Analysis of homoisoflavonoids in Ophiopogon japonicus by HPLC-DAD-ESI-MSn

The homoisoflavonoids in Ophiopogon japonicus (Thunb.) Ker-Gawler were analyzed by high-performance liquid chromatography-diode array detection-electrospray ion trap tandem mass spectrometry (HPLC-DAD-ESI-MSn). Homoisoflavonoids gave prominent [M - H]- ions by electrospray ionization monitored in the negative ion mode. They could be classified into two types depending on the fragmentation behavior of their [M - H]- ions in the ion trap mass analyzer. The [M - H]- ions of homoisoflavonoids with a saturated C2-3 bond underwent C3-9 bond cleavage to lose the B-ring, which was followed by the loss of a molecule of CO. The [M - H]- ions of homoisoflavonoids with a C2-3 double bond usually eliminated a CO molecule first, and then underwent the cleavage of C3-9 or C9-1' bonds. For homoisoflavonoids with a C-6 formyl group, however, the neutral loss of CO was the first fragmentation step; the presence of a methoxyl group at C-8 could lead to the cleavage of C-ring. No retro Diels-Alder (RDA) fragmentation characteristic for normal Flavonoids was observed. The above fragmentation rules were reported for the first time, and were implemented for the analysis of homoisoflavonoids in O. japonicus. The CHCl3-MeOH extract was separated on a Zorbax Extend-C18 column, eluting with a acetonitrile-0.3% acetic acid gradient. A total of 18 homoisoflavonoids, including seven new minor constituents, were identified or tentatively characterized based on the UV spectra and tandem mass spectra of the HPLC peaks.