Westerlund Stephanie A, Hoffmann Klaus H
Department of Animal Ecology I, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, Germany.
Anal Bioanal Chem. 2004 Jun;379(3):540-3. doi: 10.1007/s00216-004-2598-x. Epub 2004 Apr 1.
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.
开发了一种简单、快速且灵敏的方法，用于通过液相色谱 - 质谱联用（LC - MS）对昆虫血淋巴中的保幼激素（JH）、JH二醇和JH酸进行常规测定。样品净化包括用甲醇/异辛烷（1:1，v/v）沉淀蛋白质、离心以及部分蒸发有机溶剂。由于JH与血淋巴中的载体蛋白结合，因此进行了结合蛋白（BP）测定，以确保在沉淀过程中JH被去除。JH化合物在C(18)柱（ReproSil - Pur ODS - 3）上通过水和甲醇的梯度洗脱在不到22分钟内分离，并通过电喷雾质谱进行分析。由于昆虫血淋巴中Na(+)含量高，主要形成M + Na。JH的检测限和定量限分别为6 pg和20 pg，JH二醇的检测限和定量限分别为8 pg和25 pg。为了证明该方法对不同昆虫目的适用性，分析了地中海田蟋蟀（Gryllus bimaculatus）、草地贪夜蛾（Spodoptera frugiperda）、豌豆蚜（Acyrthosiphon pisum）和一种蚂蚁（Myrmicaria eumenoides）的血淋巴样品。
