1

A Validated Method for the Analysis of 142 Pesticide Residues Using Atmospheric Pressure GC Coupled with Tandem Quadrupole Mass Spectrometry L. Cherta,1 T. Portolés,1 J. Beltran,1 E. Pitarch,1 J.G.J. Mol,2 F. Hernández,1 D. Roberts,3 and R. Rao3

1 Research Institute for Pesticides and Water, University Jaume I, Castellón, Spain2 RIKILT Institute of Food Safety, Wageningen University and Research Centre, Wageningen, The Netherlands 3 Waters Corporation, Manchester, UK

IN T RO DU C T IO N

Pesticides are widely used in agricultural activity across the globe and residues of

these pesticides in food products destined for human consumption can be a major

food safety risk. Pesticide residues are high on the list of consumer concerns and

consequently laboratories are tasked to screen samples for as many pesticides as

possible in a single analysis within an appropriate timescale. Most countries have

clearly defined regulations governing pesticide residues. Legislation imposes

Maximum Residue Limits (MRLs) for pesticide residues in food commodities

requiring analytical techniques that are sensitive, accurate, and robust.

Multi-residue analysis is challenging due to the low limits of detection required

to achieve MRL compliance for a diverse range of pesticides in a wide range of

food commodities. There are currently in excess of 1000 pesticides known to

be in use, and laboratories are under increasing pressure to increase the scope

of the analytical methods for routine monitoring purposes.

Various technologies are used to meet this challenge, the most common being

Liquid Chromatography (LC) and Gas Chromatography (GC) coupled to tandem

quadrupole mass spectrometry. Implementation of these techniques allows the

laboratory to cover a range of compounds with varying chemistries as required by

legislation. In GC/MS/MS the traditional ionization mode used is Electron Impact

(EI). This is a relatively “hard” ionization method and results in a high degree

of analyte fragmentation, which compromises the selectivity and sensitivity of

the MS/MS measurement. Atmospheric Pressure Gas Chromatography (APGC) is

a “soft” ionization technique resulting in less fragmentation and subsequently

increasing the sensitivity and selectivity of MS/MS methods.1 The APGC source

is readily interchangeable with the electrospray (ESI) source enabling a single

platform to be used for the analysis of both GC- and LC-amenable pesticides.

In this application note we describe the development and validation of a

multi-class method for the routine determination of 142 pesticide residues in

various fruit and vegetable matrices. A more detailed description of the method

and of the results achieved can be found in the referenced paper.2

WAT E R S SO LU T IO NS

DisQuE™ QuEChERS, AOAC Method

Sample Preparation Kit, Pouches

Atmospheric Pressure Gas

Chromatography (APGC)

Xevo® TQ-S

TargetLynx Application Manager

K E Y W O R D S

Pesticides, QuEChERS extracts,

Atmospheric Pressure Gas

Chromatography, MS/MS

A P P L I C AT IO N B E N E F I T S ■■ Routine quantification of 142 pesticide

residues in QuEChERS extracts of fruit and

vegetables using an ionization mode that

provides enhanced sensitivity

■■ Analysis of LC and GC compounds

on a single MS platform

■■ Fast and easy processing of data using

TargetLynx™ Application Manager

2A Validated Method for the Analysis of 142 Pesticide Residues

E X P E R IM E N TA L

GC conditions

GC system: 7890A GC

Column: DB5-MS

30 m x 0.25 mm

x 0.25 µm film

Carrier gas: He, 2 mL/min

Temp gradient: Initial 70 °C for 1 min

15 °C/min to 150 °C,

10 °C/min to 300 °C,

hold 3 min

Total run time: 30 min

Injector temp.: 280 °C

Injection type: Pulsed splitless

Pulse time: 1 min

Pulse pressure: 240 kPa

Injection volume: 1 µL

Make-up gas: N2 at 300 mL/min

Transfer line temp.: 310 °C

MS conditions

MS system: Xevo TQ-S

Mode: API +

Corona : 1.8 µA

Cone gas: 170 L/Hr

Aux gas: 250 L/Hr

Source temp.: 150 °C

A vial of water was added to the source to promote

protonation. Data were processed using TargetLynx

Application Manager. TargetLynx, an option with

Waters MassLynx® Software that quickly generates

results from acquired LC/MS and GC/MS data,

permitting accurate quantification and review

of results, including evaluation of data quality

and analyte confirmation.

Sample preparation

Fortified orange, carrot, and tomato samples were used to evaluate the linearity,

recovery, precision, selectivity, limits of detection (LODs), and limits of

quantification (LOQs). Locally purchased apple, lettuce, and courgette (zucchini)

samples were additionally analyzed to test the method applicability. Sample

preparation was carried out using the DisQuE QuEChERS, AOAC Method Sample

Preparation Kit, Pouches, Part No. 176002922, that is designed specifically

for the QuEChERS procedure described in the AOAC official method 2007.01.2

QuEChERS is a simple sample preparation technique suitable for multi-residue

pesticide analysis in a diverse variety of food and agricultural products. Following

extraction, 50 μL of the extract (acetonitrile) was transferred into a 2-mL vial and

diluted with 300 μL of hexane and 150 μL of acetone. For accurate quantification

eight matrix-matched standards were prepared2 to cover the range 0.1 to

100 ng/mL (equivalent to 1 to 1000 μg/kg in the sample). These were prepared

for each sample matrix as follows: after the cleanup step, 50 μL of the acetonitrile

extract obtained from a blank sample were mixed with 250 μL of hexane, 150 μL

of acetone, and 50 μL of the pesticide standard solution in hexane at adequate

concentration to obtain a calibration range of 0.1 to 100 ng/mL (corresponding

to 1 to 1000 μg/kg in sample). All samples were analyzed using the Waters®

Xevo TQ-S with the APGC source and a 7890A GC.

3A Validated Method for the Analysis of 142 Pesticide Residues

R E SU LT S A N D D IS C U S S IO N

The first step in the method development process was the optimization of three MRM’s for each target pesticide.

Due to the soft ionization characteristics of APGC, the protonated molecule or molecular ion, base peak of

the spectrum, could be chosen as the precursor ion in most cases. Examples of APGC mass spectra compared

to EI are shown in Figure 1. The goal was to select three sensitive MRM transitions for each compound so

that confident identification and quantification of the 142 analytes could be achieved. The high sensitivity

achievable using APGC-MS/MS allowed a 10-fold dilution of the QuEChERS extract, thereby strongly reducing

the matrix load onto the column.

Figure 1. Comparison of spectra generated by APGC (top) and EI (bottom) showing enhanced intensity of the molecular ion in the APGC spectra. EI spectra exhibit extensive fragmentation.

Fluvalinate

m/z50 75 100 125 150 175 200 225 250 275 300 325 350 375 400

%

0

100

m/z50 75 100 125 150 175 200 225 250 275 300 325 350 375 400

%

0

100

252.1

286.1

287.1

162.1

144.1127.0117.155.0

72.1 115.173.1

238.1

186.1

173.1 199.1219.5240.1

Metolachlor

m/z50 100 150 200 250 300 350 400 450 500 550 600 650

%

0

100

m/z50 100 150 200 250 300 350 400 450 500 550 600 650

%

0

100

208.1

505.1

506.1

182.1

250.1

252.1

[M+H]+ [M+H]+

503.1 284.1

Missing molecular ion Missing molecular ion

APGC APGC

EI EI

4A Validated Method for the Analysis of 142 Pesticide Residues

Linearity was studied in the range 0.1 to 100 ng/mL using pure solvent standard solutions and injecting in

triplicate. The regression coefficients (R2) were greater than 0.99 for all compounds over the range tested. To

ensure accurate quantification, and to account for any enhancement/suppression due to matrix effects, matrix

matched calibration standards were used. The LODs obtained for all compounds were low and are summarized

in Figure 2. The majority of these ranged between 0.01 and 1 μg/kg in the three matrices studied with only

a few higher than 1 μg/kg. Figure 3 shows four examples, with signal-to-noise (S/N) ratios calculated for the

lowest matrix-matched standard in the various matrices.

120%

100%

80%

60%

40%

20%

0%

0.01

-0.1

0.1-

1

1.0-

10

>10

0.01

-0.1

0.1-

1

1.0-

10

>10

0.01

-0.1

0.1-

1

1.0-

10

>10

Orange Tomato Carrot

Freq

uenc

y

LODs (µg/kg)

LODs (µg/kg)

Frequency

Accumulated %

70

60

50

40

30

20

10

0

cm TOM 0.1 ppb

Time15.00 16.00 17.00

%

0

100

124: MRM of 3 Channels AP+ 378.7 > 342.7 (Endrin)

2.08e4S/N:PtP=24.60

15.8615.64 16.30

cm ZN 0.1 ppb

Time5.00 5.50 6.00 6.50 7.00 7.50 8.00

%

0

100

32: MRM of 3 Channels AP+ 220.8 > 108.9 (Dichlorvos)

6.43e5S/N:PtP=30.78

cm lechuga 0.1 ppb

Time14.00 15.00 16.00

%

0

100

130: MRM of 3 Channels AP+ 404.6 > 322.7 (alpha endosulfan)

1.34e415.05

cm NAR 0.1 ppb

Time12.00 13.00 14.00

%

0

100

91: MRM of 3 Channels AP+ 321.8 > 124.8 (Chlorpyriphos methyl)

3.19e5

S/N:PtP=196.14

Figure 2. Limits of Detection (LODs) for all 142 pesticides across the three matrices. The bar chart (left axis) shows the number of pesticides with an LOD at a particular concentration range. The line graph (right axis) shows the cumulative percentage of pesticides across the concentration ranges.

Figure 3. Examples of sensitivity in matrix for four pesticides: endrin, alpha endosulfan, chlorpyriphos methyl, and dichlorvos at 0.1 ppb.

5A Validated Method for the Analysis of 142 Pesticide Residues

The selectivity of the MRM transitions chosen from the APGC spectra was observed to be excellent as the

GC/MS/MS chromatograms did not show interferences for any of the pesticides investigated in this study.

An important consideration for the method was to satisfy regulatory criteria regarding ion ratios. For

pesticides in the EU the current guideline (SANCO/12571/2013) indicates that the ion ratio in samples

should be within 30% of that of the reference value. It was found that in general the ion ratios for the different

concentrations of the standards was very consistent, with RSDs <10% in most cases, even when an abundance

of the qualifier ion was much lower compared to the quantifier ion .

The developed method was applied to the analysis of real samples with three types of locally purchased

orange, tomato, and carrot samples analyzed and expanded to include three types of apple, lettuce, and

courgette. A combined total of 43 different pesticides were identified in all of the samples, most at levels

well below 0.01 mg/kg and below the EU MRL levels. Figure 4 shows examples of the positive findings in

various matrices with confirmatory MRM transitions.

MR NARANJA 2

Time10.00 10.50 11.00 11.50 12.00 12.50 13.00

%

0

100

10.00 10.50 11.00 11.50 12.00 12.50 13.00

%

0

100

10.00 10.50 11.00 11.50 12.00 12.50 13.00

%

0

100

38: MRM of 3 Channels AP+ 229.9 > 124.9 (Dimethoate)

1.17e5Area

11.243306

38: MRM of 3 Channels AP+ 229.9 > 198.8 (Dimethoate)

1.07e5Area

11.233010

38: MRM of 3 Channels AP+ 229.9 > 170.9 (Dimethoate)

3.43e4Area

11.231049

q/Q(St) = 0.832 q/Q (S) = 0.910

9% ✔

q/Q(St) = 0.635q/Q (S) = 0.687

8% ✔

q/Q(St) = 0.259q/Q (S) = 0.317

22 ✔

q/Q(St) = 0.119q/Q (S) = 0.141

18% ✔

Dimethoate <LOQ

9 :;

;

:

<=

;

MR calabacin 4

Time11.50 12.00 12.50 13.00 13.50 14.00 14.50

%

0

100

11.50 12.00 12.50 13.00 13.50 14.00 14.50

%

0

100

11.50 12.00 12.50 13.00 13.50 14.00 14.50

%

0

100

69: MRM of 3 Channels AP+ 280 > 220 (Metalaxyl)

6.93e5Area

13.0723380

69: MRM of 3 Channels AP+ 280 > 160 (Metalaxyl)

4.20e5Area

13.0816057

69: MRM of 3 Channels AP+ 280 > 192 (Metalaxyl)

1.00e5Area

13.083308

Metalaxyl <LOQ

E

0

E

E

E

Figure 4. Examples of ion ratios of confirmatory MRM transitions for dimethoate (left) and metalaxyl (right) at low level, below the LOQ. These compounds are challenging with traditional EI methods.

The sensitivity and performance of the Xevo TQ-S with APGC currently exceeds existing regulations related

to pesticide residue analysis. This additional sensitivity enables samples to be diluted and therefore reducing

matrix interferences and minimizing the amount injected on column. This is turn has major benefits for system

cleanliness and reduces instrument maintenance requirements.

Waters Corporation 34 Maple Street Milford, MA 01757 U.S.A. T: 1 508 478 2000 F: 1 508 872 1990 www.waters.com

References

1. T Portolés, L Cherta, J Beltran, A Gledhill, F Hernández. Enhancing MRM Experiments in GC/MS/MS Using APGC. Waters Application Note No. 720004772en, August, 2013.

2. L Cherta, T Portolés, J Beltran, E Pitarch, J G J Mol, F Hernández. Application of Gas Chromatography (Triple Quarupole) Mass Spectrometry with Atmospheric Pressure Chemical Ionization for the Determination of Multi-Class Pesticides in Fruit and Vegetables. J Chrom A. Nov 1; 1314: 224-40 (2013).

CO N C LU S IO NS■■ A method based on QuEChERS extraction/clean up and

APGC-MS/MS analysis for the determination of 142 pesticides

has been presented.

■■ Excellent sensitivity and selectivity was achieved by the APGC

source and the soft ionization allowed the quasi-molecular ion

to be used as the precursor ion.

■■ LODs generally ranged from 0.01 to 1 μg/kg in orange,

tomato, and carrot matrices. This increased sensitivity

allowed for the dilution of sample extracts and thereby

reducing matrix effects and GC maintenance.

■■ The validation results demonstrate that the method

is applicable to the quantitative routine residue analysis

of 142 GC-amenable pesticides.

■■ This method was successfully applied to the analysis of real

samples and 43 different pesticides were identified and

confirmed using ion ratios. None of the detected pesticides

exceeded their EU MRL.

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