Fatty acid picolinyl esters

Kurkiewicz, S. Dzierzewicz, Z. Wilczok,T. Dworzanski, J. P. GC/MS determination of fatty acid picolinyl esters by direct Curie-point pyrolysis of whole bacterial cells. J. Am. Soc. Mass Spectrom. 2003,14, 58-62. 

Table 4.5 GC-MS analysis of fatty acid picolinyl esters of borage oil...

Fig. 7 Separation of picolinyl ester derivatives of the fatty acids of cod liver oil by HPLC in the reverse phase mode.

Wretensjo, I. and Svensson, L. (1990) Gas chromatographic-mass spectrometric identification of the fatty acids in borage oil using the picolinyl ester derivatives. J. Chromatogr., 521, 80-97. 

Dobson, G. and Christie, W.W. (1996) Structural analysis of fatty acids by mass spectrometry of picolinyl esters and dimethyloxazoline derivatives. TrAC Trends Anal. Chem., 15 (3), 130-7. 

Fatty Acid Analysis of the Isolated Labyrinthulids. After 14 d of culture, square pieces of agar culture (almost 2 cm long) were cut off, weighed, and dried at 105°C for 3 h. The dried samples were methylated directly with 10% HCl in methanol. The resultant methyl esters were applied to a gas-liquid chromatograph (GLC) (Shimadzu GC-17A, Kyoto, Japan), equipped with a TC-70 capillary column (GL Science Inc., Tokyo, Japan) under temperature programming (170-220°C at 2°C/min increment). The amounts of total fatty acid and each fatty acid were quantified using arachidic acid (20 0) as the internal standard. The LCPUFA level of the methyl ester sample was raised by urea fractionation, and then picolinyl ester of the LCPUFA was prepared. 

In their studies of the characterization of the picolinyl esters of epoxides of polyunsaturated fatty acids, Balazy and Nies (21) prepared the picolinyl esters via unstable imidazolide intermediates, which were generated by reaction of the epoxy acids with 1,1 -carbonyldiimidazole. For the epoxides, a method using acid chlorides as intermediates (22) and thionyl choride as reagent was not recommended because of the instability of the epoxides in acidic solution. However, using oxalyl chloride as reagent for formation of the acid chloride (Scheme 1) is a viable route and was used in the formation of derivatives of ene-yne acids (19). Reaction with HMP yielded the desired picolinyl ester (Scheme 2). The method of Balazy and Nies (21) as well as the oxalyl chloride—based acid chloride method of Christie (19) are given. 

New acid chloride-based preparation of picolinyl esters. A solution of HMP in CH2CI2 (20 mg/mL) is prepared and stored over anhydrous sodium sulfate. The solution is stable in a refrigerator for 1 mo. The stoek solution of HMP in CH2CI2 (0.5 mL) is added to the freshly prepared acid chloride (synthesis of the fatty aeid ehloride, see procedure for DMOX derivatives), eooled in an iee bath, and the mixture is then left to warm up to room temperature for 1 h (a white preeipitate may form). The solvent is added in a stream of nitrogen isohexane (5 mL) is added, followed by 0.5% aqueous sodium bicarbonate. The mixture is shaken thoroughly and then the solvent layer is removed by Pasteur pipette. It is dried via a small sodium sulfate column and concentrated under nitrogen. If necessary, the product can be purified by Florisil chromatography as described above. 

Harvey, D.J., Picolinyl Esters as Derivatives for the Structural Determination of Long Chain Branched and Unsaturated Fatty Acids, Biomed. Mass Spectrom. 9 33-38 (1982). 

Derivatization of Fatty Acids Derivatized fatty acids have been analyzed to improve ionization and fragmentation characteristics. The FAB spectra of these derivatives are structurally more informative, and fragmentations predominantly occur via CRF processes. One such example is the application of aminoethyl-triphenylphosphonium (AETPP) bromide derivatives to characterize fatty acids structurally by FAB-MS/MS [12]. Other examples include derivatives of aminon-aphthalenesulfonic acid (ANSA), aminobenzenesulfonic acid (ABSA), picolinyl ester, yV-methyl-2-alkylimidazoline (MIM), and dimethyl- (DMAE) and trimethy-laminoethyl (TMAE) esters [8, and references therein], ESI-MS/MS spectra of underivatized and ANSA derivatives of docosahexaenoic acid are compared in Figure 12.8. 

As methyl esters, GC separation from other fatty acids can be achieved on polar capillary columns (Fig. 5.3 Christie, Brechany and Shukla, 1989) and would probably be adequate on non-polar columns, as this is possible for dimethyloxazoline (DMOX) derivatives (Zhang et al, 1989). If necessary, prior isolation of cyclic monoenoic and dienoic fractions, separated from straight-chain saturates, monoenes and dienes, may be obtained by means of silver-ion HPLC (Christie, Brechany and Shukla, 1989). In this way, minor components were concentrated for subsequent GC-MS analysis as the pico-linyl (3-hydroxymethylpyridinyl) esters, and the possibility of inadequate resolution from straight-chain esters on non-polar columns, necessary for eluting these relatively involatile derivatives, was avoided. Presumably the use of modern high-temperature polar phases for GC-MS would eliminate possible resolution problems with picolinyl esters. 

Figure 5.4 Mass spectrum (70 eV) of a picolinyl ester derivative of 13-cyclopent-2-enyl-tridec-6-enoic (gorlic) acid. M" = molecular ion. Redrawn from Christie, W. W., Brechany, E. Y. and Shukla, V. K. S., Analysis of seed oils containing cyclopentenyl fatty acids by combined chromatographic procedures,... 

Alcohols can be derivatized to nicotinates, equivalent to picolinyl esters of fatty acids. A preliminary study involving GC-MS of the nicotinoyl derivative of l,3-di(9,10-methyleneoctadecanoyl)glycerol showed a prominent odd-numbered ion due to fragmentation through the ring of one of the... 

Figure 5.6 Mass spectrum (25 eV) of picolinyl ester derivative of 9,10-cyclopropaneoctadecanoic (dihydrosterculic) acid. Redrawn from Harvey, D. J., Picolinyl derivatives for the characterization of cyclopropane fatty acids by mass spectrometry, Biomed. Mass Spectrum., 11 (4), 187-92, 1984.

Similarly, the mass spectra of CLA picolinyl esters are inferior to those of DMOX derivatives. Typically in the mass spectra of picolinyl esters of unsaturated fatty acids, there are gaps of 26 amu between ions corresponding to fragments containing n- and... 

The fatty acid composition of Agathis robusta seeds was determined from the analysis of extracts that were converted to their picolinyl esters and separated on a 20°C Cg column (ELSD) using an acetonitrile mobile phase [1042], Elution was complete in 55 min. The following fatty acids were foimd 18 4 and 20 5 co-ehited 18 3 and 20 4 co-eluted 18 2 and 20 3 co-eluted 18 1 16 0 and 18 0. A 1 mg sample size generated reasonable peak intensities. 

Figure 4.1. Derivatives of fatty acids, (a) pyrrolidide (b) picolinyl ester (c) trimethylsilylether (d) isopropylidene derivative (e) butylboronate derivative (f) mercuric acetate adduct (g) dimethyidisulfide...

Nearly all analysts then are going to make use of polar stationary phases for the major proportion of their work. Any attempt at a comprehensive account of what has been achieved with columns of this type would be only slightly more readable than a telephone directory. I have therefore elected to show what can be accomplished with a "standard" 25 m WCOT column of fused silica coated with Carbowax 20M , and with a similar column coated with a highly polar phase CP-Sil 84 . As the first substrate for analysis, pig testis (obtained from an abattoir) lipids were selected, as the fatty acids have been well-characterised and have been used as an external standard in the Hormel Institute for some years [392,393] they contain a wide range of fatty acids of the (n-6) series, encountered typically in animal tissues. The second substrate is cod liver oil (obtainable from any pharmacy), which has also been well-characterised and is used as an external standard in the analysis of lipids of marine origin [15] it contains many different fatty acids, and especially those of the (n-3) family. Both of these materials were used by the author in studies of the efficacy of picolinyl ester derivatives of fatty acids for identification by GC-mass spectrometry (see Chapter 7), so all the main components have been identified unequivocally [173,184]. 

After fractionation, some relationships between various components are immediately apparent. Both the chain-length and the number of double bonds in each fatty acid derivative in the chromatograms are ascertained, while relative retention times can be correlated with those of constituents of the unfractionated material. By careful measurement of ECL values, it is then frequently possible to assign double bond positions. In this sample, the identities of nearly fifty different fatty acids in the fractions were confirmed by GC-mass spectrometry following conversion to the picolinyl ester derivatives (see Chapter 7). 

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