A rapid quantitative assay for juvenile hormones and intermediates in the biosynthetic pathway using gas chromatography tandem mass spectrometry

Abstract

A method for rapid quantitation of insect juvenile hormones (JH) and intermediates in the biosynthetic pathway, both in vitro and in vivo (hemolymph and whole body), has been developed using GC–MS/MS. This method is as simple as the radiochemical assay (RCA), the most commonly used method for measurement of JH biosynthesis in vitro, without need for further purification and derivatization, or radioactive precursors or ligands. It shows high sensitivity, accuracy and reproducibility. Linear responses were obtained the range of 1–800 ng/mL (approximately 4–3000 nM). Recovery efficiencies for farnesol, farnesal, methyl farnesoate and JH III were approximately 100% in vitro and over 90% in vivo, with excellent reproducibility at three different spike levels. Titer of JH III in the hemolymph was relatively low at day 0 (adult female emergence) (79.68 ± 5.03 ng/mL) but increased to a maximum of 1717 ng/mL five days later. In whole body, JH III quantity reached a maximum on day 4 (845.5 ± 87.9 ng/g) and day 5 (679.7 ± 164.6 ng/g) and declined rapidly thereafter. It is in agreement with the hemolymph titer changes and biosynthetic rate of JH in vitro. Comparison with the results of inhibition of JH biosynthesis by two known inhibitors (allatostatin (AST) mimic H17 and pitavastatin) using RCA and GC–MS/MS, showed that there was little difference between the two methods In contrast to other methods, the present method with GC–MS/MS can be used to elucidate the mechanism of inhibition by inhibitors of JH biosynthesis without any derivatization and purification. This method is applicable to screening of JH inhibitors and the study of inhibitory mechanisms with high sensitivity and accurate quantification. It may also be useful for the determination of JH titer in other Arthropods.