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A sensitive, specific, accurate and precise LC/MS/MS method was developed for the simultaneous measurement. of dexamethasone and corticosterone in rat plasma. The method was extended to dexamethasone analysis in rat plasma ultrafiltrate and fetal tissues. Samples were processed using SPE involving Oasis HLB cartridges, which offered complete extraction recovery for the analytes. Samples were subsequently analyzed using LC/MS/MS. A structurally related corticosteroid, prednisolone, was used as the internal standard. Using a 500 mu L plasma sample, limits of quantification of 0.2 and 2.0 ng/mL were achievable for dexamethasone and corticosterone. This level of sensitivity allowed characterization of maternal/fetal dexamethasone profiles after administration of multiple doses of dexamethasone sodium phosphate to rats. However, this sensitivity was not satisfactory for corticosterone during pharmacokinetic studies involving dexamethasone due to its strong adrenosuppressive effect. This led us to investigate the suitability of a commercially available radioimmunoassay kit, which through extensive testing and minor modifications was found to offer extremely sensitive, specific, accurate and precise analysis of corticosterone. Knowledge of the steroid profiles captured using these highly sensitive analytical tools may potentially help in the optimization of corticosteroid therapy during pregnancy. Copyright (c) 2007 John Wiley & Sons, Ltd.