©2014 Waters Corporation

INTRODUCTION

Pesticides are widely used in the production of fruit and

vegetables across the globe. Governments, food

producers and food retailers have a duty to ensure they

are not present in final products for consumption. Most

countries have regulations governing pesticide residues in

food. For pesticides in food products, legislation imposes

Maximum Residue Limits (MRLs) which lead to the

requirement for analytical techniques that are sensitive,

selective and reproducible. Multi-residue pesticide analysis

is challenging due to the low limits of detection required

in a diverse range of food commodities. As there are

currently in excess of 1000 pesticides in use, laboratories

are under increasing pressure to broaden the range of

pesticides determined in ever shorter turnaround times.

Therefore, the analytical methods they employ need to

use efficient but low selectivity sample preparation

methods combined with high selectivity and sensitivity

MS/MS methods. Typically, this analysis is carried out

using a dedicated GC-MS/MS system with an EI source.

As shown by Portoles et al1, EI causes extensive

fragmentation of some pesticides leading to poor

sensitivity and selectivity. APGC is a soft ionization

technique which generates high relative and absolute

abundance molecular ions resulting in highly sensitive and

selective MRM transitions. Furthermore, the APGC source

is interchangeable with the LC electrospray source

enabling a single MS instrument to be used for the

analysis of both LC and GC amenable pesticides.

In this study, we demonstrate sensitive, accurate and

repeatable results for the analysis of pesticides in

QuEChERS extracts of strawberry, pear and spinach below

the regulatory limits.

Quantitative Analysis of Pesticides in QuEChERs Extracts Using APGC/MS/MS Douglas Stevens1, Dominic Roberts2 , Ramesh Rao2 1Waters Corporation, 34 Maple Street, Milford, MA 01757, USA. 2Waters Corporation, Altrincham Road, Wilmslow, UK.

METHODS

Strawberry, pear and spinach samples were

homogenized using a domestic food blender. The

samples were then extracted using the QuEChERS

(CEN method 15662 DisQuE #186004831) protocol to

generate blank matrix extract in acetonitrile. A nine

point calibration range from 0 to 50 ng/mL

(equivalent to µg/kg) was prepared by addition of a

mixed pesticide standard in acetonitrile to each

matrix. To test the repeatability at low concentration,

each matrix was fortified with the pesticide mix at 1

µg/kg (1ppb final concentration in aliquot). A

deuterated internal standard, chrysene-d12, was

added to give a fixed concentration of 2 ng/mL to

each vial prior to analysis and was used as an

injection standard to correct for injection volume

variation. All standards were analyzed in triplicate and

the low level spike in each matrix was analyzed ten

times using the Waters® Xevo TQ-S with the APGC

source using the conditions described below.

GC Conditions

MS Conditions

References

1. Portoles, Tania, Laura Cherta, Joaquim Beltran, and Felix Hernandez. "Improved gas chromatography–tandem mass spectrometry determination of pesticide residues making use of

atmospheric pressure chemical ionization." Journal of Chromatography A 1260 (2012): 183-192

2. Young, Michael, Tran, Kim Van, Shia, Jeremy C. “Multi-Residue

Pesticide Analysis in Ginseng Powder”. Waters application note

#720005006EN (2014)

3. Giroud, Barbara, Antoine Vauchez, Emmanuelle Vulliet, Laure Wiest, and Audrey Bulete. "Trace level determination of pyrethroid and neonicotinoid insecticides in beebread using

acetonitrile-based extraction followed by analysis with ultra-high-performance liquid chromatography–tandem mass spectrometry."

Journal of Chromatography A 1316 (2013): 53-61

RESULTS & DISCUSSION

Analysis of 20 GC amenable pesticides, difficult to analyze

in EI due to excessive fragmentation, was performed

using positive ion MRM mode. By varying source

conditions either charge exchange or protonation can be

selected for an APGC analysis. For the analysis of

pesticides, protonation provides more efficient ionization

than charge exchange. Therefore, a vial of water was

added to the source to promote protonation. The MRM

transitions with optimized cone voltages and collision

energies are shown in Table 1. Two transitions were

monitored for each pesticide to increase method

specificity. The high intensity of the precursor/molecular

ion generated by APGC makes it possible to use specific

and sensitive MRM transitions. In contrast, many pesticide

MRM transitions used with EI MS/MS use lower m/z, less

specific fragment ion as the precursor. The inherent

specificity provided by use of the molecular ion as the

precursor in an MRM transition over the use of a fragment

ion results in more confident detection of lower levels of

analytes even in these complex matrices prepared with a

simplified, generic sample preparation technique.

Table 1. Summary of the 20 pesticides analyzed, MRM

conditions and method performance results

Since the sensitivity of this system is well beyond

regulatory requirements, a practical application of this

performance is to dilute samples, thereby, further

reducing matrix effects on chromatography and

minimizing the amount of material injected on

column. This in turn reduces the frequency of column

trimming, extends the useful life of the column and

increases the interval between source cleanings which

is already measured in months. The net effect of all of

these factors is increased up-time and utilization for

the system.

CONCLUSION APGC on Xevo TQ-S is sensitive, accurate and

reproducible for pesticides that are difficult to analyze using conventional EI GC/MS/MS

Soft ionization provided by this technique produces

abundant molecular ions for selective and sensitive MRM transitions

Routine and sensitive multi-residue pesticide

analysis of QuEChERS extracts from fruit and vegetables, using the same workflow used for LC/MS/MS analysis of pesticides, is possible with this system

System can covert between GC and LC operation in

minutes allowing comprehensive analysis of both GC and LC amenable pesticides on a single instrument

Figure 2. Typical matrix matched calibration curve for

endosulfan sulphate in strawberry matrix

Figure 3. Mean calculated concentration of pesticides spiked

at 1 μg/kg in 3 different food matrices (n=10)

Table 2. Mean concentration of each pesticide (n=10) in the

three sample matrices

Each sample type, including matrix matched stan-

dards and replicates, was analyzed on three different

days. Figure 2 shows a typical calibration curve and

residuals plot for endosulfan sulphate generated from

the triplicate injection of the matrix matched calibra-

tion standard in strawberry extract. The response is

linear from 0.05 to 50 ng/mL with a correlation coeffi-

cient R2 of 0.994. All of the residuals are less than

15% demonstrating excellent linearity and repeatabil-

ity. The limits of detection and linearity achieved for

all 20 pesticides are summarized in Table 1. The limits

of detection ranged from 0.01 to 0.5 ng/mL with ex-

cellent linearity (R2 >0.99) for all. This demonstrates

that the method can easily achieve the regulatory

limits and is applicable to routine quantitative analy-

sis.

To assess the accuracy and precision of the method

each sample matrix was spiked at 1 µg/kg (10 times

below the blanket MRL of 10 µg/kg) and ten replicate

injections made. The concentration of each pesticide

was calculated using matrix matched calibration

curves. Table 2 shows the mean calculated concentra-

tions for each pesticide in all three samples matrices.

The accuracy of the method is excellent with all

measured concentrations within 5% of the true con-

centration.

The %RSD for all pesticides is also shown to be very

good at < 5%. This demonstrates that the method is

precise, accurate and reproducible across different

sample matrices analyzed on different days. The sen-

sitivity and overall performance characteristics of

APGC on Xevo TQ-S currently exceeds existing regu-

lations related to pesticide residue analysis.

Figure 1. Photo of UPLC and APGC on Xevo TQ-S

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