegetables

fruit

and

dry

products

-LRB-

after

addition

of

water

-RRB-

-LSB-

-RSB-

It

is

also

suitable

for

products

with

a

high

fat

content

because

of

the

solubility

of

fat

in

ethyl

acetate

pesticides

are

released

and

extracted

efficiently

The

extract

obtained

is

compatible

with

gel-permeation

chromatography

-LRB-

GPC

-RRB-

the

clean-up

procedure

most

suitable

for

this

type

of

sample

Ethyl

acetate

is

very

suitable

for

GC

analysis

It

has

good

wettability

in

GC

-LRB-

pre

-RRB-

columns

this

is

of

benefit

for

solvent

trapping

of

the

most

volatile

analytes

which

is

required

for

refocusing

after

injection

Its

vapor

pressure

and

expansion

volume

during

evaporation

also

favor

large-volume

injection

Finally

it

is

compatible

with

all

GC

detectors

The

same

extract

can

also

be

used

for

LC

analysis

after

a

solvent

change

into

e.g.

methanol

-LSB-

-RSB-

as

is

done

for

acetone-based

methods

also

-LSB-

-RSB-

Table

from

literature

Conditions

typically

used

in

ethyl

acetate-based

multi-residue

analysisSample

-LRB-

g

-RRB-

AdditionEtAc

-LRB-

mL

-RRB-

-LRB-

g

-RRB-

Extr.Phase

separationRe-extr

Evap

reconst

-LRB-

aliquot/to

mL

-RRB-

Clean-upEvaporation

-LRB-

from/to

mL

-RRB-

Final

extr

g

mL

-LRB-

μL

-RRB-

Analysis

GC

-LSB-

-RSB-

GC

NPD

-LSB-

-RSB-

-LRB-

dilute

-RRB-

GC

settle

-LSB-

-RSB-

mL

-LRB-

wheat

-RRB-

-LSB-

-RSB-

All

-LSB-

-RSB-

-LRB-

ENV

-RRB-

GC

ITD

-LSB-

-RSB-

clean-up

Cartridge

water

abs

Polymer

dry

MS

GC

-LSB-

-RSB-

GC

FPD

LC

g

MS

-LSB-

-RSB-

-LRB-

vac

-RRB-

All

cyclohexaneGPCEluate

GC

TOF-MS

GC

-LSB-

-RSB-

-LSB-

-RSB-

g

-LRB-

°C

-RRB-

F/cotton

wool

IS

GC

TOF-MS

-LRB-

DMI

-RRB-

-LSB-

-RSB-

settle

or

centrifuge

LC

MS

-LSB-

-RSB-

if

pH

LC

MS

-LSB-

-RSB-

mL

mol

L

MeOH

MS

-LSB-

-RSB-

mL

mol

L

All

MeOH

-LSB-

-RSB-

All

MS

-LSB-

-RSB-

Yes

All

-LSB-

-RSB-

ace/hexSPE

SAX/PSA

All

GC

-LSB-

-RSB-

-LSB-

-RSB-

MS

-LSB-

-RSB-

-LRB-

syringe

-RRB-

GC

-LSB-

-RSB-

g

-LRB-

°C

-RRB-

F

IS

GC

-LSB-

-RSB-

mL

-LRB-

barley

-RRB-

ACN

GC

TOF-MS

LC

MS

-LSB-

-RSB-

mL

mol

L

phosphate

GC

GCB/PSA

GC

MSThis

workLC

-LRB-

MeOH/water

-RRB-

LC

MS

MSaEthyl

acetate

cyclohexane

bAdditional

SPE

clean-up

step

with

Florisil

EtAc/Hex

mL

evap

to

mLT

Turrax

B

blender

S

shaking

F

filtration

MeOH

methanol

ACN

acetonitrile

ace

acetone

hex

hexane

Although

multi-residue

methods

based

on

ethyl

acetate

extraction

have

been

used

for

more

than

years

and

continue

to

be

used

in

many

laboratories

-LRB-

they

are

for

example

the

official

methods

in

Sweden

and

Spain

and

are

also

commonly

used

in

the

Netherlands

UK

Czech

Republic

Japan

and

China

-RRB-

the

methods

described

in

the

literature

frequently

include

steps

that

make

them

in

our

opinion

unnecessary

laborious

Such

steps

include

repeated

extraction

filtration

clean-up

steps

involving

GPC

for

non-fatty

matrices

column

chromatography

or

solid

phase

extraction

-LRB-

SPE

-RRB-

manifolds

and

evaporative

concentration

Typical

examples

are

given

in

Table

It

will

be

shown

in

this

paper

that

most

of

the

laborious

steps

can

be

replaced

by

more

efficient

alternatives

repeated

extraction

is

not

required

an

aliquot

is

taken

after

settling

or

centrifugation

rather

than

filtration

use

of

GCB

instead

of

GPC

for

removal

of

chlorophyll

use

of

dispersive

SPE

instead

of

classical

SPE

for

clean-up

-LRB-

analogous

to

an

acetonitrile-based

method

-LSB-

-RSB-

-RRB-

and

injection

of

larger

volumes

into

the

GC

instead

of

manual

evaporative

concentration

The

objective

of

the

work

discussed

in

this

paper

was

to

update

and

improve

the

ethyl

acetate-based

multi-residue

method

for

pesticides

in

vegetables

and

fruit

in

respect

of

straightforwardness

robustness

and

ease

and

speed

of

sample

and

extract

handling

Aspects

studied

include

dispersive

clean-up

using

combined

GCB/PSA

the

possibility

of

preventing

unacceptable

adsorption

of

planar

pesticides

by

GCB

by

addition

of

toluene

and

large-volume

-LRB-

μL

-RRB-

injection

in

GC

The

method

has

been

validated

for

pesticides

and

degradation

products

which

are

analyzed

by

GC

MS

or

LC

MS

MS.

For

the

latter

the

initial

raw

extract

was

used

and

injected

after

a

solvent

change

to

methanol

water

The

suitability

of

the

method

as

a

multi-residue

multi-matrix

method

is

evaluated

by

use

of

analytical

quality-control

data

generated

during

months

for

pesticides

and

degradation

products

for

over

different

matrices

including

less

common

and

exotic

crops

Results

obtained

for

proficiency

test

samples

during

three

years

are

also

presented

Experimental

Chemicals

and

reagents

Pesticide

reference

standards

were

obtained

from

C.N.

Schmidt

-LRB-

Amsterdam

The

Netherlands

-RRB-

For

GC

MS

a

mixed

stock

solution

containing

pesticides

-LRB-

Table

concentration

mg

L

for

each

pesticide

-RRB-

was

obtained

from

Alltech

Grace

-LRB-

Breda

The

Netherlands

-RRB-

The

full

chemical

names

of

the

metabolites

of

phenmedipham

and

pyridate

are

methyl

N

-LRB-

-RRB-

carbamate

and

respectively

Solvents

were

from

J.T.

Baker

-LRB-

ethyl

acetate

Resi-analysed

Deventer

The

Netherlands

-RRB-

Labscan

-LRB-

toluene

Pestiscan

-RRB-

and

Rathburn

-LRB-

methanol

-RRB-

Anhydrous

sodium

sulfate

ammonium

formate

potassium

dihydrogen

phosphate

disodium

hydrogen

phosphate

acetic

acid

and

diethylene

glycol

-LRB-

all

p.A.

quality

-RRB-

were

from

Merck

Water

was

purified

by

use

of

a

MilliQ

reagent-water

system

-LRB-

Millipore

-RRB-

Bondesil

primary

secondary

amine

-LRB-

PSA

μm

-RRB-

was

obtained

from

Varian

-LRB-

Middelburg

The

Netherlands

-RRB-

and

GCB

-LRB-

graphitized

carbon

black

-RRB-

was

purchased

as

Supelclean

ENVI-carb

-LRB-

mesh

Supelco

Zwijndrecht

The

Netherlands

-RRB-

For

GC

MS

in

addition

to

the

mixed

stock

solution

individual

stock

solutions

of

other

pesticides

were

prepared

in

ethyl

acetate

From

these

additional

mixed

solutions

were

prepared

in

ethyl

acetate

For

LC

MS

MS

analysis

individual

stock

solutions

were

prepared

in

methanol

Mixed

solutions

were

prepared

from

the

individual

stock

solutions

and

diluted

with

methanol

The

mixed

solutions

were

used

for

fortification

of

samples

and

for

preparation

of

matrix-matched

standards

The

extraction

solvent

was

a

solution

of

internal

standard

-LRB-

mg

L

antor

-LRB-

diethatyl-ethyl

-RRB-

-RRB-

in

ethyl

acetate

Matrix-matched

standards

were

prepared

by

addition

of

mixed

solutions

to

control

sample

extracts

Dilution

of

the

sample

extract

with

mixed

solution

was

never

more

than

Instrumentation

GC

MS

analysis

GC

MS

analysis

was

performed

with

a

model

Top

GC

equipped

with

a

Best

PTV

-LRB-

programmed

temperature

vaporizer

-RRB-

injector

an

autosampler

and

a

Voyager

mass

spectrometer

-LRB-

Interscience

Breda

The

Netherlands

-RRB-

The

instrument

was

controlled

by

Masslab

software

The

injector

was

equipped

with

a

mm

i.d.

liner

with

porous

sintered

glass

on

the

inner

surface

The

GC

was

equipped

with

a

m

mm

i.d.

μm

film

column

and

a

m

precolumn

-LRB-

same

as

the

analytical

column

connected

by

means

of

a

press-fit

connector

-RRB-

For

PTV

injection

in

solvent-vent

mode

μL

was

injected

at

μL

s

The

solvent

was

vented

at

°C

in

min

using

a

split

flow

of

mL

min

The

split

valve

was

then

closed

and

the

analytes

retained

in

the

liner

were

transferred

to

the

GC

column

by

ramping

the

temperature

at

s

to

°C

Total

transfer

time

was

min

after

which

the

split

was

re-opened

Helium

was

used

as

carrier

gas

at

constant

flow

-LRB-

mL

min

-RRB-

The

oven

temperature

was

maintained

at

°C

for

min

after

injection

then

programmed

at

min

to

°C

which

was

maintained

for

min

The

transfer

line

to

the

MS

was

maintained

at

°C

Mass

spectrometry

was

performed

with

electron-impact

-LRB-

EI

-RRB-

ionization

-LRB-

electron

energy

eV

-RRB-

at

a

source

temperature

of

°C

Data

were

acquired

in

full-scan

mode

-LRB-

m/z

-RRB-

after

a

solvent

delay

of

min

until

min

Scan

time

and

inter-scan

delay

were

and

s

respectively

resulting

in

scans

s

The

detector

potential

was

V.

Masslab

software

-LRB-

Interscience

The

Netherlands

-RRB-

and

an

Excel

macro

developed

in-house

were

used

for

data

handling

and

quantitative

data

evaluation

LC

MS

MS

analysis

LC

was

performed

with

an

Agilent

model

instrument

comprising

degas-unit

pump

autosampler

and

column

oven

A

mm

mm

i.d.

guard

column

-LRB-

Phenomenex

-RRB-

and

a

mm

mm

i.d.

LC

column

-LRB-

Aqua

μm

Phenomenex

-RRB-

were

coupled

to

a

triple-quadrupole

mass

spectrometer

-LRB-

model

or

Applied

Biosystems

Nieuwerkerk

a/d

Yssel

The

Netherlands

-RRB-

Analyst

and

later

were

used

for

instrument

control

and

data

handling

Additional

data

processing

was

performed

using

an

Excel

macro

developed

in-house

Compounds

were

separated

by

elution

with

a

gradient

prepared

from

methanol

water

mol

L

ammonium

formate

solution

-LRB-

component

A

-RRB-

and

methanol

water

mol

L

ammonium

formate

solution

-LRB-

component

B

-RRB-

The

composition

was

changed

from

A

to

B

in

min

and

was

then

isocratic

until

min

The

composition

was

then

changed

back

to

A

in

min

and

the

column

was

re-equilibrated

for

min

before

the

next

injection

The

flow

rate

was

mL

min

which

was

introduced

into

the

MS

without

splitting

The

injection

volume

was

μL

and

μL

for

the

and

respectively

Data

were

acquired

in

multiple-reaction-monitoring

-LRB-

MRM

-RRB-

mode

Electrospray

ionization

-LRB-

ESI

-RRB-

-LRB-

called

turbo

ion

spray

for

the

instruments

used

-RRB-

mass

spectrometry

was

performed

in

positive-ion

mode

For

the

the

nebulizer

gas

turbo

gas

and

curtain

gas

were

and

arbitrary

units

-LRB-

a.u.

-RRB-

respectively

The

ion-spray

potential

was

V.

Nitrogen

was

used

as

collision

gas

-LRB-

psi

-RRB-

For

the

the

nebulizer

gas

and

curtain

gas

were

and

a.u.

and

the

turbo

gas

was

L

min

The

ion

spray

potential

was

V.

Nitrogen

was

used

as

collision

gas

-LRB-

psi

-RRB-

For

both

instruments

the

pause

time

was

ms.

The

dwell

times

for

the

pesticide

transitions

varied

between

and

ms.

The

precursor

and

product

ions

and

the

collision

energy

-LRB-

data

for

-RRB-

for

each

pesticide

or

degradation

product

are

listed

in

Table

In

the

acquisition

method

one

transition

for

each

pesticide

was

measured

All

transitions

were

acquired

in

one

time

window

The

total

cycle

time

was

s

resulting

in

data

points

across

the

peak

To

measure

the

second

transition

a

second

method

was

created

and

run

if

confirmation

was

needed

Sample

preparation

Vegetable

and

fruit

samples

were

taken

from

batches

of

samples

as

received

from

the

food

industry

and

trade

for

routine

multi-residue

analysis

After

removal

of

stalks

caps

stems

etc.

as

prescribed

by

Annex

I

-LSB-

-RSB-

an

amount

corresponding

at

least

to

the

minimum

size

of

laboratory

samples

-LRB-

usually

kg

-LSB-

-RSB-

-RRB-

was

homogenized

in

a

large-scale

Stephan

food

cutter

A

subsample

-LRB-

g

-RRB-

was

weighed

into

a

centrifuge

tube

Fortification

was

performed

at

this

stage

Phosphate

buffer

-LRB-

pH

mol

L

mL

-RRB-

and

extraction

solution

-LRB-

ethyl

acetate

with

internal

standard

mL

-RRB-

were

then

added

Just

before

Turrax

extraction

anhydrous

sodium

sulfate

-LRB-

g

-RRB-

was

added

After

Turrax

extraction

-LRB-

min

-RRB-

the

tubes

were

centrifuged

-LRB-

sets

of

four

-RRB-

For

GC

MS

analysis

Eppendorf

cups

were

prefilled

with

mg

PSA

and

mg

GCB

To

avoid

a

weighing

step

scoops

were

made

in-house

for

this

purpose

Their

accuracy

was

established

to

be

mg

-LRB-

n

-RRB-

For

clean-up

mL

extract

and

mL

toluene

were

added

to

the

cup

with

the

SPE

materials

The

cups

were

then

closed

and

the

samples

were

vortex

mixed

for

s

and

centrifuged

-LRB-

up

to

at

one

time

-RRB-

One

aliquot

was

transferred

to

an

autosampler

vial

with

insert

and

a

second

aliquot

was

transferred

to

an

autosampler

vial

and

stored

under

refrigeration

as

back-up

extract

The

calculated

amount

of

initial

sample

in

the

final

extract

was

g

mL

For

LC

MS

MS

analysis

the

initial

extract

-LRB-

mL

for

the

and

mL

for

the

-RRB-

was

transferred

to

a

disposable

glass

tube

After

addition

of

a

solution

of

diethylene

glycol

in

methanol

-LRB-

μL

-RRB-

the

extract

was

evaporated

to

dryness

under

a

gentle

flow

of

nitrogen

gas

at

°C

-LRB-

up

to

tubes

in

a

heater

block

-RRB-

The

residue

was

reconstituted

in

methanol

-LRB-

mL

and

mL

for

the

and

respectively

-RRB-

by

use

of

vortex

mixing

and

ultrasonication

-LRB-

min

-RRB-

The

extract

was

then

diluted

with

component

A

After

centrifugation

one

aliquot

was

transferred

to

an

autosampler

vial

with

insert

and

a

second

aliquot

was

transferred

into

an

autosampler

vial

and

stored

under

refrigeration

as

back-up

extract

The

final

extract

concentration

was

g

mL

and

g

mL

for

the

and

respectively

For

dry

products

-LRB-

e.g.

cereals

-RRB-

g

was

weighed

and

mL

water

was

added

After

soaking

for

h

samples

were

processed

as

described

above

A

larger

amount

of

extract

was

taken

for

evaporation

to

compensate

for

the

reduced

amount

of

sample

processed

and

to

bring

the

final

extract

concentration

to

g

mL

With

the

final

method

one

person

can

process

samples

in

eight

hours

Here

processing

includes

specific

preparation

before

homogenization

-LRB-

i.e.

removal

of

caps

from

strawberries

etc.

-RRB-

homogenization

of

the

samples

extraction

cleaning

the

Turrax

between

samples

clean-up

for

GC

MS

and

solvent

switch

for

LC

MS

MS

i.e.

from

laboratory

sample

to

ready-to-inject

solutions

in

autosampler

vials

Quantification

GC

MS

For

each

pesticide

the

concentrations

were

calculated

for

two

diagnostic

ions

In

previous

validation

work

-LRB-

not

published

-RRB-

using

the

same

software

it

was

found

that

for

most

pesticides

automatic

integration

and

repeatability

of

response

were

better

when

peak

height

rather

than

area

was

used

Peak

height

was

therefore

used

with

few

exceptions

-LRB-

e.g.

pesticides

prone

to

tailing

for

example

-RRB-

All

responses

were

normalized

to

the

response

of

the

internal

standard

-LRB-

antor

-RRB-

One-point

calibration

was

performed

using

a

fixed

matrix-matched

standard

-LRB-

tomato

see

Results

and

discussion

section

-RRB-

at

a

level

corresponding

to

five

times

the

LOQ

The

linearity

of

the

plot

of

MS

response

against

concentration

was

verified

periodically

over

the

range

to

mg

kg

For

most

pesticides

linearity

was

adequate

-LRB-

relative

response

within

of

the

calibration

standard

-RRB-

up

to

at

least

mg

kg

LC

MS

MS

The

internal

standard

-LRB-

antor

-RRB-

was

evaluated

qualitatively

only

to

confirm

injection

of

the

sample

extract

Because

of

unpredictable

and

varying

matrix

effects

for

several

of

the

matrices

included

in

this

work

normalization

against

the

internal

standard

was

not

considered

feasible

For

each

sample

matrix

that

was

fortified

a

matrix-matched

standard

was

also

prepared

by

spiking

the

final

extract

of

the

corresponding

control

sample

Peak

area

was

used

for

quantification

One-point

calibration

was

performed

using

the

matrix-matched

standard

at

a

level

corresponding

to

five

times

the

LOQ

Linearity

of

the

MS

response

against

concentration

was

verified

periodically

over

the

range

to

mg

kg

For

most

pesticides

the

relationship

was

linear

-LRB-

relative

response

within

of

the

calibration

standard

-RRB-

up

to

at

least

mg

kg

Validation

Initial

method

validation

was

performed

in

accordance

with

EU

guidelines

-LSB-

-RSB-

Two

times

five

portions

of

the

homogenized

sample

were

spiked

with

a

mixture

of

pesticides

at

a

low

level

-LRB-

mg

kg

or

lower

-RRB-

and

at

a

level

ten

times

higher

Together

with

two

unfortified

control

portions

of

the

sample

they

were

processed

and

analyzed

as

outlined

above

Additional

method-performance

data

were

acquired

by

analyzing

fortified

samples

concurrently

with

each

batch

of

samples

The

spike

level

-LRB-

mg

kg

for

most

pesticides

-RRB-

was

five

times

the

LOQ

With

each

batch

different

products

were

selected

as

much

as

possible

In

the

compilation

the

emphasis

was

on

products

which

are

less

frequently

reported

in

the

literature

to

challenge

the

applicability

of

the

method

as

a

multi-matrix

method

For

this

purpose

samples

were

not

pre-screened

for

absence

of

pesticides

and

consequently

occasionally

recoveries

could

not

be

determined

because

of

the

relatively

high

levels

incurred

Such

results

were

eliminated

from

the

data

set

Spectrophotometric

measurement

of

removal

of

chlorophyll

For

evaluation

of

the

removal

of

chlorophyll

by

GCB

and

comparison

with

GPC

a

lettuce

extract

was

prepared

by

extracting

g

lettuce

with

mL

ethyl

acetate

after

addition

of

g

anhydrous

sodium

sulfate

As

a

reference

mL

ethyl

acetate

was

added

to

mL

of

this

extract

to

bring

the

extract

concentration

to

g

mL

For

dispersive

SPE

mg

GCB

was

added

to

sets

of

duplicate

tubes

and

mL

extract

was

added

to

all

tubes

Solvent

was

then

added

to

four

sets

of

tubes

set

one

mL

ethyl

acetate

set

two

mL

ethyl

acetate

and

mL

toluene

-LRB-

i.e.

toluene

-RRB-

set

three

mL

toluene

-LRB-

toluene

-RRB-

and

set

four

mL

xylene

-LRB-

xylene

-RRB-

The

extracts

were

vortex

mixed

and

centrifuged

For

GPC

clean-up

mL

lettuce

extract

was

injected

on

to

a

cm

mm

i.d.

Biobeads

column

with

ethyl

acetate

cyclohexane

as

eluent

The

fraction

collected

was

such

that

at

least

of

the

pyrethroids

were

recovered

-LRB-

fraction

from

mL

-RRB-

The

eluate

was

first

concentrated

by

rotary

evaporation

at

°C

to

approximately

mL

then

transferred

to

a

tube

for

further

concentration

under

nitrogen

gas

to

mL

Final

extract

concentration

before

and

after

clean-up

was

always

g

mL

Aliquots

of

the

extracts

were

transferred

to

a

cuvet

for

spectrophotometric

analysis

at

nm

If

required

the

extracts

were

diluted

with

ethyl

acetate

to

bring

absorption

within

the

linear

range

The

amount

of

chlorophyll

in

the

uncleaned

extract

was

defined

as

For

calibration

purposes

the

uncleaned

extract

was

diluted

and

times

with

ethyl

acetate

and

a

calibration

plot

was

constructed

Chlorophyll

remaining

after

clean-up

was

determined

from

the

decrease

in

absorption

at

nm

compared

with

the

absorption

of

the

uncleaned

lettuce

extract

Results

and

discussion

Monitoring

of

residues

in

fresh

produce

for

the

food

industry

especially

trade

and

retail

calls

for

rapid

turnaround

preferably

within

one

or

two

days

This

means

sample

preparation

must

be

rapid

and

straightforward

With

regard

to

cost

and

waste

consumption

of

solvents

and

reagents

should

be

low

At

the

same

time

EU

directives

with

regard

to

sample

definition

-LRB-

-LSB-

-RSB-

-RRB-

and

laboratory

sample

size

-LRB-

-LSB-

-RSB-

-RRB-

for

residue

analysis

should

be

respected

This

means

for

example

that

that

a

total

of

kg

grapes

-LRB-

after

removal

of

stalks

-RRB-

five

whole

melons

or

kg

strawberries

-LRB-

after

removal

of

caps

-RRB-

must

be

processed

The

actual

analysis

is

performed

on

a

subsample

of

the

laboratory

sample

after

appropriate

comminution

The

more

thorough

the

comminution

the

smaller

the

subsample

can

be

and

the

lower

the

amount

of

solvent

needed

for

extraction

It

has

furthermore

been

reported

that

for

well

homogenized

samples

extraction

by

vortex

mixing

or

shaking

instead

of

high-speed

blending

-LRB-

Turrax

-RRB-

suffices

for

effective

extraction

-LSB-

-RSB-

although

there

is

still

some

debate

on

this

matter

-LSB-

-RSB-

Homogenization

For

homogenization

there

are

several

possibilities

Food

choppers

or

kitchen

blenders

are

often

used

Very

thorough

homogenization

can

be

achieved

with

the

latter

but

it

is

not

possible

to

process

the

entire

laboratory

sample

at

once

For

this

reason

large-scale

food

choppers

are

more

suited

With

such

devices

homogeneity

is

not

always

optimum

as

can

be

observed

with

e.g.

tomatoes

for

which

small

pieces

of

skin

drift

in

the

soup

obtained

after

homogenization

Subsampling

of

very

small

amounts

is

therefore

not

acceptable

after

this

procedure

because

the

subsample

would

be

insufficiently

representative

of

the

original

sample

More

thorough

homogenization

can

be

achieved

after

addition

of

dry-ice

or

liquid

nitrogen

-LRB-

cryogenic

homogenization

-RRB-

This

procedure

is

recommended

when

reducing

the

subsample

for

analysis

to

g

This

procedure

is

more

laborious

however

because

it

involves

cutting

the

sample

into

pieces

freezing

the

sample

-LRB-

usually

overnight

-RRB-

cryogenic

comminution

then

dissipation

of

the

dry-ice

or

liquid

nitrogen

before

further

processing

or

storage

It

also

puts

higher

demands

on

the

cutter

-LRB-

blades

-RRB-

and

requires

additional

precautions

for

the

operators

-LRB-

protection

against

low

temperatures

and

noise

-RRB-

Cryogenic

comminution

has

been

recommended

for

some

pesticides

because

it

reduces

their

degradation

during

this

step

-LSB-

-RSB-

In

recent

years

the

food

trade

and

retail

have

been

intensifying

their

residue-monitoring

programs

and

require

analytical

data

before

harvest

before

accepting

an

assignment

or

before

releasing

their

products

from

distribution

centers

to

supermarkets

For

fresh

produce

this

means

there

is

a

much

pressure

on

laboratories

for

rapid

turnaround

-LRB-

h

-RRB-

This

is

difficult

to

achieve

when

the

analysis

involves

overnight

freezing

for

cryogenic

comminution

Thus

for

reasons

of

ease

and

speed

it

was

decided

to

retain

the

current

procedure

ambient

homogenization

of

the

entire

laboratory

sample

by

use

of

a

large

scale

food

cutter

-LRB-

thus

accepting

the

consequence

that

for

a

limited

number

of

pesticides

the

concentration

found

might

be

an

underestimate

-RRB-

Because

of

non-optimum

homogenization

with

the

food

cutter

subsamples

should

not

be

too

small

and

further

comminution

is

required

for

efficient

extraction

of

systemic

pesticides

This

can

be

achieved

during

extraction

by

use

of

an

Ultra

Turrax

We

have

previously

established

the

minimum

size

of

subsample

that

did

not

negatively

affect

the

repeatability

of

the

analysis

This

was

done

with

samples

which

contained

residues

For

subsamples

-LRB-

n

-RRB-

of

and

g

the

relative

standard

deviation

-LRB-

RSD

-RRB-

was

below

for

several

pesticide

matrix

combinations

For

pear

leaves

-LRB-

regarded

as

a

difficult

matrix

to

homogenize

-RRB-

containing

bromopropylate

phosalone

and

tolylfluanide

it

was

observed

that

the

RSD

increased

from

to

when

the

amount

of

subsample

was

reduced

from

g

to

g.

From

this

it

was

concluded

that

with

our

procedure

g

was

the

minimum

required

amount

of

subsample

pH

adjustment

In

the

ethyl

acetate-extraction

procedure

analytes

are

extracted

and

partitioned

between

water

-LRB-

from

the

matrix

itself

or

added

water

for

dry

crops

-RRB-

and

ethyl

acetate

in

one

step

For

basic

and

acidic

compounds

the

partitioning

can

be

affected

by

pH

which

can

vary

substantially

with

the

matrix

Because

the

same

extract

is

to

be

used

not

only

for

GC

MS

but

also

for

LC

MS

MS

-LRB-

after

changing

the

solvent

to

methanol

-RRB-

which

preferably

should

also

include

analysis

of

basic

and

acidic

pesticides

control

of

pH

was

regarded

as

necessary

A

pH

of

approximately

was

chosen

as

compromise

for

efficient

extraction

of

basic

and

acidic

compounds

Although

acidic

pesticides

were

not

included

in

this

work

data

in

the

literature

-LRB-

for

barley

without

pH

adjustment

i.e.

non-acidic

conditions

-LSB-

-RSB-

-RRB-

indicate

they

are

extracted

into

ethyl

acetate

For

pH

adjustment

others

have

used

sodium

hydroxide

-LSB-

-RSB-

or

sodium

hydrogen

carbonate

-LSB-

-RSB-

-LRB-

Table

-RRB-

A

disadvantage

of

this

is

that

the

amount

of

salt

needed

depends

on

the

acidity

of

the

sample

Addition

of

too

much

will

result

in

a

high

pH

and

possible

degradation

of

base-sensitive

pesticides

To

keep

the

method

as

straightforward

as

possible

the

pH

was

adjusted

using

a

solution

of

concentrated

phosphate

buffer

-LRB-

mol

L

mL

-RRB-

A

solution

was

preferred

over

addition

of

solid

salts

because

this

enabled

use

of

a

dispenser

and

eliminated

additional

weighing

of

the

salts

The

buffer

resulted

in

appropriate

pH

adjustment

for

most

matrices

although

there

were

exceptions

for

example

lemon

and

lime

Extraction

The

two

conditions

most

relevant

to

extraction

efficiency

are

the

sample-to-solvent

ratio

and

addition

of

salt

which

in

ethyl

acetate-based

multi-residue

methods

has

always

been

sodium

sulfate

The

amount

of

ethyl

acetate

-LRB-

in

mL

-RRB-

relative

to

the

amount

of

sample

-LRB-

in

g

-RRB-

is

typically

at

least

This

ratio

has

been

used

for

many

years

-LRB-

Table

-RRB-

It

results

in

good

extraction

efficiency

and

is

practical

with

regard

to

achieving

phase

separation

and

avoidance

of

emulsions

To

avoid

sacrificing

decades

of

method

history

no

attempts

were

made

to

reduce

the

ratio

to

do

so

might

also

adversely

affect

recovery

and/or

complicate

phase

separation

Larger

amounts

-LRB-

as

used

by

several

other

laboratories

Table

-RRB-

result

in

greater

solvent

consumption

and

more

dilute

extracts

In

previous

work

-LSB-

-RSB-

it

has

been

shown

that

the

efficiency

of

extraction

of

polar

pesticides

improves

with

the

amount

of

salt

added

When

mL

ethyl

acetate

and

g

sample

were

used

g

sodium

sulfate

was

sufficient

to

obtain

recoveries

of

or

better

even

for

very

polar

and

highly

water-soluble

compounds

for

example

acephate

and

methamidophos

Because

these

recoveries

were

obtained

with

a

single

extraction

it

was

found

unnecessary

to

perform

repeated

extraction

as

some

laboratories

are

doing

-LSB-

-RSB-

For

addition

of

the

sodium

sulfate

an

automatic

salt-dispenser

coupled

to

a

balance

as

is

used

in

our

laboratory

or

a

scoop

was

found

to

be

very

convenient

The

extraction

procedure

involves

successive

addition

of

buffer

extraction

solution

-LRB-

ethyl

acetate

with

internal

standard

-RRB-

and

sodium

sulfate

to

the

centrifuge

tube

containing

the

sample

after

which

the

pesticides

are

extracted

and

partitioned

in

one

step

using

a

Turrax

During

this

step

the

subsample

is

further

comminuted

for

efficient

extraction

of

the

pesticides

from

the

matrix

Vortex

mixing

shaking

or

sonication

were

regarded

as

less

efficient

for

subsamples

that

were

homogenized

in

a

large-scale

food

cutter

under

ambient

conditions

but

this

was

not

investigated

partly

because

a

variety

of

samples

containing

residues

would

be

required

to

do

so

in

an

appropriate

manner

It

was

noted

from

the

literature

that

filtration

is

often

performed

to

separate

the

solid

pellet

from

the

liquid

Again

there

is

no

real

need

for

this

step

which

involves

additional

glassware

and

occasionally

rinsing

-LRB-

diluting

-RRB-

of

the

extract

For

many

samples

a

clear

ethyl

acetate

extract

is

obtained

after

settling

if

not

the

tubes

can

be

centrifuged

This

is

no

more

laborious

than

filtration

and

does

not

involve

additional

glassware

Because

the

same

Turrax

is

used

for

several

samples

carry-over

is

an

aspect

to

be

considered

Between

samples

the

Turrax

is

cleaned

first

by

rinsing

with

water

by

means

of

a

flow-through

beaker

then

by

brief

immersing

in

two

beakers

containing

ethyl

acetate

Using

this

procedure

carry-over

was

tested

by

analyzing

a

blank

after

a

sample

that

had

been

fortified

at

mg

kg

Carry-over

was

less

then

indicating

that

the

straightforward

cleaning

procedure

was

sufficient

to

avoid

cross-contamination

up

to

mg

kg

when

setting

reporting

limits

not

lower

than

mg

kg

GC

MS

analysis

Clean-up

In

ethyl

acetate-based

multiresidue

methods

either

no

clean-up

or

GPC

clean-up

is

performed

This

has

hardly

changed

over

the

years

-LRB-

Table

-RRB-

In

contrast

with

acetone

and

acetonitrile-based

methods

in

which

SPE

is

commonly

employed

this

has

been

reported

only

occasionally

for

ethyl

acetate-based

methods

Obana

et

al.

-LSB-

-RSB-

used

a

cartridge

packed

with

layers

of

water-absorbing

polymer

and

GCB

Sharif

et

al.

-LSB-

-RSB-

described

a

clean-up

using

SAX/PSA

but

the

scope

of

the

method

was

restricted

to

organochlorine

and

organophosphorus

pesticides

Zhang

et

al.

-LSB-

-RSB-

used

a

clean-up

based

on

Florisil

and

achieved

adequate

recovery

of

many

pesticides

but

not

the

more

polar

organophosphorus

pesticides

It

has

been

stated

that

in

GC

analysis

with

use

of

highly

selective

detectors

for

example

MS

MS

no

clean-up

is

required

even

when

injecting

mg

equivalent

of

matrix

-LRB-

green

bean

tomato

pepper

cucumber

marrow

egg

plant

and

water

melon

-LSB-

-RSB-

-RRB-

Other

laboratories

experienced

problems

with

contamination

of

the

GC

inlet

and

tried

to

solve

this

by

automatic

exchange

of

liner

inserts

-LSB-

-RSB-

This

is

in

agreement

with

our

experience

that

injection

of

mg

matrix

equivalent

especially

for

leafy

vegetables

does

result

in

rapid

deterioration

of

system

performance

because

of

accumulation

of

non-volatile

material

in

the

inlet

This

makes

the

system

less

robust

and

frequent

exchange

of

the

liner

-LRB-

daily

-RRB-

and

GC

pre

column

-LRB-

weekly

-RRB-

is

required

Another

problem

encountered

with

injection

of

the

uncleaned

extracts

was

a

shift

in

the

retention

times

of

pesticides

relative

to

that

of

the

calibration

standard

for

some

sample

extracts

This

shift

was

insufficiently

corrected

by

automatic

adjustment

of

retention

times

relative

to

that

of

the

internal

standard

Typically

shifts

were

in

the

range

min

and

were

most

abundant

for

the

azole

pesticides

Such

shifts

can

complicate

automatic

peak

assignment

during

data-handling

When

data

acquisition

is

performed

in

a

non-continuous

mode

-LRB-

e.g.

selected-ion

monitoring

or

MS

MS

-RRB-

such

shifts

also

increase

the

risk

of

pesticides

shifting

from

their

acquisition

window

For

injection

of

relatively

large

amounts

of

matrix

-LRB-

e.g.

mg

-RRB-

in

GC

analysis

clean-up

for

removal

of

bulk

co-extractants

is

therefore

regarded

as

a

prerequisite

for

robust

analysis

of

a

wide

variety

of

vegetable

and

fruit

matrices

For

vegetables

and

fruit

matrices

chlorophyll

-LRB-

MW

-RRB-

and

other

pigments

for

example

carotenoids

-LRB-

e.g.

β-carotene

MW

-RRB-

are

typical

bulk

co-extractants

Most

of

these

compounds

are

of

low

volatility

and

are

not

apparent

as

interferences

in

the

chromatograms

they

do

however

accumulate

in

the

liner

of

the

GC

and

eventually

have

an

adverse

effect

on

transfer

of

analytes

to

the

column

and/or

on

peak

shape

Because

of

its

high

molecular

weight

chlorophyll

can

be

removed

by

GPC

A

disadvantage

is

that

the

extract

is

strongly

diluted

and

reconcentration

by

rotary

evaporation

is

almost

inevitable

when

LODs

of

mg

kg

are

required

Such

a

step

would

contribute

substantially

to

overall

sample-preparation

time

Although

a

very

efficient

on-line

combination

of

GPC

and

GC

MS

was

described

recently

-LSB-

-RSB-

avoiding

GPC

whenever

possible

would

be

even

more

straightforward

Solid-phase

extraction

is

an

alternative

clean-up

procedure

which

involves

less

dilution

and

is

less

laborious

Even

more

efficient

is

SPE

in

the

so-called

dispersive

mode

as

described

by

Anastassiades

et

al.

-LSB-

-RSB-

Here

the

solid

phase

is

simply

added

to

the

extract

thereby

avoiding

typical

SPE

procedures

such

as

conditioning

sample

transfer

elution

and

evaporative

reconcentration

The

pesticides

partition

between

the

solid

phase

and

the

solvent

and

after

vortex

mixing

and

centrifugation

the

supernatant

is

ready

for

analysis

Two

stationary

phases

graphitized

carbon

black

-LRB-

GCB

-RRB-

and

phases

with

amino

functionality

have

been

shown

to

be

particularly

effective

for

removing

co-extracted

material

from

the

raw

extract

while

not

removing

most

of

the

pesticides

this

makes

them

very

suitable

for

wide-scope

methods

-LSB-

-RSB-

Initially

a

method

was

envisaged

using

SPE

column

clean-up

with

GCB

because

for

leafy

vegetables

this

was

found

to

be

the

only

sufficiently

effective

alternative

to

GPC

After

the

publication

on

dispersive

SPE

-LSB-

-RSB-

it

was

decided

to

investigate

this

approach

thus

sacrificing

some

clean-up

potential

-LRB-

as

has

been

reported

in

the

literature

-LSB-

-RSB-

-RRB-

for

ease

and

speed

GCB

is

well

known

to

adsorb

planar

molecules

including

chlorophyll

and

other

pigments

but

also

pesticides

with

planar

functionality

In

acetonitrile-based

methods

toluene

-LRB-

typically

-RRB-

is

often

added

to

the

eluent

to

desorb

these

pesticides

also

from

the

SPE

column

-LSB-

-RSB-

One

of

the

objectives

of

this

work

was

to

investigate

the

possibility

of

using

GCB

in

a

dispersive

clean-up

step

without

unacceptable

losses

of

planar

pesticides

First

we

investigated

which

pesticides

dissolved

in

ethyl

acetate

are

adsorbed

by

GCB

A

somewhat

arbitrary

mg

mL

GCB

phase

was

added

to

standard

solutions

After

vortex

mixing

and

centrifugation

the

solution

was

analyzed

by

GC

MS

-LRB-

pesticides

-RRB-

and

after

changing

the

solvent

to

methanol

by

LC

MS

MS

-LRB-

another

pesticides

-RRB-

and

the

responses

were

compared

with

those

from

untreated

standard

solutions

For

pesticides

-LRB-

-RRB-

adsorption

was

observed

-LRB-

Table

-RRB-

In

addition

to

the

pesticides

included

in

this

test

it

is

known

from

the

literature

-LSB-

-RSB-

that

chinomethionate

furametpyr

and

pyraclofos

are

also

adsorbed

by

GCB

-LRB-

from

acetone

cyclohexane

-RRB-

Table

adsorbed

by

GCBaStrong

adsorption

-LRB-

rec

-RRB-

Medium

adsorption

-LRB-

rec

-RRB-

Not

consistentMeasured

by

GC

MS

ChlorothalonilAzinphos-ethylPhosmet

CyprodinilAzinphos-methylProchloraz

FenazaquinChlorpyrifos-methylPyrazophos

HexachlorobenzeneDicloranTrifluralin

MepanipyrimEPN

PentachloroanilineFenamiphos

PhosalonePhorate

PyrimethanilQuintozene

QuinoxyfenMeasured

by

LC

MS

MS

CarbendazimFenpyroximate

ClofentezineFlufenoxuron

DesmediphamTricyclazole

DiflubenzuronTriflumuron

FlucycloxuronThiophanate-methyl

Hexaflumuron

Phenmedipham

Pymetrozine

ThiabendazoleaPesticides

in

ethyl

acetate

mg

GCB

mL

solventrec.

recovered

To

investigate

how

much

toluene

is

required

to

prevent

adsorption

of

planar

pesticides

by

GCB

in

dispersive

SPE

the

partitioning

experiment

was

repeated

with

standard

solutions

of

or

toluene

in

ethyl

acetate

This

was

done

for

the

GC

MS

pesticide

mixture

only

As

is

apparent

from

Fig.

even

toluene

dramatically

improved

recovery

With

toluene

recovery

of

all

pesticides

was

higher

than

It

should

be

noted

that

this

experiment

with

standard

solutions

is

the

worst

case

For

real

samples

chlorophyll

and

carotenoids

will

also

affect

the

distribution

in

favor

of

the

pesticides

in

solution

Use

of

of

toluene

further

improved

recovery

only

slightly

Twenty

percent

was

regarded

as

optimum

with

regard

to

distribution

and

ease

of

solvent

elimination

in

large-volume

injection

-LRB-

see

below

-RRB-

In

addition

to

toluene

two

alternative

analogues

benzene

and

xylene

were

also

considered

Benzene

was

not

tested

because

it

could

not

be

used

in

routine

practice

because

of

its

carcinogenic

properties

-LRB-

although

it

would

have

been

favorable

with

regard

to

solvent

elimination

-RRB-

Xylene

was

tested

in

a

similar

way

as

toluene

Results

obtained

for

hexachlorobenzene

and

chlorothalonil

by

use

of

the

two

solvents

are

compared

in

Fig.

Slight

but

consistently

better

recovery

was

obtained

with

xylene

recovery

could

now

be

obtained

for

all

pesticides

Because

of

its

greater

volatility

however

toluene

was

finally

selected

Fig.

of

the

amount

-LRB-

-RRB-

of

toluene

in

ethyl

acetate

on

recovery

of

pesticides

adsorbed

by

GCB

-LRB-

mg

mL

-RRB-

hcb

hexachlorobenzene

pca

pentachloroaniline

ctn

chlorothalonil

mep

mepanipyrim

cypr

cyprodinil

pyri

pyrimethanil

fena

fenazaquin

quin

quinoxyfen

pyra

pyrazophos

epn

EPNFig

of

toluene

and

xylene

as

additives

for

preventing

adsorption

of

planar

pesticides

by

GCB

in

dispersive

SPE

Obviously

toluene

is

also

likely

to

affect

adsorption

of

chlorophyll

and/or

carotenoids

and

might

reduce

the

effectiveness

of

clean-up

To

investigate

this

a

lettuce

extract

was

prepared

the

dispersive

clean-up

experiments

were

performed

with

different

amounts

of

toluene

and

removal

of

chlorophyll

was

verified

Visually

it

was

clearly

apparent

that

despite

addition

of

toluene

the

intense

green

color

turned

light

yellow

indicating

that

chlorophyll

was

removed

to

a

large

extent

To

enable

more

quantitative

evaluation

the

extracts

were

also

measured

with

a

spectrophotometer

at

nm

For

comparison

the

same

extracts

were

also

cleaned

by

GPC

The

results

are

presented

in

Table

Without

toluene

chlorophyll

was

very

effectively

removed

Absorption

at

nm

was

reduced

by

Toluene

as

expected

reduced

adsorption

of

chlorophyll

but

removal

was

still

or

after

addition

of

or

toluene

in

ethyl

acetate

respectively

Similar

to

observations

with

the

planar

pesticides

adsorption

was

reduced

slightly

more

by

use

of

xylene

than

by

use

of

toluene

With

GPC

chlorophyll

removal

was

It

should

be

noted

here

that

the

elution

window

was

relatively

wide

to

include

pyrethroids

within

the

scope

of

the

method

The

elution

windows

for

chlorophyll

-LRB-

and

carotenoids

-RRB-

partially

overlap

those

for

pyrethroids

as

has

also

been

reported

by

others

-LSB-

-RSB-

From

these

experiments

it

can

be

concluded

that

chlorophyll

has

more

affinity

than

the

planar

pesticides

for

GCB

In

dispersive

SPE

toluene

effectively

prevents

unacceptable

adsorption

of

planar

pesticides

while

to

a

large

extent

maintaining

its

cleaning

properties

in

respect

of

chlorophyll

Dispersive

GCB

not

only

enables

much

faster

chlorophyll

removal

it

is

also

more

effective

when

including

pyrethroids

in

the

scope

of

the

method

For

non-fatty

vegetable

and/or

fruit

matrices

therefore

GPC

is

not

required

and

dispersive

GCB

clean-up

is

a

much

faster

alternative

without

sacrificing

scope

Table

of

chlorophyll

by

dispersive

SPE

-LRB-

GCB

-RRB-

and

GPCClean-up

procedureChlorophyll

removal

-LRB-

-RRB-

Dispersive

SPE

ethyl

SPE

toluene

in

ethyl

SPE

toluene

in

ethyl

SPE

xylene

in

ethyl

-LRB-

fraction

incl

pyrethroids

-RRB-

extract

lettuce

g

mL

Dispersive

SPE

mg

GCB

mL

GPC

wide

scope

elution

window

i.e.

including

pyrethroids

The

GCB

clean-up

enabled

continuous

injection

of

extracts

of

leafy

vegetables

without

rapid

system

deterioration

With

some

matrices

however

-LRB-

e.g.

plums

grapefruit

-RRB-

retention

time

shifts

were

still

observed

In

addition

depending

on

the

matrix

quite

intensive

interferences

could

be

observed

in

the

GC

MS

TIC

chromatograms

Further

clean-up

by

PSA

complementing

the

GCB

clean-up

by

removing

compounds

such

as

organic

acids

and

sugars

by

hydrogen

bonding

was

therefore

investigated

To

keep

sample

clean-up

as

straightforward

and

rapid

as

possible

focus

was

on

a

combined

dispersive

GCB/PSA

clean-up

After

the

outcome

of

the

GCB

experiments

partitioning

of

the

pesticides

and

co-extractants

will

be

between

PSA

and

ethyl

acetate

toluene

Because

no

information

was

available

about

the

distribution

of

pesticides

between

these

two

phases

this

was

obtained

by

analyzing

pesticide

standards

in

ethyl

acetate

toluene

with

and

without

PSA

Preliminary

experience

with

dispersive

PSA

clean-up

revealed

that

with

some

matrices

-LRB-

e.g.

cereals

-RRB-

mg

mL

did

not

result

in

complete

elimination

of

interfering

compounds

-LRB-

e.g.

fatty

acids

-RRB-

typically

removed

by

PSA

Partitioning

with

a

much

larger

amount

of

adsorbent

-LRB-

mg

mL

-RRB-

was

therefore

also

studied

With

mg

mL

losses

of

were

observed

for

sixteen

pesticides

most

probably

as

a

result

of

adsorption

although

the

possibility

of

degradation

induced

by

the

basic

nature

of

the

PSA

material

could

not

be

fully

excluded

The

findings

were

confirmed

by

the

experiment

with

mg

PSA

mL

-LRB-

Table

-RRB-

The

pesticides

for

which

interaction

with

PSA

was

observed

all

had

a

C

=O

or

P

=O

group

in

common

-LRB-

except

for

chlorothalonil

-RRB-

Our

findings

are

not

in

full

agreement

with

those

of

Anastassiades

et

al.

-LSB-

-RSB-

who

did

not

observe

losses

as

a

result

of

using

PSA

For

this

there

can

be

two

explanations

In

our

experiment

adsorption

was

tested

with

standard

solution

rather

than

matrix

Co-extractants

in

matrix

are

likely

to

compete

with

the

pesticides

during

adsorption

Second

with

our

method

the

organic

phase

-LRB-

ethyl

acetate

toluene

-RRB-

is

less

polar

than

the

acetonitrile

phase

this

could

result

in

a

stronger

interaction

between

the

polar

functionality

of

the

pesticides

and

amino

functionality

of

PSA

From

our

results

it

became

clear

that

with

regard

to

the

amount

of

PSA

the

more

the

better

does

not

apply

Another

observation

was

that

a

hump

appeared

in

the

TIC

chromatogram

after

a

injection

of

solvent

mixed

with

mg

PSA

mL

This

hump

which

eluted

between

and

min

consisted

of

many

peaks

and

a

variety

of

masses

Cleaning

of

the

PSA

by

washing

with

ethyl

acetate

-LRB-

mL

for

g

-RRB-

then

drying

by

rotary

evaporation

eliminated

this

contamination

without

affecting

the

clean-up

properties

To

keep

the

method

straightforward

mg

PSA

mL

was

used

as

default

and

the

material

was

not

cleaned

before

use

Table

of

pesticides

by

PSAPesticideRecovery

-LRB-

-RRB-

partitioning

with

ethyl

acetate

mg

mL

and

LC

MS

MS

analysisbAfter

partitioning

with

ethyl

acetate

toluene

mg

PSA

mL

and

GC

MS

analysis

The

clean-up

proved

effective

at

reducing

retention

time

shifts

As

an

example

for

a

plum

extract

without

clean-up

the

retention

times

of

pesticides

-LRB-

out

of

-RRB-

were

shifted

by

more

than

min

compared

with

the

calibration

standard

After

clean-up

this

occurred

for

three

pesticides

only

With

other

matrices

also

shifts

were

reduced

but

for

some

matrices

-LRB-

herbs

e.g.

parsley

-RRB-

deviations

were

still

quite

common

As

an

illustration

of

the

removal

of

co-extractants

from

the

ethyl

acetate

extract

-LRB-

or

in

fact

from

the

ethyl

acetate

toluene

extract

-RRB-

by

dispersive

GCB/PSA

clean-up

GC

MS

total

ion

current

chromatograms

of

extracts

obtained

with

and

without

clean-up

are

shown

in

Fig.

The

most

apparent

differences

are

indicated

Several

abundant

matrix

peaks

are

removed

or

strongly

reduced

For

lettuce

the

overall

background

level

between

and

min

was

also

reduced

This

clearly

visible

clean-up

was

mainly

caused

by

the

PSA

material

With

GCB

alone

differences

between

cleaned

and

uncleaned

were

much

less

apparent

The

main

benefit

of

GCB

was

prevention

of

rapid

build

up

of

non-volatile

material

-LRB-

chlorophyll

-RRB-

in

the

liner

which

enables

prolonged

use

of

the

system

without

maintenance

Experience

with

method

for

more

than

three

years

and

analysis

of

over

vegetable

and

fruit

samples

shows

that

on

average

the

liner

must

typically

be

replaced

weekly

-LRB-

after

injections

iprodion

dimethipin

and

chlorfenapyr

are

the

first

for

which

response

is

lost

-RRB-

Further

GC

MS

maintenance

consists

in

replacement

of

pre-column

once

of

twice

a

month

The

GC

column

is

replaced

approximately

twice

a

year

The

source

of

the

MS

is

cleaned

once

a

month

Fig.

MS

chromatograms

Overlay

total

ion

chromatograms

-LRB-

TICs

-RRB-

obtained

after

μL

injection

of

an

extract

of

mandarin

-LRB-

top

-RRB-

and

lettuce

-LRB-

bottom

-RRB-

without

-LRB-

higher

peaks

-RRB-

and

with

clean-up

In

a

continuing

search

for

even

further

simplification

of

sample

preparation

the

possibility

of

combined

extraction

and

dispersive

SPE

clean-up

in

one

step

was

investigated

For

two

matrices

-LRB-

lettuce

and

mandarin

fortified

with

pesticides

triplicate

experiments

-RRB-

the

solid

phase

materials

-LRB-

GCB/PSA

relative

amounts

similar

to

previous

experiments

-RRB-

were

added

directly

to

the

centrifuge

tube

containing

the

sample

sodium

sulfate

and

the

extraction

solvent

-LRB-

to

which

toluene

had

been

added

-RRB-

After

Turrax

extraction

and

centrifugation

the

extract

was

ready

for

injection

into

the

GC

Recovery

was

compared

with

that

obtained

by

use

of

dispersive

clean-up

after

separation

of

the

ethyl

acetate

extract

from

the

sample

mixture

As

could

be

seen

from

the

color

of

the

extract

-LRB-

the

lettuce

extract

was

almost

colorless

-RRB-

the

GCB

remained

effective

Adsorption

of

chlorophyll

is

based

on

planarity

-LRB-

shape

-RRB-

rather

than

polarity

and

therefore

this

will

occur

from

both

the

aqueous

and

the

organic

phases

As

was

to

be

expected

the

same

was

not

true

for

PSA

The

presence

of

water

prevented

adsorption

of

co-extractants

with

a

hydroxyl

group

i.e.

almost

identical

GC

MS

total-ion

chromatograms

were

obtained

from

extracts

which

were

not

cleaned

and

from

those

cleaned

in

the

centrifuge

tube

Pesticide

recovery

obtained

after

use

of

successive

or

simultaneous

dispersive

SPE

clean-up

was

very

similar

although

recovery

of

some

pesticides

in

the

combined

approach

was

too

high

because

of

co-elution

of

interferences

The

final

method

therefore

used

successive

extraction

and

dispersive

SPE

clean-up

Large-volume

injection

GC

MS

analysis

of

sample

extracts

was

performed

in

full-scan

mode

This

enables

detection

of

any

GC

amenable

pesticide

Because

system

LOQ

for

a

quadrupole

mass

spectrometer

in

full-scan

mode

is

limited

conservatively

estimated

at

pg

mg

matrix

equivalent

must

be

introduced

into

the

GC

to

reach

a

target

LOQ

of

mg

kg

With

an

extract

concentration

of

g

mL

this

means

μL

must

be

introduced

into

the

GC

Off-line

tenfold

evaporative

concentration

and

then

μL

injection

could

also

be

performed

but

this

would

involve

clean-up

of

larger

volumes

of

extract

the

risk

of

loss

of

the

volatile

pesticides

-LRB-

e.g.

dichlorvos

-RRB-

and

an

additional

step

in

sample

preparation

Although

large-volume

injection

in

GC

is

a

well

established

technique

-LSB-

-RSB-

many

routine

laboratories

are

still

reluctant

to

apply

it

if

they

do

the

volume

is

often

restricted

to

μL

Such

volumes

can

be

accommodated

in

liners

with

a

frit

or

even

in

empty

-LRB-

baffled

-RRB-

liners

when

injection

speed

is

carefully

adjusted

For

larger

volumes

there

is

a

risk

of

flooding

-LSB-

-RSB-

i.e.

that

extract

is

lost

as

liquid

through

the

split

exit

To

prevent

this

liners

can

be

packed

with

a

variety

of

materials

Packing

materials

often

have

the

disadvantage

of

a

large

surface

area

with

active

sites

however

resulting

in

degradation

and/or

adsorption

of

thermo

labile

and/or

polar

pesticides

problems

can

also

be

encountered

with

splitless

transfer

of

higher

boiling

pesticides

-LRB-

e.g.

deltamethrin

-RRB-

from

the

liner

to

the

GC

column

Other

disadvantages

can

be

a

pressure

drop

over

the

liner

-LRB-

slows

down

solvent

elimination

-RRB-

and

liner-to-liner

variability

requiring

re-optimization

of

the

solvent-elimination

process

after

liner

replacement

A

means

of

by-passing

the

disadvantages

of

packed

liners

while

still

achieving

accommodation

of

μL

of

liquid

was

described

in

by

Staniewski

and

Rijks

-LSB-

-RSB-

They

developed

a

liner

with

a

sintered

porous

glass

bed

on

the

inner

surface

wall

of

the

liner

The

liquid

is

retained

in

the

porous

glass

bed

The

potentially

active

glass

surface

area

is

relatively

small

compared

with

the

materials

in

packed

liners

The

gas

flow

is

not

obstructed

because

the

centre

of

the

liner

is

empty

This

enables

efficient

solvent

vapor

removal

during

solvent

elimination

and

efficient

transfer

of

analytes

to

the

analytical

column

during

splitless

injection

after

solvent

elimination

Since

the

early

such

liners

have

been

commercially

available

for

PTV

injectors

from

several

suppliers

and

since

then

our

laboratory

has

implemented

μL

as

default

injection

volume

for

ethyl

acetate

After

the

development

of

the

dispersive

GCB

clean-up

the

solvent

to

be

introduced

into

the

GC

contained

toluene

which

might

effect

the

processes

involved

in

large-volume

injection

differently

from

ethyl

acetate

Because

toluene

does

not

evaporate

azeotropically

with

ethyl

acetate

and

is

less

volatile

it

will

be

the

main

solvent

left

at

the

end

of

the

evaporation

process

Injection

of

μL

toluene

in

ethyl

acetate

means

that

μL

toluene

is

introduced

The

PTV

used

in

this

work

was

equipped

with

a

mm

i.d.

porous

glass

bed

liner

that

could

hold

approximately

μL

within

the

zone

that

is

appropriately

heated

during

splitless

transfer

Up

to

this

volume

there

is

no

need

for

optimization

of

injection

speed

To

obtain

information

about

splitless

transfer

of

the

last

few

microliters

of

toluene

after

solvent

elimination

cold

splitless

injections

of

and

μL

of

standards

in

toluene

were

performed

Even

with

volumes

peak

distortion

-LRB-

fronting

peak

shape

-RRB-

was

observed

for

pesticides

of

medium

volatility

With

μL

injections

peak

shape

was

good

and

for

several

pesticides

even

better

than

for

ethyl

acetate

On

injection

of

μL

standard

in

ethyl

acetate

toluene

in

the

solvent-vent

mode

no

peak

distortion

was

observed

indicating

that

less

then

μL

toluene

remained

in

the

injector

after

the

solvent-vent

step

As

observed

earlier

with

large-volume

injection

of

ethyl

acetate

the

vent

time

-LRB-

here

set

at

s

using

an

initial

PTV

temperature

of

°C

-RRB-

was

not

at

all

critical

even

for

the

most

volatile

pesticide

-LRB-

dichlorvos

-RRB-

Venting

for

or

s

did

not

dramatically

affect

responses

or

peak

shape

of

the

pesticides

In

our

experience

this

phenomenon

is

typical

for

porous

glass

bed

liners

and

contributes

to

the

robustness

of

the

method

Validation

of

GC

MS

method

In

the

past

a

method

based

on

simple

ethyl

acetate

extraction

followed

by

direct

GC

MS

analysis

of

the

raw

extract

-LSB-

-RSB-

had

been

validated

for

concentrations

in

the

range

mg

kg

The

modified

method

described

here

involved

a

dispersive

clean-up

step

large-volume

injection

and

injection

of

ten

times

more

matrix

into

the

GC

Re-validation

was

therefore

required

and

focused

on

method

performance

at

low

concentrations

This

was

done

using

lettuce

as

matrix

The

validation

set

consisted

of

two

control

samples

five

fortifications

at

mg

kg

and

five

fortifications

at

a

level

ten

times

higher

Over

pesticides

were

included

in

the

validation

procedure

The

results

are

presented

in

Table

For

the

mg

kg

concentration

range

the

EU

criteria

-LRB-

recovery

RSD

or

for

and

mg

kg

respectively

-LSB-

-RSB-

-RRB-

were

met

for

of

the

pesticides

included

in

the

validation

At

a

level

a

factor

of

ten

lower

-LRB-

fortification

in

the

mg

kg

range

for

most

pesticides

-RRB-

pesticides

could

still

be

detected

and

for

most

-LRB-

-RRB-

of

these

recovery

and

RSDs

were

acceptable

For

many

pesticides

S/N

ratios

were

surprisingly

good

and

background-corrected

mass

spectra

often

contained

sufficient

diagnostic

ions

-LRB-

or

were

even

recognizable

mass

spectra

-RRB-

to

enable

identity

confirmation

as

is

illustrated

in

Fig.

The

limits

of

detection

defined

as

S/N

for

one

favorable

diagnostic

ion

for

each

pesticide

were

determined

on

the

basis

of

the

signals

from

the

low

fortification

levels

and

the

average

noise

observed

in

duplicate

control

samples

The

LOD

was

at

or

below

mg

kg

for

pesticides

between

and

mg

kg

for

pesticides

between

and

mg

kg

for

pesticides

between

and

mg

kg

for

pesticides

and

higher

for

four

pesticides

Table

MS

re-validation

data

for

pesticides

in

lettuce

PesticidetR

-LRB-

min

-RRB-

m/z

-LRB-

quant

-RRB-

Level

-LRB-

mg

kg

-RRB-

Rec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

Level

-LRB-

mg

kg

-RRB-

Rec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

LOD

-LRB-

mg

kg

-RRB-

1Acephate10.451360.0263540.2575890.0062Acrinathrin22.062890.018118150.1789490.0033Aldrin16.582650.003139250.0319420.0024Atrazine14.172150.00291210.0189870.0025Azinphos-methyl21.641600

Benzoylurea

-LRB-

deg

-RRB-

Chlordane

Chlordane

Chloroaniline

Cinerin-118.671500.0538430.5289360.04131

Cyfluthrin

Cyfluthrin

DDE

o

p

DDE

p

p

DDT

o

p

DDT

p

p

Dicofol

-LRB-

as

DCBP

-RRB-

Diflubenzuron

-LRB-

deg

-RRB-

Flufenoxuron

-LRB-

deg

-RRB-

Fluvalinate

Lufenuron

-LRB-

deg

-RRB-

-LRB-

deg

-RRB-

Phenylphenol

Piperonyl

TDE

o

p

TDE

p

p

Teflubenzuron

-LRB-

deg

-RRB-

aBenzoylurea

-LRB-

deg

-RRB-

Amount

for

which

S/N

or

in

the

event

of

an

interfering

peak

the

average

peak

height

for

fortified

sample

-LRB-

n

-RRB-

should

be

times

the

average

peak

height

for

control

sample

-LRB-

n

-RRB-

Fortification

level

below

LOD

as

defined

aboveUnderlined

values

are

outside

EU

criteria

for

method

validationFig

MS

extracted-ion

chromatograms

obtained

from

lettuce

with

-LRB-

upper

traces

-RRB-

and

without

fortification

with

pesticides

and

the

corresponding

mass

spectra

-LRB-

upper

reference

spectra

lower

background-corrected

spectra

from

the

sample

-RRB-

a

b

mg

kg

disulfoton

-LRB-

m/z

-RRB-

c

d

mg

kg

fipronil

-LRB-

m/z

-RRB-

e

f

mg

kg

biphenyl

-LRB-

m/z

-RRB-

This

initial

validation

clearly

showed

it

is

possible

to

introduce

mg

of

matrix

equivalent

of

generic

extracts

obtained

after

ethyl

acetate

extraction

of

leafy

vegetables

Adequate

quantitative

data

are

obtained

for

most

of

the

pesticides

at

levels

of

mg

kg

or

even

below

Detection

limits

were

usually

well

below

mg

kg

after

full-scan

acquisition

with

a

single-quadrupole

MS.

This

means

that

for

most

pesticides

at

the

target

LOQ

of

mg

kg

-LRB-

i.e.

the

lowest

maximum

residue

limit

set

in

the

EU

for

vegetables

and

fruit

-RRB-

the

signal-to-noise

ratio

is

adequate

for

reliable

automatic

integration

of

peaks

and

that

confirmation

of

identity

of

the

pesticide

is

possible

from

its

mass

spectrum

or

at

least

one

or

two

other

diagnostic

ions

Pesticides

that

did

not

meet

the

EU

criteria

for

quantitative

analysis

and/or

for

which

relatively

high

LODs

were

obtained

included

many

compounds

known

to

be

troublesome

in

GC

analysis

because

of

to

their

high

polarity

or

thermal

lability

Typical

examples

are

acephate

cyromazine

dicofol

-LRB-

screened

for

as

its

degradation

product

dichlorobenzophenone

-RRB-

dimethoate

imazalil

metaldehyde

methamidophos

methiocarb

omethoate

and

the

benzoylureas

-LRB-

measured

as

one

common

and

one

compound-specific

degradation

product

-RRB-

The

relatively

low

recovery

of

the

polar

organophosphorus

pesticides

-LRB-

acephate

methamidophos

and

omethoate

-RRB-

can

be

attributed

to

the

GC

measurement

and

not

to

poor

extraction

efficiency

as

was

apparent

from

LC

MS

MS

analysis

of

samples

using

the

same

extraction

technique

-LRB-

see

section

LC

MS

MS

analysis

-RRB-

For

several

other

polar

or

labile

pesticides

adequate

quantitative

data

were

obtained

during

this

initial

validation

but

from

previous

experience

and

the

results

obtained

after

implementation

of

the

method

it

was

clear

that

for

such

compounds

LC

based

analysis

is

more

robust

than

GC

MS

analysis

Typical

examples

include

carbaryl

carbofuran

clofentezin

monocrotophos

and

oxydemeton-methyl

Analytical

quality-control

data

from

routine

GC

MS

analysis

The

initial

validation

data

are

continuously

being

supplemented

by

performance

data

generated

as

part

of

the

analytical

quality-control

during

routine

analysis

of

the

samples

to

gain

insight

into

reproducibility

robustness

recovery

and

selectivity

with

other

matrices

For

this

with

each

analytical

batch

one

of

the

samples

submitted

for

routine

analysis

was

spiked

with

pesticides

at

five

times

the

target

LOQ

level

-LRB-

i.e.

samples

were

spiked

with

mg

kg

of

most

of

the

pesticides

-RRB-

A

compilation

was

made

of

recovery

data

from

a

period

of

months

which

included

analysis

of

approximately

different

vegetable

and

fruit

commodities

Given

the

wide

variety

of

commodities

matrix-matched

calibration

is

quite

tedious

and

would

substantially

increase

the

number

of

standard

solutions

to

be

analyzed

in

the

GC

sequence

It

was

therefore

decided

to

select

one

relatively

simple

matrix

-LRB-

tomato

-RRB-

as

default

for

matrix-matched

calibration

i.e.

recoveries

for

all

commodities

were

calculated

against

the

tomato-matrix

standard

For

each

pesticide

calculations

were

performed

for

two

diagnostic

ions

All

together

this

resulted

in

approximately

values

According

to

the

current

EU

guideline

on

quality

control

in

pesticide

residue

analysis

-LSB-

-RSB-

the

recovery

obtained

during

routine

analysis

should

be

within

An

overview

of

the

percentage

of

recovery

values

within

or

outside

the

criterion

for

a

wide

variety

of

matrices

is

presented

in

Table

With

such

large

number

of

pesticides

-LRB-

or

actually

diagnostic

ions

-RRB-

and

matrices

one

failing

combination

or

more

occurred

for

most

matrices

There

are

several

causes

for

this

Main

reasons

for

recovery

below

could

be

poor

extraction

efficiency

or

incomplete

transfer

of

the

pesticides

to

the

GC

column

-LRB-

e.g.

adsorption

and/or

degradation

in

a

contaminated

inlet

-RRB-

Higher

recovery

may

occur

when

a

compound

from

the

matrix

generates

the

same

diagnostic

ion

as

a

pesticide

and

co-elutes

with

that

pesticide

-LRB-

i.e.

detection

was

not

selective

-RRB-

Another

reason

could

be

that

the

matrix

effect

induced

in

the

GC

inlet

-LSB-

-RSB-

for

a

pesticide

in

a

particular

matrix

is

more

pronounced

than

that

in

the

tomato-based

calibration

standard

Table

of

percentage

of

recovery

valuesa

within

or

outside

the

EU

criterion

-LSB-

-RSB-

after

GC

MS

analysis

MatrixPercentage

of

all

recovery

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

choi

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

puree

-LRB-

processed

-RRB-

tops

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

puree

-LRB-

processed

-RRB-

-LRB-

red

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

puree

-LRB-

processed

-RRB-

choi

-LRB-

-RRB-

leaves

-LRB-

-RRB-

-LRB-

-RRB-

pea

-LRB-

-RRB-

fruit

-LRB-

-RRB-

leaves

-LRB-

-RRB-

pea

-LRB-

-RRB-

-LRB-

-RRB-

pepper

-LRB-

-RRB-

fruit

-LRB-

-RRB-

leaves

-LRB-

-RRB-

puree

-LRB-

processed

-RRB-

tops

-LRB-

-RRB-

leaves

-LRB-

-RRB-

pulp

-LRB-

processed

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

pepper

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

berry

-LRB-

physalis

-RRB-

at

mg

kg

-LRB-

mg

kg

for

pesticides

-RRB-

Calculated

for

pesticides

two

diagnostic

ions

each

against

a

standard

prepared

in

blank

tomato

extract

The

pesticides

included

are

listed

in

Table

Failing

pesticide

matrix

combinations

were

most

abundant

for

herbs

kale

sweetcorn

and

golden

berry

for

which

up

to

of

recovery

values

-LRB-

calculated

using

the

two

diagnostic

ions

for

each

pesticide

-RRB-

were

outside

the

range

These

products

contain

larger

amounts

of

co-extractants

than

most

other

vegetables

and

fruits

which

may

result

in

insufficient

detection

selectivity

enhanced

response

as

a

result

of

a

matrix

effect

-LRB-

more

shielding

of

active

sites

in

the

inlet

-RRB-

and

contamination

of

the

inlet

For

this

type

of

product

more

selectivity

e.g.

by

use

of

MS

MS

would

be

beneficial

Such

detection

is

also

more

sensitive

than

single

quadrupole

full-scan

detection

and

would

enable

reduction

in

the

amount

of

matrix

introduced

thus

reducing

build

up

of

contamination

Overall

when

data

for

all

QC

samples

were

included

recovery

was

acceptable

for

of

the

diagnostic

ions

measured

On

the

basis

of

the

same

data

an

overview

by

pesticide

is

presented

in

Table

For

each

pesticide

two

diagnostic

ions

from

the

full-scan

data

were

integrated

and

concentrations

were

calculated

In

routine

practice

however

the

most

convenient

way

of

reviewing

the

data

is

by

using

one

and

the

same

diagnostic

ion

for

each

pesticide

irrespective

the

matrix

On

the

basis

of

the

data

set

obtained

-LRB-

nearly

pesticide

matrix

combinations

-RRB-

the

most

favorable

of

the

two

diagnostic

ions

i.e.

the

ion

for

which

the

highest

number

of

recoveries

within

was

obtained

was

assigned

as

the

Quan

ion

-LRB-

default

quantification

ion

-RRB-

By

using

this

ion

acceptable

recoveries

were

obtained

for

of

pesticides

matrix

combinations

This

also

means

that

or

in

absolute

figures

of

the

pesticide

matrix

combinations

did

not

meet

the

criterion

of

these

failing

combinations

could

be

accepted

after

use

of

the

alternative

ion

for

which

calculations

were

also

performed

automatically

during

data

processing

Low

recoveries

-LRB-

-RRB-

for

both

diagnostic

ions

were

obtained

for

of

pesticide

matrix

combinations

High

recoveries

-LRB-

-RRB-

were

obtained

for

of

the

combinations

For

this

latter

group

manual

evaluation

of

other

ions

if

available

and

sufficiently

abundant

could

further

increase

the

number

of

acceptable

recoveries

Because

this

is

a

time-consuming

process

it

was

not

done

routinely

In

the

event

of

deviating

recovery

assessment

of

the

results

to

be

reported

was

based

on

visual

evaluation

of

the

extracted

ion

chromatograms

of

the

two

diagnostic

ions

at

least

On

the

basis

of

on

the

findings

it

was

then

concluded

the

pesticide

could

not

be

determined

in

that

specific

matrix

or

only

at

higher

levels

Table

over

all

matrices

-LRB-

GC

MS

analysis

-RRB-

PesticideQuan

ion

m/zQual

ion

m/zFortification

level

-LRB-

mg

kg

-RRB-

QCs

matrices

-LRB-

see

Table

-RRB-

Both

diagn

ions

One

of

diagn

ions

Both

diagn

ions

Both

diagn

ions

Average

recov

-LRB-

-RRB-

Quan

ionRSD

-LRB-

-RRB-

Acrinathrin

Cyhalothrin

DDE

p

p

DDT

o

p

DDT

p

p

Fluvalinate

Phenylphenol

TDE

p

p

Total

of

It

should

be

noted

that

the

above

evaluation

applies

to

a

level

five

times

the

reporting

level

which

was

set

at

mg

kg

or

the

LOQ

if

higher

than

mg

kg

At

lower

levels

interferences

may

have

a

larger

effect

and

consequently

more

frequent

deviations

from

the

criterion

-LRB-

most

probably

-RRB-

may

be

observed

For

higher

levels

the

opposite

would

be

true

Pesticides

for

which

low

recoveries

-LRB-

-RRB-

were

frequently

obtained

-LRB-

of

QC

samples

-RRB-

included

iprodione

and

p

p

DDT

-LRB-

degradation

in

inlet

-RRB-

dimethomorph

-LRB-

polar

relatively

non-volatile

could

be

troublesome

in

splitless

transfer

-RRB-

pentachloroanisole

pentachloroaniline

and

mepanipyrim

-LRB-

no

clear

explanation

but

probably

related

to

the

dispersive

SPE

clean-up

-RRB-

There

were

no

indications

for

poor

extraction

efficiency

High

recovery

-LRB-

-RRB-

frequently

occurred

for

etridiazole

methidathion

mevinphos

phosmet

phosalone

phosphamidone

and

endosulfan-alpha

-LRB-

times

out

of

QC

samples

often

in

herbs

and

peas

-RRB-

This

was

attributed

to

matrix

effects

and

interferences

Overall

the

pesticides

that

failed

most

frequently

-LRB-

times

out

of

-RRB-

during

routine

analytical

quality

control

were

-LRB-

in

descending

order

-RRB-

etridiazole

iprodione

methidathion

pentachlorothioanisole

mevinphos

phosmet

p

p

DDT

mepanipyrim

phosalone

phosphamidon

biphenyl

dichlorvos

spirodiclofen

pentachloroaniline

deltamethrin

tau-fluvalinate

and

pyrazophos

These

would

be

the

most

relevant

for

inclusion

in

alternative

methods

for

example

GC

MS

MS

or

LC

MS

MS.

Average

recovery

and

RSD

were

calculated

for

pesticide

matrix

combinations

that

passed

the

acceptable

recovery

criterion

The

results

are

included

in

Table

Average

recovery

was

usually

close

to

and

RSDs

approximately

For

the

pesticides

known

to

be

adsorbed

by

GCB

systematically

lower

average

recovery

-LRB-

-RRB-

was

obtained

which

is

in

agreement

with

the

results

obtained

during

method

development

These

comprehensive

data

show

that

with

a

relatively

inexpensive

single-quadrupole

MS

detector

in

full-scan

mode

it

is

possible

to

obtain

reliable

quantitative

data

down

to

the

mg

kg

level

or

even

lower

for

a

wide

range

of

pesticides

in

a

wide

variety

of

matrices

after

generic

rapid

sample

preparation

based

on

extraction

with

ethyl

acetate

Unified

calibration

based

on

a

tomato-matrix

standard

is

furthermore

a

feasible

approach

One

should

however

be

aware

there

are

also

limitations

and

that

some

pesticide

matrix

combinations

can

not

be

determined

in

the

mg

kg

range

and

that

for

other

pesticides

calibration

against

the

corresponding

matrix

instead

of

tomato

is

required

to

bring

quantitative

results

within

the

AQC

criteria

especially

for

MRL

violations

when

more

stringent

criteria

apply

The

data

also

reveal

that

the

only

way

to

gain

full

insight

into

analyte

recovery

and

method

selectivity

with

a

wide

variety

of

matrices

is

by

performing

analytical

quality

control

on

all

pesticides

which

are

reported

rather

than

on

a

subset

as

is

suggested

in

the

EU

guideline

-LSB-

-RSB-

A

subset

will

suffice

for

demonstration

of

adequate

sample

preparation

and

injection

but

will

not

reveal

limitations

in

the

selectivity

of

GC

MS.

GC

single-quadrupole

MS

remains

an

effective

tool

for

routine

GC

analysis

of

pesticide

residues

For

many

vegetable

and

fruit

matrices

there

is

no

real

need

to

change

to

more

advanced

-LRB-

and

expensive

-RRB-

MS

techniques

for

example

MS

MS

-LRB-

which

has

limited

scope

-RRB-

or

accurate

mass

TOF-MS

-LRB-

which

has

a

limited

dynamic

range

-RRB-

Use

of

such

equipment

would

be

justified

for

more

complex

matrices

and

when

low

μg

kg

LOQs

are

required

for

example

analysis

of

some

pesticides

in

baby

food

LC

MS

MS

analysis

Clean-up

The

ethyl

acetate

extraction

procedure

is

also

appropriate

for

many

pesticides

not

amenable

to

GC

analysis

-LSB-

-RSB-

Typically

no

clean-up

is

performed

-LRB-

Table

-RRB-

One

reason

for

this

is

that

with

regard

to

chromatographic

performance

LC

columns

tend

to

be

more

tolerant

of

injection

of

bulk

matrix

than

GC

columns

In

our

experience

continual

injection

of

mg

equivalent

of

vegetable

and

fruit

extracts

does

not

result

in

deterioration

of

chromatographic

performance

or

unacceptable

contamination

of

the

ion

source

-LRB-

the

system

used

here

was

an

-RRB-

In

LC

MS

co-extracted

matrix

does

have

an

effect

on

the

response

however

by

interfering

with

the

ionization

process

This

results

in

suppression

-LRB-

sometimes

enhancement

-RRB-

of

the

response

to

a

pesticide

in

a

matrix

compared

with

that

in

a

solvent

standard

-LSB-

-RSB-

and

complicates

quantification

of

pesticides

in

the

samples

The

possibility

of

reducing

matrix

effects

by

use

of

dispersive

SPE

clean-up

was

investigated

in

a

similar

way

as

for

GC

First

the

effectiveness

of

the

clean-up

step

was

investigated

by

addition

of

mg

GCB

and

mg

PSA

to

mL

raw

extract

of

a

mixed

spinach

grape

onion

sample

-LRB-

g

-RRB-

Seventy

pesticides

-LRB-

the

ones

in

Table

with

in

the

MS-MS

column

-RRB-

were

added

after

clean-up

and

analyzed

by

LC

MS

MS.

The

response

was

compared

with

that

of

solutions

of

equal

concentration

in

the

raw

extract

and

a

solvent

standard

Clean-up

increased

the

number

of

pesticides

for

which

no

pronounced

matrix

effect

-LRB-

less

than

suppression

or

enhancement

-RRB-

was

observed

from

to

Several

of

the

pesticides

-LRB-

Tables

and

-RRB-

were

adsorbed

by

the

SPE

material

however

Although

adsorption

by

the

GCB

could

have

been

avoided

or

reduced

by

addition

of

toluene

-LRB-

although

less

practical

when

changing

from

extraction

solvent

to

methanol/water

-RRB-

it

was

concluded

that

PSA

was

not

compatible

with

a

generic

method

for

pesticides

amenable

to

LC

MS

MS.

It

was

therefore

decided

not

to

include

a

clean-up

step

for

LC

MS

MS

analysis

and

to

use

the

initial

raw

ethyl

acetate

extract

Another

reason

for

not

further

pursuing

clean-up

in

LC

MS

MS

analysis

was

that

the

sensitivity

of

current

triple-quadrupole

instruments

enables

injection

of

only

small

amounts

of

matrix

into

the

LC

MS

MS

system

-LRB-

e.g.

mg

-RRB-

while

still

achieving

the

desired

limits

of

quantification

Experiments

showed

that

tenfold

dilution

of

g

mL

extracts

increased

the

number

of

pesticides

for

which

no

pronounced

matrix

effect

occurred

from

to

and

from

to

for

cucumber

and

cabbage

respectively

Table

MS

MS

settings

and

performance-validation

characteristicsPesticidetr

-LRB-

min

-RRB-

Precurs.Prod

ion

ion

mg

kg

mg

kg

mg

kg

mg

kg

MSMatrixRec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

Rec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

MatrixRec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

Rec

-LRB-

-RRB-

RSD

-LRB-

-RRB-

Abamectinea

desmethyl

cucumberLett

adductbNa

adductcLOQ

level

mg

kg

level

mg

kg

Routine

experience

with

LC

MS

MS

analysis

for

over

four

years

both

with

the

-LRB-

mg

matrix

-RRB-

and

the

-LRB-

mg

matrix

-RRB-

has

shown

that

injection

of

uncleaned

extracts

does

not

result

in

special

maintenance

requirements

The

source

is

cleaned

with

a

tissue

daily

The

LC

column

typically

lasts

for

months

Changing

the

solvent

Because

ethyl

acetate

is

less

suitable

for

direct

injection

in

reversed

phase

LC

the

solvent

was

changed

Because

only

small

amounts

of

the

raw

extract

need

to

be

evaporated

-LRB-

less

than

mL

in

the

final

method

-RRB-

and

evaporation

blocks

enable

simultaneous

evaporation

of

many

-LRB-

typically

-RRB-

extracts

this

step

adds

little

to

the

overall

sample-preparation

time

Changing

the

solvent

was

even

regarded

as

advantageous

It

resulted

in

more

freedom

in

selection

of

the

final

solvent

to

be

injected

into

the

LC

which

can

be

critical

for

very

polar

compounds

-LRB-

e.g.

in

acetonitrile-based

extraction

methods

injection

of

acetonitrile

easily

leads

to

band-broadening

for

methamidophos

-RRB-

It

is

also

easier

to

compensate

for

the

smaller

amount

of

sample

processed

for

dry

crops

-LRB-

because

of

the

need

for

addition

of

water

-RRB-

by

evaporating

a

larger

amount

of

the

ethyl

acetate

extract

In

previous

work

-LSB-

-RSB-

a

small

amount

of

a

diethylene

glycol

-LRB-

added

as

solution

in

methanol

-RRB-

was

added

because

this

was

found

to

facilitate

reconstitution

thereby

improving

recovery

for

some

pesticide

matrix

combinations

It

was

also

shown

that

the

evaporation

step

did

not

require

special

attention

and

that

continuing

the

process

for

another

half

hour

after

completion

of

evaporation

of

the

solvent

did

not

affect

recovery

The

same

procedure

was

therefore

used

here

without

re-evaluating

the

real

need

for

it

Reconstitution

was

performed

by

first

dissolving

in

methanol

-LRB-

ultrasonication

-RRB-

and

then

dilution

with

LC

mobile

phase

component

A.

Validation

of

LC

MS

MS

method

The

LC

MS

MS

method

was

validated

in

three

separate

studies

one

using

the

with

injection

of

mg

matrix

equivalent

and

the

other

two

using

the

with

injection

of

mg

matrix

equivalent

A

total

of

pesticides

and

degradation

products

were

included

In

contrast

with

the

full-scan

acquisition

in

GC

MS

in

LC

MS

MS

data

were

acquired

for

a

fixed

limited

set

of

pesticides

Although

many

pesticides

from

the

GC

MS

method

can

also

be

analyzed

by

LC

MS

MS

emphasis

was

on

pesticides

that

were

not

or

less

amenable

to

GC

analysis

Recovery

based

on

matrix-matched

calibration

and

repeatability

were

evaluated

at

the

and

mg

kg

level

for

vegetable

and

fruit

matrices

the

results

are

listed

in

Table

Although

acceptable

performance

data

were

obtained

for

most

of

the

pesticides

low

recovery

and/or

high

variability

were

observed

for

some

Among

these

were

compounds

that

were

also

reported

as

troublesome

by

other

workers

using

alternative

multi-residue

methods

e.g.

asulam

-LSB-

-RSB-

Low

recovery

could

be

partly

attributed

to

poor

extraction

efficiency

-LRB-

asulam

hymexazol

and

in

orange

propamocarb

-RRB-

or

degradation

during

sample

preparation

-LRB-

cycloxydim

sethoxydim

profoxydim

tepraloxydim

dichlofluanide

tolylfluanide

thiodicarb

thiophanate-methyl

and

in

lettuce

disulfoton

and

furathiocarb

-RRB-

The

degradation

seems

to

be

related

to

the

change

of

solvent

as

is

apparent

from

comparison

of

GC

MS

and

LC

MS

MS

validation

data

for

dichlofluanide

tolylfluanide

and

disulfoton

Fortunately

for

many

of

these

the

degradation

products

formed

are

also

part

of

the

residue

definition

and

are

included

in

the

method

Indeed

elevated

recovery

was

observed

for

the

degradation

products

when

determined

in

the

same

validation

set

as

the

parent

compound

In

the

analysis

therefore

degradation

is

not

necessarily

a

problem

because

the

results

-LRB-

expressed

as

defined

in

the

residue

definition

-RRB-

have

to

be

summed

In

routine

analytical

quality

control

-LRB-

see

below

-RRB-

the

data

were

evaluated

this

way

Analytical

quality-control

data

from

routine

LC

MS

MS

analysis

In

the

same

way

as

for

GC

MS

analysis

the

initial

validation

data

are

continually

being

supplemented

by

performance

data

generated

as

part

of

analytical

quality

control

during

routine

analysis

of

samples

With

each

set

of

analytical

samples

at

least

one

was

fortified

with

the

full

quantitative

suite

-LRB-

i.e.

pesticides

and

degradation

products

-RRB-

at

the

mg

kg

level

A

compilation

was

made

from

all

the

data

generated

over

a

period

of

months

which

included

data

for

more

than

one

hundred

vegetable

and

fruit

matrices

A

limited

number

of

dry

matrices

-LRB-

flour

milk

powder

-RRB-

were

also

included

in

the

set

The

data

were

evaluated

for

one

transition

for

each

pesticide

using

the

and

injection

of

mg

equivalent

of

matrix

-LRB-

μL

of

a

g

mL

extract

-RRB-

Examples

of

typical

extracted

ion

chromatograms

are

shown

in

Fig.

Fig.

extracted

ion

chromatograms

obtained

by

LC

MS

MS

analysis

of

vegetable

and

fruit

extracts

-LRB-

calibration

standard

in

mango

matrix

pg

μL

corresponding

to

mg

kg

-RRB-

For

all

fortified

samples

the

matrix

effect

was

also

established

by

analyzing

the

corresponding

matrix-matched

standard

at

the

same

level

as

in

the

extract

of

the

fortified

sample

against

a

solvent

standard

Suppression

-LRB-

or

enhancement

-RRB-

of

up

to

was

regarded

as

acceptable

for

quantification

The

number

of

compounds

for

which

the

response

in

matrix

relative

to

that

in

solvent

was

between

and

is

given

in

Table

for

each

matrix

Whereas

for

beetroot

asparagus

and

kangkung

little

or

no

matrix

effects

exceeding

were

observed

such

effects

were

much

more

common

for

herbs

and

citrus

fruits

Table

of

matrix

effects

and

recoverya

within

or

outside

the

EU

criterion

-LSB-

-RSB-

after

LC

MS

MS

analysis

NMatrix

effectsn

Recovery

Pesticides

PesticidesRel

resp

suppr

enhanc.Calc

using

solvent

stdCalc

using

matrix-matched

Corn

syrup

-LRB-

-RRB-

syrup

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

leaves

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

crinkley

-LRB-

-RRB-

-LRB-

-RRB-

leaves

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

leaves

-LRB-

-RRB-

leaves

-LRB-

-RRB-

-LRB-

feed

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

leaves

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

black

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

-LRB-

-RRB-

herb

QC

of

total

at

mg

kg

-LRB-

higher

for

seven

pesticides

-RRB-

The

pesticides

included

are

listed

in

Table

is

the

total

number

of

individual

compounds

-LRB-

pesticides

and

metabolites

-RRB-

added

to

the

matrixn

is

the

total

number

of

pesticides

added

to

the

matrix

Compounds

belonging

to

the

same

residue

definition

counted

as

one

In

contrast

with

GC

for

which

matrix

effects

are

mainly

caused

by

shielding

of

active

sites

in

the

inlet

and

were

to

some

extent

predictable

-LRB-

in

relation

to

the

matrix

load

injected

and

the

lability

and/or

polarity

of

analyte

-RRB-

in

LC

MS

MS

matrix

effects

are

much

less

predictable

Although

they

do

depend

on

the

amount

of

matrix

introduced

into

the

system

and

also

tend

to

be

more

abundant

in

complex

-LRB-

aromatic

-RRB-

matrices

it

can

not

be

readily

predicted

for

which

pesticides

the

effects

occur

For

this

reason

use

of

one

matrix-matched

standard

as

representative

calibrant

for

a

whole

range

of

commodities

which

worked

reasonably

well

in

GC

MS

analysis

was

not

feasible

in

LC

MS

MS

analysis

Consequently

critical

evaluation

of

the

matrix

effect

was

required

if

unacceptable

suppression

occurred

there

was

no

alternative

to

quantification

by

use

of

the

appropriate

matrix-matched

calibration

standard

or

when

not

available

by

standard

addition

Recovery

of

the

pesticides

from

the

fortified

samples

was

calculated

relative

to

that

from

a

solvent

standard

and

a

matrix-matched

standard

and

tested

against

the

criterion

for

evaluation

of

routine

analytical

quality-control

samples

-LSB-

-RSB-

A

total

of

more

than

recovery

values

were

evaluated

Without

matrix-matched

calibration

acceptable

recovery

was

obtained

for

of

the

pesticides

Deviating

recoveries

were

usually

too

low

mainly

because

of

ion

suppression

as

is

apparent

from

the

results

obtained

from

determination

of

recovery

using

matrix-matched

calibration

for

which

met

the

criterion

Concentrating

on

performance

at

the

pesticide

level

-LRB-

Table

-RRB-

enables

easy

identification

of

troublesome

pesticides

All

compounds

belonging

to

the

same

residue

definition

were

summed

-LRB-

according

to

the

residue

definition

-RRB-

and

counted

as

one

thereby

compensating

for

possible

conversion

during

sample

pretreatment

This

way

the

low

recovery

of

dichlofluanide

and

the

corresponding

high

recovery

of

DMSA

were

acceptable

for

most

matrices

because

recovery

for

the

sum

met

the

criterion

Pesticides

for

which

multi-matrix

analysis

under

fixed

conditions

was

less

favorable

included

asulam

bifenazate

cyromazine

furathiocarb

propamocarb

pymetrozine

and

thiocyclam

-LRB-

low

recovery

because

of

varying

extraction

efficiency

and/or

degradation

-RRB-

As

already

observed

during

validation

the

method

was

also

less

suitable

for

cycloxydim

profoxydim

sethoxydim

and

tepraloxydim

For

these

compounds

recovery

was

too

high

possibly

because

of

degradation

in

the

calibration

standard

used

for

preparation

of

the

matrix-matched

standards

Table

over

all

matrices

-LRB-

LC

MS

MS

-RRB-

ACQ

samples

Recov

Recov

Recov

Average

recov

-LRB-

-RRB-

aRSD

-LRB-

-RRB-

and

RSD

for

recoveries

within

rangeMatrix-matched

calibration

mg

kg

for

most

pesticides/metabolitesBold

indicates

pesticides

including

metabolites

that

are

part

of

residue

definition

if

appropriate

Averaging

acceptable

recoveries

reveals

there

is

some

bias

because

the

values

are

mostly

approximately

-LRB-

in

contrast

with

the

GC

MS

data

for

which

the

average

was

approximately

-RRB-

It

was

noted

that

for

dry

crops

relatively

low

recovery

-LRB-

typically

between

-RRB-

was

obtained

for

all

pesticides

The

cause

is

not

clear

This

bias

can

also

be

seen

in

tables

in

other

papers

-LRB-

barley

-LSB-

-RSB-

soya

grain

-LSB-

-RSB-

-RRB-

Independent

evaluation

of

method

performance

by

proficiency

testing

From

results

obtained

over

the

years

from

participation

in

proficiency

tests

an

additional

and

independent

verification

of

method

performance

could

be

made

The

data

are

summarized

in

Table

and

clearly

show

that

good

quantitative

data

were

consistently

obtained

from

both

GC

MS

and

LC

MS

MS

with

method

performance

good

-LRB-

Z-score

-RRB-

times

doubtful

-LRB-

Z

-RRB-

three

times

and

never

poor

It

also

shows

that

the

calibration

approach

-LRB-

one-point

calibration

tomato-matrix

standard

for

GC

and

matrix-matched

standard

for

LC

-RRB-

is

fit-for-purpose

Table

from

the

analysis

of

Fapas

-LRB-

series

-RRB-

proficiency

test

samples

-LRB-

-RRB-

SamplePesticideMRMSpike

level

added

-LRB-

μg

kg

-RRB-

Inter-lab

result

-LRB-

μg

kg

-RRB-

TNO

result

-LRB-

μg

kg

-RRB-

Z-score

TNO

AppleFenpropathrinGC

TetradifonGC

TriazofosGC

VinchlozolinGC

CucumberIprodioneGC

MethomylLC

MS

ThiabendazoleLC

MS

PearCarbendazimLC

MS

Dodinenot

in

ImazalilLC

MS

MelonChlorprophamGC

ChlorpyrifosGC

DimethoateLC

MS

PirimicarbLC

MS

TomatoAzoxystrobinGC

MSNot

BifenthrinGC

MSNot

BuprofezinGC

MSNot

MSNot

ProcymidoneGC

MSNot

GrapefruitDiazinonGC

MSNot

HeptenophosGC

MSNot

MalathionGC

MSNot

MethidathionGC

MSNot

LettuceBromopropylateGC

DimethoateLC

MS

OxadixylGC

PenconazoleGC

AppleChlorfenvinphosGC

ChlorpyrifosGC

MethamidophosLC

MS

MonocrotophosLC

MS

OmethoateLC

MS

TrifluralinGC

BasilKresoxim-methylGC

ProcymidoneGC

PropyzamideGC

VinclozolinGC

TomatoAzoxystrobinGC

BupirimateGC

QuinalphosGC

LemonDiazinonGC

FenitrothionGC

MetalaxylGC

LettuceCarbendazimLC

MS

lambda

CyhalothrinGC

MetalaxylGC

AppleDiphenylamineGC

PropargiteGC

TetradifonGC

Sweet

pepperDichloranGC

MecarbamGC

MethamidophosLC

MS

Conclusions

The

ethyl

acetate-based

multi-residue

method

has

been

modified

to

meet

today

's

demands

in

respect

of

ease

and

speed

of

sample

preparation

For

GC

MS

analysis

combined

GCB/PSA

dispersive

clean-up

enables

prolonged

injection

of

vegetable

and

fruit

extracts

-LRB-

mg

matrix

equivalent

-RRB-

without

maintenance

Retention

time

shifts

induced

by

some

matrices

compared

with

the

calibration

standard

are

reduced

by

the

clean-up

procedure

Interferences

are

partially

removed

resulting

in

cleaner

-LRB-

extracted

ion

-RRB-

chromatograms

The

last

two

benefits

aid

correct

automatic

peak

assignment

and

confirmation

Addition

of

toluene

during

dispersive

clean-up

prevented

unacceptable

adsorption

of

planar

pesticides

by

GCB

yet

removal

of

chlorophyll

and

other

pigments

was

still

sufficient

Use

of

liners

with

a

sintered

porous

glass

bed

on

the

inner

wall

makes

μL

injection

non-critical

and

robust

In

GC

use

of

a

universal

matrix-matched

standard

-LRB-

tomato

-RRB-

is

a

feasible

means

of

compensating

for

the

matrix

effects

of

many

other

vegetable

and

fruit

samples

For

most

pesticides

LOQs

of

mg

kg

can

be

obtained

by

GC

MS

with

full-scan

acquisition

The

same

initial

extract

-LRB-

i.e.

without

any

clean-up

-RRB-

can

be

used

for

LC

MS

MS

analysis

after

changing

the

solvent

to

methanol

water

LC

MS

MS

is

relatively

tolerant

of

injection

of

matrix

despite

the

absence

of

any

clean-up

no

special

maintenance

was

required

Matrix-induced

suppression

was

observed

for

several

matrices

however

especially

herbs

and

citrus

and

must

be

evaluated

for

all

pesticide-matrix

combinations

In

contrast

with

the

GC

based

method

use

of

a

universal

matrix-matched

standard

to

compensate

for

matrix

effects

was

not

feasible

Evaluation

of

analytical

quality

control

data

for

pesticides

and

degradation

products

in

over

one

hundred

matrices

showed

that

at

the

mg

kg

level

recovery

was

acceptable

for

-LRB-

LC

MS

MS

-RRB-

and

-LRB-

GC

MS

-RRB-

of

all

pesticide

matrix

combinations

It

also

revealed

that

the

method

fails

in

the

other

because

of

lack

of

specificity

-LRB-

mostly

in

GC

MS

-RRB-

or

because

of

poor

extraction

efficiency

and/or

degradation

-LRB-

LC

MS

MS

-RRB-

The

only

way

to

identify

these

limitations

is

by

thorough

and

continual

evaluation

of

the

quantitative

performance

of

the

method

for

all

the

pesticides

-LRB-

rather

then

a

representative

subset

-RRB-

in

all

the

matrices

foods/beverages
gc-ms
lc-ms/ms
multi-residue analysis