Accurate Analysis of Tricarboxylic Acid Cycle Metabolites and Anion Components in Hemocytes by IC-CD/ESI-MS for Quantifying Insecticide Impairment on Cellular Immunity in Mythimna separata.

Insecticides are more broadly known to affect insect cellular immunity, but the components in hemocytes and their response to insecticide stress are still unknown. In this paper, a method based on trifluoroacetic acid extraction, followed by IC-CD/ESI-MS analysis, was developed to simultaneously determine tricarboxylic acid (TCA) cycle metabolites and anion components in hemocytes from Mythimna separata larvae. Validation gave excellent selectivity, recovery (88.7-107.6%), linear correlation (r2 > 0.9961), precision (<3.89%), LOD (0.002-0.006 mg/L), LOQ (0.006-0.020 mg/L), and a short chromatographic run. The method was verified by administration of 4-((3-chloro-4-fluorophenyl)amino)-7-methoxyquinazolin-6-yl 3-(1,3-dioxoiso-indolin-2-yl) propanoate (QDP) or emamectin benzoate (EMB) to hemocytes in vitro and larvae in vivo. TCA metabolites including citrate, α-ketoglutarate, fumarate, malate, and oxaloacetate, and anions including acetate, oxalate, chloride, carbonate, and sulfate were identified and clearly separated. QDP and EMB showed a biphasic dose effect on TCA metabolites, and the contrary hormesis paralleled the different actions of QDP and EMB. The inhibition or improvement of cellular immunity depended on the QDP concentration. In conclusion, a highly sensitive, reliable, and robust method was developed, enabling the monitoring of hemocyte immunity by the quantification of TCA metabolites and anion components in minute hemocyte samples.