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A specific and sensitive liquid chromatography-electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1 alpha-hydroxyvitamin D-3 (1 alpha OHD3) in rat plasma. A new ferrocene-based Cookson-type reagent, 4-ferrocenylmethyl-1,2,4-triazoline-3,5-dione (FMTAD), designed and synthesized to be highly sensitive to vitamin D analogs in EST, considerably improved the detection limit with 250 fg (359 amol)/injection. 1 alpha OHD3 in rat plasma was extracted with acetonitrile and then purified using Oasis HLB 96-well plates. After the precolumn derivatization with FMTAD, samples were subjected to LC/ESI-MS/MS employing a column-switching system. This method achieved a lower limit of quantitation of 5 pg from 0.1-mL plasma aliquots and 200-fold sensitivity of that without derivatization. The calibration curve (0.05-15 ng/mL) exhibited acceptable linearity (r > 0.9966), intraassay precision ranged from 3.8 to 9.6%, interassay precision ranged from 3.0 to 17.0%, and accuracy was within 81.4-112.0%. This FMTAD derivatization method is considered very useful for determination of vitamin D analogs in EST and applicable for biological samples. (C) 2005 Elsevier Inc. All rights reserved.