乳腺癌患者血清雌激素及其代谢产物的液相色谱串联质谱检测方法的建立及应用  

Establishment and application of a liquid chromatography-tandem mass spectrometry method for the detection of serum estrogens and its metabolites in breast cancer patients

作　　者：郝志翔 钟雅男 徐吟雪 印晓星 张蓓[3] 周雪妍 HAO Zhi-xiang;ZHONG Ya-nan;XU Yin-xue;YIN Xiao-xing;ZHANG Bei;ZHOU Xue-yan(Jiangsu Key Laboratory of New Drug Research and Clinical Pharmacy,School of Pharmacy,Xuzhou Medical University,Xuzhou 221004,China;Department of Pharmacy,Xuzhou First People’s hospital,Xuzhou 221000,China;Department of Obstetrics and Gynecology,Xuzhou Central Hospital,Xuzhou 221009,China)

机构地区：[1]徐州医科大学药学院江苏省新药研究与临床药学重点实验室,徐州221004 [2]徐州市第一人民医院药学部,徐州221000 [3]徐州市中心医院妇产科,徐州221009

出　　处：《药物分析杂志》2022年第10期1739-1753,共15页Chinese Journal of Pharmaceutical Analysis

基　　金：江苏省高等学校自然科学研究重大项目(18KJA350002);江苏省自然科学基金面上项目(SBK2018021825);徐州市推动科技创新专项资金项目(KC18044)。

摘　　要：目的:建立稳定可靠的液相色谱串联质谱(LC-MS/MS)方法,检测雌激素雌酮(E_(1))、雌二醇(E_(2))及其代谢产物2-甲氧基雌酮(2-MeOE_(1))、4-甲氧基雌酮(4-MeOE_(1))、2-甲氧基雌二醇(2-MeOE_(2))、4-甲氧基雌二醇(4-MeOE_(2))、16α-羟基雌酮(16α-OHE_(1))、2-羟基雌酮(2-OHE_(1))、4-羟基雌酮(4-OHE_(1))、2-羟基雌二醇(2-OHE_(2))、4-羟基雌二醇(4-OHE_(2))共11个目标分析物的浓度水平,观察乳腺癌患者血清雌激素稳态的变化规律。方法:利用丹磺酰氯作为衍生化试剂,对待测物进行衍生化处理,采用Agilent ZORBAX Extend-C_(18)色谱柱(100 mm×2.1 mm,1.8μm),以含0.1%甲酸的乙腈溶液和含0.1%甲酸的水溶液为流动相,梯度洗脱,流速0.3 mL·min^(-1),柱温为25℃,进样量30μL。质谱采用ESI离子源,多反映监测(MRM)的正离子扫描方式进行定量检测雌激素及其代谢产物共11个目标分析物(E_(1)、E_(2)、2-MeOE_(1)、4-MeOE_(1)、2-MeOE_(2)、4-MeOE_(2)、16α-OHE_(1)、2-OHE_(1)、4-OHE_(1)、2-OHE_(2)和4-OHE_(2))。从特异性、线性及范围、定量限、准确度和精密度、提取回收率、基质效应和稳定性等方面对建立的分析方法进行验证。用本法对41例乳腺癌患者和25例健康志愿者的血清样本进行雌激素及其代谢产物检测,比较2组间雌激素及其代谢产物的差异。结果:LC-MS/MS法可同时检测人血清中11个目标分析物的浓度,在标准曲线范围内线性良好;批内和批间精密度<15%;准确度绝对值<15%;提取回收率均>85%;基质效应在85%~115%;稳定性良好。用本方法进行临床血清雌激素及其代谢产物检测,测得对照组中E_(1)、E_(2)及其代谢产物2-MeOE_(1)、4-MeOE_(1)、2-MeOE_(2)、4-MeOE_(2)、16α-OHE_(1)、2-OHE_(1)/4-OHE_(1)、2-OHE_(2)、4-OHE_(2)的浓度分别为(0.101±0.006)(0.170±0.012)(1.078±0.104)(0.086±0.008)(0.064±0.000)(0.115±0.051)(0.071±0.006)(0.132±0.023)(0.237±0.020)和(0.225±0.027)nmol·L^(-1),乳腺癌患者组相应的Objective:To establish a stable and reliable liquid chromatography-tandem mass spectrometry(LC-MS/MS)method to detect the concentration levels of 11 target analytes,including estrogen estrone(E_(1)),estradiol(E_(2))and their metabolites 2-methoxyestrone(2-MeOE_(1)),4-methoxyestrone(4-MeOE_(1)),2-methoxyestradiol(2-MeOE_(2)),4-methoxyestradiol(4-MeOE_(2)),16α-hydroxyestrone(16α-OHE_(1)),2-hydroxyestrone(2-OHE_(1)),4-hydroxyestrone(4-OHE_(1)),2-hydroxyestradiol(2-OHE_(2))and 4-hydroxyestradiol(4-OHE_(2))and observe the changes of serum estrogen homeostasis in breast cancer patients.Methods:Using dansyl chloride as a derivatization reagent,the derivazation of the analytes was carried out on an Agilent ZORBAX Extend-C_(18)column(100 mm×2.1 mm,1.8μm)witha mobile phase consisting of 0.1%formic acid acetonitrile solution and 0.1%formic acid aqueous solution at a flow rate of 0.3 mL·min^(-1)by gradient elution.The column temperature was 25℃,and the injection volume was 30μL.ESI ion source was used for mass spectrometry.The positive ion scan with multiple reaction monitoring(MRM)mode was used for quantitative detection of estrogens and their metabolites for a total of 11 target analytes(E_(1),E_(2),2-MeOE_(1),4-MeOE_(1),2-MeOE_(2),4-MeOE_(2),16α-OHE_(1),2-OHE_(1),4-OHE_(1),2-OHE_(2)and 4-OHE_(2)).The established analytical method was validated in terms of specificity,linearity and range,limit of quantification,accuracy and precision,extraction recovery,matrix effect and stability.To compare the difference of estrogens and its metabolites between the two groups,the serum samples from 41 breast cancer patients and 25 healthy volunteers were detected by using this method.Results:The LC-MS/MS method described above can simultaneously detect the concentration of 11 target analytes in human serum.The linearity was good in the range of standard curve.The intra-and inter-run precisions were good with RSD<15%;accuracy was good with|RE|<15%;extraction recoveries were all>85%;matrix effect in 85%-115%.The stabilities were a

关 键 词：乳腺癌 血清 原型雌激素 羟基化雌激素 甲氧基化雌激素 雌激素代谢 液相色谱串联质谱 

分 类 号：R917[医药卫生—药物分析学]