Poster Reprint

ASMS 2018
MP-264

Analysis of glyphosate, AMPA

and 7 other polar pesticides in
food and water by SAX
chromatography with MS/MS
detection

Jerry Zweigenbaum, Tarun Anumol, and

Derick Lucas
Agilent, Wilmington, Delaware, USA
 Introduction Experimental

Glyphosate is the active ingredient in the popular Sample Preparation

herbicide Roundup® and is used throughout the
world. Glyphosate is a broad-spectrum systemic At least 2 g of sample is weighed to a 15 mL VWR
herbicide. It is an organophosphorus compound, plastic centrifuge tube. The sample is then extracted
specifically a phosphonate. Recently, its safe use has following the QuPPE method where high water
come into question. This has heightened the demand content samples (<80%) are extracted with an equal
for a sensitive method at the low ppb level for food amount of acidified methanol and lower water content
and even lower levels for environmental water samples have added water to equal the amount of
analysis. Reliable sample preparation and analysis is acidified methanol.1
needed to routinely analyze glyphosate and its major
metabolite, aminomethylphosphonic acid (AMPA).
However, glyphosate and its metabolite’s high polarity Instrumental Conditions
can be quite challenging not only extraction from food
but in its analysis. Of concern is the affinity of these
compounds to stainless steel and other surfaces
making system-to-system reproducibility difficult.
This shows the performance of the extraction of
glyphosate, its major metabolite and seven other
metabolites and polar pesticides.

Polar Pesticides Analyzed

O O
HO HO
NH P OH P
OH
H2N
O OH Figure 1. Agilent 1260 Infinity Bio-inert Quaternary LC
Glyphosate m/z 168→63/150 Aminomethylphosphonic acid (AMPA) with 6495A Triple Quadrupole LC/MS System.
m/z 110 →63/79
O NH2
O
HPLC Condition
H3C P OH
PH Column: Shodex HILIC VT50 2D
OH HO O CH3
O
Glufosinate m/z 180 →134/63 Fosetyl (Al+3 salt) m/z 109 →79/63 Injection volumn 10 uL
Mobile Phase Initial

O
A:50% UHP H2O, B: 10% NH4HCO3, C: 40% Acetonitrile
Gradient
O
HN CH3 Time A B C
HO HO P 6.50 min 40.0 % 50.0 % 10.0 %
OH
P OH OH 10.00 min 15.0 % 85.0 % 0.0 %
O
CH3 O 18.00 min 0.0 % 100.0 % 0.0 %
Flow 0.2 mL/min
N-acetylglufosinate m/z 222 →136/59 2-hydroxyethyphosphonic acid
(HEPA) m/z 125 →95/79 MS Conditions
ESI negative
Source Parameters
O Value (-)
O
Cl HO O Gas Temp (°C) 140
P
HO P Gas Flow (l/min) 18
OH 30
H3C OH Nebulizer (psi)
SheathGasHeater 375
Ethephon m/z 145 →107 3-methylphosphinicoproprionic acid (MPPA)
SheathGasFlow 12
m/z 143 → 107 m/z 151 →133/63
Capillary (V) 3000
HO N VCharging 500
N
Ion Funnel Parameters
OH Neg High Pressure RF 110
Neg Low Pressure RF 60
maleic hydrazide m/z 111 →82/42

2
 Results and Discussion

Chromatographic Separation of standards in water and in QuPPE extraction solvent

Figure 2 shows the separation and sensitivity obtained with a standard in water. There is excellent separation of AMPA
and fosetyl. However, the same separation is altered by dissolving the standard in the extraction solvent. Figure 3
shows AMPA and fosetyl much closer together while MPPA and ethephon actually switch in elution.

x10 4 MPPA: -ESI MRM Frag=380.0V [email protected] (151.0 -> 63.2) std25ppb_40ACN_1.d
1 2 2
1.6

1.55

1.5 20.007

1.45

Ethephon
1.4

1.35

1.3
1.25

1.2

1.15
Baseline separation of each with good peak shape
1.1

1.05

0.95

0.9

0.85

0.8
0.75

0.7
9.742
0.65

0.6 Fosetyl N-acetyl

0.55

0.5 Maleic Glyphosate MPPA

0.45

0.4

0.35
hydrazide 18.245
20.441

0.3
0.25
AMPA HEPA
19.524
0.2 12.351

Glufosinate
0.15

0.1 11.132 19.048

10.243
0.05
9.765 10.535 11.541 19.282
0
4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
Counts vs. Acquisition Time (min)

Figure 2. LC/MS/MS chromatogram of nine polar pesticides and metabolites of 10 ppb standard in water .

MPPA

Fosetyl

Glyphosate
Maleic N-acetyl
Glufosinate
Ethephon
AMPA HEPA

Figure 3. LC/MS/MS chromatogram of nine polar pesticides and metabolites of 10 ppb standard in 50:50
water:acidified methanol (QuPPE extraction solvent).

3
 Results and Discussion

Need to separation fosetyl and AMPA

Figure 4 shows the MRM chromatograms for fosetyl and AMAP. A distinct peak appears in both AMPA MRMs that
exactly aligns with fosetyl. Figure 5 shows the fragmentation for the product ions of both compounds.2 Because the
fragments contain no carbon, the C13 isotope for fosetyl is producing the same transitions as AMPA and thus the two
must be chromatographically separated.
x10 3 AMPA: -ESI MRM Frag=380.0V [email protected] (110.0 -> 63.1) STD25.d
2 10.267 2
2.5
O O
2
HO
1.5

1
AMPA 110 → 63 P - P
-
O
0.5 9.204
10.722 11.272 H2N HO O CH3
0

m/z 110
x10 3 AMPA: -ESI MRM Frag=380.0V [email protected] (110.0 -> 79.2) STD25.d
2.25 2
2
10.278 2
m/z 109
AMPA m/z 110 → m/z 79
1.75
1.5
1.25
1
0.75 O
0.5
9.215
0.25 10.839
11.096
0

x10 4 Fosetyl: -ESI MRM Frag=380.0V [email protected] (109.0 -> 63.1) STD25.d
2.25 2 2
P - m/z 79
2
9.205

O O
Fosetyl m/z 109→ m/z 63
1.75
1.5
1.25
1
0.75
0.5
0.25
0

x10 3
2 2
Fosetyl: -ESI MRM Frag=380.0V [email protected] (109.0 -> 79.0) STD25.d
2
O
m/z 63
9.205

Fosetyl m/z 109 → m/z 79

1.8
1.6
1.4 -
1.2
1
P
0.8
0.6
O
0.4
0.2 14.392
0
3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 10.5 11 11.5 12 12.5 13 13.5 14 14.5 15 15.5 16 16.5 17 17.5 18 18.5 19 19.5 20 20.5 21 21.5
Counts vs. Acquisition Time (min)

Figure 4. Transitions for AMPA and fosetyl. Figure 5. Fragmentation of AMPA and fosetyl.

Conclusions

Method for direct analysis of polar pesticides provides

good recovery and detection for high water content low
protein foods
• Not shown but recovery for strawberry, orange, bell
pepper, spinach, and peach is greater than 80%. Corn
is about 50%.

Figure 6. Calibration curve for glyphosate from 1 to 100 • Using all PEEK from injection loop to spray provides
ppb repeatable results even though sample and standard
solvent impact chromatography, QuPPE extraction
provides good reproducibility among different foods
• LOQs for high water content foods is below 10 ppb
Glyphosate AMPA
10 ppb Spike 10 ppb Spike References

1. Anastassiades, M., et al. "Quick method for the analysis of numerous highly polar
pesticides in foods of plant origin via LC-MS/MS involving simultaneous extraction
with methanol (QuPPe-Method)." EU Reference Laboratory for pesticides requiring
Single Residue Methods (EURL-SRM) (2016).

2. Goodwin, Lee, et al. "Tandem mass spectrometric analysis of glyphosate,

glufosinate, aminomethylphosphonic acid and methylphosphinicopropionic acid."
Rapid communications in mass spectrometry 17.9 (2003): 963-969.

Figure 7. Quantitation transitions for glyphosate and

AMPA extracted from strawberry at 10 ppb

For Research Use Only. Not for use in diagnostic procedures.

This information is subject to change without notice.

© Agilent Technologies, Inc. 2018