A method based on high performance liquid chromatography coupled with electronspray ionization/triple quadrupole mass spectrometry(HPLC/QqQ-MS) has been established for the determination of 20-hydroxyecdysone in the haemolymph of Portunus trituberculatus and in the muscle of crustacean (Portunus trituberculatus, Palaernon carincauda Holthuis and Oratosquilla oratoria ).Homobrassinolide was utilized as internal standard. The mass spectrometry was performed in positive mode, using precursor ions at m/z 445.2 and 459.4 as quantitative ions for 20-hydroxyecdysone and 22(S), 23 (S)-homobrassinolide, respectively. The results showed that 20-hydroxyecdysone and 22 (S), 23(S)-homobrassinolide were separated with high efficiency in the selective reaction monitoring (SRM) mode on Hypersil Gold C18column. Linearity of the method was good with correlation coefficients(R 2) of 0. 9978 in the range of 0-50 μg/L. The detection limit was 0.03 μg/L. The recoveries varied from 91.8％ to 99.2％ for the haemolymph and 85.7％ to 91.2％ for the muscle with the relative standard deviations(RSDs) from 7.3％ and 7.1％, respectively. Using this method, 20-hydroxyecdysone in the haemolymph of Portunus trituberculatus collected in Ningbo China, has been determined with content ranged from 2.43 to 2.8( μg/L; the concentration of 20-Hydroxyecdysone in the muscle of crustaceans( Portunus trituberculatus , Palaernon carincauda Holthuis and Oratosquilla oratoria) were determined from 4.8 to 9. 6 ng/g. The method is a powerful tool for comprehensive analysis of biological sample with exclusive selectivity and excellent sensitivity.%在三疣梭子蟹(Portunus trituberculatus)血淋巴中加入高油菜素内酯作为内标,用乙腈去除蛋白,采用高效液相色谱-三重四级杆质谱(HPLC-QqQ MS)对20-羟基蜕皮酮进行分析;对3种甲壳动物(-三疣梭子蟹、脊尾白虾(PaJaemon carincaua'a Holthuis)和口虾蛄(Oratosquilla oratoria))的肌肉则采用甲醇提取、正己烷脱脂,加入高油菜素内酯作为内标进行分析.在电喷雾电离源正离子模式下,分别取m/z 445.2和109.1作为蜕皮激素和内标的定量目标离子进行检测.结果表明,蜕皮激素和内标在Hypersil Gold C18色谱柱上得到良好分离,在O～50μg/L范嗣内线性相关系数为0.9978,方法的检出限为0.03μg/L.三疣梭子蟹血淋巴中蜕皮激素回收率达91.8%～99.2%,相对标准偏差小于7.1%;3种甲壳动物肌肉中蜕皮激素回收率达85.7%～91.2%,相对标准偏差小于7.3%.对采自浙江宁波3批三疣梭子蟹血淋巴及三疣梭子蟹、脊尾白虾和口虾蛄的肌肉进行测定,三疣梭子蟹血淋巴中游离20-羟基蜕皮酮平均浓度在2.43～2.86μg几范围内,而3种甲壳动物肌肉中20-羟基蜕皮酮平均含量在4.8～9.6 ng/g范围内.
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