Development of an accurate and sensitive assay for 2-methoxyestradiol using derivatization and liquid chromatography-tandem mass spectrometry.

2-Methoxyestradiol (2ME) is involved in the pathogenesis of preeclampsia and antitumor activity. In addition to its low concentration in healthy human serum, presence of isomers makes quantification of 2ME for clinical research and laboratory medicine difficult. The objective of this study was to develop a highly sensitive and accurate method for quantifying 2ME using LC-MS/MS combined with derivatization with 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F). This approach significantly enhanced the detectability of 2ME in positive electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and enabled the chromatographic separation of 2ME from isomeric metabolites possessing a methoxy group, including 4-methoxyestradiol, 3-O-methyl 2-hydroxyestradiol, and 3-O-methyl 4-hydroxyestradiol (3M4OH). Although the derivatized 2ME and 3M4OH were closely eluted under the optimized LC conditions, the different fragmentation patterns of these isomers during MS/MS allowed their distinction. The lower limit of quantification for 2ME was 2.5 pg/mL, indicating a satisfactory sensitivity. These findings demonstrated that this LC-MS/MS method combined with the MPDNP-F derivatization can provide accurate and highly sensitive quantification of 2ME.