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.
Trennung von Triglyceriden nach Kettenlänge und Sättigungsgrad durch HPLC
Die HPLC last sich in der routinemasigen Fettanalytik sinnvoll einsetzen. Fur konstantes Arbeiten sind Salen mit chemisch gebundenen C18-Gruppen besonders geeignet. Fettsaure-methylester ergeben kritische Paare, die sich aber bei Verwendung von Elutionsmitteln auf Acetonitrilbasis auftrennen lassen. Auch die Trennung von Olsaure und Elaidinsaure ist so zu erreichen. Triglyceride lassen sich bei der GC nur nach der Zahl der C-Atome trennen, wahrend bei der HPLC auch die Doppelbindungen einen grosen Einflus auf die Retentionszeiten haben. Dies tritt besonders bei Verwendung von Elutionsmitteln auf Nitrilbasis hervor. Das Prinzip, nach dem diese Trennungen erfolgen, wurde durch kapillargaschromatographische Analyse der HPLC-Fraktionen ermittelt. Weiterhin werden Hinweise zur Vermeidung von technischen Schwierigkeiten beim Einsatz von Differentialrefraktometern gegeben. 
 
 
 
Separation of Triglycerides According to Chain Length and Degree of Saturation by HPLC 
 
 
 
In routine fat analysis HPLC is efficiently used. For constant work columns with chemically bound C18-groups are especially suitable. There are critical pairs of fatty acid-methylesters which however can be separated by means of elution agents basing on acetonitril. Oleic acid and elaidic acid can be separated by this way, too. By GLC triglycerides can only be separated according to the number ob C-atoms, whereas in HPLC double bounds have a great influence on the retention time, too. This is especially evident if elution agents, basing on nitrils, are used. The separation principle was determined by capillar gas chromatographic analysis of the HPLC fractions. Further on hints are given to avoid technical difficulties in the use of differential refractometers.
Synthesis of 1-sn-lysophosphatidylcholine and mixed acid 1-sn-phosphatidylcholine.
An investigation of the biochemical effects of L-lysophosphatidylcholine (1-acyl-sn-glycerol-3-phosphorylcholine) requires as a control for its detergent action the metabolically inert, unnatural enantiomer, D-lysophosphatidylcholine (3-acyl-sn-glycerol-1-phosphorylcholine). The latter can be easily prepared in milligram quantities from the commercially available DL-phosphatidylcholine. For this purpose rac-1,2-dipalmitoyl-sn-glycerol-3-phosphorylcholine is solubilized with the aid of one-half its weight of taurocholate and hydrolyzed with phospholipase A2 to completion and the intact residual 2,3-dipalmitoyl-sn-glycerol-1-phosphorylcholine recovered by chromatography. The pure D-phosphatidylcholine is then subjected to methanolysis in the presence of octylamine and the product isomerized with glacial acetic acid. The 3-palmitoyl-sn-glycerol-1-phosphorylcholine thus obtained is chromatographically pure, possesses correct optical rotation and NMR spectrum, and is resistant to hydrolysis by phospholipase A2 following reacylation to the diacylphosphatide. Acylation of the 1-sn-lysophosphatide provides a convenient method for preparation of mixed acid 1-sn-phosphatidylcholines which are not otherwise available.