3′, 5′ – Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger that is involved in many cellular functions and biological processes. In several cell types, cholera toxin will increase the level of cAMP, which mediates toxic effects on cells. In this context, we have developed a fast and simple method based on extraction with 5% trichloroacetic acid (TCA) and quantitation with liquid chromatography-mass tandem spectrometry (LC-MS/MS) for measuring cAMP in cells. A main feature of the LC-MS method was employing a reversed phase C18 column (2.1 mm × 50 mm, 1.6 µm particles) compatible with a 100% aqueous mobile phase, providing retention of the highly polar analyte. Isocratic separations allowed for fast subsequent injections. Negative mode electrospray ionization detection was performed with a triple quadrupole (QqQ)MS. cAMP was extracted from cell samples (~10⁶ cells per well) and spiked with a labelled internal standard, using 200 µL of 5% TCA. The extraction solvent was fully compatible for direct injection onto the reversed phase column. After 10 min incubation, the supernatant was removed, and 10 µL of the supernatant was directly analysed by LC-MS. The method was characterized by the simplicity of the extraction, and the speed (3 min retention time of cAMP), sensitivity (250 pg/mL detection limit), and selectivity (separation from interferences e.g. isomeric compounds) of the LC-MS method, and could be used for quantitation of cAMP in the range 1–500 ng/mL cell extract.

3'，5'–环状单磷酸腺苷（cAMP）是一种普遍存在的第二信使，涉及许多细胞功能和生物学过程。在几种细胞类型中，霍乱毒素会增加cAMP的水平，从而介导对细胞的毒性作用。在这种情况下，我们已经开发了一种快速简单的方法，该方法基于5％三氯乙酸（TCA）的提取和液相色谱-串联质谱（LC-MS / MS）的定量方法来测量细胞中的cAMP。LC-MS方法的主要特点是采用反相C18色谱柱（2.1 mm×50 mm，1.6 µm颗粒），与100％水性流动相兼容，可保留高极性分析物。等度分离允许快速后续注射。负模式电喷雾电离检测使用三重四极杆（QqQ）MS进行。从细胞样品中提取cAMP（〜10 ⁶每孔细胞数），并用200 µL 5％TCA加标有标记的内标。萃取溶剂与直接注入反相色谱柱完全相容。温育10分钟后，除去上清液，并通过LC-MS直接分析10μL上清液。该方法的特点是萃取简单，LC-MS方法的速度（cAMP的保留时间为3分钟），灵敏度（检测限为250 pg / mL）和选择性（与干扰物（例如，异构体分离）），可用于定量1–500 ng / mL细胞提取物中的cAMP。