Normal-Phase LC-MS-Based Approaches

In the most common application of this separation mode, components are separated according to the number and nature of the polar functional groups (e.g., ester bonds, phosphate, hydroxyl, and amine groups) in lipid molecules. Since the head group of an individual lipid class predominantly determines the polar interactions with stationary phase, normal-phase HPLC separates a lipid extract solution into the lipid classes rather than into molecular species. 

A solvent system consisting of chloroform and methanol or hexane and isopropanol with or without addition of a small volume of water is commonly used for this mode to separate lipid classes. These solvent systems can be readily compiled with an ESI ion source. Unfortunately, due to the toxicity of chloroform, inclusion of this solvent in LC-MS is seldom. The use of inorganic salts is incompatible with MS, a small amount of organic salt or acid may be used as a modifier. Isocratic elution by using a mobile phase with a constant composition may be used for a certain lipid class, but gradient elution in which the polarity of the mobile phase is increased at a controlled rate affords greater versatility. 

For example, Hermansson et al. [63] employed a diol-modified silica column (250 X1.0 mm, 5-pm particles) and used an isocratic elution with a mobile phase comprising hexane-isopropanol-water-formic acid-triethylamine (628 348 24 2 0.8 v/v). The mass spectrometric detection was in the negative-ion mode, in which either [M-H] or [M-i-HCOO] molecular ions were monitored. The investigators separated most of the common lipid classes and developed a method with MS detection to automatically identify and quantify over 100 lipid species through two-dimensional mapping of elution time and the masses of lipid species (i.e., an SIM approach). 

In another example of normal-phase LC-MS analysis of human and monkey plasma lipids, a Luna Silica column (150 X 2.0 mm, 3-pm particle size) was employed with a linear gradient from mobile phase A (chloroform-methanol-ammonium hydroxide, 89.5 10 0.5) to B (chloroform-methanol-ammonium hydroxide-water, 55 39 0.5 5.5) [68]. Mass spectra were recorded in both positive-ion and negative-ion ESI modes with an MRM method. PC, SM, ceramide, and GluCer were monitored in the positive-ion mode and PE, PI, PS, PG, PA, and ganglioside M3 were measured in the negative-ion mode. A total of 153 lipid species were determined in the study. 

Overall, when LC-MS with a normal-phase column is employed for lipid analysis, the SIM approach is the likely choice for MS detection due to a short elution time of an individual lipid class. Although the MRM approach could be successfully applied, great efforts on setting up the ion transitions are needed. It appears that the data-dependent analysis approach is less favored, also likely due to the limited elution time to perform product-ion analysis of all lipid species of a class. 

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