雌激素與雌激素代謝物經多項研究指出與乳癌的發生息息相關，這類小分子中，高活性的兒茶酚雌激素會形成具醌類(quinone)結構的致癌性物質，經檢測這類物質會與DNA上的鳥嘌呤(Guanine, Gua)及腺嘌呤(Adenine, Ade)形成鍵結，造成DNA的斷裂，並產生突變，最後可能導致癌症發生。所以人體內的雌激素代謝物濃度是一潛在的生物性指標。
目前已知人類血清中的血清白蛋白上的離胺酸(Lysine, K)、半胱胺酸(Cysteine, C)和組胺酸(Histidine, H)會與兒茶酚雌激素進行共價性鍵結，其中以雌激素代謝物4-hydroxyestradiol(4-OHE2)反應性特別高，透過先前本實驗室的研究結果指出，低濃度時主要鍵結在C34上。而我們添加多種雌激素到血清中，包含Estrone (E1)、Estradiol (E2)、Estriol (E3)、4-hydroxyestradiol (4-OHE2)、16α-hydroxyestradiol (16α-OHE1)、4-methoxyestradiol (4-MeOE2)、Estrone-3-glucuronide (E1-3G)、Estrone-3-sulfate (E1-3S)，4-hydroxyestrone-1-N7Guanine (DNA-adduct)、Ethinylestradiol (EE2)、4-hydroxy Ethynylestradiol (4-OHEE2) 共11種經不同時間的反應測試，結果得知除4-OHE2外大部分雌激素代謝物會穩定存在於血清當中。所以為了精確比較人體內的雌激素代謝物的含量，我們使用乙腈沉澱蛋白質並利用QuEChERS萃取方式成功的從同一血清樣品內獲取雌激素代謝物與蛋白鍵結分子。搭配自製的微米級毛細管管柱與奈米噴灑式游離源，其雌激素代謝物的偵測極限更可達到0.5 - 25 ppt，且只需微量注射體積即可達到很好的靈敏度。我們成功的對血清中的雌激素代謝物進行直接偵測，精簡了樣品前處理的過程，例如:衍生化，不但能縮減準備時間且可避免實驗中雌激素代謝物的損失，使其更加接近人體內雌激素代謝物的含量。
在C34胜肽方面，我們進行固相萃取條件的優化，提供了較適合的沖提條件，以利減少樣品基值對其分析上的干擾。

Estrogens and their bioactive metabolites play key roles in regulating diverse processes in health and disease. Among these small molecules, the highly active catechol estrogens (CEs) can form quinone structure, which reacts with DNA leading to apurinic sites. These sites represent premutagenic lesions. Consequently, the concentration of endogenous estrogen metabolites in body is a potential biomarker.
It has already known that Lysine (K), Cysteine (C) and Histidine (H) on human serum albumin (HSA) could conjugate with CEs. Especially 4-hydroxyestradiol (4-OHE2) has high reactivity. According to previous research results in our lab, the C34 was the major adduction site at low concentration. A variety of estrogen metabolites (EMs) was spiked to blood serum to test the reactivity, including Estrone (E1), Estradiol (E2), Estriol (E3), 4-hydroxyestradiol (4-OHE2), 16α-hydroxyestradiol (16α-OHE1), 4-Methoxyestradiol (4-MeOE2), Estrone-3-glucuronide (E1-3G), Estrone-3-sulfate (E1-3S),4-hydroxyestrone-1-N7Guanine (DNA-adduct), Ethinylestradiol (EE2), 4-hydroxy ethynylestradiol (4-OHEE2). Except for 4-OHE2, it was found that most of EMs were stably existed in blood serum. Therefore, in order to accurately evaluate the content of EMs in human body, Acetonitrile protein precipitation was combined with QuEChERS extraction method to obtain free EMs and protein-conjugated estrogen metabolites from single serum sample. With a self-made micro flow-rate column and Nano-electrospray source, the detection limit of EMs could reach 0.5 – 25 ppt, and only need a small amount of injection volume. We successfully detected the EMs in blood serum directly and simplified the sample preparation process, which not only reduced the preparation time but also avoid the loss of EMs in experiment. Make the result closer to the content of EMs in the human body.
For the C34 peptide, the solid phase extraction (SPE) method is applied to remove contaminants from peptides and provide an effective cleanup procedure.

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