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Determination of glyphosate levels in breast milk samples from Germany by LC-MS/MS and GC-MS/MS Angelika Steinborn, Lutz Alder, Britta Michalski, Paul Zomer, Paul Bendig, Sandra Aleson Martinez, Hans G.J. Mol, Thomas Class, and Nathalie Costa-Pinheiro J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05852 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016
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Journal of Agricultural and Food Chemistry
Determination of glyphosate levels in breast milk samples from Germany by LC-MS/MS and GC-MS/MS
Angelika Steinborna*, Lutz Aldera, Britta Michalskia, Paul Zomerb, Paul Bendigc, Sandra Aleson Martinezc, Hans G. J. Molb, Thomas J. Classc, Nathalie Costa Pinheirod a
Federal Institute for Risk Assessment, Department of Pesticides Safety, Max-
Dohrn-Str. 8-10, 10589 Berlin, Germany b
RIKILT Wageningen UR, Natural Toxins and Pesticides, Akkermaalsbos 2, 6708
WB Wageningen, The Netherlands c
PTRL Europe, Helmholtzstr. 22, 89081 Ulm, Germany
d
Governmental Institute of Public Health of Lower Saxony, Roesebeckstr. 4-6, 30449
Hannover, Germany
*Corresponding author. Phone: +4930-18412-4792; e-mail: [email protected]
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Abstract
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This study describes the validation and application of two independent analytical
3
methods for the determination of glyphosate in breast milk. They are based on liquid
4
chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-
5
tandem mass spectrometry (GC-MS/MS), respectively. For LC-MS/MS, sample
6
preparation involved an ultra-filtration followed by chromatography on an anion
7
exchange column. The analysis by GC-MS/MS involved an extraction step, clean-up
8
on a cation exchange column and derivatization with heptafluorobutanol and
9
trifluoroacetic acid anhydride. Both methods were newly developed for breast milk
10
and are able to quantify glyphosate residues at concentrations as low as 1 ng/mL.
11
The methods were applied to quantify glyphosate levels in 114 breast milk samples,
12
which had been collected from August to September of 2015 in Germany. The
13
mothers participated at their own request and thus do not form a representative
14
sample. In none of the investigated samples were glyphosate residues above the
15
limit of detection found.
16 17
Keywords
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Glyphosate, breast milk, residues, LC-MS/MS, GC-MS/MS
19
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Introduction
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Glyphosate (N-(Phosphonomethyl)glycine) is among the most frequently used active
22
ingredients of plant protection products worldwide. It is applied as a non-selective
23
herbicide for pre-emergence weed control as well as for desiccation treatment pre-
24
harvest. The use of glyphosate might lead to residues in food, especially if applied
25
shortly before harvest. In the European Union (EU), maximum residue levels (MRLs)
26
have been established for glyphosate, which are set for most plant and animal
27
commodities at the limit of quantification (LOQ) of 0.1 mg/kg and 0.05 mg/kg,
28
respectively. MRLs are up to 20 mg/kg for barley, oats, sorghum, sunflower seeds
29
and soybeans, and 10 mg/kg for wheat, rye, linseed, rapeseed, mustard seed, cotton
30
seed, lentils, peas and lupins.1 These food items make up an important part of
31
human and animal diets and thus might lead to intake of small amounts of glyphosate
32
by both humans and livestock. Further exposure of humans to glyphosate might be
33
due to direct exposure during and shortly after its application in agriculture (operator,
34
worker, bystander and/or resident exposure).
35
Glyphosate findings in urine have been reported in the literature for farmers and their
36
families as well as for patients in cases of acute intoxication.2-4 Published data
37
indicated that positive findings of glyphosate in human urine are quite common and
38
result from different exposure or intake pathways.5,6
39
In April 2014, a non-peer reviewed report was published, in which glyphosate in
40
breast milk of American mothers was detected in 3 out of 10 samples ranging from
41
76 to 166 ng/mL.7
42
Because of the high media response concerning the potential health risks for breast-
43
fed children, 16 breast milk samples from Germany were analysed for glyphosate in
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June 2015. The unpublished and non-peer reviewed results reported glyphosate
45
levels between 0.2 and 0.4 ng/mL in all 16 samples.8 Page 3 ACS Paragon Plus Environment
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In both surveys, the concentration of glyphosate in milk samples was determined by
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enzyme-linked immunosorbent assay (ELISA). A 96 well microtiter plate competitive
48
assay was used for detection and quantification of glyphosate levels in breast milk of
49
American mothers. The detection limit of the assay was given as 75 µg/L in milk.7
50
Information on suitability of the ELISA for breast milk as well as the documentation of
51
validation results was not provided for the study from Germany.8 The studies both
52
had methodological shortcomings. The analytical results have not been confirmed by
53
an independent method. The number of samples was quite low and details on study
54
participants were not reported.
55
The quantification of glyphosate residues is very challenging because of the highly
56
polar and amphoteric nature of the molecule, the low molecular weight, the high
57
water solubility and the lack of chromophores. For these reasons, glyphosate is one
58
of the few pesticides which are not amenable to the multi-residue methods typically
59
employed in pesticide residue analysis.
60
Several extraction procedures have been reported in the literature involving
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extraction with water, water-methanol or water-acetonitrile mixtures followed by LC-
62
MS/MS measurement.9-11
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Kacynski and Lozowicka12 tested different extraction solvents for their ability to
64
extract glyphosate and its metabolite aminomethyl phosphonic acid (AMPA) from
65
rape seed. The recoveries were between 82% and 93% using water or acidified
66
water. For liquid samples, e.g. beer, wine, tap water, surface water, groundwater,
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direct injection of samples without preliminary extraction steps is also reported.9,13
68
Raina-Fulton14 reviewed analytical methods for residue analysis of glyphosate and
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AMPA. Most of the published liquid chromatographic methods for glyphosate are
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based on derivatization of the molecule followed by reversed phase HPLC separation
71
and mass spectrometric or fluorescence detection. Preferred procedures for Page 4 ACS Paragon Plus Environment
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derivatization depend on the detection method and include reaction with
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fluorenylmethoxycarbonyl (FMOC) or o-phthalaldehyde-2-mercaptoethanol.10,15
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In contrast, the direct chromatographic separation without previous derivatization is
75
possible by using anion-exchange columns or Hydrophilic Interaction Liquid
76
Chromatography (HILIC), since these stationary phases are able to retain polar
77
compounds.11,16-18
78
The EU reference laboratory (EURL) for residues of pesticides, which is responsible
79
for single residue methods (SRM) (CVUA Stuttgart, Germany), developed a method
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for the analysis of highly polar pesticides.19 The residues are extracted by an
81
acidified methanol-water mixture. For glyphosate, different detection modules
82
involving liquid chromatographic separation on an anion exchange column or porous
83
graphitic carbon column are described.
84
Application of liquid chromatographic methods was reported for the quantification of
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glyphosate residues in environmental matrices, food matrices and human urine and
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serum samples.
87
Determination of glyphosate by gas chromatography followed by mass spectrometric
88
detection (GC-MS) requires the derivatization of the phosphorous acid moiety, the
89
carboxyl group and the secondary amine prior to analysis. Two different
90
derivatization approaches are described in the literature involving either
91
trialkysilylation20 or simultaneous acylation and esterification. The latter method was
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tested successfully in five laboratories for corn grain, soya forage and walnut meat.21
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The lowest tested concentration was 0.05 mg/kg.
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Breast milk is a challenging matrix due to its very complex nature. It is an aqueous
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mixture of carbohydrates, proteins and fat. The composition varies individually and
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over the lactation period. Typically, the content of proteins is in the range of 0.8%-
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0.9%. The fat content is in the range of 3%-5% and the carbohydrate content, mainly Page 5 ACS Paragon Plus Environment
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present as lactose, is in the range of 6.9%-7.2%.22 Thus, analytical methods
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developed for watery matrices cannot be directly transferred to breast milk. An
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essential step prior to the analysis of glyphosate in breast milk is the separation of
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the proteins and fat by suitable separation steps.
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Recently, Ehling & Reddy23 published the application of LC-MS/MS method with
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previous derivatization of the glyphosate with FMOC on different nutritional
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ingredients derived from herbicide-tolerant soybean, corn and sugar beet as well as
105
breast milk. The authors reported an LOQ of 5 ng/g for milk samples, which is
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approximately 10 fold higher compared to the reported glyphosate levels of up to 0.4
107
ng/mL in breast milk samples from Germany.8
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The aim of the work was the development and validation of two independent
109
analytical principles for the quantification of glyphosate in breast milk samples. The
110
method should validate for the highest sensitivity as possible. The first method is
111
based on LC-MS/MS without derivatization. The second method is based on
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derivatization with trifluoroacetic acid anhydride (TFAA) and heptafluorobutanol
113
(HFB) followed by GC-MS/MS determination of derivatives. The two newly developed
114
methodologies were employed to analyze 114 breast milk samples collected from
115
German breast-feeding women for residues of glyphosate. The results of these
116
analyses are reported.
117 118
Material and Methods
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Collection of breast milk samples
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Breast milk samples were collected in August and September 2015 by the
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Governmental Institute of Public Health of Lower Saxony (Niedersächsisches
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Landesgesundheitsamt, Hannover, Germany) and by the Bavarian Authority for
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Health and Food Safety (Bayerisches Landesamt für Gesundheit und Page 6 ACS Paragon Plus Environment
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Lebensmittelsicherheit, Erlangen, Germany). Since 1999, mothers from Lower
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Saxony can send in their breast milk for analysis of selected pesticides (e.g.
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organochlorine pesticides).24 In the framework of this program, additional samples
127
were collected for a definite period of time for this study. Breast milk samples from
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Bavaria were collected on a voluntary basis for the analysis of glyphosate. All
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participants signed a declaration of consent concerning the use of their samples for
130
further scientific purposes. Participating mothers had not been selected by random
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sampling. Moreover, there are no restrictions for participating in the monitoring
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program (e.g. relating to age, point of sampling during lactation period, etc.). The milk
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samples for this study were collected and stored in polypropylene tubes, which
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remained frozen during storage and shipment. In total 114 milk samples were
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analyzed. The participants completed a self-administered questionnaire. Information
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on sample collection, biometric data and self-reported pesticide exposure of the
137
participants is given in Table 1.
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The questionnaire also asked for the place of residence and the jobs practiced in the
139
last 10 years. 38 participants declared the use of chemical insecticides, herbicides or
140
wood preservatives. At least one participant has worked in a residue analytical
141
laboratory and used pesticide standards regularly.
142
Twenty of the 114 breast milk samples were divided each into two subsamples to
143
allow the parallel analysis by LC-MS/MS and GC-MS/MS.
144 145
Fortification of breast milk samples used for performance tests
146
For performance tests a homogenous sample of breast milk was prepared and
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spiked with different amounts of a glyphosate standard solution (LGC Standards,
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Wesel, Germany) having a concentration of 10 µg/mL in water.
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28 stored breast milk samples from a previous study of the Governmental Institute of
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Public Health of Lower Saxony were pooled. Using this pooled sample, four aliquots
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of 100 mL were fortified with glyphosate resulting in concentrations of 0.5 ng/mL, 1
152
ng/mL, 3 ng/mL, and 5 ng/mL. Moreover, an additional aliquot of the pooled sample
153
served as control sample (blank sample). All five ‘performance’ samples were divided
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into two subsamples and analyzed in parallel with both methods. These samples
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served as independent quality control. It has to be noted that analysts were not
156
informed about this additional quality test. The labelling of these ‘performance’
157
samples did not differ from other samples of this study.
158 159
LC-MS/MS analysis
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Chemicals and Apparatus
161
Reference compound and internal standard (13C215N labelled glyphosate) were
162
purchased from LGC Standards. Methanol was obtained from Actu-All Chemicals
163
(Oss, The Netherlands), acetic acid and citric acid from Merck (Darmstadt, Germany)
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and dimethylamine 40% solution from Sigma-Aldrich (Zwijndrecht, The Netherlands).
165
The water used was purified by a Milli-Q system from Merck Millipore (Tullagreen,
166
Ireland).
167
The 30 kDa molecular weight cut-off filter used for sample preparation (Amicon Ultra
168
4 Centrifugal filter, 30000 NMWL) was purchased from Merck Millipore, the LC
169
column (Dionex Ionpac AS 11 (2x250 mm) and the AG-11 guard column (2x50 mm)
170
from Thermo Fischer Scientific (Breda, The Netherlands). For centrifugation, a Z-513
171
centrifuge from Hermle Labortechnik (Wehingen, Germany) was used. The
172
syringeless filter devices Mini-Uniprep (PTFE filter, 0.45 µm) from Whatman, (GE
173
Healthcare, Eindhoven, The Netherlands) were used as LC vials. The LC-MS/MS
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system consisted of a Nexera UHPLC system from Shimadzu (Kyoto, Japan) and a
175
5500 Q-Trap system from Sciex (Concord, ON, Canada).
176 177
Sample preparation and measurement
178
For extraction of glyphosate the use of acidified methanol/water has been
179
described.19 However, in contrast to published results for plant materials, repeated
180
experiments using this extraction showed no or insufficient findings of both labeled
181
and native glyphosate in milk samples. Spiking of an extract with glyphosate did
182
produce a signal, so matrix effects could be eliminated as the source of the problem.
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An alternative approach involving removal of fat by centrifugation and proteins by
184
ultrafiltration in one step through centrifugal filtration using a molecular weight cut-off
185
filter (30 kDa) was found to be suitable. For the samples from this study the
186
procedure was as follows: to 3 mL of sample, 30 µL of internal standard solution
187
containing 1000 ng/mL 13C215N glyphosate was added to obtain a level of 10 ng/mL.
188
After mixing, the sample was transferred to the top part of the cut-off filter tube. The
189
filter was centrifuged at 5000 g (corresponding to 3500 rpm) for 20 min. 500 µL of the
190
filtrate was then transferred to the LC filter vial, the solution was filtered and the vial
191
was used for measurement. After this procedure, one mL of final extract contained
192
the glyphosate residue of one mL breast milk.
193
The LC-MS/MS measurement was based on a method developed by the EURL –
194
SRM. 19 In brief: 25 µL of standard solution or filtrate were injected onto an anion
195
exchange LC column. Glyphosate was eluted from the column using a gradient of (A)
196
water and (B) water containing 1 mM citric acid and brought to a pH of 11 by addition
197
of dimethyl amine solution. Gradient elution was performed: 100% A from 0 to 2 min;
198
linear to 25% B in 5.5 min; then linear to 50% B in 2.5 min; this was held for 4 min;
199
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the next injection. The total run time (including injection) was 22.5 min. A flow rate of
201
0.4 mL/min was used. A column temperature of 40 °C was maintained while the
202
temperature of the samples in the autosampler was 12 °C. For detection, the 5500
203
Qtrap system was used in triple-quad mode. The MS/MS transitions and the
204
transition specific parameters that were used are provided in Table 2.
205
All three transitions were measured using a declustering potential of -75 V, an
206
entrance potential of -10 V and a dwell time of 50 ms. The Turbospray source was
207
used in negative electrospray mode using the following parameters: Curtain gas 20
208
arbitrary units; Collision gas Medium; IonSpray –4000 V; Temperature 400 °C;
209
IonSpray gas 1: 40 and IonSpray gas 2: 50 arbitrary units.
210
Calculations were performed using the ratio of the peak areas of the quantifier
211
transition of glyphosate and the internal standard.
212 213
GC-MS/MS analysis
214
Chemicals and Apparatus
215
Reference compound and internal standard (13C215N labelled glyphosate) were
216
purchased from Dr. Ehrenstorfer (Augsburg, Germany). Methanol HPLC grade, ethyl
217
acetate and dichloromethane were purchased from LGC Standards (Wesel,
218
Germany). Acetic acid, glacial 100% was purchased form Merck (Darmstadt,
219
Germany). Anion exchange resin (Dowex 50WX8 hydrogen form, 200-400 mesh),
220
citral (95%), 2,2,3,3,4,4,4-heptafluoro-1-butanol (98%), trifluoroacetic acid anhydride
221
99%, water (Chromasolv® for HPLC) and hydrochloric acid 37% and 10 N were
222
purchased from Sigma-Aldrich (Seelze, Germany). The solution for cation exchange
223
clean-up (CAX solution) was prepared by mixing of 800 mL HPLC grade water, 13.5
224
mL 10 N HCl solution and 200 mL methanol.
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The derivatization reagent was prepared by mixing 1 volume fraction 2,2,3,3,4,4,4-
226
heptafluoro-1-butanol and 1 volume fractions trifluoroacetic acid anhydride. This
227
solution was prepared fresh on a daily basis.
228
The columns for anion exchange were empty Poly-Prep columns (Bio Rad
229
Laboratories Inc., Hercules, CA, USA). Vortex mixer REAX from Heidolph
230
Instruments (Schwabach, Germany) was used. Heating block Reacti-Therm III #TS-
231
18824 heating module, evaporator Reacti-Vap III #TS-18826 evaporation unit and
232
vacuum concentrator Express SpeedVac concentrator SC250 from Thermo Fisher
233
Scientific (San Jose, CA, USA) were used. A HS 601 D flatbed shaker was obtained
234
from IKA (Staufen, Germany). A Rotanta 460 centrifuge from Hettich (Tuttlingen,
235
Germany) was used. The 0.45 µm Nylon filter units (Chromafil AO-45/15 MS 15 mm)
236
were purchased from Macherey-Nagel (Düren, Germany).
237
The GC-MS/MS system consisted of a Thermo Trace GC Ultra equipped with TriPlus
238
liquid Autosampler, split/splitless injector and MS detector TSQ Quantum with triple
239
quadrupole (Thermo Fisher Scientific). The GC column Optima 5HT, 30 m length,
240
0.25 mm internal diameter and 0.25 μm film thickness was purchased from
241
Macherey-Nagel.
242 243
Sample preparation and measurement
244
The extraction procedure was based on the method by Alferness and Iwata25 and
245
was adapted to reach a lower limit of quantification.
246
A 2 mL milk sample was extracted with 3.75 mL of 0.6% acetic acid for 10 min on a
247
flatbed shaker with 200 rpm. Taking into account a water content of 87% for breast
248
milk26, the obtained volume of aqueous phase containing glyphosate residues is 5.5
249
mL. Then the resulting mixture was centrifuged for 5 min at 3220 g (4000 rpm). A 2
250
mL aliquot of the supernatant liquid was removed and transferred in a 15 mL PP Page 11 ACS Paragon Plus Environment
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tube. 2 mL dichloromethane were added. The sample was shaken for 2 min and
252
centrifuged for 5 min at 3220 g (4000 rpm). A 1 mL aliquot of the supernatant liquid
253
was filtered using a 0.45 µm Nylon filter unit.
254
A cation exchange clean-up was performed using disposable Bio-Rad Poly-Prep
255
columns filled with 1.72 g (equivalent to 2 mL filling volume) of AG 50W-X8 resin (H+
256
form). Before use, the columns were washed with 10 mL of water (Chromasolv® for
257
HPLC). A 0.55 mL aliquot of the filtered extract (corresponding to 0.2 mL breast milk)
258
and 0.100 mL of internal standard (20 ng/mL) were added to the cation exchange
259
column and were eluted until the liquid level reached the top of the resin. Co-
260
extractives were eluted by adding 2.0 mL of CAX solution. Both eluates were
261
discarded. Glyphosate residues were eluted with 12.5 mL of the CAX solution. All
262
elutions were performed using gravity. The eluate was collected in a 15 mL PP tube,
263
and evaporated to dryness using a vacuum concentrator at 60 °C and 60 mbar. The
264
residues were dissolved in 1.0 mL of the CAX solution. It is expected that extraction
265
and clean-up in acidic solvents allows the decomplexation of all cationic glyphosate
266
complexes.27
267
A 1.5 mL aliquot of the derivatization reagent was added to 2 mL vials which were
268
sealed and placed in the heating block. The block was cooled to a temperature of -20
269
°C before proceeding and after adding sample extract. A 0.05 mL aliquot of the
270
redissolved eluate (corresponding to 0.01 mL breast milk) was drawn into the
271
disposable pipet tip and then dispensed under the surface of the chilled reagent.
272
After 5 min, the derivatization of analyte is started by heating the reaction vial to 92-
273
97 °C for 1 h. After allowing vials to cool, the excess of derivatization reagent was
274
removed by evaporation to dryness. The derivatization step and the structure of the
275
derivative were described by Alferness and Iwata.25 Briefly, the carboxylic and
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phosphonic acid group were esterified to the corresponding heptafluorobutyl ester
277
and the amine function was derivatized to the trifluoroacetyl derivative.
278
The residues were redissolved in 0.2 mL of ethyl acetate containing 0.2 mL/L citral
279
and later concentrated to a final volume of 20 µL. The addition of citral to the injection
280
solvent was made to reduce adsorption of the analytes in the inlet and the GC
281
column. Thus, the peak shape and the sensitivity of the method was improved.25
282
Following this procedure, one mL of final extract contained the glyphosate residue of
283
0.5 mL breast milk.
284
These final extracts were analyzed by gas chromatography with tandem mass
285
spectrometric detection (GC-MS/MS). 4.0 µL of the extracts were injected splitless.
286
The injector temperature was 280 °C. Oven temperature program was held at 80 °C
287
for 1.5 min, ramped then with 10 °C/min to 180 °C, ramped with 30 °C/min to 300 °C
288
and was held for 2.8 min. Helium was used as carrier gas with a constant flow rate of
289
1 mL/min. The expected retention time for the glyphosate derivative was 9.1 min. The
290
temperature of the ion source was 280 °C. Electron impact (EI) energy was 70 eV
291
and emission current was 50 µA. The MS/MS transitions and the transition specific
292
parameters are provided in Table 2.
293
Calculations were performed using the ratio of the peak areas of the quantifier
294
transition of glyphosate derivative and the internal standard derivative. Calibration
295
solutions were prepared by volumetric dilution of a glyphosate stock solution in a
296
solution containing 20 ng/mL internal standard. The dilutions were made in CAX
297
solution. 0.05 mL aliquots of these calibration solutions were derivatized as described
298
for the breast milk extracts. Concentration of the derivatized calibration solutions
299
ranged from 0.01 to 10 ng/mL. The concentration of the internal standard in the final
300
extract was always 5 ng/mL.
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Results and Discussion
303
For method development the advantages and disadvantages of different extraction
304
procedures have to be weighed in order to achieve the best method performance.
305
For evaluation of the methods the requirements of the EU guidance document for
306
quality control and validation procedure 28 were considered. The guidance applies to
307
laboratories involved in the official control of pesticide residues in food and feed in
308
the EU. Briefly, a quantitative analytical method has to be validated with respect to
309
sensitivity/linearity, specificity, trueness (bias), precision, and robustness. Matrix
310
effects should be assessed. The average recovery (trueness) of a minimum of five
311
spiked samples per fortification level should be in a range of 70-120%. The
312
repeatability (relative standard deviation) should be < 20% for each analyte. The
313
LOQ is established as the lowest fortification level with an acceptable mean recovery
314
and an acceptable relative standard deviation.
315
In our work, the method development is clearly focussed on reaching the lowest
316
quantification limit in breast milk with still sufficient method performance. From farm
317
animal metabolism studies with radiolabelled glyphosate a very low transfer into
318
muscle, milk and fat was observed.29 Consequently, if glyphosate concentrations
319
occur at all in breast milk, they are expected to be low. Therefore, the LOQ of the
320
method should achieve at least the published LOQ of Ehling & Redding23 of 5 ng/g
321
for milk measured by LC-MS/MS. Unpublished results of the analysis of glyphosate in
322
urine by GC-MS/MS indicated that this method might be at least similarly sensitive.
323
According to published results, a derivatization of glyphosate residues should
324
improve the detection in LC-MS/MS. On the other hand, after derivatization several
325
additional clean-up steps might be required to remove the excess of derivatization
326
chemicals. Considering this aspect, it was preferred to forgo a derivatization step for
327
the determination by LC-MS/MS. Page 14 ACS Paragon Plus Environment
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Prior to the LC-MS/MS analysis removal of fat by centrifugation and proteins by using
329
a 30 kDa cut-off filter was necessary to prevent contamination of the system. The LC-
330
MS/MS method was validated for glyphosate in accordance with the requirements of
331
the EU guidance document for quality control and validation procedure.28 Recovery
332
and precision of glyphosate were determined for 6 or 7 replicates at two fortification
333
levels. The linearity of the system was tested by injecting eight standards in water in
334
a concentration range from 0 to 50 ng/mL. A linear relationship between
335
concentration and the ratio of the peak area of glyphosate and its internal standard
336
was observed. The coefficient of determination was greater than 0.99. All calibration
337
points were within 20% of the theoretical value. The quantification was performed
338
using single-point calibration which is acceptable if the response of the analyte in the
339
samples is close to the response in the standard.28
340
The lower level (1 ng/mL) demonstrated sufficient recovery and precision. This level
341
is considered as the LOQ of the LC-MS/MS method. Possible matrix effects were
342
corrected by use of the stable isotope labelled internal standard 13C215N glyphosate.
343
At a concentration of 0.5 ng/mL, a signal to noise ratio of approximately 4 is obtained.
344
This concentration is considered as the limit of detection of the LC-MS/MS method.
345
Chromatograms of blank milk samples and fortified samples are provided in Figure 1.
346
It is clearly visible at the 1 ng/mL level that the signal to noise ratios for both
347
quantifier and qualifier transitions are well above three.
348
For GC-MS/MS determination, extraction with acidified water was combined with
349
clean-up on a cation exchange column to remove interfering natural compounds
350
present in breast milk. Since glyphosate is too polar for gas chromatography, a
351
derivatization of all polar groups (the phosphorous acid moiety, the carboxyl group
352
and the secondary amine) prior to analysis by heptafluoro-1-butanol and
353
trifluoroacetic acid anhydride was chosen. The validation data for the GC-MS/MS Page 15 ACS Paragon Plus Environment
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354
method determined in accordance with the requirements of the relevant EU guidance
355
document28 are given in Table 3.
356
The calibration was performed with freshly prepared derivatives of eight glyphosate
357
standard solutions in the concentration range from 0.01 to 10 ng/mL. All standard
358
solutions contained the internal standard at a level of 5 ng/mL. The coefficient of
359
determination was always equal to or greater than 0.9980.
360
Considering the sensitivity of the instrument, the method would have allowed the
361
detection of glyphosate residues at a level as low as 0.02 ng/mL (instrumental
362
detection limit). However, significant blank values were detected in all samples, the
363
reagent blank and all calibration standards. To identify the source of this glyphosate
364
interference, all solvents and most reagents were tested (i.e. ultrapure water from
365
three different sources, all components of CAX solution and extraction solution).
366
However, a clear origin of the interference was not detected. Therefore, it is assumed
367
that one of the derivatization agents produced the blank signal. It was not possible to
368
check this hypothesis within the scope of this study. It is noteworthy that there was
369
no detectable interference of the mass transition monitored for the internal standard
370
in all derivatization blanks.
371
Since this interference could not be eliminated, all results obtained by GC-MS/MS
372
had to be corrected for (reagent) blank interferences. A set of reagent blanks (at least
373
4 samples) were analyzed within each set of breast milk samples. The average
374
measured blank values ranged from 0.2 to 0.6 ng/mL. The relative standard
375
deviations of blank values in the sample sets ranged from 19% to 33%. Considering
376
the blank values from the derivatization reagent, the LOQ of the GC-MS/MS method
377
is 1 ng/mL. Chromatograms of reagent blank and spiked milk samples are given in
378
Figure 2.
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379
The recovery and precision data of both methods obtained during method validation
380
are provided in Table 3.
381
Notwithstanding the interference problem of the GC-MS/MS method, both analytical
382
methods were able to measure the occurrence and level of glyphosate residues in
383
breast milk from German women with an LOQ of 1 ng/mL. The availability of two
384
validated methods offered the chance to confirm positive results, if this would be
385
required.
386
In total, 114 different breast milk samples were analyzed for glyphosate. 75 samples
387
were analyzed by LC-MS/MS only. Because of the lower performance of the second
388
method, only 19 samples were analyzed exclusively by GC-MS/MS. Further 20 milk
389
samples were analyzed by both methods.
390
In addition to these 114 samples, five samples for the performance test were
391
analyzed by both LC-MS/MS and GC-MS/MS: four breast milk samples which were
392
spiked in advance with glyphosate and one control sample. Glyphosate was
393
identified by LC-MS/MS in all samples containing glyphosate. The recoveries for the
394
LC-MS/MS method were 110%, 97% and 102% for the spiking level of 1 ng/mL, 3
395
ng/mL and 5 ng/mL, respectively.
396
In the sample spiked at 0.5 ng/mL, glyphosate could still be detected by the LC-
397
MS/MS method. An ion chromatogram of this sample is shown in Figure 3. Due to
398
the interference problem in GC-MS/MS, no clear detection of glyphosate was
399
possible at this level.
400
The recoveries for the GC-MS/MS method were 70%, 70% and 54% for the spiking
401
level of 1 ng/mL, 3 ng/mL and 5 ng/mL, respectively. Generally, the GC-MS/MS
402
method tended to result in lower concentrations, probably due to the correction for
403
the procedural reagent blank values. The bias of the GC-MS/MS method is higher
404
compared to the LC-MS/MS method. This might be due to dilution steps with very Page 17 ACS Paragon Plus Environment
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405
small volume. The concentration step to yield the final volume might result in a partial
406
loss of the glyphosate derivative.
407
Nevertheless, both methods are able to quantify glyphosate residues in breast milk at
408
or above a concentration of 1 ng/mL. Because of the lack of significant blank values
409
in the LC-MS/MS method, residues of glyphosate higher than 0.5 ng/mL are still
410
detectable by this method. In none of the 114 analyzed breast milk samples, apart
411
from the spiked samples, was glyphosate detected.
412
An LC-MS/MS and a GC-MS/MS method have been newly developed for the
413
detection of glyphosate in breast milk. Both methods have been fully validated and
414
are suitable for the determination of glyphosate with an LOQ of 1 ng/mL. The LC-
415
MS/MS method furthermore allows detection of glyphosate at or above a level of 0.5
416
ng/mL. The LC-MS/MS method is much faster than the GC-MS/MS method, thus
417
making it suitable for higher sample throughput.
418
Summarizing the results, the positive findings of glyphosate in breast milk of
419
American women7 could not be confirmed by our results. In none of the 114 breast
420
milk samples collected from German women in August and September 2015 was
421
glyphosate found within the detection limitations of the analytical methods.
422
Available data from farm animal studies on glyphosate with non-labelled material
423
support these results. They provide no indication of a significant carry-over into fatty
424
tissues or milk even at high dosing levels.29
425 426
Acknowledgments
427
It is gratefully acknowledged that Dr. Magnus Jezussek from the Bavarian Authority
428
for Health and Food Safety (Bayerisches Landesamt für Gesundheit und
429
Lebensmittelsicherheit, Erlangen, Germany) provided Bavarian breast milk samples
430
for the study. Moreover, the authors would like to thank Renè Huppmann and Roland Page 18 ACS Paragon Plus Environment
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Journal of Agricultural and Food Chemistry
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Suchenwirth from the Governmental Institute of Public Health of Lower Saxony
432
(Niedersächsisches Landesgesundheitsamt, Hannover, Germany) for sampling and
433
logistics of the Lower Saxony breast milk samples.
434 435
References
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Union L 96/1, 05.04.2013.
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Acquavella, J.F.; Alexander, B.H.; Mandel, J.S.; Gustin, C.; Baker, B.;
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Jayasumana, C.; Gunatilake, S.; Siribaddana, S. Simultaneous exposure to
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Krüger, M.; Schledorn, P.; Schrödl, W.; Hoppe, H.-W.; Lutz, W. Detection of
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chromatography-tandem mass spectrometry determination of underivatized
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glyphosate in rice, maize and soybean. J. Chromatogr. A 2013, 1313, 157-165.
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Determination of glyphosate and its metabolite AMPA (aminomethylphosphonic acid)
Hao, C.; Morse, D.; Morra, F.; Zhao, X.; Yang, P.; Nunn, B. Direct aqueous
Arkan, T.; Molnar-Perl, I. The role of derivatization techniques in the analysis
Botero-Coy, A.M.; Ibañez, M.; Sancho, J.V.; Hernández, F. Direct liquid
Kacyński P.; Lozowicka, B.; Liquid chromatographic determination of
Nagatomi, Y.; Yoshioka, T.; Yanagisawa, M.; Uyama, A.; Mochizuki, N.
Raina-Fulton, R. A review of methods for the analysis of orphan and difficult
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Rapid determination of glyphosate, glufosinate, bialaphos, and their major
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metabolites in serum by liquid chromatography-tandem mass spectrometry using
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hydrophilic interaction chromatography. J. Chromatogr. A 2011, 1218, 3635-3680.
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alternative and fast method for determination of glyphosate and
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aminomethylphosphonic acid (AMPA) residues in soybean using liquid
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chromatography coupled with tandem mass spectrometry. Rapid Commun. Mass
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Spectrom. 2009, 23, 1029-1034.
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glyphosate and its major metabolite, aminomethylphosphonic acid, in fruits and
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vegetables by mixed-mode hydrophilic interaction/weak anion-exchange liquid
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chromatography coupled with electrospray tandem mass spectrometry. J.
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Chromatogr. A 2013, 1272, 90-99.
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plant origin via LC-MS/MS involving simultaneous extraction with methanol (QuPPe-
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Method), published the website of the EURL for single residue methods, version 8.1,
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March 2015, http://www.crl-
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(accessed December 7, 2015)
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Pinho, G.P. Study of the reaction derivatization glyphosate and
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aminomethylphosphonic acid (AMPA) with N,O-Bis(trimethylsilyl)trifluoroacetamide.
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Am. J. Anal. Chem. 2013, 4, 647-652.
Yoshioka, N.; Asano, M.; Kuse, A.; Mitsuhashi, T.; Nagasaki, Y.; Ueno, Y.
Martins-Junior, H.A.; Lebre, D.T.; Wang, A.Y.; Pires, M.A.F.; Bustillos, O.V. An
Chen, M.-X.; Cao, Z.-Y.; Jiang, Y.; Zhu, Z.-W. Direct determination of
Quick method for the analysis of numerous highly polar pesticides in foods of
Catrinck, T.C. P.G.; Aguiar, M.C.S.; Dias, A.; Silvério F.O.; Fidêncio, P.H.; de
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Alferness, P.L.; Wiebe, L.A. Determination of glyphosate and
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aminomethylphosphonic acid in crops by capillary gas chromatography with mass-
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selective detection: collaborative study. J. AOAC Int. 2001, 84, 823-846.
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acid in nutritional ingredients and milk by derivatization with
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fluorenylmethyloxycarbonyl chloride and liquid chromatography-mass spectrometry.
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J. Agric. Food Chem. 2015, 63, 10562-10568.
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http://www.nlga.niedersachsen.de/download/90400/Das_Muttermilchuntersuchungs-
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_programm_des_NLGA_von_1999-2012.pdf (only in German) (accessed December
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7, 2015).
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(25) Alferness, P.L.; Iwata, Y. Determination of glyphosate and
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(aminomethyl)phosphonic acid in soil, plant and animal matrices, and water by
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capillary gas chromatography with mass-selective detection, J. Agric. Food Chem.
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1994, 42, 2751-2758.
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on Dietary Reference Values for Water, EFSA Journal 2010, 8(3),1459
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Available online:
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http://www.efsa.europa.eu/sites/default/files/scientific_output/files/main_documents/1
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459.pdf (accessed December 7, 2015).
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(27) Freuze, I.; Jadas-Hecart, A.; Royer, A.; Communal, P.-Y. Influence of
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complexation phenomena with multivalent cations on the analysis of glyphosate and
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aminomethyl phosphonic acid in water. J. Chromatogr. A 2007, 1175, 197-206.
Jenness, R. The composition of human milk. Semin Perinatol. 1979, 3, 225-
Ehling, S.; Reddy, T.M. Analysis of glyphosate and aminomethylphosphonic
EFSA Panel on Dietetic Products, Nutrition, and Allergies: Scientific Opinion
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Guidance document on analytical quality control and validation procedures for
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pesticide residues in food and feed, SANCO/12571/2013, November 19 2013, rev. 0,
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http://ec.europa.eu/food/plant/pesticides/guidance_documents/docs/qualcontrol_en.p
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df (accessed December 7, 2015).
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(29) EFSA, 2014. Renewal Assessment Report Glyphosate,
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http://dar.efsa.europa.eu/dar-web/provision (accessed December 7, 2015).
540 541
Funding
542
This work was funded by the Federal Institute for Risk Assessment, Germany.
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FIGURE CAPTIONS Figure 1. LC-MS/MS chromatograms of breast milk samples obtained from method validation; From left to right: blank sample, blank spiked at 1 ng/mL glyphosate and blank spiked at 5 ng/mL glyphosate Top: Quantifier m/z 168.2>62.8 for glyphosate Bottom: Qualifier m/z 168.2>79.0 for glyphosate Figure 2. GC-MS/MS chromatograms of breast milk samples obtained from method validation From left to right: reagent blank sample; breast milk sample spiked at 1 ng/mL glyphosate; breast milk sample fortified at 5 ng/mL glyphosate Top: Quantifier m/z 612>213 for glyphosate derivative Bottom: Qualifier m/z 611>261 for glyphosate derivative Figure 3. Extracted ion chromatograms of a breast milk sample spiked with glyphosate at 0.5 ng/mL (limit of detection) Top: Quantifier m/z 168.2>62.8 for glyphosate Bottom: Internal standard C215N-glyphosate m/z 171.2>62.8
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Journal of Agricultural and Food Chemistry
Table 1. Biometric Data of Study Participants parameter samples from Bavaria, Germany number of samples 17 age of mother (years) median 32.1 range 26-39 body weight of mother (kg) median 63.0 range 54-90 duration of lactating period (weeks) median 11.0 range 3-80 self-reported exposure to 6 participants pesticides
samples from Lower Saxony, Germany 97 32.0 22-39 67.0 48-102
18 4-52 32 participants
Page 25 ACS Paragon Plus Environment
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Table 2. Transition Specific Data for the Analytical Methods product method transition precursor ion (m/z) ion (m/z) LC-MS/MS Glyphosate quantifier 168.2 62.8 Glyphosate qualifier 168.2 79.0 13 C2 15N Glyphosate 171.2 62.8 GC-MS/MS Glyphosate quantifier 612 213 Glyphosate qualifier 611 261 13 C2 15N Glyphosate 615 213 a CE = collision energy; b CXP = collision cell exit potential
Page 26 of 31
CEa (V) -32 -52 -32 25 25 15
CXPb (V) -17 -19 -17
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Journal of Agricultural and Food Chemistry
Table 3. Method Performance Characteristics of LC-MS/MS and GC-MS/MS Methods as Obtained from Spiked Samples Concurrently Analyzed with the Study Samplesa method spiking level average recovery range (%) RSD(R)b (%) (%) LC-MS/MS 1 ng/mL 99 85-128 16 (n=7) 5 ng/mL 91 83-99 7 (n=6) GC-MS/MS 1 ng/mL 84 71-102 13 (n=6) 10 ng/mL 83 73-90 8 (n=6) a for quantifier transition; b RSD(R) = within-laboratory reproducibility.
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Figure 1
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Journal of Agricultural and Food Chemistry
Figure 2 RT: 7.98 - 9.98 SM: 7G
RT: 7.92 - 9.92 SM: 7G NL: 1.07E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-163
Glyphosate_m/z_612->213 RT:8.92 AA:15901
100
90
90 80
70
70
70
50 40
R elativ e Abundance
80
60
60 50 40
60 50 40
30
30
30
20
20
20
10
10 8.10
8.30
8.0
9.20 9.37
8.53 8.69 8.5
9.0 Time (min)
9.55 9.71
10
8.12
0
9.5
8.35
8.0
8.58 8.75 8.5
9.16
9.37
9.0 Time (min)
9.58
9.80
0
NL: 3.41E3 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-163
Glyphosate_m/z_611->261 RT:8.92 AA:6367
100 90
100 90
R elative Abundance
70
R elative Abundance
80
60 50 40
30
20
20
10
10 9.26 9.41
10
9.67
8.14 8.30
0 8.0
8.5
9.0 Time (min)
9.5
NL: 1.74E4 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-293
Glyphosate_m/z_611->261 RT:9.23 AA:44646
40
20
8.53 8.69
10.06 10.0
50
30
8.30
9.86
60
30
8.07
9.5
90
70
0
9.61
9.0
100
70
40
8.87 9.02
RT: 8.23 - 10.23 SM: 7G
NL: 1.46E4 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-192
Glyphosate_m/z_611->261 RT:8.99 AA:26858
80
50
8.69
Time (min)
80
60
8.44 8.5
9.5
RT: 7.99 - 9.99 SM: 7G
RT: 7.92 - 9.92 SM: 7G
NL: 3.47E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-293
Glyphosate_m/z_612->213 RT:9.23 AA:88158
100
80
0
R elative Abundance
100
R elativ e Abundanc e
R elative Abundanc e
90
RT: 8.23 - 10.23 SM: 7G
NL: 2.65E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-192
Glyphosate_m/z_612->213 RT:8.98 AA:49806
8.0
8.48 8.5
8.85 9.0 Time (min)
9.29
9.53
9.79
9.5
0
8.44 8.5
8.72
8.94 9.10 9.0
9.43 9.5
9.62
9.81 9.96 10.0
Time (min)
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Figure 3
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Table of Contents (TOC) Graphic
Breast milk
Glyphosat e
Page 31 ACS Paragon Plus Environment
Article
Determination of glyphosate levels in breast milk samples from Germany by LC-MS/MS and GC-MS/MS Angelika Steinborn, Lutz Alder, Britta Michalski, Paul Zomer, Paul Bendig, Sandra Aleson Martinez, Hans G.J. Mol, Thomas Class, and Nathalie Costa-Pinheiro J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05852 • Publication Date (Web): 25 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016
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Page 1 of 31
Journal of Agricultural and Food Chemistry
Determination of glyphosate levels in breast milk samples from Germany by LC-MS/MS and GC-MS/MS
Angelika Steinborna*, Lutz Aldera, Britta Michalskia, Paul Zomerb, Paul Bendigc, Sandra Aleson Martinezc, Hans G. J. Molb, Thomas J. Classc, Nathalie Costa Pinheirod a
Federal Institute for Risk Assessment, Department of Pesticides Safety, Max-
Dohrn-Str. 8-10, 10589 Berlin, Germany b
RIKILT Wageningen UR, Natural Toxins and Pesticides, Akkermaalsbos 2, 6708
WB Wageningen, The Netherlands c
PTRL Europe, Helmholtzstr. 22, 89081 Ulm, Germany
d
Governmental Institute of Public Health of Lower Saxony, Roesebeckstr. 4-6, 30449
Hannover, Germany
*Corresponding author. Phone: +4930-18412-4792; e-mail: [email protected]
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1
Abstract
2
This study describes the validation and application of two independent analytical
3
methods for the determination of glyphosate in breast milk. They are based on liquid
4
chromatography-tandem mass spectrometry (LC-MS/MS) and gas chromatography-
5
tandem mass spectrometry (GC-MS/MS), respectively. For LC-MS/MS, sample
6
preparation involved an ultra-filtration followed by chromatography on an anion
7
exchange column. The analysis by GC-MS/MS involved an extraction step, clean-up
8
on a cation exchange column and derivatization with heptafluorobutanol and
9
trifluoroacetic acid anhydride. Both methods were newly developed for breast milk
10
and are able to quantify glyphosate residues at concentrations as low as 1 ng/mL.
11
The methods were applied to quantify glyphosate levels in 114 breast milk samples,
12
which had been collected from August to September of 2015 in Germany. The
13
mothers participated at their own request and thus do not form a representative
14
sample. In none of the investigated samples were glyphosate residues above the
15
limit of detection found.
16 17
Keywords
18
Glyphosate, breast milk, residues, LC-MS/MS, GC-MS/MS
19
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Journal of Agricultural and Food Chemistry
20
Introduction
21
Glyphosate (N-(Phosphonomethyl)glycine) is among the most frequently used active
22
ingredients of plant protection products worldwide. It is applied as a non-selective
23
herbicide for pre-emergence weed control as well as for desiccation treatment pre-
24
harvest. The use of glyphosate might lead to residues in food, especially if applied
25
shortly before harvest. In the European Union (EU), maximum residue levels (MRLs)
26
have been established for glyphosate, which are set for most plant and animal
27
commodities at the limit of quantification (LOQ) of 0.1 mg/kg and 0.05 mg/kg,
28
respectively. MRLs are up to 20 mg/kg for barley, oats, sorghum, sunflower seeds
29
and soybeans, and 10 mg/kg for wheat, rye, linseed, rapeseed, mustard seed, cotton
30
seed, lentils, peas and lupins.1 These food items make up an important part of
31
human and animal diets and thus might lead to intake of small amounts of glyphosate
32
by both humans and livestock. Further exposure of humans to glyphosate might be
33
due to direct exposure during and shortly after its application in agriculture (operator,
34
worker, bystander and/or resident exposure).
35
Glyphosate findings in urine have been reported in the literature for farmers and their
36
families as well as for patients in cases of acute intoxication.2-4 Published data
37
indicated that positive findings of glyphosate in human urine are quite common and
38
result from different exposure or intake pathways.5,6
39
In April 2014, a non-peer reviewed report was published, in which glyphosate in
40
breast milk of American mothers was detected in 3 out of 10 samples ranging from
41
76 to 166 ng/mL.7
42
Because of the high media response concerning the potential health risks for breast-
43
fed children, 16 breast milk samples from Germany were analysed for glyphosate in
44
June 2015. The unpublished and non-peer reviewed results reported glyphosate
45
levels between 0.2 and 0.4 ng/mL in all 16 samples.8 Page 3 ACS Paragon Plus Environment
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46
In both surveys, the concentration of glyphosate in milk samples was determined by
47
enzyme-linked immunosorbent assay (ELISA). A 96 well microtiter plate competitive
48
assay was used for detection and quantification of glyphosate levels in breast milk of
49
American mothers. The detection limit of the assay was given as 75 µg/L in milk.7
50
Information on suitability of the ELISA for breast milk as well as the documentation of
51
validation results was not provided for the study from Germany.8 The studies both
52
had methodological shortcomings. The analytical results have not been confirmed by
53
an independent method. The number of samples was quite low and details on study
54
participants were not reported.
55
The quantification of glyphosate residues is very challenging because of the highly
56
polar and amphoteric nature of the molecule, the low molecular weight, the high
57
water solubility and the lack of chromophores. For these reasons, glyphosate is one
58
of the few pesticides which are not amenable to the multi-residue methods typically
59
employed in pesticide residue analysis.
60
Several extraction procedures have been reported in the literature involving
61
extraction with water, water-methanol or water-acetonitrile mixtures followed by LC-
62
MS/MS measurement.9-11
63
Kacynski and Lozowicka12 tested different extraction solvents for their ability to
64
extract glyphosate and its metabolite aminomethyl phosphonic acid (AMPA) from
65
rape seed. The recoveries were between 82% and 93% using water or acidified
66
water. For liquid samples, e.g. beer, wine, tap water, surface water, groundwater,
67
direct injection of samples without preliminary extraction steps is also reported.9,13
68
Raina-Fulton14 reviewed analytical methods for residue analysis of glyphosate and
69
AMPA. Most of the published liquid chromatographic methods for glyphosate are
70
based on derivatization of the molecule followed by reversed phase HPLC separation
71
and mass spectrometric or fluorescence detection. Preferred procedures for Page 4 ACS Paragon Plus Environment
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72
derivatization depend on the detection method and include reaction with
73
fluorenylmethoxycarbonyl (FMOC) or o-phthalaldehyde-2-mercaptoethanol.10,15
74
In contrast, the direct chromatographic separation without previous derivatization is
75
possible by using anion-exchange columns or Hydrophilic Interaction Liquid
76
Chromatography (HILIC), since these stationary phases are able to retain polar
77
compounds.11,16-18
78
The EU reference laboratory (EURL) for residues of pesticides, which is responsible
79
for single residue methods (SRM) (CVUA Stuttgart, Germany), developed a method
80
for the analysis of highly polar pesticides.19 The residues are extracted by an
81
acidified methanol-water mixture. For glyphosate, different detection modules
82
involving liquid chromatographic separation on an anion exchange column or porous
83
graphitic carbon column are described.
84
Application of liquid chromatographic methods was reported for the quantification of
85
glyphosate residues in environmental matrices, food matrices and human urine and
86
serum samples.
87
Determination of glyphosate by gas chromatography followed by mass spectrometric
88
detection (GC-MS) requires the derivatization of the phosphorous acid moiety, the
89
carboxyl group and the secondary amine prior to analysis. Two different
90
derivatization approaches are described in the literature involving either
91
trialkysilylation20 or simultaneous acylation and esterification. The latter method was
92
tested successfully in five laboratories for corn grain, soya forage and walnut meat.21
93
The lowest tested concentration was 0.05 mg/kg.
94
Breast milk is a challenging matrix due to its very complex nature. It is an aqueous
95
mixture of carbohydrates, proteins and fat. The composition varies individually and
96
over the lactation period. Typically, the content of proteins is in the range of 0.8%-
97
0.9%. The fat content is in the range of 3%-5% and the carbohydrate content, mainly Page 5 ACS Paragon Plus Environment
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98
present as lactose, is in the range of 6.9%-7.2%.22 Thus, analytical methods
99
developed for watery matrices cannot be directly transferred to breast milk. An
Page 6 of 31
100
essential step prior to the analysis of glyphosate in breast milk is the separation of
101
the proteins and fat by suitable separation steps.
102
Recently, Ehling & Reddy23 published the application of LC-MS/MS method with
103
previous derivatization of the glyphosate with FMOC on different nutritional
104
ingredients derived from herbicide-tolerant soybean, corn and sugar beet as well as
105
breast milk. The authors reported an LOQ of 5 ng/g for milk samples, which is
106
approximately 10 fold higher compared to the reported glyphosate levels of up to 0.4
107
ng/mL in breast milk samples from Germany.8
108
The aim of the work was the development and validation of two independent
109
analytical principles for the quantification of glyphosate in breast milk samples. The
110
method should validate for the highest sensitivity as possible. The first method is
111
based on LC-MS/MS without derivatization. The second method is based on
112
derivatization with trifluoroacetic acid anhydride (TFAA) and heptafluorobutanol
113
(HFB) followed by GC-MS/MS determination of derivatives. The two newly developed
114
methodologies were employed to analyze 114 breast milk samples collected from
115
German breast-feeding women for residues of glyphosate. The results of these
116
analyses are reported.
117 118
Material and Methods
119
Collection of breast milk samples
120
Breast milk samples were collected in August and September 2015 by the
121
Governmental Institute of Public Health of Lower Saxony (Niedersächsisches
122
Landesgesundheitsamt, Hannover, Germany) and by the Bavarian Authority for
123
Health and Food Safety (Bayerisches Landesamt für Gesundheit und Page 6 ACS Paragon Plus Environment
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124
Lebensmittelsicherheit, Erlangen, Germany). Since 1999, mothers from Lower
125
Saxony can send in their breast milk for analysis of selected pesticides (e.g.
126
organochlorine pesticides).24 In the framework of this program, additional samples
127
were collected for a definite period of time for this study. Breast milk samples from
128
Bavaria were collected on a voluntary basis for the analysis of glyphosate. All
129
participants signed a declaration of consent concerning the use of their samples for
130
further scientific purposes. Participating mothers had not been selected by random
131
sampling. Moreover, there are no restrictions for participating in the monitoring
132
program (e.g. relating to age, point of sampling during lactation period, etc.). The milk
133
samples for this study were collected and stored in polypropylene tubes, which
134
remained frozen during storage and shipment. In total 114 milk samples were
135
analyzed. The participants completed a self-administered questionnaire. Information
136
on sample collection, biometric data and self-reported pesticide exposure of the
137
participants is given in Table 1.
138
The questionnaire also asked for the place of residence and the jobs practiced in the
139
last 10 years. 38 participants declared the use of chemical insecticides, herbicides or
140
wood preservatives. At least one participant has worked in a residue analytical
141
laboratory and used pesticide standards regularly.
142
Twenty of the 114 breast milk samples were divided each into two subsamples to
143
allow the parallel analysis by LC-MS/MS and GC-MS/MS.
144 145
Fortification of breast milk samples used for performance tests
146
For performance tests a homogenous sample of breast milk was prepared and
147
spiked with different amounts of a glyphosate standard solution (LGC Standards,
148
Wesel, Germany) having a concentration of 10 µg/mL in water.
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149
28 stored breast milk samples from a previous study of the Governmental Institute of
150
Public Health of Lower Saxony were pooled. Using this pooled sample, four aliquots
151
of 100 mL were fortified with glyphosate resulting in concentrations of 0.5 ng/mL, 1
152
ng/mL, 3 ng/mL, and 5 ng/mL. Moreover, an additional aliquot of the pooled sample
153
served as control sample (blank sample). All five ‘performance’ samples were divided
154
into two subsamples and analyzed in parallel with both methods. These samples
155
served as independent quality control. It has to be noted that analysts were not
156
informed about this additional quality test. The labelling of these ‘performance’
157
samples did not differ from other samples of this study.
158 159
LC-MS/MS analysis
160
Chemicals and Apparatus
161
Reference compound and internal standard (13C215N labelled glyphosate) were
162
purchased from LGC Standards. Methanol was obtained from Actu-All Chemicals
163
(Oss, The Netherlands), acetic acid and citric acid from Merck (Darmstadt, Germany)
164
and dimethylamine 40% solution from Sigma-Aldrich (Zwijndrecht, The Netherlands).
165
The water used was purified by a Milli-Q system from Merck Millipore (Tullagreen,
166
Ireland).
167
The 30 kDa molecular weight cut-off filter used for sample preparation (Amicon Ultra
168
4 Centrifugal filter, 30000 NMWL) was purchased from Merck Millipore, the LC
169
column (Dionex Ionpac AS 11 (2x250 mm) and the AG-11 guard column (2x50 mm)
170
from Thermo Fischer Scientific (Breda, The Netherlands). For centrifugation, a Z-513
171
centrifuge from Hermle Labortechnik (Wehingen, Germany) was used. The
172
syringeless filter devices Mini-Uniprep (PTFE filter, 0.45 µm) from Whatman, (GE
173
Healthcare, Eindhoven, The Netherlands) were used as LC vials. The LC-MS/MS
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174
system consisted of a Nexera UHPLC system from Shimadzu (Kyoto, Japan) and a
175
5500 Q-Trap system from Sciex (Concord, ON, Canada).
176 177
Sample preparation and measurement
178
For extraction of glyphosate the use of acidified methanol/water has been
179
described.19 However, in contrast to published results for plant materials, repeated
180
experiments using this extraction showed no or insufficient findings of both labeled
181
and native glyphosate in milk samples. Spiking of an extract with glyphosate did
182
produce a signal, so matrix effects could be eliminated as the source of the problem.
183
An alternative approach involving removal of fat by centrifugation and proteins by
184
ultrafiltration in one step through centrifugal filtration using a molecular weight cut-off
185
filter (30 kDa) was found to be suitable. For the samples from this study the
186
procedure was as follows: to 3 mL of sample, 30 µL of internal standard solution
187
containing 1000 ng/mL 13C215N glyphosate was added to obtain a level of 10 ng/mL.
188
After mixing, the sample was transferred to the top part of the cut-off filter tube. The
189
filter was centrifuged at 5000 g (corresponding to 3500 rpm) for 20 min. 500 µL of the
190
filtrate was then transferred to the LC filter vial, the solution was filtered and the vial
191
was used for measurement. After this procedure, one mL of final extract contained
192
the glyphosate residue of one mL breast milk.
193
The LC-MS/MS measurement was based on a method developed by the EURL –
194
SRM. 19 In brief: 25 µL of standard solution or filtrate were injected onto an anion
195
exchange LC column. Glyphosate was eluted from the column using a gradient of (A)
196
water and (B) water containing 1 mM citric acid and brought to a pH of 11 by addition
197
of dimethyl amine solution. Gradient elution was performed: 100% A from 0 to 2 min;
198
linear to 25% B in 5.5 min; then linear to 50% B in 2.5 min; this was held for 4 min;
199
after returning to 100% in 0.5 min the system was re-equilibrated for 7.5 min before Page 9 ACS Paragon Plus Environment
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200
the next injection. The total run time (including injection) was 22.5 min. A flow rate of
201
0.4 mL/min was used. A column temperature of 40 °C was maintained while the
202
temperature of the samples in the autosampler was 12 °C. For detection, the 5500
203
Qtrap system was used in triple-quad mode. The MS/MS transitions and the
204
transition specific parameters that were used are provided in Table 2.
205
All three transitions were measured using a declustering potential of -75 V, an
206
entrance potential of -10 V and a dwell time of 50 ms. The Turbospray source was
207
used in negative electrospray mode using the following parameters: Curtain gas 20
208
arbitrary units; Collision gas Medium; IonSpray –4000 V; Temperature 400 °C;
209
IonSpray gas 1: 40 and IonSpray gas 2: 50 arbitrary units.
210
Calculations were performed using the ratio of the peak areas of the quantifier
211
transition of glyphosate and the internal standard.
212 213
GC-MS/MS analysis
214
Chemicals and Apparatus
215
Reference compound and internal standard (13C215N labelled glyphosate) were
216
purchased from Dr. Ehrenstorfer (Augsburg, Germany). Methanol HPLC grade, ethyl
217
acetate and dichloromethane were purchased from LGC Standards (Wesel,
218
Germany). Acetic acid, glacial 100% was purchased form Merck (Darmstadt,
219
Germany). Anion exchange resin (Dowex 50WX8 hydrogen form, 200-400 mesh),
220
citral (95%), 2,2,3,3,4,4,4-heptafluoro-1-butanol (98%), trifluoroacetic acid anhydride
221
99%, water (Chromasolv® for HPLC) and hydrochloric acid 37% and 10 N were
222
purchased from Sigma-Aldrich (Seelze, Germany). The solution for cation exchange
223
clean-up (CAX solution) was prepared by mixing of 800 mL HPLC grade water, 13.5
224
mL 10 N HCl solution and 200 mL methanol.
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225
The derivatization reagent was prepared by mixing 1 volume fraction 2,2,3,3,4,4,4-
226
heptafluoro-1-butanol and 1 volume fractions trifluoroacetic acid anhydride. This
227
solution was prepared fresh on a daily basis.
228
The columns for anion exchange were empty Poly-Prep columns (Bio Rad
229
Laboratories Inc., Hercules, CA, USA). Vortex mixer REAX from Heidolph
230
Instruments (Schwabach, Germany) was used. Heating block Reacti-Therm III #TS-
231
18824 heating module, evaporator Reacti-Vap III #TS-18826 evaporation unit and
232
vacuum concentrator Express SpeedVac concentrator SC250 from Thermo Fisher
233
Scientific (San Jose, CA, USA) were used. A HS 601 D flatbed shaker was obtained
234
from IKA (Staufen, Germany). A Rotanta 460 centrifuge from Hettich (Tuttlingen,
235
Germany) was used. The 0.45 µm Nylon filter units (Chromafil AO-45/15 MS 15 mm)
236
were purchased from Macherey-Nagel (Düren, Germany).
237
The GC-MS/MS system consisted of a Thermo Trace GC Ultra equipped with TriPlus
238
liquid Autosampler, split/splitless injector and MS detector TSQ Quantum with triple
239
quadrupole (Thermo Fisher Scientific). The GC column Optima 5HT, 30 m length,
240
0.25 mm internal diameter and 0.25 μm film thickness was purchased from
241
Macherey-Nagel.
242 243
Sample preparation and measurement
244
The extraction procedure was based on the method by Alferness and Iwata25 and
245
was adapted to reach a lower limit of quantification.
246
A 2 mL milk sample was extracted with 3.75 mL of 0.6% acetic acid for 10 min on a
247
flatbed shaker with 200 rpm. Taking into account a water content of 87% for breast
248
milk26, the obtained volume of aqueous phase containing glyphosate residues is 5.5
249
mL. Then the resulting mixture was centrifuged for 5 min at 3220 g (4000 rpm). A 2
250
mL aliquot of the supernatant liquid was removed and transferred in a 15 mL PP Page 11 ACS Paragon Plus Environment
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251
tube. 2 mL dichloromethane were added. The sample was shaken for 2 min and
252
centrifuged for 5 min at 3220 g (4000 rpm). A 1 mL aliquot of the supernatant liquid
253
was filtered using a 0.45 µm Nylon filter unit.
254
A cation exchange clean-up was performed using disposable Bio-Rad Poly-Prep
255
columns filled with 1.72 g (equivalent to 2 mL filling volume) of AG 50W-X8 resin (H+
256
form). Before use, the columns were washed with 10 mL of water (Chromasolv® for
257
HPLC). A 0.55 mL aliquot of the filtered extract (corresponding to 0.2 mL breast milk)
258
and 0.100 mL of internal standard (20 ng/mL) were added to the cation exchange
259
column and were eluted until the liquid level reached the top of the resin. Co-
260
extractives were eluted by adding 2.0 mL of CAX solution. Both eluates were
261
discarded. Glyphosate residues were eluted with 12.5 mL of the CAX solution. All
262
elutions were performed using gravity. The eluate was collected in a 15 mL PP tube,
263
and evaporated to dryness using a vacuum concentrator at 60 °C and 60 mbar. The
264
residues were dissolved in 1.0 mL of the CAX solution. It is expected that extraction
265
and clean-up in acidic solvents allows the decomplexation of all cationic glyphosate
266
complexes.27
267
A 1.5 mL aliquot of the derivatization reagent was added to 2 mL vials which were
268
sealed and placed in the heating block. The block was cooled to a temperature of -20
269
°C before proceeding and after adding sample extract. A 0.05 mL aliquot of the
270
redissolved eluate (corresponding to 0.01 mL breast milk) was drawn into the
271
disposable pipet tip and then dispensed under the surface of the chilled reagent.
272
After 5 min, the derivatization of analyte is started by heating the reaction vial to 92-
273
97 °C for 1 h. After allowing vials to cool, the excess of derivatization reagent was
274
removed by evaporation to dryness. The derivatization step and the structure of the
275
derivative were described by Alferness and Iwata.25 Briefly, the carboxylic and
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276
phosphonic acid group were esterified to the corresponding heptafluorobutyl ester
277
and the amine function was derivatized to the trifluoroacetyl derivative.
278
The residues were redissolved in 0.2 mL of ethyl acetate containing 0.2 mL/L citral
279
and later concentrated to a final volume of 20 µL. The addition of citral to the injection
280
solvent was made to reduce adsorption of the analytes in the inlet and the GC
281
column. Thus, the peak shape and the sensitivity of the method was improved.25
282
Following this procedure, one mL of final extract contained the glyphosate residue of
283
0.5 mL breast milk.
284
These final extracts were analyzed by gas chromatography with tandem mass
285
spectrometric detection (GC-MS/MS). 4.0 µL of the extracts were injected splitless.
286
The injector temperature was 280 °C. Oven temperature program was held at 80 °C
287
for 1.5 min, ramped then with 10 °C/min to 180 °C, ramped with 30 °C/min to 300 °C
288
and was held for 2.8 min. Helium was used as carrier gas with a constant flow rate of
289
1 mL/min. The expected retention time for the glyphosate derivative was 9.1 min. The
290
temperature of the ion source was 280 °C. Electron impact (EI) energy was 70 eV
291
and emission current was 50 µA. The MS/MS transitions and the transition specific
292
parameters are provided in Table 2.
293
Calculations were performed using the ratio of the peak areas of the quantifier
294
transition of glyphosate derivative and the internal standard derivative. Calibration
295
solutions were prepared by volumetric dilution of a glyphosate stock solution in a
296
solution containing 20 ng/mL internal standard. The dilutions were made in CAX
297
solution. 0.05 mL aliquots of these calibration solutions were derivatized as described
298
for the breast milk extracts. Concentration of the derivatized calibration solutions
299
ranged from 0.01 to 10 ng/mL. The concentration of the internal standard in the final
300
extract was always 5 ng/mL.
301 Page 13 ACS Paragon Plus Environment
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302
Results and Discussion
303
For method development the advantages and disadvantages of different extraction
304
procedures have to be weighed in order to achieve the best method performance.
305
For evaluation of the methods the requirements of the EU guidance document for
306
quality control and validation procedure 28 were considered. The guidance applies to
307
laboratories involved in the official control of pesticide residues in food and feed in
308
the EU. Briefly, a quantitative analytical method has to be validated with respect to
309
sensitivity/linearity, specificity, trueness (bias), precision, and robustness. Matrix
310
effects should be assessed. The average recovery (trueness) of a minimum of five
311
spiked samples per fortification level should be in a range of 70-120%. The
312
repeatability (relative standard deviation) should be < 20% for each analyte. The
313
LOQ is established as the lowest fortification level with an acceptable mean recovery
314
and an acceptable relative standard deviation.
315
In our work, the method development is clearly focussed on reaching the lowest
316
quantification limit in breast milk with still sufficient method performance. From farm
317
animal metabolism studies with radiolabelled glyphosate a very low transfer into
318
muscle, milk and fat was observed.29 Consequently, if glyphosate concentrations
319
occur at all in breast milk, they are expected to be low. Therefore, the LOQ of the
320
method should achieve at least the published LOQ of Ehling & Redding23 of 5 ng/g
321
for milk measured by LC-MS/MS. Unpublished results of the analysis of glyphosate in
322
urine by GC-MS/MS indicated that this method might be at least similarly sensitive.
323
According to published results, a derivatization of glyphosate residues should
324
improve the detection in LC-MS/MS. On the other hand, after derivatization several
325
additional clean-up steps might be required to remove the excess of derivatization
326
chemicals. Considering this aspect, it was preferred to forgo a derivatization step for
327
the determination by LC-MS/MS. Page 14 ACS Paragon Plus Environment
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328
Prior to the LC-MS/MS analysis removal of fat by centrifugation and proteins by using
329
a 30 kDa cut-off filter was necessary to prevent contamination of the system. The LC-
330
MS/MS method was validated for glyphosate in accordance with the requirements of
331
the EU guidance document for quality control and validation procedure.28 Recovery
332
and precision of glyphosate were determined for 6 or 7 replicates at two fortification
333
levels. The linearity of the system was tested by injecting eight standards in water in
334
a concentration range from 0 to 50 ng/mL. A linear relationship between
335
concentration and the ratio of the peak area of glyphosate and its internal standard
336
was observed. The coefficient of determination was greater than 0.99. All calibration
337
points were within 20% of the theoretical value. The quantification was performed
338
using single-point calibration which is acceptable if the response of the analyte in the
339
samples is close to the response in the standard.28
340
The lower level (1 ng/mL) demonstrated sufficient recovery and precision. This level
341
is considered as the LOQ of the LC-MS/MS method. Possible matrix effects were
342
corrected by use of the stable isotope labelled internal standard 13C215N glyphosate.
343
At a concentration of 0.5 ng/mL, a signal to noise ratio of approximately 4 is obtained.
344
This concentration is considered as the limit of detection of the LC-MS/MS method.
345
Chromatograms of blank milk samples and fortified samples are provided in Figure 1.
346
It is clearly visible at the 1 ng/mL level that the signal to noise ratios for both
347
quantifier and qualifier transitions are well above three.
348
For GC-MS/MS determination, extraction with acidified water was combined with
349
clean-up on a cation exchange column to remove interfering natural compounds
350
present in breast milk. Since glyphosate is too polar for gas chromatography, a
351
derivatization of all polar groups (the phosphorous acid moiety, the carboxyl group
352
and the secondary amine) prior to analysis by heptafluoro-1-butanol and
353
trifluoroacetic acid anhydride was chosen. The validation data for the GC-MS/MS Page 15 ACS Paragon Plus Environment
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354
method determined in accordance with the requirements of the relevant EU guidance
355
document28 are given in Table 3.
356
The calibration was performed with freshly prepared derivatives of eight glyphosate
357
standard solutions in the concentration range from 0.01 to 10 ng/mL. All standard
358
solutions contained the internal standard at a level of 5 ng/mL. The coefficient of
359
determination was always equal to or greater than 0.9980.
360
Considering the sensitivity of the instrument, the method would have allowed the
361
detection of glyphosate residues at a level as low as 0.02 ng/mL (instrumental
362
detection limit). However, significant blank values were detected in all samples, the
363
reagent blank and all calibration standards. To identify the source of this glyphosate
364
interference, all solvents and most reagents were tested (i.e. ultrapure water from
365
three different sources, all components of CAX solution and extraction solution).
366
However, a clear origin of the interference was not detected. Therefore, it is assumed
367
that one of the derivatization agents produced the blank signal. It was not possible to
368
check this hypothesis within the scope of this study. It is noteworthy that there was
369
no detectable interference of the mass transition monitored for the internal standard
370
in all derivatization blanks.
371
Since this interference could not be eliminated, all results obtained by GC-MS/MS
372
had to be corrected for (reagent) blank interferences. A set of reagent blanks (at least
373
4 samples) were analyzed within each set of breast milk samples. The average
374
measured blank values ranged from 0.2 to 0.6 ng/mL. The relative standard
375
deviations of blank values in the sample sets ranged from 19% to 33%. Considering
376
the blank values from the derivatization reagent, the LOQ of the GC-MS/MS method
377
is 1 ng/mL. Chromatograms of reagent blank and spiked milk samples are given in
378
Figure 2.
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379
The recovery and precision data of both methods obtained during method validation
380
are provided in Table 3.
381
Notwithstanding the interference problem of the GC-MS/MS method, both analytical
382
methods were able to measure the occurrence and level of glyphosate residues in
383
breast milk from German women with an LOQ of 1 ng/mL. The availability of two
384
validated methods offered the chance to confirm positive results, if this would be
385
required.
386
In total, 114 different breast milk samples were analyzed for glyphosate. 75 samples
387
were analyzed by LC-MS/MS only. Because of the lower performance of the second
388
method, only 19 samples were analyzed exclusively by GC-MS/MS. Further 20 milk
389
samples were analyzed by both methods.
390
In addition to these 114 samples, five samples for the performance test were
391
analyzed by both LC-MS/MS and GC-MS/MS: four breast milk samples which were
392
spiked in advance with glyphosate and one control sample. Glyphosate was
393
identified by LC-MS/MS in all samples containing glyphosate. The recoveries for the
394
LC-MS/MS method were 110%, 97% and 102% for the spiking level of 1 ng/mL, 3
395
ng/mL and 5 ng/mL, respectively.
396
In the sample spiked at 0.5 ng/mL, glyphosate could still be detected by the LC-
397
MS/MS method. An ion chromatogram of this sample is shown in Figure 3. Due to
398
the interference problem in GC-MS/MS, no clear detection of glyphosate was
399
possible at this level.
400
The recoveries for the GC-MS/MS method were 70%, 70% and 54% for the spiking
401
level of 1 ng/mL, 3 ng/mL and 5 ng/mL, respectively. Generally, the GC-MS/MS
402
method tended to result in lower concentrations, probably due to the correction for
403
the procedural reagent blank values. The bias of the GC-MS/MS method is higher
404
compared to the LC-MS/MS method. This might be due to dilution steps with very Page 17 ACS Paragon Plus Environment
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405
small volume. The concentration step to yield the final volume might result in a partial
406
loss of the glyphosate derivative.
407
Nevertheless, both methods are able to quantify glyphosate residues in breast milk at
408
or above a concentration of 1 ng/mL. Because of the lack of significant blank values
409
in the LC-MS/MS method, residues of glyphosate higher than 0.5 ng/mL are still
410
detectable by this method. In none of the 114 analyzed breast milk samples, apart
411
from the spiked samples, was glyphosate detected.
412
An LC-MS/MS and a GC-MS/MS method have been newly developed for the
413
detection of glyphosate in breast milk. Both methods have been fully validated and
414
are suitable for the determination of glyphosate with an LOQ of 1 ng/mL. The LC-
415
MS/MS method furthermore allows detection of glyphosate at or above a level of 0.5
416
ng/mL. The LC-MS/MS method is much faster than the GC-MS/MS method, thus
417
making it suitable for higher sample throughput.
418
Summarizing the results, the positive findings of glyphosate in breast milk of
419
American women7 could not be confirmed by our results. In none of the 114 breast
420
milk samples collected from German women in August and September 2015 was
421
glyphosate found within the detection limitations of the analytical methods.
422
Available data from farm animal studies on glyphosate with non-labelled material
423
support these results. They provide no indication of a significant carry-over into fatty
424
tissues or milk even at high dosing levels.29
425 426
Acknowledgments
427
It is gratefully acknowledged that Dr. Magnus Jezussek from the Bavarian Authority
428
for Health and Food Safety (Bayerisches Landesamt für Gesundheit und
429
Lebensmittelsicherheit, Erlangen, Germany) provided Bavarian breast milk samples
430
for the study. Moreover, the authors would like to thank Renè Huppmann and Roland Page 18 ACS Paragon Plus Environment
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Suchenwirth from the Governmental Institute of Public Health of Lower Saxony
432
(Niedersächsisches Landesgesundheitsamt, Hannover, Germany) for sampling and
433
logistics of the Lower Saxony breast milk samples.
434 435
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This work was funded by the Federal Institute for Risk Assessment, Germany.
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FIGURE CAPTIONS Figure 1. LC-MS/MS chromatograms of breast milk samples obtained from method validation; From left to right: blank sample, blank spiked at 1 ng/mL glyphosate and blank spiked at 5 ng/mL glyphosate Top: Quantifier m/z 168.2>62.8 for glyphosate Bottom: Qualifier m/z 168.2>79.0 for glyphosate Figure 2. GC-MS/MS chromatograms of breast milk samples obtained from method validation From left to right: reagent blank sample; breast milk sample spiked at 1 ng/mL glyphosate; breast milk sample fortified at 5 ng/mL glyphosate Top: Quantifier m/z 612>213 for glyphosate derivative Bottom: Qualifier m/z 611>261 for glyphosate derivative Figure 3. Extracted ion chromatograms of a breast milk sample spiked with glyphosate at 0.5 ng/mL (limit of detection) Top: Quantifier m/z 168.2>62.8 for glyphosate Bottom: Internal standard C215N-glyphosate m/z 171.2>62.8
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Table 1. Biometric Data of Study Participants parameter samples from Bavaria, Germany number of samples 17 age of mother (years) median 32.1 range 26-39 body weight of mother (kg) median 63.0 range 54-90 duration of lactating period (weeks) median 11.0 range 3-80 self-reported exposure to 6 participants pesticides
samples from Lower Saxony, Germany 97 32.0 22-39 67.0 48-102
18 4-52 32 participants
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Table 2. Transition Specific Data for the Analytical Methods product method transition precursor ion (m/z) ion (m/z) LC-MS/MS Glyphosate quantifier 168.2 62.8 Glyphosate qualifier 168.2 79.0 13 C2 15N Glyphosate 171.2 62.8 GC-MS/MS Glyphosate quantifier 612 213 Glyphosate qualifier 611 261 13 C2 15N Glyphosate 615 213 a CE = collision energy; b CXP = collision cell exit potential
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CEa (V) -32 -52 -32 25 25 15
CXPb (V) -17 -19 -17
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Table 3. Method Performance Characteristics of LC-MS/MS and GC-MS/MS Methods as Obtained from Spiked Samples Concurrently Analyzed with the Study Samplesa method spiking level average recovery range (%) RSD(R)b (%) (%) LC-MS/MS 1 ng/mL 99 85-128 16 (n=7) 5 ng/mL 91 83-99 7 (n=6) GC-MS/MS 1 ng/mL 84 71-102 13 (n=6) 10 ng/mL 83 73-90 8 (n=6) a for quantifier transition; b RSD(R) = within-laboratory reproducibility.
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Figure 1
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Figure 2 RT: 7.98 - 9.98 SM: 7G
RT: 7.92 - 9.92 SM: 7G NL: 1.07E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-163
Glyphosate_m/z_612->213 RT:8.92 AA:15901
100
90
90 80
70
70
70
50 40
R elativ e Abundance
80
60
60 50 40
60 50 40
30
30
30
20
20
20
10
10 8.10
8.30
8.0
9.20 9.37
8.53 8.69 8.5
9.0 Time (min)
9.55 9.71
10
8.12
0
9.5
8.35
8.0
8.58 8.75 8.5
9.16
9.37
9.0 Time (min)
9.58
9.80
0
NL: 3.41E3 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-163
Glyphosate_m/z_611->261 RT:8.92 AA:6367
100 90
100 90
R elative Abundance
70
R elative Abundance
80
60 50 40
30
20
20
10
10 9.26 9.41
10
9.67
8.14 8.30
0 8.0
8.5
9.0 Time (min)
9.5
NL: 1.74E4 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-293
Glyphosate_m/z_611->261 RT:9.23 AA:44646
40
20
8.53 8.69
10.06 10.0
50
30
8.30
9.86
60
30
8.07
9.5
90
70
0
9.61
9.0
100
70
40
8.87 9.02
RT: 8.23 - 10.23 SM: 7G
NL: 1.46E4 TIC F: + p EI SRM ms2 611.000 [260.500-261.500] MS Genesis P3764TSQ-192
Glyphosate_m/z_611->261 RT:8.99 AA:26858
80
50
8.69
Time (min)
80
60
8.44 8.5
9.5
RT: 7.99 - 9.99 SM: 7G
RT: 7.92 - 9.92 SM: 7G
NL: 3.47E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-293
Glyphosate_m/z_612->213 RT:9.23 AA:88158
100
80
0
R elative Abundance
100
R elativ e Abundanc e
R elative Abundanc e
90
RT: 8.23 - 10.23 SM: 7G
NL: 2.65E4 TIC F: + p EI SRM ms2 612.000 [212.500-213.500] MS Genesis P3764TSQ-192
Glyphosate_m/z_612->213 RT:8.98 AA:49806
8.0
8.48 8.5
8.85 9.0 Time (min)
9.29
9.53
9.79
9.5
0
8.44 8.5
8.72
8.94 9.10 9.0
9.43 9.5
9.62
9.81 9.96 10.0
Time (min)
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Figure 3
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Table of Contents (TOC) Graphic
Breast milk
Glyphosat e
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