Use of high-performance liquid chromatography/electrospray ionization collision-induced dissociation mass spectrometry for structural identification of monohydroxylated progesterones.
For the structural identification of monohydroxylated progesterones synthesized by microorganisms, a method was developed using a combination of high-performance liquid chromatography and electrospray ionization collision-induced dissociation mass spectrometry (HPLC/ESI-CIDMS). The retention times and MS/MS spectra of 11 different standards at 30 eV were collected and compared. The identification of D-ring-hydroxylated progesterones (15beta-, 16alpha-, 17alpha- and 21-OH-P) using ESI-CIDMS was not possible. However, they were separated chromatographically using a 65:35 mixture of water and acetonitrile containing 0.5% acetic acid. The other hydroxylated progesterones (2alpha-, 6beta-, 7beta-, 9alpha-, 11alpha-, 11beta-, and 19-OH-P) could be identified by comparison of eight fragments. The complete separation of 11 standards was achieved chromatographically. The developed assay was evaluated by the identification of monohydroxylated progesterones produced by CYP106A2 from Bacillus megaterium ATCC 13368.
Kang
Min-Jung
MJ
Pharmaceutical and Medicinal Chemistry, Saarland University, PO Box 151150, D-66041 Saarbrücken, Germany.
Lisurek
Michael
M
Bernhardt
Rita
R
Hartmann
Rolf W
RW
D000818:Animals; D001426:Bacterial Proteins / Q000032:analysis / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism; D006900:Hydroxylation; D011374:Progesterone / Q000031:analogs & derivatives / Q000032:analysis / Q000096:biosynthesis; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D001426:Bacterial Proteins; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System