Reversed-Phase Retention Behavior of Fluorescence Labeled Phospholipids in Ammonium Acetate Buffers

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Abstract
Subcomponents of fluorescent derivatives of phosphatidylethanolamine (PE) and phosphatidylserine (PS) were resolved by reversed-phase high-performance liquid chromatography (HPLC) with mobile phases containing acetonitrile, methanol, water, and ammonium acetate. The fluorescence labeled phospholipids (PL) include N-(rhodamine B sulfonyl)-PE, N- (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) -PE, N-(5-fluoresceinthiocarbamoyl) (FL)-PE, N-(1-pyrenesulfonyl)-PE, and N-(5-dimethylaminonaphthalene-l-sulfcnyl)-PE. Among the compounds studied, FL-PE exhibited the highest degree of selectivity for component resolution. The HPLC behavior of the five PE derivatives was examined under variable concentrations of ammonium acetate. Capacity factors of the PL subcomponents increased with increasing concentrations of the acetate buffers. Incorporation of triethylamine into the mobile phase alleviated peak broadening and improved detection sensitivity of polar PL (FL-PE and PS). Fatty acid structures of molecular species in FL-PE were studied by particle beam (PB) -LC-mass spectrometry (MS). Compatibility of the HPLC method with PB-LC-MS is demonstrated.

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