The fragmentation behavior of 18 chlorogenic acids that are not substituted at position 1 has been investigated using LC-MS(4) applied to a methanolic coffee bean extract and commercial cider (hard cider). Using LC-MS(3), it is possible to discriminate between each of the three isomers of p-coumaroylquinic acid, caffeoylquinic acid, feruloylquinic acid, and dicaffeoylquinic acid, and a hierarchical key has been prepared to facilitate this process when standards are not available. MS(4) fragmentations further support these assignments, but were not essential in reaching them. The distinctive behavior of 4-acyl and 3-acyl chlorogenic acids compared with the 5-acyl chlorogenic acids is a key factor permitting these assignments. The fragmentation patterns are dependent upon the particular stereochemical relationships between the individual substituents on the quinic acid moiety. Fragmentation is facilitated by 1,2-acyl participation and proceeds through quinic acid conformers in which the relevant substituents transiently adopt a 1,3-syn-diaxial relationship. Selected ion monitoring at m/z 529 clearly indicated the presence in coffee of six caffeoylferuloylquinic acid isomers, whereas previously only two or three had been demonstrated. The hierarchical key permitted specific structures to be assigned to each of the six isomers. These assignments are internally consistent and consistent with the limited data previously available.

The fragmentation behavior of all six dicaffeoylquinic acids (diCQA) has been investigated using LC-MS(4). It is possible to discriminate between each of the isomers including those for which commercial standards are not available. For diCQA, the ease of removal of the caffeoyl residue during fragmentation is 1 approximately = 5 > 3 > 4. The distinctive fragmentation observed for the little-studied 1,4-dicaffeoylquinic acid involves elimination of the C1 caffeoyl residue, repeated dehydrations leading to the aromatization of the quinic acid moiety, and its decarboxylation. It is suggested that this process is initiated by the C1 carboxyl protonating the C5 hydroxyl in the inverted chair conformer, followed by its protonating the C1 caffeoyl residue in the favored chair conformation. The fragmentation of 1-caffeoylquinic acid is indistinguishable from that of the commercially available 5-caffeoylquinic acid, but these two isomers can be distinguished easily by their facile chromatographic resolution on reversed phase packings. The hierarchical key previously developed for characterizing chlorogenic acids has been extended to accommodate 1-caffeoylquinic acid and the 1-acyl dicaffeoylquinic acids.

LC-MS4 has been used to detect and characterize in green coffee beans 12 chlorogenic acids not previously reported in nature. These comprise three isomeric dimethoxycinnamoylquinic acids (7-9) (Mr 382), three caffeoyl-dimethoxycinnamoylquinic acids (22, 24, and 26) (Mr 544), three diferuloylquinic acids (13-15) (Mr 544), and three feruloyl-dimethoxycinnamoylquinic acids (28, 30, and 32) (Mr 558). Structures have been assigned on the basis of LC-MS4 patterns of fragmentation and relative hydrophobicity and, in the case of the dimethoxycinnamoylquinic acids, by comparison with authentic standards. Several new structure-diagnostic fragmentations have been identified for use with diacyl-chlorogenic acids, for example, m/z 299 and 255 for C4 caffeoyl, m/z 313 and 269 for C4 feruloyl, nearly equal elimination of both cinnamoyl residues for vic-3,4-diacyl, and an increasing ratio of "dehydrated" ions to "non-dehydrated" ions at MS2 with increasing methylation of those cinnamoyl residues. Possible mechanisms have been proposed to account for the fragmentations observed. The mass spectrometric resolution of six isomeric chlorogenic acids (Mr 544) in a crude plant extract by fragment-targeted LC-MS2 and LC-MS3 experiments illustrates the analytical power and advantage of ion trap mass spectroscopy.

LC-MS4 has been used to detect and characterize in green coffee beans 15 quantitatively minor p-coumaric acid-containing chlorogenic acids not previously reported in nature. These comprise 3,4-di-p-coumaroylquinic acid, 3,5-di-p-coumaroylquinic acid, and 4,5-di-p-coumaroylquinic acid (Mr 484); 3-p-coumaroyl-4-caffeoylquinic acid, 3-p-coumaroyl-5-caffeoylquinic acid, 4-p-coumaroyl-5-caffeoylquinic acid, 3-caffeoyl-4-p-coumaroyl-quinic acid, 3-caffeoyl-5-p-coumaroyl-quinic acid; and 4-caffeoyl-5-p-coumaroyl-quinic acid (Mr 500); 3-p-coumaroyl-4-feruloylquinic acid, 3-p-coumaroyl-5-feruloylquinic acid and 4-p-coumaroyl-5-feruloylquinic acid (Mr 514); and 4-dimethoxycinnamoyl-5-p-coumaroylquinic acid and two isomers (Mr 528) for which identities could not be assigned unequivocally. Structures have been assigned on the basis of LC-MS4 patterns of fragmentation. Forty-five chlorogenic acids have now been characterized in green Robusta coffee beans.

Abstract-The carbon-I3 NMR spectra of lanosta-8-en-3p-ol, lanosta-8,24-dien-3p-ol, lanosta-7,9 (1 I )-dien-3b-ol, lanostan-38-01, eupha-8-en-3,!l-ol, eupha-8,24-dien-3p-ol, ursa-12-en-3/l-ol (cc-amyrin) and oleana-12-en-3p-01 (p-amyrin) have been obtained and completely assigned. The results of this study provide chemical shift data for methyl, methylene, methine and quaternary carbon atoms in tetra-and pentacyclic triterpenoid spectra. The carbon-13 N M R spectrum of a triterpenoid provides a unique fingerprint for the molecule.

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The triterpenoids euphol and lanosterol are similar in many respects. This is illustrated by parallel reactions carried out on rings B and c of the tetracyclic nucleus and by degradations which demonstrate the presence of an isooctenyl side chain in both compounds.Lanostene and euphene are shown to have the same number of methyl groups. In the light of earlier work this demonstrates that ring D of euphol cannot be six-membered.Dehydrogenation of euphadiene affords 1 : 7 : 8-tnmethylphenanthrene ; the same degradation applied to isoeuphadiene gives 1 : 2 : 5-trimethylnaphthalene. This evidence provides strong support for the view that migration of a methyl group is involved in the conversion of euphenol into isoeuphenol.A tricyclic diketone from euphenol is shown to have two methylene groupings adjacent to the ketone groups. This observation excludes an earlier formula for euphol.The constitution and stereochemistry shown in (I ; R = OH) are advanced to explain these and earlier observations on the chemistry of euphol. THE triterpenoid alcohol euphol, a major constituent of dried euphorbia latex, was first isolated in a state of purity by Newbold and Spring (J., 1944, 249). In the last decade this compound has been the subject of detailed structural investigation. The present paper summarises new experimental work which, when combined with previous studies cited in detail below, throws considerable light on the constitutional and stereochemical problems involved. Our views on these subjects can be conveniently summarised in the expression (I; R = OH) for euphol. A preliminary communication to this effect has already appeared (Barton, SIcGhie, Pradhan, and Knight, Chenz. and Ifid., 1954, 1325).We commence our exposition by reporting work (for preliminaq communications see