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使用超高灵敏LC/MS/MS对生物体液中的脂质进行定量分析
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使用超高灵敏LC/MS/MS对生物体液中的脂质进行定量分析 |
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前沿：
Purpose: To measure biologically active hydroxy fatty acids at low concentrations using normal-phase LC/electron capture APCI-MS/MS. Methods: Lipid extracts from rat intestinal epithelial (RIE) cells are derivatized to the corresponding pentafluorobenzyl-derivatized hydroxy fatty acids. Normal phase chiral LC is used to separate (R) and (S) enantiomers of lipid metabolites. Detection is achieved with a triple quadrupole MS in selected reaction monitoring (SRM) mode after ionization by electron capture APCI.
Results: Limits of quantitation (LOQs) for hydroxyeicosatetraenoic acids (HETEs) and hydroxyoctadecadienoic acids (HODEs) were 10-25 pg/mL for pure standards and 100-200 pg/mL in cell incubation media. Enhanced-resolution SRM increased S/N by factor of 2-4 for several lipid
metabolites. Formation of an electrically conductive filament on the corona discharge needle was reduced by the post-column addition of 1:1 isopropanol/methanol. Incubation of RIE cells with aspirin showed a near equivalent concentration of 5(R)- and 5(S)-HETE, suggesting that 5-HETE is formed by a non-enzymatic pathway under these conditions.
仪器：
TSQ Quantum Ultra™ mass spectrometer
结论：
Derivatization of hydroxy fatty acids with pentafluorobenzyl bromide affords low-level detection using electron capture APCI and selected reaction monitoring. The unique enhanced-resolution SRM capability of the Finnigan TSQ Quantum increased the selectivity and sensitivity of certain PFB-derivatized lipid metabolites. To suppress the formation of a conductive filament on the discharge needle, a post-column addition of 1:1 IPA/MeOH was used in conjunction with reducing the corona discharge current. The LOQs for the lipid metabolites were 4-10 times higher in the biological cell media versus pure solvent solutions. Preliminary data from rat intestinal epithelial cell extracts incubated with aspirin showed that 5(R)- and 5(S)-HETE are produced as a racemic mixture, suggesting that 5-HETE is formed non-enzymatically under these conditions.
