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.
HAO Zhi-xiang, ZHONG Ya-nan, XU Yin-xue, YIN Xiao-xing, ZHANG Bei, ZHOU Xue-yan
. Establishment and application of a liquid chromatography-tandem mass spectrometry method for the detection of serum estrogens and its metabolites in breast cancer patients*[J]. Chinese Journal of Pharmaceutical Analysis, 2022
, 42(10)
: 1739
-1753
.
DOI: 10.16155/j.0254-1793.2022.10.07
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