Picolinyl ester

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. 

The picolinyl esters (32) permit the location of double bonds, since these give distinctive fragmentations that are characteristic of the double-bond positions. They are easily prepared and are not too polar for separation by gas-liquid chromatography (GLC). It has been confirmed that the picolinyl esters are the most useful, since they permit unequivocal identifications even with polyunsatured components. It was also demonstrated that derivatives of this type, prepared from natural mixtures, give satisfactory resolutions when subjected to GLC on capillary columns of fused silica coated with a nonpolar methylsilicone phase, for identification by mass spectrometry (MS). 

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

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

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. 

Cui, D. and Harvison, P.J., Determination of the site of glucuronidation in an N-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters, Rapid Commun. Mass Spectrom., 14(21), 1985, 2000. 

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. 

Identification of n-6 DPA as the picolinyl ester was performed by gas chromatography-mass spectrometry (Hewlett Packard 5971A, Rockeville, MD) (3). 

Fig. 5. Mass spectra (70 eV) of picolinyl ester derivatives of cyclic dienoic acids derived from a-linolenic acid in heated linseed oil (A) 10-(2 -propyl-cyclopentenyl)dec-c/s-9-enoate and (B) 9-(2 -prop-frans-1-enyl-cyclohex-c/s-4-enyl)nonanoate. Source Ref. 10.

Picolinyl (3-hydroxymethyl pyridinyl) esters as derivatives of fatty compounds for the purpose of mass spectral structure determination were originally introduced by Harvey (20). Picolinyl esters have replaced the originally developed pyrrolidides (6-9) because of easier preparation and ions of greater abundance (20). For both pyridinecarboxylates and hydroxymethyl pyridinyl esters, it was shown fliat the nitrogen atom must be in the 3-position in order to obtain spectra with peaks of suffi-... 

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. 

As an example, the mass spectrum of the picolinyl ester of oleic acid is given in Figure 2. Picolinyl esters give prominent peaks characteristic of the picolinyl moiety at m/z 92, 108, 151 (McLafferty rearrangement ion), and 164. The gap of 26 amu between m/z 234 and 260 locates the double bond in the example in Figure 2. 

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. 

A better derivative is the picolinyl ester. This derivative, under El, acquires a positive charge on the nitrogen atom rather than on the ester oxygen atoms. The stability of the pyridine ring prevents fragmentation near the charge site, but causes the nitrogen atom to act as a proton acceptor and to abstract a proton from various positions of the alkyl chain to... 

Figure 6 El (70eV) mass spectrum of the picolinyl ester of 14-methyloctadecanoic acid. Abstraction of a proton from different positions of the aiiphatic chain produces radical sites that induce chain cleavage. The position of the methyl group Is revealed by the absence of the even-mass ion at m/z 318 and the relatively high abundance of the ions at m/z 304 and 332.

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.

Until recently, a complete analysis of CFAMs from heated oils was a daunting task first, because of the complexities of the mixtures and, second, because of the difficulties in obtaining full structural information, especially with respect to double bond positions. However, recently this has been made possible by combining fractionation of a CFAM mixture (obtained by urea adduction) by silver-ion HPLC with GC-MS analysis of the picolinyl esters or DMOX derivatives of the fractions (Fig. 5.10 Christie et al, 1993 ... 

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