Department of Biological Sciences, Florida International University, Miami, Florida, United States of America.
PLoS One. 2012;7(8):e43784. doi: 10.1371/journal.pone.0043784. Epub 2012 Aug 22.
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.
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).
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 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.
The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.
保幼激素（JH）是一种倍半萜烯化合物，在昆虫的繁殖、发育和行为中起着核心作用。JH 及其前体具有亲脂性，组织中浓度低，易降解，这使得它们的定量检测变得困难。目前存在多种 JH 定量方法，但能定量检测到飞摩尔级别的方法却很少。目前应用的方法既昂贵又耗时。在本研究中，我们试图开发一种新的方法，用于准确检测和定量 JH 及其前体。
开发了一种灵敏而稳健的方法，用于定量检测生物样本中的前体法呢酸（FA）和保幼激素 III（JH III）。该方法基于用荧光标签对分析物进行衍生化，然后通过反相高效液相色谱法（HPLC）与荧光检测器（HPLC-FD）相结合进行分析。FA 的羧基与 4-乙酰氨基-7-巯基-2,1,3-苯并恶二唑（AABD-SH）衍生化。JH III 的环氧化物基团需要两步反应进行标记：用硫化钠打开环氧化物环，然后用荧光标签 4-（N，N-二甲氨基磺酰基）-7-（N-氯甲酰基-N-甲基氨基）-2,1,3-苯并恶二唑（DBD-COCl）衍生化。
本研究中开发的方法具有高灵敏度、准确性和重现性。在 10-20 到 1000 fmols 的范围内获得线性响应。JH III 的回收率超过 90%，FA 的回收率超过 98%，重现性良好。
当需要检测有限量的样品且需要进行灵敏检测和准确定量时，该方法具有应用价值。例如，埃及伊蚊的前脑神经分泌细胞、血淋巴和成年雌蚊的整体，以及黑腹果蝇的整体都可以采用这种方法。还可以针对其他各种功能基团添加荧光标签，对剩余的 JH III 前体进行标记。
