Development of an LC-MS/MS method for aromatase inhibitor screening.
Aromatase (CYP 19A1) is a key steroidogenic enzyme that catalyzes the conversion of androgen to estrogen. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for aromatase inhibitor screening was developed and validated. The substrate androstenedione was incubated with human CYP 19A1 supersomes in the presence of NADPH for 30 min, and estrone formation was determined by LC-MS/MS analysis. Cortisone was used as internal standard. The incubation mixture was extracted using a liquid-liquid extraction method with ethyl acetate. Chromatographic separation was achieved using a C18 column (3.0 × 50 mm, 2.7 μm) with a mobile phase consisting of 0.1% formic acid/acetonitrile adopting gradient elution at a flow rate of 0.4 mL/min. The mass spectrometer was operated in positive electrospray ionization mode. The precursor-product ion pairs used for multiple reaction monitoring were m/z 287→97 (androstenedione), m/z 271 → 159 (estrone), and m/z 361 → 163 (IS, cortisone). The developed method met the required criteria for the validation of bioanalytical methods. The validated method was successfully applied to evaluate aromatase inhibitory activity of plants extracts of Simaroubaceae.
Institute of Pharmaceutical Science and Technology and College of Pharmacy, Hanyang University, Gyeonggi-do, 426-791, South Korea.
Myeong Hyeon
MH
Park
In Sook
IS
Kim
Mi-Sook
MS
Dong
Hye Hyun
HH
Yoo
D001141:Aromatase / Q000737:chemistry*; D047072:Aromatase Inhibitors / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D003348:Cortisone / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D004970:Estrone / Q000737:chemistry; D006801:Humans; D059625:Liquid-Liquid Extraction; D010936:Plant Extracts; D015203:Reproducibility of Results; D029641:Simaroubaceae; D053719:Tandem Mass Spectrometry / Q000379:methods*
D047072:Aromatase Inhibitors; D010936:Plant Extracts; D004970:Estrone; D001141:Aromatase; C561410:CYP19A1 protein, human; D003348:Cortisone