a quantitative assay for the juvenile hormones and their precursors using fluorescent tags定量分析为保幼激素及其使用荧光标记前体.pdfVIP

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags Crisalejandra Rivera-Perez, Marcela Nouzova, Fernando G. Noriega* Department of Biological Sciences, Florida International University, Miami, Florida, United States of America Abstract Background: The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors. Methods: A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl). Conclusions: The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fm