Study of the fragmentation of arginine isobutyl ester applied to arginine quantification in Aedes aegypti mosquito excreta

It has been demonstrated that argininolysis and uricolysis are involved in the synthesis and excretion of urea in Aedes aegypti female mosquitoes. To further investigate the metabolic regulation of urea in female mosquitoes, it is desirable to have a rapid and efficient method to monitor arginine (Arg) concentration in mosquito excreta. Thus, a procedure currently used for the identification of Arg in urea cycle disorders in newborn babies was adapted to analyze Arg in A. aegypti excreta. The fragmentation patterns of the isobutyl esters of Arg and (15)N(2)-Arg (labeled at the guanidino group) were explored by electrospray ionization (ESI)-tandem mass spectrometry and fragmentation pathways not described before were characterized. In addition, Arg, (18)O(2)-Arg, (15)N(2)-Arg and (15)N(2)-(18)O(2)-Arg were also analyzed to elucidate some of the minor fragments in greater detail. Mosquito excreta from individual females were collected before and at different times after feeding a blood meal, mixed with (15)N(2)-Arg, an internal standard, and then derivatized as isobutyl esters. Based on the fragmentation mechanisms of Arg standards, studied by MS(2) and MS(3), Arg in the mosquito excreta was successfully analyzed by ESI-multiple reaction monitoring in a triple-quadrupole mass spectrometer. Arg excretion was monitored over a 120 h window before and after feeding female mosquitoes with a blood meal, with the maximum level of Arg excretion observed at 36-48 h post blood feeding. This method provides an efficient and rapid tool to quantify Arg in individual blood-fed mosquitoes, and can be applied to other organisms, whose small size severally limits the use of conventional biochemical analysis.