Detection of hydroxy fatty acids in biological samples using capillary gas chromatography in combination with positive and negative chemical ionization mass spectrometry

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Abstract
The most common method for use in the structural analysis of hydroxy fatty acids in biological samples is the gas chromatography‐mass spectrometry (GC‐MS) analysis of trimethylsilyl ethers of the methyl esters using electron impact ionization. A comparison of electron impact (EI) with chemical ionization mass spectrometry (CI‐MS) shows that CI‐MS is the superior technique. All ions necessary for structural analysis are observed at sufficiently high levels of intensity when methane or isobutane are used as reactant gases. The molecular weight can be determined from the ion group M+H, M‐15 and M+H−90. The ionic series M+H−n×90 enables one to determine the number of hydroxyl groups. The position of the hydroxyl groups can be derived from the fragments of the α‐cleavage of the fatty acid chain. The application of heptafluorobutyrates as derivatives for hydroxy fatty acid methyl esters shows advantages in the trace analysis of these compounds. Heptafluorobutyrates exhibit useful mass fragmentation patterns in the positive as well as in the negative CI mode. With methane as the reactant gas, M+H usually is base peak in positive mass spectra. The ionic series M+H−n×214 leads to the number of hydroxy groups in the molecule. In the negative mass spectra, M and M‐20 are indicative for the molecular weight. The ion group m/z 213, 194 and 178 at high levels of intensity is typical for heptafluorobutyrates. The advantage of the application of heptafluorobutyrates is the high sensitivity which can be obtained in trace analysis using negative MS. Heptafluorobutyrates of hydroxy fatty acids gave a 20‐fold higher response in the negative scan mode compared to that of the positive. The detection limit for heptafluorobutyrates in negative CI‐MS was on the order of 1 fg (10−15 g).