Pesticides are harmful to human health and so methods are needed to measure their amounts in foods. Many countries have adopted the Positive List System, which sets limits on the amounts of residual pesticides in foods. Mass spectrometry (MS) is commonly used to analyze hundreds of pesticides in agricultural products. The QuEChERS (quick, easy, cheap, effective, rugged, and safe) method was developed as a simple extraction and purification method for this purpose (Anastassiades et al. 2003; Lehotay et al. 2005). Since extracting pesticides from solid agricultural products is inefficient, the QuEChERS method ignores water solubility, hydrophobicity, and the functional groups of pesticides, and extracts pesticides with a medium polar organic solvent such as acetone or acetonitrile. A problem with MS is that the matrix can interfere with pesticide measurement. Although purification methods are being improved (Rutkowska et al. 2018; Lucini and Molinari 2011) and automation is being introduced, the solvent extraction process is complicated and requires large equipment.
Enzymes can be used to solubilize components in agricultural products, and can reduce the need for large equipment. However, in food analysis, which requires speed, there is no time for various enzymes to act sequentially under their optimum conditions. Kasai et al. have proposed autoclaving as a method that does not require enzymes such as pectinase to degrade the lamellar structure of plant tissues. Subsequent enzymatic digestion of the cell wall does not require much time (Kasai et al. 2006; Kasai et al. 2004). We thought that this technique of autoclaving and cell wall enzyme digestion could be used to rapidly extract pesticides from plant tissues.
Diquat and paraquat are typical bipyridinium with a wide plant spectrum (Pesticide Manual Online). They have been used for pre- and post-planting weed control in many crops such as potatoes, olives, apples, and tomatoes (Pateiro-Moure et al. 2013; European Food Safety Authority, 2015). The physical properties of diquat and paraquat, such as ease of handling, low vapor pressure, high water solubility, and high soil-binding capacity, coupled with their strong herbicidal activity, make them suitable for widespread use in agriculture (Taguchi et al. 1998; EU pesticide database). Bipyridinium herbicides are characterized by strong ionic properties and associated adsorption, and have been subjected to drastic extraction, such as prolonged reflux with sulfuric acid (Guijarro et al. 1987; Anderson and Boseley 1997; Japanese official method) or microwave extraction (Pateiro-Moure et al. 2008; Winnik et al. 2009). Recently, the Quick Polar Pesticides method (EURL-SRM QuPPe-PO Method for products of Plant Origin) has been used to extract bipyridinium herbicides using methanol containing hydrochloric acid and heated, but its extraction from lentils is reported to be insufficient. In this study, we developed a highly sensitive MS/MS detection method for bipyridinium herbicides, which were difficult to retain and detect by conventional LC-MS/MS measurements, and examined the applicability of the enzymatic solubilization method to the analysis of these herbicides.