Bile acids, mass spectra

Early studies of bile acid mass spectra were made by Bergstrom, Ryhage, and Stenhagen (3-5) and the results were used to determine the structure of Hammarsten s a-phocaecholic acid (6). Other groups applied mass spectrometry to the elucidation of structures of the closely related bile alcohols (7). At about this time combined gas chromatography-mass spectrometry instruments were developed that could be used in studies of compounds with molecular weights as high as those of bile acid derivatives (8-13). The early... 

Many of the common major fragmentations were discussed by Bergstrom, Ryhage, and Stenhagen (5). Their results have been extended, with more comprehensive discussions of bile acid mass spectra (I, 12, 15, 18, 19). The fragmentation patterns of sterol derivatives are in many respects similar to those of the bile acids discussions of sterol mass spectra are given in (1, 11, 20-23). General reviews of steroid mass spectra may be consulted (1, 24,25). 

Spectra of O-methyloximes of methyl 3,6-diketo-5a- and 3,6-diketo-5/3-cholanoates have been reported by Allen et al. (30). The O-methyloximes, prepared as described by Fales and Luukkainen (49), were found to be particularly useful derivatives since the 5a- and 5 -isomers could be separated by gas chromatography, which was not the case with the free ketones. Considerable differences in relative intensities of the peaks were noted between the spectra of the two isomers. The 5y3 compound gave a base peak at mje 138 (probably ring A) whereas the molecular ion was base peak in the spectrum of the 5a-epimer. These results show the value of O-methyloximes in mass spectrometry of ketonic bile acids. It should be recognized that partial derivative formation may occur, and that formation of syn- and o //-isomers may result in a complex mixture of products from a pure compound. 

The intensity of the peak at mje 292 depends on the stereochemistry of A/B ring junction. It is very low in the spectrum of methyl 3,7-diketo-5a-cholanoate (36) and is about 30% of the base peak in that of the 5 -isomer. The same difference is seen for the peak at mje 111. Comparisons of spectra of different 7-keto bile acid derivatives indicate that the latter ion may represent loss of side chain from the ion of mass 292. 

GC-MS analysis is used to confirm the identities of ions in the LSI-MS urine spectrum and show that the excretion of abnormal cholanoids is >20 times normal. In the case of 5 ff-reductase deficiency GC-MS analysis should show that S-oxo-A" bile acids account for >70% of the total urinary bile acid excretion. In the case of sterol 27-hydroxylase deficiency (CTX), GC-MS analysis should indicate that the major cholestane pentols in the urine are 3,7,12,22,25 and 3,7,12,23,25-pentols. (One patient has been described who had familial cholestatic liver disease associated with greatly increased urinary excretion of 5jff-cholestane-3a,7a,12a,24 S,25-pentol [see previous table]). Liquid secondary ion-tandem mass spectrometry (LSI-MS/ MS) is an alternative method to GC-MS and can rapidly confirm the identity of a number of diagnostic ions that are found in the LSI-MS spectrum of urine. These include sulphated and taurine-conjugated abnormal metabolites such as those observed in 3 ff-HSDH deficiency (32.1), 5)9-reductase deficiency (32.2), oxysterol 7a-hydroxylase deficiency (32.4) and peroxisomal disorders [13]. 

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