Glyphosate, Glufosinate and AMPA analysis in bottled and

Glyphosate, Glufosinate and AMPA analysis in bottled and

Glyphosate, Glufosinate and AMPA analysis in bottled and surface water
using Time De-Coupled Chromatography
Claude R Mallet, Pete Claise
Separation Technologies, Workflow Integration Group, Waters Corporation, 34 Maple St., Milford, MA 01757-3696 corresponding author: claude_mallet@waters.com
Abstract
Time De-Coupled Chromatography 2D mode
The popularity of glyphosate as a weed killer for crop protection is mainly due to its effectiveness against broadleaf
plants. This herbicide acts as an enzyme inhibitor and is
only active on growing plants. After absorption in soil,
glyphosate is rapidly converted to its main metabolite
(aminomethylphosphonic acid or AMPA). Due to its strong
retention characteristic, it is not typically found in ground
water, but can potentially contaminate surface waters
through soil erosion and run-offs. Glyphosate’s toxicity is
classified at level III by the EPA; as such, the herbicide is
regulated to protect public health. Due to its ionic structure, poor volatility and low molecule mass, the analysis of
glyphosate in water at low ppb is very difficult.
Glyphosate
Trap
Glufosinate
100
MRM of 6 Channels ES+
404 > 182 (glufosinate)
1.84e7
6.56
500 L injection @ 1 ppb
2:1 aqueous/ACN
%
1000 µL loop
BEH C8 – Trap aqueous no additive
BEH C18 – Aqueous /ACN mobile phase (0.5 % FA) 5 min gradient
LC D1
Detector
0
1.00
2.00
3.00
4.00
5.00
6.00
100 uL
1 ppb
Acetone
ACD on @ 5%
organic 1267
6: MRM of 1 Channel ES+
TIC (salbutamol)
1.60e5
3.89
3.93
%
%
Aqueous Std
ACD on
0
5.05
5.10
5.15
5.20
5.25
5.19
100
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
5.00
5.10
5.20
5.30
5.20
100
7.00
7.50
8.00
8.50
5.34
100
0
5.30
5.35
8: MRM of 1 Channel ES+
TIC (tripolidine)
1.97e6
6.50
organic 1223
Acetonitrile Std
ACD on
D
2.00
5.40
5.50
8: MRM of 1 Channel ES+
TIC (tripolidine)
8.08e5
9.00
9.50
8: MRM of 1 Channel ES+
TIC (enrofloxacin)
1.44e5
Sign of peak tailing
Dilution too weak
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
organic 1267
5.85
9.00
9.50
11: MRM of 1 Channel ES+
TIC (bromhexine)
4.23e6
%
%
100
%
Acetonitrile Std
ACD off
Time
5.05
5.10
5.15
5.20
5.25
5.30
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
0
5.35
5.10
5.20
5.30
5.40
0.50
5.50
Acetone
organic 1252
6: MRM of 1 Channel ES+
TIC (salbutamol)
5.66e5
4.07
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
100 uL
1 ppb
Acetone
ACD on @ 2%
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
organic 2136
100
9.00
9.50
Time
0
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
organic 1208
5.32
100
9.00
9.50
8: MRM of 1 Channel ES+
TIC (enrofloxacin)
2.49e4
100
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
0
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
organic 1252
5.85
100
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
8.36
100
12.00
8: MRM of 1 Channel ES+
TIC (enrofloxacin)
1.35e5
0
100
196
125
150
175
0
1.00
392
225
250
275
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
organic 2136
8.80
100
12.00
11: MRM of 1 Channel ES+
TIC (bromhexine)
4.27e6
0
100
%
0
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
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5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
9.00
9.50
Time
350
375
400
425
450
475
500
179
119
125
137
208
183
209
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
11.00
12.00
Time
165
150
175
200
225
360
250
275
300
325
350
405
375
400
425
450
475
500
[AMPA-FMOC + H]+
112
125
550
575
m/z
600
150
175
525
550
575
m/z
600
1: Daughters of 334ES+
1.59e7
179
0
100
525
404.0  136.0
404.0  182.0
334.0  111.8
334.0  156.0
156
0
1.00
325
1: Daughters of 404ES+
1.59e7
%
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
300
[Glufosinate-FMOC + H]+
136
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
9.00
9.50
11: MRM of 1 Channel ES+
TIC (bromhexine)
9.69e5
200
182
100
100
0.50
4.00
5.00
6.00
5.50
MRM of 6 Channels ES+
404 > 182 (glufosinate)
100
3.86e6
Calibration Range
1 ppb – 200 ppb
1/X - r2 0.993
6.00
6.50
6.23
12.8 ppb
7.00
7.50
8.00
MRM of 6 Channels ES+
392 > 214 (glyphosate)
4.81e5
Calibration Range
1 ppb – 200 ppb
1/X - r2 0.996
9.00
7.00
8.00
9.00
10.00
MRM of 6 Channels ES+
334 > 156 (ampa)
7.04e6
10.00
Time
6.57
Tap Water
< 1 ppb
1: MRM of 6 Channels ES+
404 > 182 (glufosinate)
1.21e5
0
5.00
5.50
6.00
6.50
6.29
100
< 1 ppb
7.00
7.50
8.00
1: MRM of 6 Channels ES+
392 > 214 (glyphosate)
2.55e4
200
225
250
275
300
325
350
375
400
425
450
7.93
5.50
6.00
6.50
6.39
18.4 ppb
0
5.00
475
500
525
550
575
m/z
600
5.50
7.00
7.50
8.00
MRM of 6 Channels ES+
334 > 156 (ampa)
1.18e6
Calibration Range
1 ppb – 200 ppb
1/X - r2 0.991
6.00
6.50
7.00
7.50
Time
8.00
0
5.00
100
5.50
6.00
6.50
6.42
< 1 ppb
7.00
7.50
8.00
1: MRM of 6 Channels ES+
334 > 156 (ampa)
6.40e4
0
5.00
5.50
6.00
6.50
7.00
7.50
Time
8.00
Conclusions
392.0  170.0
392.0  214.0
%
2.00
organic 2092
8.66 8.94 9.06 9.10
[Glyphosate-FMOC + H]+
214
124
0
1.00
1: Daughters of 392ES+
7.38e6
170
%
%
Low response
Potential adsorption issue
179
%
1.00
MRM Transitions
Fmoc
BEH C8 - NA
BEH C18 2.1 x 50
MeOH + FA
BEH C8 - NA
BEH C18 2.1 x 50
MeOH + FA
0.50
3.00
Surface Water
21.8 ppb
0
5.00
Final Sample Composition: 2:1 Aqueous/ACN (33 % organic solvent)
6: MRM of 1 Channel ES+
TIC (salbutamol)
5.51e5
6.97
%
%
100
Step 5: Add 500 L FMOC-Cl (1.5 mg/mL)
100
0
Time
5.00
aqueous
100 uL
1 ppb
Water
ACD on @ 5%
100
Step 7: Add 100 L HCl pH 1 – quench reaction - stable conditions
C
0
8.00
Step 6: 30 min at 60 C
Aqueous Std
ACD off
A
2.00
6.55
0
5.00
Step 4: Add 500 L Borate Buffer 5 % pH 10 – neutral conditions
0
1.00
1.00
Step 2a: Option – Add 50 L Internal Standard
Step 3: Add 5 mL ACN – optimized derivatization conditions
Oasis HLB- NA
BEH C18 2.1 x 50
ACN + FA
0.50
7.00
%
Step 2: Add 200 L HCl pH 1 – binding effect reduction
1.50
%
B
1.00
100
4.02
0
0.50
6.00
Detection Limit & Linearity
Step 1a: Option – filter or clean up with anion/cation exchanger SPE
BEH C8 - NA
BEH C18 2.1 x 50
MeOH + FA
5.00
Step 1: 10 mL water sample in 20 mL vial
Sign of peak spliting
Dilution too weak
%
100
4.00
6.39
Derivatization Method
Acetone
3.00
%
8: MRM of 1 Channel ES+
TIC (tripolidine)
3.31e6
Sample Chromatography
100
0
5.20
100
2.00
10.00
MRM of 6 Channels ES+
392 > 214 (glyphosate)
4.00e6
%
8: MRM of 1 Channel ES+
TIC (tripolidine)
1.65e6
5.21
100
Elution
9.00
%
AT column Dilutor
1.00
%
Load
Pump
0
%
Pump
Herbicide (weed killer) sold as “Round Up”
- Market since 1970
- Top seller in USA
- 185 million pounds crop protection
- 8 million pounds home garden
- EPA MCL set at 700 ppb in water
8.00
Sample Enrichment
%
Pump
%
AMPA - Glyphosate Metabolite
Injector
7.00
6.24
100
%
The analysis of glyphosate in drinking water usually requires elaborate sample extraction and clean up protocol to
minimize matrix effects. One major drawback is the high
amount of manual labor required to produce a clean extract
leading to increased operator induced error. Since glyphosate is highly soluble in water, an ion exchange chromatography is usually used for enrichment purpose. Another
drawback is the insolubility of FMOC in water, thus the derivatization agent is usually dissolved in acetonitrile. This
brings a level of complexity regarding the ratio of wateracetonitrile for optimum yield without causing a salting-out
effect. Also, with high acetonitrile level present in the sample, potential breakthrough effect can be expected during
analysis. With a time de-coupled solution, the analysis of
glyphosate and AMPA in water was performed using 3 automated and programmable sequences. The first part of the
analysis performed the conversion of glyphosate and AMPA
with the FMOC derivative. This sequence used a high pH
buffer (0.5 mL Borate buffer ph 9) to neutralize the amine
functionality, followed by the addition of the FMOC derivative (0.5 mL). The reaction gave an optimum yield after 60
minutes at 60 °C temperature. The second part of the
analysis used an automated sequence for quenching the reaction with an automated addition of 100 uL of hydrochloric
acid. The final part of the analysis used an AT-column dilution function for high volume injection of the water:acetonitrile (66:33) sample. Up to 0.5 mL of derivatized
sample was loaded onto a trap column. Several trapping
sorbents were evaluated for trapping efficiencies. A weak
reversed phase sorbent gave the best performance. The
trapped analytes were analyzed on a high resolution column
using a back flush gradient. With this automated solution,
glyphosate and AMPA were detected at low ppb level in tap
and surface water samples.
Results
-
Trace level detection (ppb)
Automated sample preparation
Rapid method development
High level of reproducibility and robustness
Better precision & accuracy (< 5 %)
Special thanks to Tom Flug (CTC analytics) for the XChange
Syringe upgrade kit
©2014 Waters Corporation v1