Quantitation of natural triacylglycerols by reversed-phase liquid chromatography with direct liquid inlet mass spectrometry.
Using acetonitrile and propionitrile as eluting solvents and reagent gases the yields of both quasi-molecular and fragment ions were found to vary with the molecular weight, degree of unsaturation and positional distribution of the fatty acids in the triacylglycerol molecule, and appropriate calibration factors were necessary for accurate quantitation. In the absence of pure structural isomers and mixed acid standards, preliminary calibration factors have been determined for total ion and specific ion current responses by comparing the peak area ratios obtained by liquid chromatography-mass spectrometry with the proportions of the molecular species known to be present in randomized oils and in natural oils of known chemical composition. Although the derived factors include both chromatographic and mass spectrometric effects and are obtained with a gradient of reagent gases, they appear to be generally applicable. It was shown that positional isomers affected the yield of the (MH-RCOOH)+ ions over a 1-3-fold range of intensities, while the nature of the fatty acid affected it over a range of 1.25-fold. After suitable calibration of the relative ion responses it was possible to determine the identities and amounts of the individual molecular species in natural fats and oils.
Strategy of glycerolipid separation and quantitation by complementary analytical techniques. Plenary lecture.
Although improved systems for chromatographic resolution continue to be developed there is good reason to believe that no single method will be capable fo complete separation of all lipid mixtures including the geometric, positional and stereochemical isomers in each molecular species. Furthermore, the chromatographic systems giving the highest resolution usually yield the least complete recoveries of components and require separate procedures of quantitation. It is therefore necessary to develop appropriate strategies that yield the required resolution as a result of consecutive application of complementary analytical techniques. At the present time, the original combination of thin-layer and gas--liquid chromatography has been joined by the combination of thin-layer and liquid, and liquid and gas--liquid chromatography with both liquid and gas--liquid chromatography being frequently coupled to mass spectrometry with computerized data processing. Internal standardization with hydrogen flame ionization provides a simple quantitative detection for gas chromatography, while mass spectrometry serves a similar purpose in liquid chromatography, although a much more extensive calibration may be required for quantitation. Special advantages for both separation and quantitation of most neutral lipid mixtures are derived from enzymic and chemical modification of the samples prior to chromatography. With imaginative work-up of samples, superior qualitative and quantitative results can frequently be obtained by appropriate combination of chromatographic techniques of limited resolving power.
HPLC of triglycerides
Triglyceride separation was investigated on a reverse phase high performance liquid chromatography (HPLC) column using two different solvent systems. Complete separation of model compounds differing by two methylene groups was achieved. Partial or complete separation was also observed in critical pairs; for example, the different types of triglycerides consisting of palmitic and oleic acids. This observation was confirmed on natural oils (coconut oil, palm kernel oil).