Analysis of isoprenoid phosphates and oligophosphates by capillary zone electrophoresis

Capillary zone electrophoresis (CZE) has been used for the first time to analyse water-soluble phosphorylated isoprenoids, key intermediates in the branched pathway of mevalonate metabolism. Following synthesis, isoprenoid phosphates and oligophosphates were isolated by flash chromatography and their purity was established using P-31 NMR spectrometry. In developing optimum conditions for CZE, several factors were considered: ionization properties of the solutes, stability of the solutes and maximum signal-to-noise ratio. At pH 8.5 in 0.3 M sodium berate buffer and monitoring UV absorbance at 200 nm, detection of farnesyl diphosphate was linear in the sub-picomol range; the limit of detection was ca. 12 fmol. For a given phosphorylated series (i.e., monophosphates, diphosphates, triphosphates), CZE cleanly resolved isoprenes of different chain length, and plots of relative mobility vs. M(r) were curvlinear. Remarkably, neryl phosphate (C10, alpha-cis-isoprene), geranyl phosphate (C10, alpha-trans-isoprene) and citronellyl phosphate (C10, alpha-saturated isoprene) were resolved. In addition, isopentenyl monophosphate (C5,Delta(3)) and dimethylallyl phosphate (C5,Delta(2)) exhibited different electrophoretic mobilities. These studies pave the way for future work on determining levels of phosphorylated isoprenoids in various tissues under conditions of altered mevalonate production.