37
Sample preparation for beer
Beer samples were also analysed using the same methodology. First, a thorough degassing of beer was undertaken by placing the beer sample in the ultrasonic bath for 15 minutes. Then 5 mL of the beer sample was mixed with 5 mL of methanol with 1% formic acid and internal standard. This sample was briefly mixed and cleaned using the SPE procedure.
LC-MS/MS method
Figure 2. LC/MS Analysis of Glyphosate, AMPA, and Glufosinate using apHera NH2 chromatographic column. Quantitative transitions are shown for each analyte. Conditions are described in the experimental section.
Sample pre-treatment
The extraction method was based on the QuPPe (Quick Polar Pesticides Method) methodology developed in the European Union (EU) for fruits and vegetables, and used water:methanol (50:50) containing 0.5% formic acid as the final extraction solvent [5]. Individual five gram samples of homogenised grain and/or cereal were weighed into 50 mL centrifuge tubes. Water (10 mL) and 100 µL of an internal standard solution (20 µg/mL of each analyte in water) were added to each vessel. The samples were then left to stand for the time period of two hours. At the conclusion of the wait period, 10 mL of methanol containing 1% v/v formic acid was added. The samples were mixed for 15 minutes on a laboratory shaker and then centrifuged. Two grains, oats and wheat, used during method development, gave significantly different extracts when examined visually. The oats produced a clear, yellow extract, while the wheat extract was cloudy and found to be difficult to filter when using syringe filters. As a result, two different sample cleanup procedures were utilised based on the grain sample under study.
Sample cleanup using SPE
For the samples that did not have particulates after extraction and centrifugation, such as oats and corn, a solid phase extraction (SPE) cleanup using Supel-Select HLB cartridges was applied, similarly to a method reported by Chamkasem and Harmon [6]. Polymeric hydrophilic-lipophilic balance or HLB SPE can be applied to broad range of analytes and used under reversed phase methodology. The SPE cleanup method in the present work is based on the chemical ‘filtration’ of the extract through the HLB cartridge when the impurities that are more hydrophobic in comparison to the analytes are retained
Table 1. MS parameters for analytes. Compound Glyphosate
Glyphosate-2-13 AMPA
AMPA-13 C,15 C,15
Quant 167.8 63 Qual 167.8 79
on the SPE phase while the more polar analytes do not retain and come through in the eluate. The HLB SPE cartridges were conditioned using 100% methanol followed by water:methanol (50:50) containing 0.5% v/v formic acid. For 1 mL SPE cartridges, 0.5 mL of the sample extract was used to pre-wet the cartridge. The eluate from this step was discarded. A second aliquot of the sample extract (0.5 mL) was loaded into the HLB cartridge. This eluate was collected and filtered through 0.2 micron filter vials with polypropylene membrane.
Sample cleanup using ultrafiltration
Ultrafiltration devices were used for cleanup of sample extracts that had particulates, such as wheat. 1 mL of the sample extract was loaded onto the Amicon ultrafiltration devices. The ultrafiltration step was performed by centrifugation for 45 minutes at 4000 rpm. The clear sample that passed through the membrane was collected and analysed. Low-absorption vials were used for this analysis to prevent loss of analytes through adsorption to the glass surface.
The analysis was done using Agilent 1200 HPLC stacked with AB Sciex QTrap 3200. The HPLC column used for this analysis was the polymer-based ion-exchange apHera™ NH2 column, which can provide stable and robust LC separations in the wise range of pH values. The column’s size was 15 cm x 4.6 mm packed with 5-micron size particles. The aqueous mobile phase used water and ammonium carbonate at pH 9 with 5% methanol. This mobile phase ensured the proper ionisation of glyphosate, which has a phosphate group in its structure, with detection under negative ESI conditions. In addition, ammonium carbonate buffer is volatile and is fully compatible with LC/MS instrumentation. The flow through HPLC column at 0.5 mL/min started with water for 2 minutes, the change to 95% 20 mM ammonium carbonate buffer (pH 9) and 5% methanol occurred at 2.1 minutes, continued for the next 10 minutes, and the column was flushed with pure water for 5 minutes at the end of the run. Column temperature was 35ºC. 60 µL injection volume was used. Table 1 lists the MS source conditions for all analytes. In addition, voltage was at -4000 V, curtain gas was at 45, source temperature at 500 ºC, Gas 1 at 50 and Gas 2 was at 60. Figure 2 presents a chromatogram of a standard injection.
Calibration curves used internal standards and were made in 50-50 methanol-water with 0.5% formic acid. Concentration range of calibration curves was from 10 ng/mL to 300 ng/mL.
N,D2 Glufosinate Glufosinate-D3
N Quant 19.8 62.9 Quant 110 63 Qual 110 81 Quant 113.9 63 Quant 180 63
Q1 Q3 DP EP CEP CE CXP -30 -30 -30 -45 -40 -40 -35
Qual 180 136 -40 Quant 182.9 63
-30
-5.5-8 -5.5 -4.5 -10 -10 -5 -6 -4
-7.5
-32 -8
-10 -10 -10 -10 -12 -12 -12
-4
-58 -28 -28 -18 -38 -56 -25 -70
-3
-12 -6 -1 0 0
-2 0
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