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(E1)、雌二醇(E2)及其代谢产物2-甲氧基雌酮(2-MeOE1)、4-甲氧基雌酮(4-MeOE1)、2-甲氧基雌二醇(2-MeOE2)、4-甲氧基雌二醇(4-MeOE2)、16α-羟基雌酮(16α-OHE1)、2-羟基雌酮(2-OHE1)、4-羟基雌酮(4-OHE1)、2-羟基雌二醇(2-OHE2)、4-羟基雌二醇(4-OHE2)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-C18色谱柱(100 mm×2.1 mm,1.8 μm),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30 μL。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(E1、E2、2-MeOE1、4-MeOE1、2-MeOE2、4-MeOE2、16α-OHE1、2-OHE1、4-OHE1、2-OHE2和4-OHE2)。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E1、E2及其代谢产物2-MeOE1、4-MeOE1、2-MeOE2、4-MeOE2、16α-OHE1、2-OHE1/4-OHE1、2-OHE2、4-OHE2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11个目标分析物的浓度分别为(0.174±0.017)(0.335±0.041)(0.958±0.080)(0.072±0.006)(0.066±0.001)(0.065±0.023)(0.097±0.007)(0.183±0.020)(0.365±0.084)和(0.366±0.082) nmol·L-1,结果表明乳腺癌患者原型雌激素(E1和E2)和羟基化雌激素(16α-OHE1,2-OHE2,4-OHE2和2/4-OHE1)的浓度显著增加,而甲氧基化雌激素(4-MeOE2)的浓度明显降低。结论: 本实验建立的血清中雌激素(E1和E2)及其代谢产物(2-MeOE1、4-MeOE1、2-MeOE2、4-MeOE2、16α-OHE1、2-OHE1、4-OHE1、2-OHE2和4-OHE2)的LC-MS/MS定量检测方法准确、快速、可靠,可作为乳腺癌患者血清雌激素及其代谢产物检测的参考方法。乳腺癌状态下,雌激素稳态失衡与乳腺癌的发生发展密切相关,雌激素可能作为乳腺癌发生发展的潜在疾病标志物。
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 (E1), estradiol (E2) and their metabolites 2-methoxyestrone (2-MeOE1), 4-methoxyestrone (4-MeOE1), 2-methoxyestradiol (2-MeOE2), 4-methoxyestradiol (4-MeOE2), 16α-hydroxyestrone (16α-OHE1), 2-hydroxyestrone (2-OHE1), 4-hydroxyestrone (4-OHE1), 2-hydroxyestradiol (2-OHE2) and 4-hydroxyestradiol (4-OHE2) 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-C18 column (100 mm×2.1 mm, 1.8 μm) 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 30 μL. 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 (E1, E2, 2-MeOE1, 4-MeOE1, 2-MeOE2, 4-MeOE2, 16α-OHE1, 2-OHE1, 4-OHE1, 2-OHE2 and 4-OHE2). 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 all within the required limits. Using this method to detect clinical serum estrogens and its metabolites, the concentrations of E1, E2 and their metabolites 2-MeOE1, 4-MeOE1, 2-MeOE2, 4-MeOE2, 16α-OHE1, 2-OHE1/4-OHE1, 2-OHE2, 4-OHE2 in the control group were (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) and (0.225±0.027) nmol·L-1 respectively, and the corresponding concentrations of 11 target analytes in the breast cancer patient group were (0.174±0.017) (0.335±0.041) (0.958±0.080) (0.072±0.006) (0.066±0.001) (0.065±0.023) (0.097±0.007) (0.183±0.020) (0.365±0.084) and (0.366±0.082) nmol·L-1 respectively. The results showed that the concentration of prototype estrogens (E1 and E2) and hydroxylated estrogens (16α-OHE1, 2-OHE2, 4-OHE2 and 2/4-OHE1) in breast cancer patients were significantly increased, while the concentration of methoxylated estrogen (4-MeOE2) was significantly reduced. Conclusion: The LC-MS/MS method was established for the quantitative detection of estrogens (E1, E2) and its metabolites (2-MeOE1, 4-MeOE1, 2-MeOE2, 4-MeOE2, 16α-OHE1, 2-OHE1, 4-OHE1, 2-OHE2 and 4-OHE2) in serumis accurate, fast and reliable in this study, which can be used as a reference method for the detection of serum estrogens and its metabolites in breast cancer patients. In the state of breast cancer, the imbalance of estrogen homeostasis isclosely related to the occurrence and development of breast cancer, and estrogen may be used as a potential disease marker for the development and progression of breast cancer.
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