高效液相色谱-串联质谱法在检测绝经后妇女血清中雌激素及其代谢物中的应用  

Application of ultra-sensitive LC-MS/MS method targeting estrogen metabolites in serum of postmenopausal women

作　　者：杨娜[1] 陈捷 李美娟 葛佳佳 张潇莉 王敏[1] 朱怀军[1] 成晓亮 顾林[2] 葛卫红[1] YANG Na;CHEN Jie;LI Meijuan;GE Jiajia;ZHANG Xiaoli;WANG Min;ZHU Huaijun;CHENG Xiaoliang;GU Lin;GE Weihong(Department of Pharmacy,Nanjing Drum Tower Hospital,The Affiliated Hospital of Nanjing University Medical School,Nanjing 210008,Jiangsu;Nanjing Maternity and Child Health Care Hospital,Nanjing 210001,Jiangsu;Nanjing Qlife Medical Technology Co.,Ltd,Nanjing 210032,Jiangsu,China)

机构地区：[1]南京大学医学院附属鼓楼医院药学部,南京210008 [2]南京市妇幼保健院妇科内分泌科,南京210001 [3]南京品生医疗科技有限公司研发中心技术部,南京210032

出　　处：《临床检验杂志》2022年第10期756-762,共7页Chinese Journal of Clinical Laboratory Science

基　　金：江苏省自然科学基金青年基金(BK20190122);江苏省研究型医院学会精益化用药科研基金(JY202109);南京药学会-常州四药医院药学科研基金(2021YX001)

摘　　要：目的 建立一种高灵敏度的高效液相色谱-串联质谱法(LC-MS/MS),分析绝经后妇女血清中包含雌酮(E1)、雌二醇(E2)及其羟基化、甲基化、葡萄糖醛酸结合/硫酸结合(-G/S)等代谢产物的水平。方法 采用叔丁基甲醚液-液萃取法,结合β-葡萄糖醛酸苷酶/芳基硫酸酯酶水解及丹磺酰氯对萃取后的血清样品进行衍生化处理。以同位素标记的雌酮-d4作为内标,采用Waters UPLCⅠ-Class/Xevo TQS液质联用系统测定。以ACQUITY UPLC BEH C18 (2.1 mm×100 mm, 1.7μm)为色谱柱,水/甲醇(含0.05%甲酸和2 mmol/L甲酸铵)为流动相,0.3 mL/min流速梯度洗脱。柱温为50℃,进样量10μL。电喷雾离子源(ESI)正离子模式下扫描,多重反应监测(MRM)下测定,分析时间7 min。结果 各类雌激素定量下限低至1~2 pg/mL,平均加标回收率在90.3%~111.3%之间。绝经后妇女血清各类雌激素总量远高于临床常规雌激素指标E2的水平,并以E1-G/S和16α-羟基雌酮结合型(16OHE1-G/S)水平相对较高。各类雌激素-G/S结合型水平均不低于其游离型水平。2-羟化和16-羟化通路为优势代谢通路。结论 该方法灵敏度高、分析时间短、特异性强,可用于临床绝经后妇女血清样本中微量雌激素及代谢物的定量分析。Objective To establish an ultra-sensitive liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for comprehensive analysis of the serum levels of estrone(E1),estradiol(E2)and downstream metabolites such as hydroxylated,methylated,glucuronidated/sulfonated(-G/S)metabolites in postmenopausal women.Methods The serum samples were prepared for derivation by tert-butyl methyl ether liquid-liquid extraction combined withβ-glucuronidase/aryl sulfatase hydrolysis and dansyl chloride derivat-ization.By using isotope-labeled estrone-D4 as internal standard,the analysis was achieved on a Waters UPLCⅠ-Class/Xevo TQS LC/MS system.The chromatographic separation was performed using ACQUITY UPLC BEH C18 column(2.1 mm×100 mm,1.7μm)at 50℃with the mobile phase of methanol-water(containing 0.05%formic acid and 2 mmol/L ammonium formate)for a gradient elu-tion at flow rate of 0.3 mL/min with injection volume of 10μL.The MS quantification was scanned in the mode of electrospray ioniza-tion source(ESI)positive ion and detected in multiple reaction monitoring(MRM)with analysis time of 7 minutes.Results The low-er limit of quantification for various estrogens reached 1-2 pg/mL in target serum.The average recovery rate was between 90.3%to 111.3%.The total amount of estrogens in the serum of postmenopausal women was much higher than that of clinical routine E2 of E1-G/S and 16α-hydroxyestrone-G/S conjugate(16OHE1-G/S)were found to be relatively high levels and the levels of each estrogen-conjugated form were all not lower than its those of free form 2-hydroxyl and 16-hydroxyl pathways were the dominant metabolic path-ways of estrogens.Conclusion The developed method showed ultra-high sensitivity,short analysis time and high specificity and could be used for the quantitative analysis of trace estrogen and its metabolites in the serum samples of postmenopausal women.

关 键 词：雌激素 高效液相色谱-串联质谱法 雌酮 雌二醇 绝经 

分 类 号：R446[医药卫生—诊断学]