Derivatization procedure of estradiol with a combination of MPDNP-F and 4-dimethylaminopyridine to generate product ion containing estradiol-skeleton for reliable determination of its serum/plasma concentrations by LC/ESI-MS/MS

The quantification of serum/plasma estradiol (E₂) is useful for the diagnosis, pathological analysis, and monitoring of the therapeutic efficacy of estrogen-dependent diseases. In this study, an improved derivatization method using 1-(2,4-dinitro-5-fluorophenyl)-4,4-dimethylpiperazinium iodide (MPDNP-F) was developed and combined with liquid chromatography/electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) for the sensitive and specific quantification of the serum/plasma E₂. In the new method, the reaction time was reduced to 15 min from 90 min (two-step reaction in the previous method) by the direct reaction of MPDNP-F with E₂ at 60°C in the presence of 4-dimethylaminopyridine (DMAP). DMAP served as the organic catalyst and had a less negative effect on the LC/ESI-MS/MS instrument compared to the non-volatile inorganic salt (NaHCO₃), which was used in the previous method. The collision-induced dissociation of the molecular cation ([M]⁺) of the resulting derivative provided a product ion containing the E₂-skeleton ([M–NO₂–H]⁺), which significantly enhanced the assay sensitivity and specificity; compared to the dansyl chloride derivatization, which is the currently most-used derivatization procedure for the LC/ESI-MS/MS assays of E₂, the MPDNP-F derivatization had significantly fewer interfering peaks and a clear and flat baseline in the serum sample analysis. The MPDNP-F derivatization–LC/ESI-MS/MS method enabled the precise and accurate quantification of E₂ even at a 5.0 pg/mL concentration (lower limit of quantification) with a small sample volume (100 μL of serum/plasma) and had a tolerance for the matrix effect. This method was also proven to serve as a more sensitive and specific alternative to the clinically used chemiluminescence enzyme immunoassay. Graphical Abstract: [Figure not available: see fulltext.]