Recovery & Sample Cleanup of Pesticides in Spinach Using Supelclean ENVI-Carb-II/PSA SPE

By: Olga Shimelis, Katherine Stenerson, Reporter US Volume 27.5

Olga Shimelis and Katherine Stenerson


The multi-residue surveillance of pesticides in agricultural/ food products is an ongoing project for regulatory agencies and industrial laboratories worldwide. Hundreds of thousands of samples are analyzed annually to meet a variety of purposes including regulatory enforcement and surveillance monitoring.

A number of procedures have been published for the extraction and analyses of multi-residue pesticides from a variety of food matrixes. Most of which involve an initial liquid-liquid extraction step, and/or solid-liquid extraction step using a water miscible solvent followed by GC analyses. Prior to GC analyses, further sample cleanup is necessary to decrease background levels for trace pesticide detection, reduce matrix-induced signal enhancement, and relieve stress on the GC system.

Supelclean™ ENVI-Carb™-II SPE is a graphitized non-porous carbon (100/140 mesh, surface area 100 m2/g) that has a strong affinity towards planar molecules, and has been quality controlled specifically for the isolation/removal of pigments (e.g. chlorophyll and carotinoids) and sterols commonly present in fruits, vegetables and other natural products.

Supelclean PSA SPE is a polymerically bonded, ethylenediamine- N-propyl phase (Figure 1) that contains both primary and secondary amines. The phase has a strong affinity and high capacity for removing up to 99% of fatty acids (oleic, palmitic and linoleic acid), organic acids, and some polar pigments and sugars when conducting multi-residue pesticide analysis in foods. By removing these interferences, it has been shown to greatly reduce matrixinduced response enhancement encountered during the GC-analyses of food products.

In this article, we offer quantitative data by demonstrating the high recovery and sample cleanup of 18 pesticides from spinach using Supelclean ENVI-Carb-II/PSA SPE and Equity®-1 GC-MS.

Figure 1 Structure of Supelclean PSA SPE

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Technology Description

Food samples are initially extracted/homogenized with a water miscible solvent (e.g. acetonitrile or acetone) in the presence of sodium chloride. The sodium chloride drives phase separation between the solvent and endogenous aqueous residues within the sample. Upon centrifugation/ filtration to remove particulate matter, the acetonitrile layer of the supernatant/filtrate is removed and dried over anhydrous sodium sulfate or magnesium sulfate. At this point, the acetonitrile extract is unsuitable for further analysis due to the high levels of endogenous interferences co-extracted with the pesticides during initial solvent extraction. SPE is necessary for further cleanup prior to GC-MS.

Supelclean ENVI-Carb-II/PSA SPE is a dual-layer SPE cartridge (Figure 2). After the initial acetonitrile extract is concentrated, it is applied to a pre-conditioned ENVI-Carb-II/ PSA SPE tube. The tube acts as a chemical filter in which pigments and sterols are retained on the ENVI-Carb-II layer (graphitized carbon), while fatty acids, organic acids, polar pigments, and sugars are trapped on the PSA (primary-secondary amine) layer. Pesticides are weakly retained on the tube and subsequently eluted with acetonitrile:toluene (3:1). Although the use of a vacuum manifold is recommended, the tubes are also amenable to gravity-driven applications.

Figure 2 Supelclean ENVI-Carb-II/PSA SPE Tube

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Experimental Approach

The performance of the SPE technology was evaluated by spiking spinach with a representative pesticide test mix. The mix represented a range of pesticide classes with varying physicochemical properties. Included within this test mix were highly polar pesticides such as carbaryl, dichlorovos, acepate, and procymidone, which are primarily analyzed by LC.

Pesticides were spiked at the level of 0.2 ppm into 10 g fresh spinach. The mixture was carefully homogenized and extracted with acetonitrile:sodium chloride. The excess of acetonitrile was evaporated to ~1 mL under nitrogen at 40 °C, and the resulting extract was loaded onto the Supelclean ENVI-Carb-II/PSA SPE tube, 500 mg/300 mg/6 mL, preconditioned with 5 mL acetonitrile:toluene (3:1). Pesticide elution was facilitated with 20 mL acetonitrile:toluene (3:1). The SPE eluate was evaporated and reconstituted with 1 mL hexane:acetone (1:1) for subsequent GC-MS analysis.

To compensate for ‘matrix effects’, matrix match standards were prepared by spiking pesticides into blank spinach extracts subjected to the SPE process.

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High Recoveries and Good Sample Cleanup

Recovery for the 18 pesticides tested averaged at 96%. Highly polar pesticides such as dichlorovos and methamidophos may be over-retained on the PSA sorbent due to secondary normal-phase (polar-polar) interactions resulting in poor recovery. At least 70% recovery was observed for all pesticides tested In this application example with the exception of acepate (60% recovery). Table 1 lists recovery values for the pesticides tested.

Table 1 Recovery of Pesticides from Spinach Using Supelclean ENVI-Carb-II/PSA SPE

Excellent sample cleanup was observed signified by sharp peaks and low background (Figure 3).

Figure 3 Pesticides Spiked into Spinach Followed by Supelclean ENVI-Carb-II/PSA SPE (28046-U)

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Dual-layer SPE cartridges provide efficient sample cleanup of food samples, decreasing background interference levels during GC-MS analysis. Recoveries for most of the more challenging polar pesticides were 70% or greater. Table 2 describes the benefits of dual-layer SPE technology for pesticide analysis in food/agricultural products.

Table 2 Benefits of Supelclean ENVI-Carb-II/PSA SPE

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