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 the perspective of PK/PD comparative analysis,it was inferred that after the intervention of acteoside,the 24 hour total testosterone of the infertility model rats could basically return to normal level,so as to improve the male reproductive function.
作者
王志
居博伟
文丽梅
杨建华
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)
出处
《西北药学杂志》
CAS
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)。
