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为分析成年花鳗鲡经多次注射鲤鱼脑垂体(carp pituitary extract,CPE,每周12 mg/kg)和人绒毛膜促性腺激素(h CG,每周300 IU/kg)后,卵巢发育成熟过程中6种性类固醇激素的含量及变化,实验采用超高效液相色谱—串联质谱(UPLC/MS/MS)联用方法对花鳗鲡性腺内这6种类固醇激素进行研究。结果发现,睾酮(testosterone,T)、孕酮(progesterone,P)、雌二醇(estradiol-17β,E2)、雌三醇(estriol,E3)、17α,20β-二羟基-4-孕烯-3-酮(17α,20β-dihydroxy-4-pregnen-3-one,DHP)、17α-羟基孕酮(17α-hydroxyprogesterone,17α-OHP)在x Bridge C18色谱柱上得到良好分离,在0~200 ng/m L的范围内线性相关系数均大于0.99,检测限为0.1~0.5 ng/g,回收率为89.00%~94.83%,相对标准差均小于20%;注射外源激素后,6种性类固醇激素在花鳗鲡卵巢中的含量及变化情况如下:第2次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(0.27±0.05)、(0.64±0.05)、0、(1.17±0.19)、0、0 ng/g;第9次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(0.73±0.13)、(1.28±0.38)、(1.27±0.27)、(0.83±0.14)、(1.50±0.59)、(0.43±0.25)ng/g;第16次注射后,T、P、E2、E3、DHP、17α-OHP的含量分别为(1.17±0.14)、(2.23±0.51)、(5.59±0.96)、(2.46±0.70)、(2.29±0.65)、(4.56±0.74)ng/g。对照组中均未检测到雌二醇和雌三醇。研究表明,雌二醇和雌三醇是花鳗鲡性腺发育的关键因素;随着性腺发育,花鳗鲡卵巢中6种性类固醇激素的含量整体呈逐步增加的趋势。另一方面,UPLC/MS/MS法能同时定量测定这6种性类固醇激素,具有灵敏、高回收率、稳定的优势。
In order to analyze the process of ovarian development and maturation after multiple injections of carp pituitary extract (CPE, 12 mg / kg weekly) and human chorionic gonadotropin (h CG, 300 IU / kg weekly) In six gonadal hormones of Eel (Anguilla anguilla) were studied by using UPLC / MS / MS. As a result, it was found that testosterone (T), progesterone (P), estradiol-17β (E2), estriol (E3), 17α, 20β-dihydroxy- 17 -hydroxy progesterone (17α-hydroxyprogesterone, 17α-OHP) was well separated on x Bridge C18 column, The linear correlation coefficients were all greater than 0.99 with the detection limits of 0.1-0.5 ng / g and the recoveries ranged from 89.00% to 94.83% with the relative standard deviations (RSDs) less than 20%. After injection of exogenous hormones, The contents and changes of steroid hormones in the ovary of Anguilla japonica were as follows: The contents of T, P, E2, E3, DHP and 17α-OHP after the second injection were 0.27 ± 0.05, 0.64 ± 0.05, (1.17 ± 0.19) and 0,0 ng / g, respectively. The contents of T, P, E2, E3, DHP and 17α-OHP after the 9th injection were 0.73 ± 0.13 and 1.28 ± 0.38, 1.27 ± 0.27, 0.83 ± 0.14, 1.50 ± 0.59 and 0.43 ± 0.25 ng / g, respectively. The contents of T, P, E2, E3, DHP and 17α-OHP after the 16th injection were 1.17 ± 0.14, 2.23 ± 0.51, 5.59 ± 0.96, 2.46 ± 0.70, 2.29 ± 0.65 and 4.56 ± 0.74 ng / g, respectively. Estradiol and estriol were not detected in the control group. The results showed that estradiol and estriol were the key factors of gonadal development in Anguilla japonica. With the development of gonads, the contents of six sex steroid hormones in the ovary of Anguilla japonica tended to increase gradually. On the other hand, the UPLC / MS / MS method can simultaneously determine these six sex steroid hormones, with the advantages of high sensitivity, high recovery and stability.