AB SCIEX Detection of Underivatized Glyphosate and

Detection of Underivatized Glyphosate and Similar Polar Pesticides in Food of Plant Origin by LC-MS/MS Stephen Lock1 and Hermann Unterluggauer2 1 AB SCIEX Warrington (UK) 2 Austrian Agency for Health and Food Safety (AGES GmbH), Innsbruck (Austria)

Introduction Glyphosate is a common broad-spectrum systemic herbicide used widely to kill weeds especially annual broadleaf weeds and grasses known to compete with crops. Usually Glyphosate, as it is very polar, undergoes FMOC derivatization by reacting the native glyphosate with fluorenylmethyloxycarbonyl chloride (FMOC-Cl) before analysis. This derivatization step complicates the analysis and there is a growing need for a method which can detect not only Glyphosate (and it´s major metabolite AMPA) but also Glufosinate and similar highly polar compounds, in their underivatized states. In addition a simplified approach to sample extraction using either QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) or a solvent extraction would be beneficial. Here we present initial data using a new LC-MS/MS method which combines the use of a HILIC type chromatography on an AB SCIEX LC/MS/MS system to detect underivatized glyphosate and other polar pesticides which have been spiked in different food matrices. A simple solvent extraction has been used and initial data will be presented to show how applicable this approach is to food analysis.

OH

HO

HO

OH

OH NH

H2N

P

O

P O

O Glyphosate

OH

AMPA

H3C

OH

O

OH O

P Cl

O

NH2 Gluf osinate

Ethephon

Sample Preparation For linearity and sensitivity tests calibration standards were prepared in 50/50 methanol/water from concentrations 1 to 500 ng/mL. Matrix samples were prepared by spiking the polar pesticides into 50% aqueous methanol extracts of onion, wheat, rice and grapes prepared as per the QuPPe (Quick Polar Pesticides) Method from the EU Reference Laboratories for 1 Residues of Pesticides. These extracts were then diluted 5x with 50% aqueous methanol before injection to reduce possible matrix effects. LC

HO

P

Experimental

The LC system used for this analysis was a ShimadzuXR LC system consisting of two Shimadzu LC20AD pumps, SIL 20AC autosampler, and a CTO20A column oven. The analyses were performed at 50ºC on an Obelisc N phase HPLC column. An injection volume of 50 µL was used using the gradient separation as shown in Table 1 where mobile phase A was acidified 85% acetonitrile/water (85/15) containing ammonium acetate and mobile phase B was acidified water containing ammonium acetate. The gradient used is shown in Table 1.

p1

Table 1. Gradient conditions used for separation Step

Time

Flow (mL/min)

A (%)

B (%)

XIC of -MRM (11 pairs): 168.000/63.000 Da ID: Glyphosate 3 ...

Max. 1.8e4 cps.

XIC of -MRM (11 pairs): 180.000/62.900 Da ID: Glufosinate 1 ...

6.82

0

2

1.2

100

0

1.6e4

2.2e4

Glyphosate

100

0

1.8e4

2

3.6

1.2

0

100

1.6e4 1.2e4 Intensity, cps

1.2

Intensity, cps

3

Glufosinate

2.0e4

1.4e4

1

Max. 2.3e4 cps.

6.01

1.8e4

1.0e4 8000.0

1.4e4 1.2e4 1.0e4 8000.0

6000.0

6000.0

3

8

1.2

0

100

4000.0 4000.0 2000.0

4 5

8

1.5

8.2

0

1.5

0.0

100

100

2000.0 4.0

6.5 7.0 7.5 Time, min XIC of -MRM (11 pairs): 109.923/62.900 Da ID: AMPA 2 from ...

100

1.2

0

100

0

5.5

6.0

8.0

8.5

9.0

Max. 4.2e4 cps.

4.29

14

1.2

100

0

7.0

7.5

8.0

8.5

9.0

Max. 5.7e4 cps.

7.43 5.5e4

AMPA

Ethephon

5.0e4 4.5e4

3.0e4

4.0e4

2.5e4 2.0e4 1.5e4

8

0.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 Time, min XIC of -MRM (11 pairs): 143.000/106.900 Da ID: Ethephon 2 f...

9.5

Intensity, cps

13.8

1.5

Intensity, cps

7

13.5

5.0

4.2e4 4.0e4

0

3.5e4

6

4.5

3.5e4 3.0e4 2.5e4 2.0e4 1.5e4

1.0e4

1.0e4 5000.0 0.0

5000.0 1.5

2.0

2.5

3.0

3.5

4.0 4.5 Time, min

5.0

5.5

6.0

6.5

7.0

7.5

0.0

4.5

5.0

5.5

6.0

6.5

7.0 7.5 Time, min

8.0

8.5

9.0

9.5

10.0

Figure 1. Injection of a 10 ng/mL standard

MS/MS ®

The analyses were performed on an AB SCIEX QTRAP 5500 LC/MS/MS system using the Turbo V™ source operated in electrospray ionization and negative polarity with an IonSpray (IS) voltage of -4500 V. The curtain gas was set at 35 psi, nebulizer gas (Gas 1) set at 60 psi, drying gas (Gas 2) set at 70 psi, CAD gas set at medium, and the temperature set at 650ºC. The MRM transitions used as well as the retention times for the compounds are shown in Table 2. Each MRM was monitored with a dwell time of 50 ms.

Table 2. LC-MS/MS parameters for the analyzed compounds Compound AMPA

Ethephon

Glufosinate

Glyphosate

Retention Q1 (amu) Q3 (amu) time (min) 4.3

7.4

6.0

6.8

DP (V)

CE (V)

110

79

-60

-24

110

63

-60

-26

143

79

-45

-26

143

107

-45

-12

180

63

-60

-66

180

95

-60

-24

168

79

-110

-54

168

63

-110

-32

Results and Discussion Figure 1 shows a typical chromatogram obtained from an injection of a 10 ng/mL standard of all studied pesticides. The monitoring of two transitions per compound also allows compound identification using the MRM ratio.

Figure 2 and Table 3 shows typical calibration lines obtained the target pesticides. The response over the range tested, 1 to 500 ng/mL, was linear with a 1/x weighting. All accuracy values were well in between 80 and 120%. The sensitivity of the different pesticides is also shown in Table 3. All pesticides were easily identified and quantified at the maximum residue limits (MRL) set by EU and CODEX Alimentarius where limits for most fruit and vegetables are 0.1 2, 3 mg/kg. The extra sensitivity also allowed dilution of sample extract to minimize possible matrix effects.

Table 3. Linearity with a 1 /x weighting (1 to 500 ng/mL range) and signal-to-noise (S/N*) from a 1 ng/mL standard injected Compound AMPA

Ethephon

Glufosinate

Glyphosate

MRM transition

Linear fit (r value)

S/N at 1 ng/mL

110/79

0.999

131

110/63

0.999

234

143/79

0.979

58

143/107

0.984

155

180/63

1.000

88

180/95

0.999

47

168/79

0.999

52

168/63

0.999

102

* S/N values were calculated in MultiQuant™ software

p2

XIC of -MRM (11 pairs): 168.000/63.000 Da ID: Glyphosate 3 ...

AMPA

Max. 6.4e4 cps.


Glyphosate

9.00e4

6.0e4 5.5e4

7.00e4

5.0e4

6.59 Intensity, cps

Intensity, cps

Glufosinate

6.5e4

8.00e4

Max. 7.4e4 cps.

5.83

7.4e4 7.0e4

1.00e5

6.00e4 5.00e4 4.00e4

4.5e4 4.0e4 3.5e4 3.0e4 2.5e4

Ethephon

3.00e4

2.0e4 1.5e4

Area

2.00e4

1.0e4 1.00e4 0.00


4.0

4.5

5.0

5.5

6.0

8.0

8.5

9.0

0.0

9.5

6.0 6.5 Time, min XIC of -MRM (11 pairs): 143.000/106.900 Da ID: Ethephon 2 f...

Max. 1.4e5 cps.

4.28

1.4e5

3.5

4.0

4.5

5.0

5.5

7.0

7.5

8.0

8.5

9.0

Max. 1.7e5 cps.

7.21

1.7e5 1.6e5

1.3e5

Glufosinate

7.55

5000.0

AMPA

1.2e5 1.1e5

Ethephon

1.4e5 1.2e5

1.0e5 Intensity, cps

Intensity, cps

9.0e4 8.0e4 7.0e4 6.0e4

1.0e5 8.0e4 6.0e4

5.0e4 4.0e4

Glyphosate

4.0e4

3.0e4 2.0e4

2.0e4

1.0e4 0.0

1.5

2.0

2.5

3.0

3.5

4.0 4.5 Time, min

5.0

5.5

6.0

6.5

7.0

0.0

7.5

4.5

5.0

5.5

6.0

6.5

7.0 7.5 Time, min

8.0

8.5

9.0

9.5

10.0

Figure 3. 100 μg/kg spike into rice extract diluted 5x with acetonitrile Figure 2. Calibration lines (2 MRM transitions each) for analyzed polar pesticides from 1 to 500 ng/mL XIC of -MRM (11 pairs): 168.000/63.000 Da ID: Glyphosate 3 ...

Max. 2.4e4 cps.


3.5e4

The method was then applied to spiked matrices. Figures 3 and 4 show that all the different polar pesticides, spiked at the level of the EU MRL (0.1 mg/kg), can be detected in different matrices.

6.5e4

Glyphosate

Max. 7.1e4 cps.

5.77

7.0e4

Glufosinate

6.0e4 5.5e4

3.0e4

5.0e4 6.35


Intensity, cps

2.5e4 2.0e4 1.5e4

4.0e4 3.5e4 3.0e4 2.5e4 2.0e4

1.0e4

1.5e4 1.0e4

5000.0

5000.0 0.0


The S/N data is also shown in Table 4 for the results of spiking experiments in four different matrices. What can also be seen is that even with a 5x dilution of the food extract matrix effects can be observed as there is a slight shift in retention times and some suppression / enhancement was also observed. For that reason it is recommended to use isotopically labeled standards for quantitation.

4.0

4.5

5.0

5.5

6.0

8.0

8.5

9.0

3.21 3.49 4.24 4.39 4.71 5.11 5.43 6.33 6.54 7.06 0.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Time, min XIC of -MRM (11 pairs): 143.000/106.900 Da ID: Ethephon 2 f...

9.5

Max. 8.5e4 cps.

4.24

8.5e4

7.65 7.77 8.118.61 8.0 8.5 9.0

7.5

Max. 1.2e5 cps.

7.14 1.20e5

8.0e4

AMPA

7.0e4

Ethephon

1.10e5 1.00e5 9.00e4


Intensity, cps

8.00e4 5.0e4 4.0e4 3.0e4

7.00e4 6.00e4 5.00e4 4.00e4 3.00e4

2.0e4

2.00e4 1.0e4 1.00e4 0.0

1.5

2.0

2.5

3.0

3.5

4.0 4.5 Time, min

5.0

5.5

6.0

6.5

7.0

7.5

0.00

4.5

5.0

5.5

6.0

6.5

7.0 7.5 Time, min

8.0

8.5

9.0

9.5

10.0

Figure 4. 100 μg/kg spike into grape extract diluted 5x with acetonitrile

Table 4. Signal-to-noise (S/N*) observed from 0.1 mg/kg spikes into different matrices indicating matrix suppression and enhancement even after sample dilution. AMPA

Ethephon

Glufosinate

Glyphosate

110/79

110/63

143/79

143/107

180/63

180/95

168/79

168/63

Rice

2900

5483

998

998

3058

653

911

3110

Onion

761

1379

281

2514

2310

373

249

1312

Grapes

1187

2344

133

1149

2892

440

534

1799

Wheat

1636

3117

174

1014

3062

557

588

2708

* S/N values were calculated in MultiQuant™ software

p3

Table 5. Recoveries observed from 100 μg/kg spikes into different matrices without the use of any internal standards. This shows the need for internal standards or matrix matched calibration lines to counter matrix effects which lead to recoveries varying with matrix. AMPA

Ethephon

Glufosinate

Glyphosate

Rice

151%

159%

148%

156%

Onion

48%

243%

99%

92%

Grapes

110%

167%

145%

95%

Wheat

106%

213%

155%

155%

Data was processed using MultiQuant™ software version 2.1 with the ‘Multicomponent’ query. Query files are customizable commands to perform custom querying of the result table. The ‘Multicomponent’ query automatically calculates and compares MRM ratios for compound identification and highlights concentrations above a user specified level. An example of the results and peak review after running the query file is shown in Figure 5.

Summary This study has clearly demonstrated that Glyphosate and other polar pesticides can be detected at low levels in their underivatized state using a highly sensitive LC-MS/MS system, ® like the AB SCIEX QTRAP 5500, and separation using a new HILIC type LC column. The detection of these compounds is quick even in the non-optimal acidic mobile phase conditions and is additionally only possible due to the ability of the Turbo V™ source to deal with highly aqueous solvents at high flow rates in excess of 1 mL/min. This means that FMOC derivatization or lengthy ion chromatography is no longer needed. All the compounds were identified and quantified using two MRM transitions at 0.1 mg/kg after 5x dilution of QuPPe extraction. However, matrix effects were observed so in routine analysis it is recommended that matrix matched calibration standards or ideally heavy labeled internal standards are used.

References 1

2

3

http://www.crlpesticides.eu/library/docs/srm/meth_QuPPe.pdf Regulation (EC) ‘concerning the placing of plant protection products on the market’ No 1107/2009 Commission Regulation (EU) ‘regards maximum residue levels’ No 441/2012

Figure 5. Automatic reporting of pesticides using the ‘Multicomponent’ query in MultiQuant™ software: the query calculates MRM ratios and flags samples with MRL violations.

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