Rapid quantification of juvenile hormones and their metabolites in insect haemolymph by liquid chromatography-mass spectrometry (LC-MS).

A simple, fast and sensitive method was developed for routine determination of juvenile hormone (JH), JH diols and JH acids in insect haemolymph, by liquid chromatography-mass spectrometry (LC-MS). Sample clean-up involves the precipitation of proteins by methanol/isooctane (1:1, v/v), centrifugation and partial evaporation of the organic solvents. Since JH is bound to a carrier protein in the haemolymph, a binding protein (BP) assay was performed to ensure JH is removed during precipitation. The JH compounds were separated on a C(18) column (ReproSil-Pur ODS-3) by gradient elution with water and methanol in less than 22 min and analysed by electrospray mass spectrometry. Due to the high abundance of Na(+) in insect haemolymph, [M+Na](+) is primarily formed. The limit of detection and quantification was 6 and 20 pg for JHs, and 8 and 25 pg for JH diols, respectively. To demonstrate the applicability of the method to different insect orders, haemolymph samples from the Mediterranean field cricket ( Gryllus bimaculatus), the fall armyworm ( Spodoptera frugiperda), the pea aphid ( Acyrthosiphon pisum) and an ant species ( Myrmicaria eumenoides) were analysed.