Reversed-phase ultrahigh-performance liquid chromatography–mass spectrometry (UHPLC–MS) is the method of choice for lipidomic profiling of blood plasma. For comprehensive screening, lipids need to be screened in both positive and negative electrospray mode. We here show that optimal results for increased lipidome coverage are only obtained if two different mobile-phase modifier systems are used, as opposed to using a single common modifier for both ionization modes. Specifically, ammonium acetate and ammonium formate were tested with and without acidifiers on standards representing 16 lipid classes, as well as on 164 blood plasma lipids from 11 lipid classes. Optimal coverage was obtained using 10 mM ammonium formate in ESI(+) and 10 mM ammonium acetate in ESI(−), both without acidification. Importantly, detection of free fatty acids was suppressed at more than 50-fold lower signal intensity if ammonium formate or formic acid as acidifier was used in ESI(−), whereas cholesteryl esters were hampered by up to 10-fold lower peak heights when ammonium acetate was used under ESI(+) conditions. Using charged surface hybrid C18 columns had slight advantages for lipids detected in blood plasma extracts, except for phosphatidic acids for which bridged ethylene hybrid C18 columns showed clear advantages.
- Blood plasma
- Liquid chromatography–mass spectrometry
- Mobile phase
ASJC Scopus subject areas
- Clinical Biochemistry
- Endocrinology, Diabetes and Metabolism