Measurement of estradiol, estrone, and testosterone in postmenopausal human serum by isotope dilution liquid chromatography tandem mass spectrometry without derivatization.

BACKGROUND: A high-throughput, sensitive, specific, mass spectrometry-based method for quantitating estrone (E1), estradiol (E2), and testosterone (T) in postmenopausal human serum has been developed for clinical research. The method consumes 100µl human serum for each measurement (triplicates consume 300µl) and does not require derivatization. We adapted a commercially available 96-well plate for sample preparation, extraction, and introduction into the mass spectrometer on a single platform. METHODS: Steroid extraction from serum samples and mass spectrometer operational parameters were optimized for analysis of estradiol and subsequently applied to other analytes. In addition to determining the limit of detection (LOD) and limit of quantitation (LOQ) from standard curves, a serum LOQ (sLOQ) was determined by addition of known steroid quantities to serum samples. Mass spectrometric method quantitative data were compared to results using a state-of-the-art ELISA (enzyme-linked immunosorbent assay) using stored serum samples from menopausal women. RESULTS: The LOD, LOQ, sLOQ was (0.1pg, 0.3pg, 1pg/ml) for estrone, (0.3pg, 1pg, 3pg/ml) for estradiol, and (0.3pg, 1pg, 30pg/ml) for testosterone, respectively. Mass spectrometry accurately determined concentrations of E2 that could not be quantified by immunochemical methods. E1 concentrations measured by mass spectrometry were in all cases significantly lower than the ELISA measurements, suggesting immunoreactive contaminants in serum may interfere with ELISA. The testosterone measurements broadly agreed with each other in that both techniques could differentiate between low, medium and high serum levels. CONCLUSIONS: We have developed and validated a scalable, sensitive assay for trace quantitation of E1, E2 and T in human serum samples in a single assay using sample preparation method and stable isotope dilution mass spectrometry.

Five- and Six-Coordinate Group 4 Compounds Stabilized by beta-Ketiminate and Diketiminate Ligands: Syntheses and Comparisons between Solid-State and Solution Structures.

The preparation and reaction chemistry of beta-diketiminate titanium and zirconium complexes is described. Amine elimination reactions work well for introducing Tolnacnac or Tolnacac to the metal centers (TolnacnacH = 2-(p-tolylamino)-4-(p-tolylimino)-2-pentene; TolnacacH = 4-p-toluidinopent-3-en-2-one). In certain cases, the iminium salt of the diketimine can be used to circumvent the unfavorable reaction kinetics. Salt elimination reactions starting from group 4 metal halides and beta-diketiminate lithium reagents are the most versatile method for introducing beta-diketiminate ligands to the metal. For (beta-diketiminate)MCl(3) compounds (M = Ti, Zr) eta(5)- and eta(2)-coordination modes can be controlled by modifying the diketiminate ligands. Several structures of five- and six-coordinate metal complexes were solved by X-ray diffraction methods. Five-coordinate metal complexes adopt both trigonal bipyramidal and square pyramidal geometries, and the six-coordinate metal complexes possess pseudooctahedral metal centers. For (Tolnacnac)(2)ZrX(2) (X = Cl, OR, NMe(2)) the activation parameters for Lambda/Delta conversion have been probed by dynamic NMR and are consistent with a Bailar-twist mechanism. At a common temperature, the isomerization rates follow the order Cl > OR > NMe(2).