Liquid chromatography mass spectrometric analysis of ouabainlike factor inbiological fluid

Ouabainlike factor (OLF), assayed as ouabainlike immunoreactivity (OLI), is a probable endogenous digitalislike factor (EDLF). Liquid chromatography/m ass spectrometry (LC/MS) is one of the most highly sensitive tools for obta ining structural information regarding low-molecular weight materials in a target compound, and to measure the concentrations of these materials. We h ave previously reported that OLI can be isolated from the culture supernata nt of the rat pheochromocytoma cell line, PC12, by several reverse-phase ch romatography and LC/MS techniques. The present study was performed to chara cterize OLF from biological fluids such as plasma and culture supernatant o f PC12 cells by LC/MS. The previous applications of LC/MS to OLI in plasma have been limited to structural identification at the final stages of isola tion, in which the starting volume of plasma has been over 10 l. In the pre sent study, we tried to minimize the volume of plasma, and to develop a new preclearing step to gain adequate LC/MS characterization using MS/MS analy sis. The plasma was acidified, and OLI was purified by ODS column chromatog raphy. OLI in chromatographic fractions from plasma was assayed by a sensit ive enzyme-linked immunosorbent assay for ouabain. After Sep-Pak treatment and two rounds of ODS column chromatography, OLI was identified from 80 ml of plasma. The structure of the purified OLI was identical to authentic oua bain and digoxin, as assessed by LC/MS. In conclusion, we identified the ch emically or structurally clarified ouabain and digoxin as the circulating f orm in plasma by LC/MS,