Rapid Cleanup of Spinach Extracts Using Gravity Elution SPE Prior to Pesticide Analysis

By: Katherine K. Stenerson and Jennifer Claus, Reporter US Volume 31.1

Prior to analysis for pesticide residues, extracts of highly pigmented foods, such as spinach, require a cleanup step for removal of matrix materials. Failure to do so can lead to ion suppression in LC-MS, inlet contamination in GC-MS, and column contamination for both techniques. A dual-layer SPE tube containing graphitized carbon and aminopropyl on silica (NH2) is often used.

  • Carbon removes pigments and sterols
  • NH2 removes fatty acids, polar pigments, organic acids, and sugars

Gravity Elution

Sample processing can be done either by applying a vacuum to pull the sample/solvents through the SPE tube, or by allowing gravity to pull the sample/solvents through. Gravity elution has the advantage of allowing more contact time between the extract and the sorbents. However, vacuum elution is more widely practiced due to limitations in sorbent particle technology.

Spherical Particles

Current dual-layer SPE tubes contain granular materials. These materials have variations in particle size and shape, which can lead to slower flow, and more inconsistency in flow characteristics between tubes. Fines, which also contribute to flow and consistency issues, can be formed during manufacture and/or shipping due to the friable nature of granular materials.

A new dual-layer SPE tube, Supel™ Sphere Carbon/NH2, was developed that uses spherical materials for both the carbon and NH2 layers, providing fast, more consistent flows compared to tubes containing granular material. This allows gravity elution (instead of vacuum elution) to be applied, resulting in more contact time between the extract and the sorbents.

In this work, Supel Sphere Carbon/NH2 tubes were compared to traditional tubes containing granular materials for flow characteristics, removal of matrix interferences and pesticide recovery for spinach extracts. The physical difference between spherical and granular materials is illustrated in Figure 1.

Spherical vs. Granular Materials

Figure 1. Spherical vs. Granular Materials

Experimental

Extraction and cleanup procedures derived from two published methodologies1,2 are summarized in Table 1. Replicates of unspiked and spiked (5 ng/g) spinach samples were prepared, and allowed to sit for one hour prior to extraction. After extraction, extracts were split for cleanup using a tube containing spherical materials or a tube containing granular materials. Following cleanup, analysis was performed by GC-MS/SIM using large volume injection (LVI).

Quantitation was done using matrix-matched standards. The list of compounds evaluated included organophosphorus, organochlorine, acidic and hydrophobic pesticides.

Table 1. Extraction and Cleanup Procedures

  1. Place 10 g of homogenized spinach (spike added if a spiked replicate) and 10 mL of acetonitrile (Product No. 34481) into a 50 mL centrifuge tube (Product No. 55248-U), then shake for one minute.
  2. Add content of an Acetate Extraction Tube (Product No. 55234-U), then shake for one minute.
  3. Centrifuge at 3,200 rpm for five minutes.
  4. Transfer the supernatant to a 12 mL tube containing 1 g of anhydrous magnesium sulfate (Product No. 63135).
  5. Shake for one minute, then centrifuge at 3,200 rpm for five minutes.
  6. Transfer 5 mL of the supernatant to a glass test tube and evaporate to 1 mL (at 40 °C).
  7. Add 250 µL of toluene (Product No. 34494).
  8. Condition a Supel Sphere Carbon/NH2 Tube (Product No. 54283-U) or a traditional tube with 10 mL of acetonitrile:toluene (75:25).
  9. Add sample extract from step 7, start eluent collection immediately.
  10. Gravity elute tube with 20 mL of acetonitrile:toluene (75:25) and collect all eluent.

Flow Characteristics

The average time required for 25 mL of extraction solvent (75:25 acetonitrile:toluene) to be gravity eluted through each tube type is displayed in Figure 2. The range (slowest and fastest) of the five replicates is depicted by the vertical lines. Average flow times for the granular materials were similar, while flow through the Supel Sphere Carbon/NH2 tubes was 50% faster. In addition, the Supel Sphere tube exhibited the best flow reproducibility between cartridges.

Average Timed Gravity Elution of 25 mL Solvent (n = 5)

Figure 2. Average Timed Gravity Elution of 25 mL Solvent (n = 5)

Removal of Matrix Interference

Full scan GC-MS runs of two extracts after cleanup are shown in Figure 3. The chromatogram of the extract when a Supel™ Sphere Carbon/NH2 tube was used is notably cleaner. A cleaner background will translate to greater sensitivity and less instrument contamination, leading to longer periods of acceptable GC-MS performance before system maintenance is required.

Extracts of Spiked Spinach (full scan mode, same Y-axis)

Figure 3. Extracts of Spiked Spinach (full scan mode, same Y-axis)

Conditions
column
: SLB®-5ms, 20 m x 0.18 mm I.D., 0.36 µm (Product No. 28576-U); oven: 70 °C (2 min), 15 °C/min to 325 °C (5 min); inj. temp.: Programmed, 60 °C (0.28 min), 600 °C/min to 325 °C (5 min); detector: MS, SIM mode; carrier gas: helium, 1 mL/min constant; injection: 10 µL LVI, PTV solvent vent, rapid injection speed; split vent; flow: 100 mL/min (5 psi) until 0.28 min, 60 mL/min at 2.78 min; liner: 4 mm I.D., split/splitless type, single taper FocusLiner™ design (wool packed) (Product No. 28795)

Pesticide Recovery

Table 2 compares average recoveries and %RSD values for three spiked samples after cleanup using Supel Sphere Carbon/NH2 tubes and traditional tubes containing granular materials. As shown, recovery values when Supel Sphere Carbon/NH2 tubes were used were similar or better for most pesticides. Reproducibility was also better for most pesticides.

Table 2. Average % Recovery (%RSD), n=3

Analyte Supel Sphere Tube
(Spherical Material)
Traditional Tube
(Granular Material)
Trifluralin 81 (2) 78 (3)
α-BHC 83 (1) 75 (10)
Thiometon 81 (5) 75 (3)
Hexachlorobenzene 43 (6) 55 (5)
Simazine 82 (2) 81 (4)
Quintozene 74 (8) 71 (6)
γ-BHC 84 (5) 80 (7)
Tolclofos-methyl 83 (4) 80 (3)
Heptachlor 72 (3) 69 (9)
Malathion 83 (11) 90 (5)
Metolachlor 83 (4) 82 (2)
Chloropyrifos 84 (3) 70 (4)
Endosulfan I 84 (8) 66 (11)
Oxadiazon 79 (3) 83 (3)
Ethion 81 (1) 79 (5)
Triazophos 73 (1) 83 (2)
4,4'-DDT 67 (3) 67 (4)
Phosmet 81 (3) 82 (4)
Methoxychlor 71 (4) 80 (3)
Coumaphos 79 (2) 79 (3)
Cyfluthrin isomer 66 (5) 71 (14)
Cypermethrin isomer 88 (5) 72 (8)
Deltamethrin 76 (7) 63 (36)

Conclusion

Gravity elution may be preferred to vacuum elution for SPE as it allows a greater extract-sorbent contact time. When applied to the cleanup of spinach extracts and compared to traditional tubes containing granular materials, Supel Sphere Carbon/NH2 SPE tubes exhibited superior and more consistent flow, removed as much or more background, and resulted in improved recovery and %RSD values for most pesticides.

Legal Information

SLB is a registered trademark of Sigma-Aldrich Co. LLC
Supel is a trademark of Sigma-Aldrich Co. LLC
FocusLiner is a trademark of SGE Analytical Science Pty Ltd.

Materials

     

 References

  1. Multiresidue Method for Agricultural Chemicals by GC/MS (Agricultural Products), Analytical Methods for Residual Compositional Substances of Agricultural Chemicals, Feed Additives, and Veterinary Drugs in Food: Syoku-An No. 0124001; Department of Food Safety, Japanese Ministry of Health, Labour and Welfare: January 24, 2005.
  2. Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate. AOAC Official Method 2007.01.

 

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