Direct detection of phenylephrine 3-O-sulfate in LS180 human intestinal cells using a novel hydrophilic interaction liquid chromatography (HILIC) assay

- Direct simultaneous quantitation of phenylephrine and its sulfate metabolite.
- Hydrophilic analytes: novel sugaring-out liquid-liquid extraction method.
- Hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection.
- Validated assay applied to study kinetics of phenylephrine sulfation.

The efficacy of phenylephrine (PE) is controversial due to its extensive pre-systemic metabolism through sulfation to form phenylephrine-3-O-sulfate (PES). Hence quantitation of PES is important in order to study the metabolism of PE. There are no published methods available for direction detection of PES. We have developed and validated a hydrophilic interaction liquid chromatography (HILIC) method for the direct detection of PES and simultaneous detection of PE to study the enzyme kinetics and metabolism of PE to enable approaches to reduce the presystemic metabolism of PE. This is the first method which facilitates direct detection of PES and simultaneous detection of PE using a zwitterionic HILIC column with improved sensitivity in a single short run. The observed quantitative ranges of our method for PE and PES were 0.39-200 μM and 0.0625-32 μM (respectively) with a run time of 6.0 min. The method was applied to the determination of PE and PES in LS180 human intestinal cell line, recombinant enzymes and human intestinal cytosol (HIC).