A novel GC-MS methodology to evaluate aromatase activity in human placental microsomes: a comparative study with the...

Estrogens are key factors in the development of the estrogen receptor-positive (ER⁺) breast cancer. Estrogens, estrone (E₁), and estradiol (E₂) production is achieved by aromatase, a cytochrome P450 enzyme that has androgens, androstenedione (AD), and testosterone (T) as substrates. Nowadays, third-generation aromatase inhibitors (AIs) are considered the gold-standard treatment for ER⁺ breast cancer in postmenopausal women as well as in premenopausal women with ovary ablation. Aromatase activity assessment still relies on radiometric assays that are expensive, hazardous, and non-environmentally friendly. Thus, in order to overcome these disadvantages, a new methodology was developed to evaluate aromatase activity, based on dispersive liquid-liquid microextraction (DLLME) followed by gas chromatography-mass spectrometry (GC-MS). The enzymatic reaction was carried out in human placental microsomes, using AD as substrate, and the anti-aromatase activity was measured by determining the conversion percentage of AD into E₁ (ratio E₁/AD) using isotopic analogues as internal standards. The method showed good linearity (r² = 0.9908 for AD and 0.9944 for E₁), high accuracy (more than 74% for AD and more than 66% for E₁), high extraction efficiency, and good intra-day and inter-day precision (below 14%, 4 levels). In this work, the IC₅₀ values of the third-generation AIs, anastrozole, letrozole, and exemestane, obtained from the radiometric assay are also compared, and similar IC₅₀ values are described. This method is a good alternative to the current radiometric assay, being fast and sensitive with a good extraction efficiency, accuracy, and recovery. In addition, it may be applied for the evaluation of the anti-aromatase activity of new potential AIs. Graphical abstract.