Page 32 - Volume 2,Issue 2, July 2009

Anagnostopoulos

et al.

78

boscalid, bromuconazole, buprofezin, car-

baryl, carbendazim, carbofuran, carbofuran

3-hydroxy, clofentezine, cymoxanil, deme-

ton-S-methyl, demeton-S-methyl sulfoxide,

dimethomorph, fenamidone, fenbucon-

azole, fenhexamid, fenoxycarb, fenpyroxi-

mate, fenthion, fosthiazate, hexaconazole,

imidacloprid,

indoxacarb,

iprovalicarb,

mepanipyrim,

methiocarb,

methiocarb

sulfone, methiocarb sulfoxide, methom-

yl, methoxyfenozide, monocrotophos, my-

clobutanil, oxamyl, prochloraz, profenofos,

pyraclostrobin, pyrimethanil, quinoxyfen,

spinosad (A), spiroxamine, tebuconazole,

tebufenozid, tebufenpyrad, tetraconazole,

thiabendazole, thiacloprid, thiamethoxam,

thiodicarb, triadimefon, triadimenol, triflox-

ystrobin and vamidothion.

Acetone, dichloromethane, and petro-

leum ether of pesticide grade analysis were

used for the extraction procedure. Metha-

nol and water, LC-MS grade, were used for

the preparation of stock and working stan-

dard solutions. All solvents were obtained

from Lab Scan (Ireland).

2. Preparation of stock standard solu-

tions

Stock standard solutions at 1000 μg/ml

were prepared in acetone for each of the

56 pesticides and stored at -20

o

C. A sin-

gle composite standard mixture was pre-

pared by combining aliquots of each stock

solution and diluting them with a metha-

nol/water (30:70 v/v) mixture to obtain a fi-

nal concentration of 1 mg/ml. Blank peach

samples were spiked at 0.01, 0.05 and 0.5

mg/kg by adding appropriate volumes of

the composite standard mixture. Working

standard mixture solutions for measure-

ment were prepared in blank peach ex-

tract, previously analysed for the absence of

peaks interfering with the peaks of the an-

alytes. Calibration curves were constructed

from injections of matrix-matched calibra-

tion standards in blank matrix of peach in

methanol/water (30:70 v/v) at eight concen-

trations, within the range of 0.01–0.75 mg/

ml (i.e. 0.01–0.025–0.05–0.075-0.1-0.25-0.5-

0.75 μg/ml) for all pesticides.

For the preparation of the blank extract,

the sample extraction procedure men-

tioned below under paragraph “Sample ex-

traction” was followed. The only difference

was that at the final step the blank extract

was taken in 3 ml of MeOH. An aliquot of 1

ml was evaporated to dryness by a stream of

N

2

and 1 ml of a standard solution of the de-

sired concentration, prepared in methanol/

water (30:70 v/v), was added. Prior to injec-

tion in the chromatographic system, the fi-

nal solution was filtered through a disposa-

ble PTFE syringe filter, 0.45 μm in diameter.

3. Liquid chromatography

The liquid chromatographic (LC) system

used consisted of two Varian Prostar 210

pumps. Chromatographic separation was

achieved using a Polaris C

18

5 μm particle

size, 2 mm x 50 mm analytical column from

Varian, at a flow rate of 250 μl/min with mo-

bile phases consisting of methanol/water

(10:90 v/v) – 1mM ammonium formate (sol-

vent A) and methanol/water (90:10 v/v) –

1mM ammonium formate (solvent B). A gra-

dient programwas used consisting of 90%of

solvent A and 10% of solvent B, ramped lin-

early over the course of 14 minutes to 100%

of solvent B. This composition was held for 6

additional minutes before returning to the

initial condition. The column was re-equili-

brated for 10 min at the initial mobile phase

composition. The total run-time was 30 min.

The injection volume was 5 μl and in order

to avoid carry-over, the autosampler was

purged with a mixture of methanol/water

(50:50 v/v) before sample injection.

4. Mass spectrometry

Detection was achieved using a tri-

ple quadrupole mass spectrometer (Varian

model 1200L) equippedwith an electrospray

ionization interface operating in the positive

mode. Typical source parameters were as fol-

lows: capillary voltage and collision cell en-

ergy varied depending on the precursor ion,

as shown in Table 2, source temperature was

set at 250

o

C and drying gas temperature at

200

o

C. Nitrogen, generated from high purity

generator, was used as drying gas and neb-