UPLC-MS/MS法测定毛蕊花糖苷干预不育症模型大鼠的睾酮水平  

Determination of the testosterone levels in infertile rats treated with acteoside by UPLC-MS/MS

作　　者：王志[1] 居博伟 文丽梅 杨建华[1] WANG Zhi;JU Bowei;WEN Limei;YANG Jianhua(Department of Pharmacy,the First Affiliated Hospital of Xinjiang Medical University,Urumqi 830054,China;Department of Pharmacy,the Fifth Affiliated Hospital of Xinjiang Medical University,Urumqi 830011,China)

机构地区：[1]新疆医科大学第一附属医院药学部,乌鲁木齐830054 [2]新疆医科大学第五附属医院药学部,乌鲁木齐830011

出　　处：《西北药学杂志》2024年第3期21-27,共7页Northwest Pharmaceutical Journal

基　　金：国家自然科学基金项目(编号:82160772);新疆维吾尔自治区科技厅科技创新领军人物后备人选项目(编号:2019XS14);新疆维吾尔自治区自然科学基金项目(编号:2022D01C260)。

摘　　要：目的建立超高效液相色谱-串联质谱法(ultra high performance liquid chromatography tandem mass spectrometry,UPLCMS/MS)检测毛蕊花糖苷干预不育症模型大鼠血浆睾酮的含量,测定不育症模型大鼠血浆24 h睾酮含量,并借助药动学理论定量评价干预效果。方法大鼠含药血浆以睾酮-C3为内标,用液液萃取法,以12000 r·min^(-1)离心10 min,取上清液,5μL进样分析。以Acquity UPLCⅢBEH C_(18)(50 mm×2.1 mm,1.7μm)为色谱柱;以甲醇为流动相A、5 mL·L^(-1)甲酸溶液为流动相B进行梯度洗脱,洗脱程序:流速为0.2 mL∙min^(-1);柱温为35℃;样品室温度为4℃;运行时间为10 min;质量检测器使用ESI离子源,正模式检测,睾酮及内标睾酮-C3用于定量的母→子离子对分别为m/z 289.2→97.0、m/z 291.4→101.1。借助药动学软件分析,获取空白组和毛蕊花糖苷干预组大鼠体内24 h睾酮曲线下面积(area under the curve,AUC)数据。结果睾酮质量浓度在1~1000 ng·mL^(-1)范围内呈良好的线性关系。该方法日内、日间精密度RSD值均<10.84%,日内、日间准确度RE均<10.03%,样品提取回收率为95.82%~99.17%,置于进样器于4℃冰箱及长期冷冻稳定性RSD值均<10.63%。测定结果显示,毛蕊花糖苷干预组药峰浓度(peak concentration,C_(max))为7.92 ng·mL^(-1),空白组C_(max)为10.26 ng·mL^(-1)。与空白组比较,毛蕊花糖苷干预组大鼠体内睾酮24 h总AUC相对平稳。结论建立了UPLC-MS/MS测定不育症模型大鼠血浆睾酮含量的方法。从PK/PD角度比较分析推测出,给予毛蕊花糖苷干预后,不育症模型大鼠的24 h总体睾酮可基本恢复正常水平,从而发挥改善大鼠雄性生殖功能的作用。Objective To establish an ultra-high performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS)method for the detection of plasma testosterone level in infertility model rats treated with acteoside(ACT),to determine the 24-hour plasma testosterone level of infertility model rats,and to evaluate the intervention effect quantitatively by pharmacokinetic theory.Methods Testosterone C3 as the internal standard,the plasma was centrifuged at 12000 r·min^(-1) for 10 minutes by liquid-liquid extraction method.The supernatant was taken and 5μL was injected for analysis.The determination was performed on an Acquity UPLⅢBeh C_(18)(50 mm×2.1 mm,1.7μm)column.Gradient elution was performed with methanol as mobile phase A and 5 mL·L^(-1) formic acid solution as mobile phase B.The flow rate was 0.2 mL·min^(-1),the column temperature was 35℃,and the sample room temperature was 4℃.The running time was 10 min with ESI ion source in positive mode.The quantitative precursor to fragment ion pairs of testosterone and internal standard testosterone-C3 were m/z 289.2→97.0 and m/z 291.4→101.1,respectively.By means of pharmacokinetic software analysis,the 24-hour testosterone AUC data of rats in blank group and ACT intervention group were obtained.Results The mass concentration of testosterone showed a good linear relationship in the range of 1—1000 ng∙mL^(-1).The method precision RSDs of intra-day and inter-day were<10.84%,and the method accuracy REs of intra-day and interday were<10.03%.The recoveries of sample extraction were 95.82—99.17%.The RSDs of long-term freezing stability were<10.63%when the samples were stored in the 4℃refrigerator.The results showed that in the intervention group,the C_(max) was 7.92 ng·mL^(-1).The C_(max) of the blank group was 10.26 ng·mL^(-1).Compared with the blank group,the 24-hour total AUC of testosterone in the ACT intervention group was unchanged.Conclusion A method for the determination of plasma testosterone in infertile rats by UPLC-MS/MS was established.From th

关 键 词：毛蕊花糖苷 不育症 睾酮 评价 超高效液相色谱-质谱联用法 

分 类 号：R927.2[医药卫生—药学]