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浊点萃取结合UPLC-MS/MS法测定气道炎症模型豚鼠灌胃射干提取物后血浆中LTB4的浓度
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| 篇名： | 浊点萃取结合UPLC-MS/MS法测定气道炎症模型豚鼠灌胃射干提取物后血浆中LTB4的浓度 |
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| 摘要： | 目的：建立测定气道炎症豚鼠血浆中白三烯B4（LTB4）浓度的方法，考察给予射干提取物后其对豚鼠血浆中LTB4浓度的影响，为阐明射干提取物抗炎止咳的作用机制提供参考。方法：将30只豚鼠随机分为空白组、模型组和射干提取物组，每组10只。除空白组外，其余2组豚鼠采用烟熏+合胞病毒滴鼻法制造气道炎症模型。造模结束后次日，射干提取物组豚鼠灌胃给药（10 mg/kg射干提取物，以野鸢尾黄素计），空白组和模型组豚鼠灌胃等体积生理盐水，每天给药1次，连续给药7 d，末次给药后收集血浆样品。分别考察表面活性剂Triton X-114浓度、盐酸浓度、盐酸加入量、平衡温度和平衡时间对LTB4提取回收率的影响（以44.5 g/L的牛血清白蛋白生理盐水溶液替代空白血浆），确定最优浊点萃取工艺进行生物样品处理。然后以吲哚美辛为内标，采用超高效液相色谱-串联质谱（UPLC-MS/MS）法测定血浆中LTB4浓度。色谱条件：色谱柱为ACQUITY UPLC BEH C18，流动相为0.2％甲酸水-乙腈（40 ∶ 60，V/V），流速为0.4 mL/min，柱温为40 ℃，进样量为5 μL；MS条件：电喷雾离子源，正离子模式下扫描，LTB4用于定量的离子为[M+H]+质荷比（m/z）335.219 4→195.099 8，吲哚美辛为[M+H]+ m/z 356.083 6→312.084 2。结果：浊点萃取最优条件为Triton X-114浓度5％，加入0.3 mol/L盐酸50 μL调至酸性、平衡温度50 ℃，平衡时间20 min。在该含量测定条件下方法学验证结果均符合相关要求。与空白组比较，模型组豚鼠血浆中LTB4浓度显著升高（P＜0.05）；与模型组比较，射干提取物组豚鼠血浆中LTB4浓度显著降低（P＜0.05）。结论：本研究中样品处理方法绿色环保，含量检测方法灵敏度高、准确性好，适用于血浆中LTB4浓度的测定；射干提取物可使气道炎症豚鼠血浆中升高的LTB4浓度得到显著回调。 |
| ABSTRACT： | OBJECTIVE： To establish the method for determining the concentration of leukotriene B4 （LTB4） in plasma of airway inflammatory guinea pigs， investigate the effects after giving Belamcanda chinensis extract on plasma concentration of LTB4 in guinea pigs and provide reference for clarifying the mechanism of anti-inflammatory and antitussive effects of B. chinensis extract. METHODS： Totally 30 guinea pigs were randomly divided into blank group， model group and B. chinensis extract group， with 10 guinea pigs in each group. Except for blank group， airway inflammation model was induced by smokation+syncytial virus intranasal method in other 2 groups. At next day after modeling， guinea pigs were given relevant medicine （10 mg/kg B. chinensis extract， calculated by irigenin） intragastrically in B. chinensis extract group； blank group and model group were given constant volume of normal saline intragastrically， once a day， for consecutive 7 d， and the plasma samples were collected after last administration. Then， the effects of surface active agent Triton X-114 concentration， hydrochloric acid concentration， hydrochloric acid amount. Equilibrium temperature and equilibrium time on extraction recovery rate of LTB4 were investigated （using 44.5 g/mL BAS saline solution instead of blank plasma）. The optimal cloud point extraction technology was determined for biological sample treatment. Using indometacin as internal standard， UPLC- MS/MS method was used to determine the concentration of LTB4 in plasma. Chromatographic conditions： the chromatographic column is ACQUITY UPLC BEH C18， the mobile phase is 0.2％ formic acid-acetonitrile （40 ∶ 60，V/V）， flow rate is 0.4 mL/min， column temperature is 40 ℃， sample volume is 5 μL. MS condition： Electrospray ion source, positive ion mode scanning, and the LTB4 used for the [M+H]+ mass ratio (m/z) 335.219 4→195.099 8， Indomethacin is [M+H]+ m/z 356.083 6→312.084 2. RESULTS： The optimal cloud point extraction condition included Triton X-114 concentration 5％， added into 0.3 mol/L hydrochloric acid 50 μL adjusted to acid and equilibrium temperature 50 ℃， for 20 min. The results of methodology validation were all in line with the content determination requirements. Compared with blank group， the plasma concentration of LTB4 in guinea pigs was increased significantly in model group （P＜0.05）. Compared with model group， the plasma concentration of LTB4 in guinea pigs was decreased significantly in B. chinensis extract group （P＜0.05）. CONCLUSIONS： The method of sample processing is environmental friendly， and the content determination method is high sensitivity and accuracy， and applicable for determination of LTB4 in plasma samples. B. chinensis extract can significantly regulate the increase of LTB4 concentration in plasma of guinea pigs with airway inflammation. |
| 期刊： | 2018年第29卷第15期 |
| 作者： | 杨瑞，邸子真，吴怡，陶弘武 |
| AUTHORS： | YANG Rui，DI Zizhen，WU Yi，TAO Hongwu |
| 关键字： | 浊点萃取；超高效液相色谱-串联质谱法；射干提取物；白三烯B4；豚鼠 |
| KEYWORDS： | Cloud point extraction； UPLC-MS/MS； Belamcanda chinensis extract； Leukotriene B4； Guinea pig |
| 阅读数： | 242 次 |
| 本月下载数： | 12 次 |
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