The typical pesticide residue set at DuPont contains eight samples and usually takes an eight-hour workday to complete. The goal of this study is to take advantage of recent developments in sample extraction with a new laboratory grinder–mill and improved instrument sensitivity and selectivity using liquid chromatography–tandem mass spectrometry (LC–MS-MS) to increase the number of samples analyzed per day by a factor of three. The methodology conforms to the U.S. Environmental Protection Agency (EPA) OPPTS (Office of Prevention, Pesticides and Toxic Substances) Residue Chemistry Test Guidelines — OPPTS 860.1340 Residue Analytical Methods (1). These were developed by the EPA as guidance for developing analytical methods to be submitted to the agency for review under federal regulations and dictate that average recoveries must be 70–120% with good reproducibility.

Sample Preparation

Traditional Pesticide Analysis Using Liquid Chromatography

For the analysis of cymoxanil, the acetonitrile aliquot is back extracted with hexane, concentrated, diluted with water, and passed through a conditioned strong anion exchange solid-phase extraction (SPE) cartridge stacked on top of a conditioned carbon black SPE cartridge. Cymoxanil passes through the strong anion exchange cartridge and is retained on the carbon black cartridge. It is then selectively eluted off the cartridge, and the concentrated cymoxanil residues are dissolved in a hexane–ethyl acetate mixture. The resulting solutions are passed through silica SPE cartridges. Cymoxanil is retained, selectively eluted, and concentrated into a methanol–water mixture adjusted to pH 3. Samples are filtered and analyzed by high performance liquid chromatography (HPLC) with UV detection.

Sample Preparation for Detection by LC–MS

There are approximately 20 clean-up steps in the AMR 3705-95 procedure, which limits the number of samples to 8–10 per day. A modification to this procedure, laid out in Dupont 13753, Analytical Method for the Determination of Cymoxanil and its Metabolites in Leafy Vegetables Using LC/MS" (3), significantly reduces the number of clean-up steps. In this procedure, samples are extracted using an acetonitrile–water mixture as described in DuPont Report No. AMR 3705 9, Revision 2. For cymoxanil, sodium chloride is added to an aliquot to separate the aqueous phase from the organic phase. The aqueous phase is discarded, and the acetonitrile layer containing cymoxanil is passed through a strong anion exchange SPE column. The extract is then further cleaned up using a hexane liquid–liquid extraction followed by an Envi Carb (Supelco, Bellefonte, Pennsylvania) SPE column. Cymoxanil is not retained on either of these columns, so the eluent is passed directly into an LC–MS system for analysis. Based upon the sensitivity and selectivity of LC–MS, the number of sample cleanup steps is reduced to approximately 10, allowing for the analysis of up to 16 samples per day.

Novel Approach to Sample Preparation

To further reduce the clean-up steps, a new sample preparation technique was evaluated that used a Geno/Grinder Model 2000 laboratory mill (Spex SamplePrep, Metuchen, New Jersey). This is a laboratory mill or grinder specifically designed for vigorous vertical shaking of deep-well plates. It originally was designed to prepare plant tissue for extractions of nucleic acid, protein, and other constituents by shaking the tissue, steel balls, and a buffering agent together in each well of a titer plate. The application of this tool can be expanded to include micro organisms when small silica beads are used instead of steel grinding balls. Microbes also can be disrupted in standard 96-well plates as opposed to deep well plates. Sample material that can be prepared includes bacteria, yeasts, molds, seeds, stems, roots, leaves, and certain animal tissue. Because the vertical shaking motion of the equipment is so strong, many seeds and other forms of plant tissue can also be pulverized dry with the help of one or two grinding balls per well.