Ferrocene-based Diels–Alder derivatization for the determination of 1α-hydroxyvitamin D 3 in rat plasma by high-performance liquid chromatography–electrospray tandem mass spectrometry

• Published in: Analytical biochemistry Vol. 346; no. 1; pp. 158 - 166
• Main Authors: Murao, Naoaki, Ishigai, Masaki, Sekiguchi, Nobuo, Takahashi, Tadakatsu, Aso, Yoshinori
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.11.2005
• Subjects: 1αOHD 3; Derivatization; Ferrocene-based Cookson-type reagent; FMTAD; LC/ESI-MS/MS; Validation; Ferrocene-based Cookson-type reagent; Validation; 1αOHD 3; Derivatization; FMTAD; LC/ESI-MS/MS
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2005.08.002

A specific and sensitive liquid chromatography–electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method has been developed for the determination of 1α-hydroxyvitamin D 3 (1αOHD 3) 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 ESI, considerably improved the detection limit with 250 fg (359 amol)/injection. 1αOHD 3 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 ESI and applicable for biological samples.