超高效液相色谱-质谱联用技术检测兔房水中糖皮质激素

Quantitative detection of glucocorticoids by ultra high performance liquid chromatography-mass spectrometry in aqueous humor of rabbit

目的应用超高效液相色谱-质谱联用技术（ultra high performance liquid chromatography-mass spectrometry,UPLC/MS）建立同时检测兔房水中可的松、皮质醇及皮质酮的方法。方法 24只新西兰白兔随机分为3组：对照组：玻璃体内注射0.1mL必施灌注液;曲安奈德组（TA组）：玻璃体内注射0.1mL（4mg）TA;地塞米松组（DEX组）：隔日（共15次）球结膜下注射0.1mL（0.5mg）地塞米松。在注射前,注射后1d、1周、2周、3周和4周检测兔眼眼压。在注射前和注射后第29天抽取房水,应用UPLC/MS检测可的松、皮质醇以及皮质酮的含量。结果注射后1d,TA组眼压（36.6±4.1）mmHg（1kPa=7.5mmHg）较对照组（15.3±2.5）mmHg明显上升,差异有显著统计学意义（P〈0.01）;首次注射后2周,DEX组眼压（20.7±3.6）mmHg较对照组（14.6±1.8）mmHg明显上升,差异也有显著统计学意义（P〈0.01）;TA组和DEX组造模成功。在建立的标准曲线中,可的松、皮质醇在0.16~100.00μg.L-1的浓度范围内线性良好（r〉0.998）,最低检测下限均为0.16μg.L-1;皮质酮最低检测下限为10.00μg.L-1。注射后TA组房水中可的松含量为（1.87±0.23）μg.L-1、皮质醇为（2.13±0.68）μg.L-1、皮质酮为（25.5±5.9）μg.L-1,测定含量均明显高于DEX组的（1.36±0.47）μg.L-1、（1.98±0.55）μg.L-1、（18.3±3.1）μg.L-1和对照组（均低于最低检测下限）,差异均有显著统计学意义（均为P〈0.01）。结论采用UPLC/MS可建立同时定量检测兔房水中可的松、皮质醇和皮质酮含量的方法,其灵敏度高,线性关系好。

Objective To detect cortisone,cortisol and corticosterone spontaneously by ultra high performance liquid chromatography-mass spectrometry（UPLC/MS）in the aqueous humor（AH）of rabbits.Methods Twenty-four rabbits were divided into 3 groups：control group with intravitreal injection of 0.1 mL Bishi perfusate,triamcinolone acetonide（TA）group with intravitreal injection of 0.1 mL（0.4 mg）TA and dexamethasone（DEX）group with sub-conjunctival injection of 0.1 mL（0.5 mg）DEX on every other day.Intraocular pressure（IOP）was measured before injection,at 1 day,1 week,2 weeks,3 weeks and 4 weeks after injection.AH was withdrawn before injection and at the 29th day after injection.Concentrations of cortisone,cortisol and corticosterone were tested by UPLC/MS.Results At 1 day after injection,IOP was（36.6±4.1）mmHg（1 kPa=7.5 mmHg）in TA group,and obviously higher than（15.3±2.5）mmHg in control group,there was significant difference（P0.01）.At 2 weeks after the first injection,IOP was（20.7±3.6）mmHg in DEX group,and higher than（14.6±1.8）mmHg in control group,there was significant difference（P0.01）.Models were successfully made in TA group and DEX group.In the standard curve,cortisone and cortisol had better linear relations between concentrations of 0.16 μg·L-1 to 100 μg·L-1（r0.998）,and the lowest detection for concentration was 0.16 μg·L-1.The lowest detection for concentration was 10 μg·L-1 for corticosterone.After injection,concentration of cortisone was（1.87±0.23）μg·L-1,concentrations of cortisol were（2.13±0.68）μg·L-1 and（25.5±5.9）μg·L-1 in TA group.The detected concentrations were obviously higher than（1.36±0.47）μg·L-1,（1.98±0.55）μg·L-1 and（18.3±3.1）μg·L-1 in DEX group and control group（all were lower than the lowest detect limit）,there were obvious differences（all P0.01）.Conclusion The concentration of cortisone,cortisol and corticosterone in AH of rabbit could be detected by UPLC/MS spontaneously,with good linearity and high sensitivity.