The novel and rapid assay presented here combines high-performance liquid chromatography and electrospray ionisation tandem mass spectrometry (HPLC-ESI-MS/MS) to directly measure and quantify the
CoA esters of 3alpha,7alpha,12alpha-trihydroxy- and 3alpha,7alpha-dihydroxy-5beta-cholestan-26-oic
acid (THCA and DHCA). The latter are converted inside peroxisomes to the primary
bile acids, cholic and chenodeoxycholic
acids, respectively. Prior to MS/MS,
esters were separated by reversed-phase HPLC on a C(18) column using an isocratic mobile phase (
acetonitrile/water/
2-propanol) and subsequently detected by multiple reaction monitoring. For quantification, the
CoA ester of
deuterium-labelled 3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic
acid (d(4)-CA) was used as internal standard. To complete an assay took less than 8 min. To verify the validity of the assay, the effect of peroxisomal
proteins on the efficacy of extraction of the
CoA esters was tested. To this end, variable amounts of the
CoA esters were spiked with a fixed amount of either intact peroxisomes or peroxisomal matrix
proteins and then extracted using a solid-phase extraction system. The
CoA esters could be reproducibly recovered in the range of 0.1-4 micromol l(-1) (linear correlation coefficient R(2) > 0.99), with a detection limit of 0.1 micromol l(-1). In summary, electrospray ionization tandem mass spectrometry combined with HPLC as described here proved to be a rapid and versatile technique for the determination of
bile acid CoA esters in a mixture with peroxisomal
proteins. This suggests this technique to become a valuable tool in studies dealing with the multi-step biosynthesis of
bile acids and its disturbances in disorders like the
Zellweger syndrome.