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
[1] SHI Y, ZHAO Y, ZHANG Y, et al. AFF3 upregulation mediates tamoxifen resistance in breast cancers[J].J Exp Clin Cancer Res, 2018, 37(1):254
[2] HANKER AB, SUDHAN DR, ARTEAGA CL. Overcoming endocrine resistance in breast cancer[J].Cancer Cell, 2020, 37(4):496
[3] RODGERS KM, UDESKY JO, RUDEL RA, et al. Environmental chemicals and breast cancer: an updated review of epidemiological literature informed by biological mechanisms[J].Environ Res, 2018, 160:152
[4] HUANG J, SUN J, CHEN Y, et al. Analysis of multiplex endogenous estrogen metabolites in human urine using ultra-fast liquid chromatography-tandem mass spectrometry: a case study for breast cancer[J].Anal Chim Acta, 2012, 711:60
[5] CASTAGNETTA LA, GRANATA OM, TRAINA A, et al. Tissue content of hydroxyestrogens in relation to survival of breast cancer patients[J].Clin Cancer Res, 2002, 8(10):3146
[6] DENVER N, KHAN S, HOMER NZM, et al. Current strategies for quantification of estrogens in clinical research[J].J Steroid Biochem Mol Biol, 2019, 192:105373
[7] ZACS D, PERKONS I, BARTKEVICS V. Determination of steroidal oestrogens in tap water samples using solid-phase extraction on a molecularly imprinted polymer sorbent and quantification with gas chromatography-mass spectrometry(GC-MS)[J].Environ Monit Assess, 2016, 188(7):433
[8] CRUZ G, BARRA R, GONZÁLEZ D, et al. Temporal window in which exposure to estradiol permanently modifies ovarian function causing polycystic ovary morphology in rats[J].Fertil Steril, 2012, 98(5):1283
[9] LI XS, LI S, KELLERMANN G. Simultaneous determination of three estrogens in human saliva without derivatization or liquid-liquid extraction for routine testing via miniaturized solid phase extraction with LC-MS/MS detection[J].Talanta, 2018, 178:464
[10] VAN DER BERG C, VENTER G, VAN DER WESTHUIZEN FH, et al. Development and validation of LC-ESI-MS/MS methods for quantification of 27 free and conjugated estrogen-related metabolites[J].Anal Biochem, 2020, 590:113531
[11] SON HH, YUN WS, CHO SH. Development and validation of an LC-MS/MS method for profiling 39 urinary steroids(estrogens, androgens, corticoids, and progestins)[J].Biomed Chromatogr, 2020, 34(2):e4723
[12] YUAN TF, LE J, CUI Y, et al. An LC-MS/MS analysis for seven sex hormones in serum[J].J Pharm Biomed Anal, 2019, 162:34
[13] XU X, ROMAN JM, ISSAQ HJ, et al. Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry[J].Anal Chem, 2007, 79(20):7813
[14] ZHAO Y, BOYD JM, SAWYER MB, et al. Liquid chromatography tandem mass spectrometry determination of free and conjugated estrogens in breast cancer patients before and after exemestane treatment[J].Anal Chim Acta, 2014, 806:172
[15] KULKOYLUOGLU-COTUL E, ARCA A, MADAK-ERDOGAN Z. Crosstalk between estrogen signaling and breast cancer metabolism[J].Trends Endocrinol Metab, 2019, 30(1):25
[16] THAKARE R, CHHONKER YS, GAUTAM N, et al. Quantitative analysis of endogenous compounds[J].J Pharm Biomed Anal, 2016, 128:426
[17] KARNOVSKY A, LI S. Pathway analysis for targeted and untargeted metabolomics[J].Methods Mol Biol, 2020, 2104:387
[18] RODRÍGUEZ-MORATÓ J, POZOÓJ, MARCOS J. Targeting human urinary metabolome by LC-MS/MS: a review[J].Bioanalysis, 2018, 10(7):489
[19] YUE W, SANTEN RJ, WANG JP, et al. Genotoxic metabolites of estradiol in breast: potential mechanism of estradiol induced carcinogenesis[J].J Steroid Biochem Mol Biol, 2003, 86(3-5):477
[20] KE Y, BERTIN J, GONTHIER R, et al. A sensitive, simple and robust LC-MS/MS method for the simultaneous quantification of seven androgen-and estrogen-related steroids in postmenopausal serum[J].J Steroid Biochem Mol Biol, 2014, 144 Pt B:523
[21] QIN F, ZHAO YY, SAWYER MB, et al. Column-switching reversed phase-hydrophilic interaction liquid chromatography/tandem mass spectrometry method for determination of free estrogens and their conjugates in river water[J].Anal Chim Acta, 2008, 627(1):91
[22] ZHOU X, ZHAO Y, WANG J, et al. Resveratrol represses estrogen-induced mammary carcinogenesis through NRF2-UGT1A8-estrogen metabolic axis activation[J].Biochem Pharmacol, 2018, 155:252
[23] ZHOU X, ZHENG Z, XU C, et al. Disturbance of mammary UDP-glucuronosyltransferase represses estrogen metabolism and exacerbates experimental breast cancer[J].J Pharm Sci, 2017, 106(8):2152
[24] ZHAO F, HAO Z, ZHONG Y, et al. Discovery of breast cancer risk genes and establishment of a prediction model based on estrogen metabolism regulation[J].BMC Cancer, 2021, 21(1):194
[25] LIU F, GE S, SU M, et al. Electrochemiluminescence device for in-situ and accurate determination of CA153 at the MCF-7 cell surface based on graphene quantum dots loaded surface villous Au nanocage[J].Biosens Bioelectron, 2015, 71:286
