Steroid hormones like estradiol are excreted in animals as aggregates - a form presenting no hormonal activity. It has however been reported that upon removal of water, they disassemble to release the free active form, which has prompted efforts to characterize such aggregates. In this application, estradiol and its metabolic by-products, i.e. 11 compounds including estrone and a variety of estroriol glucuronides were analyzed with the polymer-based Asahipak NH2P-40 2D column in HILIC mode with LC/MS/MS detection.
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Sample : 0.1 ppm each, 5 μL
1. Estrone -3-sulfate
2. β-Estradiol -3-sulfate
3. Estriol -3-sulfate
4. Dehydroepiandrosterone -3-sulfate
5. Estrone -3-glucuronide
6. β-Estradiol -17-glucuronide
7. β-Estradiol -3-glucuronide
8. Estriol -3-glucuronide
9. Estriol -16-glucuronide
10. Dehydroepiandrosterone -3-glucuronide
11. Androsterone -3-glucuronide

Column    : Shodex Asahipak NH2P-40 2D (2.0 mm I.D. x 150 mm)
Eluent    : A; 10 mM NH4OH CH3CN
            B; 10 mM NH4OH H2O
Gradient  : Linear gradient; 
            (B %) 22 % (0 - 5 min), 22 - 70 % (5 - 10 min), 70 % (10 - 15 min)
Flow rate : 0.2 mL/min
Detector  : ESI-MS/MS (Polarity : Negative)
Column oven : 40 °C

Data courtesy of Mr. Atsushi Yamamoto, Osaka City Institute of Public Health and Environmental Sciences