Page 33 - Volume 2,Issue 2, July 2009

LC-MS/MS multiresidue method in peaches

79

ulizing gas, with pressures set at 18 and 55

psi, respectively. For the operation in MS/MS

mode, Argon 99.999% (Messer) was used as

collision gas at a pressure of 1.5 mTorr. The

multiple reaction monitoring experiments

were conducted with a dwell time of 100

msec, except for aldicarb, hexaconazole and

hexythiazox for which the dwell time was

set at 300, 200 and 300 msec, respectively.

For instrument control, data acquisition and

processing, Varian MS Workstation software

version 6.8 was used.

5. Sample extraction

The extraction was based on the acetone–

dichloromethane–petroleum ether multiresi-

due method developed by the Dutch Minis-

try of Public Health, Welfare and Sport (16).

The use of acetone (a water-miscible sol-

vent) provides the extraction of the analytes

without extracting the larger lipid volumes

that later cause emulsions and interferenc-

es. With the addition of immiscible to water

solvents, such as dichloromethane and pe-

troleum ether, the analytes under study are

extracted in the organic phase without ad-

ditional co-extractives that were previously

in the acetone phase.

The sample processing, according to

the applied method, was the following (7,

16): an aliquot of 15±0.15 g of the previous-

ly homogenized sample was weighted into

a 250 ml PTFE centrifuge bottle (Nalgene,

Rochester, NY) and 30 ml of acetone were

added and stirred for 1 min in an ultra-tur-

rax homogenizer at 15,000 rpm. Then, 30

ml of dichloromethane and 30 ml of petro-

leum ether were added and the mixture was

stirred again for 1 min. The sample was cen-

trifuged at 4,000 rpm for 5 min, 15 ml of the

supernatant liquid were transferred in a flat-

bottom long necked flask and evaporated

to dryness in a water bath at 65–70

o

C and 3

ml of methanol/water (30:70 v/v) were add-

ed as follows: 0.9 ml of methanol were add-

ed in the flask, the extract was placed in an

ultrasonic bath for 30 sec and transferred

into a 3 ml volumetric flask; the flat-bottom

long necked flask was rinsed twice with 1 ml

of water, the resulting solution was trans-

ferred into the 3 ml volumetric flask and wa-

ter was added to the mark. The final extract

was placed in an ultrasonic bath for 30 sec

and was then transferred into a vial with a

Teflon stopper. The final extract was filtered

through a disposable PTFE syringe filter, 0.45

μm in diameter and 5 μl of the final extract

were injected in the chromatographic sys-

tem. The MS/MS acquisition method includ-

ed the monitoring of one transition, that of

the quantification, for each compound, as

shown in Table 2.

6. Confirmation

The confirmation of positive findings

was achieved by a second injection under

the chromatographic conditions described

above. Individual MS/MS acquisition meth-

ods, including both transitions of Table 2 for

each separate analyte, were developed and

used for confirmation purposes. Then, an in-

dependent confirmation injection was per-

formed for every positive sample. Confirma-

tion included retention times of standard

and sample acceptable tolerances and the

ion ratios of qualifier and quantifier ions, as

they are referred in the Document No SAN-

CO/2007/3131 (5). The retention time of the

analyte in the sample extract must match

that of the matrix-matched calibration stan-

dard with a tolerance of ±2.5%.

Results and discussion

1. Pesticide and parameter selection

The ionization of the 56 pesticides and

metabolites in positive electrospray ion

mode was studied. Table 2 shows the pre-

cursor ions used for data acquisition, the

transitions used for quantification and qual-

ification, the capillary voltage and collision

cell energy for each transition, segments

(time windows) and the retention times of

the analytes in which the transitions were

scanned. Pesticides and metabolites were

ionized in the form of [M+H]

+

.

Tandem mass spectrometry (MS/MS)

provides a powerful confirmatory tool for

pesticide residue analysis because it dis-