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 (r
2 > 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.