Analytes Derived from Oils and Fats

Triglycerides have been separated in various fats and oils using both leversed-phase and normal-phase systems [425]. Alcohols have been used effectively as the low-volume mobile phase modifier for NP work (the reader is referred to Chapters 5 and 6 for details). 

The hydroperoxide by-products of the reaction of unsaturated triacylglycerol (TAG) (e.g., stearoyl oleoyl stearoyl, palmitoyl palmitoyl oleoyl) with t-butyl hydroperoxide were studied on a Cjg column (ELSD, drift tube 85°C) using a 30-min 80/20 - 20/80 methanol/IPA gradient [426]. Up to 16 peaks.were generated in the product chromatogram. Electrospir MS was used to identify some of the peaks. Common products included TAG hydroperoxide, epoxide, and combined peroxide/epoxide products. 

Twelve phosphatidylcholine molecular species (e.g., 18 2/20 4, 16 0/20 4, 18 0/20 4, 18 0/18 2) were obtained from lever microsomes and separated on two 250 X 4.6 mm Cjg columns in series (ELSD, N2 nebulizer at 1.8L/min, drift tube T = 100°C). Good resolution was achieved with 45-min 40/48/ 12/040/42/8/8 (at 15min hold 30min) hexane/lPA dichloromethane/water gradient [428]. 

Markello et al. [433] developed a separation on a silica column that gave class separation of nonpolar lipids, glycolipids, phospholipids, and sphingolipids in 60 min. This work was based on the seminal work of Christie [434, 435] and used a silica column and a complex 60-min hexane/THF (99/1)— chloroform/IPA (1/4)- -IPA/water (1/1) gradient (ELSD). Results were used to assist in the diagnosis of metabolic disease in humans. Retention times for all 13 classes of compounds (fatty acids, PE, PI, PS, PC, ceramides, etc.) were tabulated. The authors noted that retention of CE, TAG and phytol was extremely sensitive to the water level in the mobile phase (i.e., creation of a hydrated and deactivated silica surface). The authon claim that the key here was the use of a saturation column (a column placed in the solvent flow path prior to the injector) and an extended column re-equilibration period after each analysis. 

The activity of ceramidase was monitored through its action on ceramide. Ceramide was derivatized with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole, NBD) and the resulting NBD-derivatized ceramide, sphingomyelin, and free fatty acids were separated on a C g column (A = 455 nm, ex 530 nm, em) using an 85/15/0.15 methanol/water/H3P04 mobile phase [436]. Baseline resolution was achieved and complete elution in 15 min. 

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