In this example, an in vitro metabolite
generation reaction produced a mixture of busiprone metabolites.
SepNMR was used to rapidly and easily isolate the 4th major metabilite. The chromtogram below shows the incubation mixture where the peak eluting at about 6.5 minutes is of interest.
Experimental:
Incubate700 ug of
buspirone with human microsomes in 1ml at 37 C
5 mg/ml HLM
3 mM NADPH
Quench reaction after 2 hours with formic/HFBA stop
Spin down and remove supernatant
Dry down to approximately 400 ul with nitrogen at 40C
Inject 80 ul of solution onto C8 with standard method
Chromatogram of human microsomal incubation of busiprone showing metabolites generated
Parent elutes at 7.3 min where metabolite of interest elutes at 6.5 minutes
Chromatogram of human microsomal incubation of busiprone showing metabolites generated
Red trace is the original injection, blue trace is the reinjected trapped peak
Trapped peak (blue ) is free of any material other than the targeted metabolite peak eluting at 6.5 minutes
Zoomed chromatogram of human microsomal incubation of busiprone showing metabolites generated
Red trace is the original injection, blue trace is the reinjected trapped peak
Blue trace free of any material other than the targeted metabolite peak eluting at 6.5 minutes
Recovery, based on comparing area of red (original sample) and blue (isolated trapped peak), is estimated at > 90%
Estmated mass of buspirone metabolite is 5 ug (assuming similar extinction coeffiecient as buspirone at 220 nm and an absorbance of 835 mAU)
CapNMR data of SepNMR isloated (blue) peak from above
Top spectrum: Buspirone Parent
Bottom spectrum: 1D1H of Buspirone Metabolite
60 min acquisition
ca 1 ug of metabolite in flow cell based on A220
OH modification localized to position 13,20 based these data
CapNMR data of SepNMR isloated buspirone metabolite
