Simultaneous Measurement of 17β-Estradiol, 17α-Estradiol and Estrone by GC-Isotope Dilution MS/MS.

A convenient GC-MS/MS-based method was developed for the simultaneous measurement of 17β-estradiol, 17α-estradiol and estrone using liquid-liquid extraction, a single-step derivatization with N-(trimethylsilyl)imidazole and the corresponding deuterated estrogens as internal standards. Separation of these estrogens was achieved on a 50% phenyl polysilphenylene-siloxane bonded phase column. MS/MS response factors for the derivatized analytes and their corresponding internal standards were found to be practically identical. Therefore, analyte concentrations could be determined by multiplying the measured analyte to internal standard ion-current ratio with known molar concentration of the corresponding deuterated internal standards. Assay accuracies, determined from the analyses of quality control samples obtained by spiking known concentrations of analytes into charcoal-stripped human serum, were in the -11% to +14% range. Limits of quantitations were between 13 pg/mL and 21 pg/mL from this biological medium.