Spinach Matrix Removal Capacity of CarbonX for QuEChERS compared to GCB
Graphitized Carbon Black (GCB) is generally added to QuEChERS dispersive solid phase extraction (dSPE) kits when the food extract is highly pigmented. This is due to the fact that pigments and sterols cause fouling of the liner and column as well as decreased signal to noise when quantitating samples on GC/MS and LC/MS/MS systems. CarbonX for QuEChERS greatly reduces the losses of planar pesticides observed with GCB sorbents. In addition, the CarbonX for QuEChERS has improved baselines and therefore improved matrix removal capacity relative to GCB.
Reagents and Chemicals
All reagents and solvents were high-performance liquid chromatography (HPLC) grade or better. Methanol (MeOH) was obtained from Sigma Aldrich, Acetonitrile (ACN) was obtained from J&W, and glacial acetic acid (HAc) was obtained from Ampresco. QuEChERS AOAC dSPE tubes for pigmented samples containing GCB were purchased from a leading supplier.
Solutions and Standards
A 1% acetic acid (HAc) in ACN solution was prepared by adding 10 mL of HAc to 1 L of ACN. The 18 pesticide mix stock solution (2 mg/mL) was made in MeOH and stored at -20 °C. This solution was diluted to a spiking solution (5 µg/mL in MeOH) for use in the method.
The sample preparation procedure includes sample comminution, extraction, and partitioning followed by dispersive SPE clean-up. The spinach (~35g) was homogenized by freezing with liquid nitrogen and grinding in a Cuisinart DCG-12BC for approximately 30 seconds. Two 15 g (±0.1 g) amounts of homogenized spinach sample were placed into two 50 mL centrifuge tubes. 150 µL of the spiking solution was added to one tube which was vortexed for 15 seconds. Next, 15 mL of 1% HAc in ACN was added to each tube, followed by an extract packet containing 6g of MgSO4 and 1.5 g NaAcetate. (United Science P/N 1605165160). The tubes were capped tightly, and hand-shaken vigorously for 1 minute. The tubes were then centrifuged at 4000 rpm for 5 minutes. Next, the ACN extracts were decanted into separate containers. The volume of ACN extracts (about 10 mL from each tube) were enough for multiple sample tests when using 2 mL dispersive SPE (dSPE) tubes. For this experiment, 2 mL AOAC pigmented QuEChERS tubes were used from United Science (P/N 1605040110) and a leading supplier Technologies.
The dry tubes were vortexed for a few seconds before adding the 1 mL of spiked extract to prevent possible agglomerates. 1 mL of spiked extract was added to each of 3 tubes: The CarbonX for QuEChERS Tube, the original method GCB tube, and the modified GCB with toluene tube. To the modified GCB tube 375 µL of toluene was also added. The dSPE tubes were tightly capped and vortexed for 1 min. The tubes were then centrifuged at 5,000 rpm for 2 min. The original method samples were then transferred directly into auto-sampler vials for GC/MS injection. From the modified method sample 825 µL were transferred to another tube, dried under nitrogen, and reconstituted with 600 µL of 1% HAc in ACN. Finally the modified sample was put into an auto-sampler vial for GC/MS injection.
Recovery and Reproducibility
The recovery and reproducibility were evaluated by spiking 18 pesticide standards in the comminuted spinach sample at levels of 50 ng/g. These samples were quantitated against a matrix standard. The standard was prepared by removing the pigment through dSPE with the CarbonX for QuEChERS Pigmented Samples tube, followed by spiking of the resulting matrix blank. The analysis was performed in replicates of three (n = 3).
An Agilent 5890 GC/MS was used for this study.
Inlet liner: Agilent Ultra Inert splitless single taper wool p/n 51903163
Carrier gas: Helium
Inlet pressure: 19.6 psi (constant pressure mode) during run
Inlet temperature: 250 ºC
Injection volume: 1.0 µL
Purge flow to split vent: 30 mL/min at 0.75 min
Oven temperature program: 70 ºC (1 min), 50 ºC/min to 150 ºC (0 min), 6 ºC/min to
200 ºC (0 min), 16 ºC/min to 280 ºC (6 min)
Column: Agilent J&W HP-5MS Ultra Inert 15 m × 0.25 mm, 0.25 µm (p/n 19091S-431UI)
MSD (Agilent 5973)
Mode: SIM (refer to Table 1 for settings in detail) and Scan (50-550 m/z)
Source, quad, transfer
line temperature: 230 ºC, 150 ºC and 280 ºC respectively
Solvent delay: 2.30 min
Multiplier voltage: Auto-tune voltage
Comparison of Scan Mode Baselines
Figure 1. Scan mode (50 to 550 m/z) comparison of GCB (top) and CarbonX for QuEChERS (bottom).
Figure 2. Scan mode (50 to 550 m/z) of modified GCB toluene method (top) and CarbonX for QuEChERS (bottom).
It is clear that there is significantly less material eluting from the matrix cleaned up with CarbonX for QuEChERS, especially early on in the chromatogram. When the modified GCB with toluene method is run, to elute the planar pesticides that CarbonX for QuEChERS elutes without the need for toluene, the later eluting contaminants also show up.
Figure 3. SIM mode comparison of CarbonX for QuEChERS (top), GCB (middle), and modified GCB Toluene method (bottom).
Once again it is clear that the lower baseline with CarbonX for QuEChERS leads to better resolved peaks than GCB, either using the original or modified toluene method. This improved baseline is concurrent with excellent recoveries, as seen below.
Figure 4. Recovery percentages of 18 GC amenable pesticides by CarbonX for QuEChERS (blue) GCB with the original method (purple), and GCB with the modified toluene method (yellow). Note the overly high recoveries for phenylphenol for GCB, as well as the low recoveries for planar pesticides Chlorothalonil, Folpet, and Coumaphos.
CarbonX for QuEChERS gives excellent recoveries of 18 GC/MS amenable pesticides after cleanup of pigments without the need for the addition of toxic solvents such as toluene. In addition, the baseline seen both in Scan and SIM mode is an improvement over the baseline obtained by GCB cleanup.
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Table 1 – SIM acquisition parameters used for the analysis of 18 pesticides by GC/MS