38 May / June 2019
employed. The resulting signals were found to have higher variability in comparison to the samples cleaned using SPE.
Glyphosate in beer
No analytes were found in the tested beer. Thus, the beer sample was spiked at 50 ppb with glyphosate and glufosinate and analysed. The results are presented in Table 2. In this work further analysis of beer samples was not attempted due to the limited instrumental detection limit.
Figure 3. Organic Oatmeal Sample, Blank and Spiked at 100 ppb Glyphosate. Results Method development
While multiple columns and mobile phase conditions were tested during method development for detection of underivatised glyphosate using LC-MS/MS, the final chromatographic method used an anion exchange column. It allowed for retention of compounds under the basic mobile phase conditions after direct injection of samples in 50-50 water-methanol extraction solvent. No further dilution or solvent exchange was required for the samples which resulted in convenient and fast analysis method. The elution off the HPLC column was performed by increasing the pH of the mobile phase to 9.2 using 20 mM ammonium carbonate buffer and introducing small amount of methanol (up to 5% methanol in the mobile phase). Multiple injections of samples extracted from multiple matrices did not result in significant shifts in the retention time indicating the ruggedness of this LC method.
Sample preparation was performed using fast extraction with methanol. SPE was the first choice for sample cleanup as the sample can be simply passed through the cartridge. Since difficulty was encountered with undissolved co-extracted particulates in wheat samples, ultrafiltration cleanup was employed. The sample cleanup by both methods was acceptable for LC/MS analysis, injection of multiple extracted samples did not result in the shift of retention times or decrease in the analyte’s signals.
Results from spiked grain samples
Oatmeal and wheat samples that were labelled ‘organic’ were used during method development. The samples were analysed
for glyphosate and related compounds. The compounds were not found to be present in these samples (Figure 3). Consequently, the samples were spiked with analytes and analysed using the proposed method. The results of method development using spiked organic samples are shown in Table 2. In the spiked oatmeal samples, all three analytes were detected and quantified at 100 ppb, and in the wheat flour samples glyphosate and glufosinate were detected and quantified at 100 ppb. Wheat flour was not spiked with AMPA. Accuracy of the method was measured as the % recovery of the known spiked amounts. For glyphosate, the recovery values ranged from 118% to 125%, while for glufosinate they were between 105% and 112%. For AMPA in oatmeal sample recovery was at 118%. The method for wheat samples produced slightly higher uncertainties for both glyphosate and glufosinate, up to 19% RSD. The sample preparation method for wheat used only ultrafiltration and SPE cleanup was not
Table 2. Method development results after spiking 100 ppb into cereal/grains labelled ‘organic’ and 50 ppb into beer.
Analyte/Matrix Glyphosate N=6
Oatmeal
Whole wheat 125 Beer
118 AMPA 5
19 21
118 - -
15 - -
Glufosinate
Recovery (%) % RSD Recovery (%) % RSD Recovery (%) % RSD 125
105 105 112
Table 3. Analysis results in non-organic grains and cereals Analyte/Matrix
Glyphosate N=3 Oatmeal
White wheat flour Infant rice cereal Infant oat cereal
Infant mixed cereal Beer (Lager)
8 5
16 7
Identification and quantitation of glyphosate in cereals
The results of glyphosate analysis in cereals using the proposed methods are presented in Table 3. These samples were purchased in the grocery store, were not labelled ‘organic’ and were not spiked with standards. Internal standards were used as described in the experimental section. The samples of instant oatmeal (Figure 4) and wheat white flour contained significant incurred amounts of glyphosate, 1.2 and 0.8 ppm, respectively. Infant rice cereal had very low levels of glyphosate. The levels were close to the limit of instrument sensitivity in that matrix and the resulting RSD was high. Infant oat cereal contained glyphosate at 1.1 ppm and infant mixed cereal was found to contain glyphosate at 0.25 ppm. Glufosinate was not found in any cereal products. AMPA was found only in instant oatmeal at low levels.
AMPA
0.06 1.1
0.25 ND
NT=not tested, ND=not detected
12 12 4 5
0.04 NT ND NT NT ND
Glufosinate
Found, ppm % RSD Found, ppm % RSD Found, ppm 1.2 0.8
27 ND ND ND ND ND ND
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