Comparison of radioimmunoassay and liquid chromatography tandem mass spectrometry for determination of juvenile hormone titers

This paper compares the results of juvenile hormone (JH) titer determinations in two insect species, Melanoplus sanguinipes, a migratory grasshopper, and Acyrthosiphon pisum, the pea aphid, using a chiral-specific JH radioimmunoassay (RIA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), after extraction of JH with either hexane or isooctane–methanol.We compared results of JH titer determinations done on extracts of M. sanguinipes hemolymph taken from animals flown to exhaustion in tethered flight tests or unflown controls and from whole body extracts of A. pisum raised at two different temperatures. In each case the two different treatments experienced by the experimental animals were expected to result in widely differing JH titers. Methoprene and precocene II were used as internal standards. Samples were split and titers determined simultaneously with both the LC-MS/MS and RIA procedures. Unambiguous detection of JH III by LC-MS/MS was done by identification of its specific parent ion and its mass fingerprint (m/z 289, 267, 249, 235, 217, and 189). We conclude that isooctane–methanol-extracted JH samples can be accurately analyzed by LC-MS/MS, but not by RIA without further separation of JH from contaminating lipids. Hexane extracted JH samples from hemolymph can be analyzed accurately by both RIA and LC-MS/MS. However, the RIA results from whole body extracts of aphids reared at two different temperatures were initially obscured with excess lipids even when hexane was the extraction solvent. Thus samples were further purified by Waters Sep-Pak C18 column, but contaminating phospholipids continued to cause problems with the RIA assay.The detection limit of JH III standard for RIA was 13.75±2.39 pg whereas that for LC-M/MS was 8.25±1.44 pg in our experimental conditions.