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.
作者
杨娜
陈捷
李美娟
葛佳佳
张潇莉
王敏
朱怀军
成晓亮
顾林
葛卫红
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)
出处
《临床检验杂志》
CAS
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)
关键词
雌激素
高效液相色谱-串联质谱法
雌酮
雌二醇
绝经
estrogens
liquid chromatography tandem-mass spectrometry
estrone
estradiol
menopause
