8 7 6 _s a4 E -3 2 0 ANALYSIS OF WOOL-WAX EXTRACT 16 18 20 chain length 22 Iii Sphingosine □ Dihydrosphingosine 24 Figure 4. Quantification at a molecular level of the ceramides II (mg/g) of wool-wax extract. b 35 30 25 6 a Time ( (min) 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 chain length 31 Figure 5. Medium-polarity compounds. (a) TLC-FID chromatogram of the p-TLC fraction E. Compound identification: 5, free acids 6, alcohols 7, sterols. (b) Quantification at a molecular level of the fatty alcohols (mg/g) of wool-wax extract. medium-chain fatty acids and the other for the monomodal distribution of the long- chain fatty acids (26). Fatty alcohols present in fractions D, E, F, and G were characterized by [M-CH 3 r and [CH 2 =O + Si(CH 3 ) 3 } (m/z 103). Their molecular weight ranged from 258 to 566. They

32 JOURNAL OF COSMETIC SCIENCE showed a bimodal distribution pattern, with a maximum at C 20 -C21 and another one at C 26 -C 27 . In Figure Sa, a TLC-FID chromatogram of fraction E is shown, and in Figure Sb, as an example of the potential of the combination of TLC-FID and GC-MS, fatty alcohol chemical class quantification is now completed at the molecular level. In these fractions, potential anticarcinogenic compounds ( 4) were found, but further work is needed to reveal their identity. Cholesterol, the most abundant sterol, was present in fractions C, D, and E, and des- mosterol, a minor compound, was found in fractions C and D. Ions [M.+} (m/z 458 and m/z 368), corresponding to water elimination and the formation of a double bond in position 3 were characteristic of cholesterol, and [M.+} (m/z 456) of desmosterol. Also, medium-polarity compounds, such as hydroxycholesteryl esters, were identified, eluting within fractions F and G. This chemical class of high molecular weight was monitored using the diagnostic ion (m/z 456), corresponding to the loss of the acid chain with the formation of a double bond. The components of this chemical class are not yet fully characterized. Low-polarity lipids At the limit between medium-polarity compounds and low-polarity compounds, tri- acylglycerols were identified in fractions G, H, and I. Nevertheless, their presence was confirmed with the ions [M.+}, [M-CH 3 r, and [M-Cncoor, and also several frag- ments from the acid moieties were observed as [C n =or. Due to their high molecular weights, these compounds were not fully characterized. Two ester functional groups, less polar than triacylglycerols, were identified in the fractions J and K as the aliphatic diesters. This chemical class was confirmed by GC- EIMS ions [C n =O+} and also by the pseudomolecular ions [M+NH 4 +}, obtained by GC-PCIMS. Due to their high molecular weight, these compounds were not fully characterized. Finally, monoesters, either aliphatic or steroidal, eluting in fraction K were identified as the most apolar compounds (Figure 6a). These chemical classes appeared as a single peak in TLC-FID, and thus GC-MS was needed in order to identify their components. Furthermore, the steryl esters, cholesteryl, lanosteryl, and dihydrolanosteryl esters have been identified by their sterol moiety, using electron impact ions at m/z 368, 393, and 395, respectively, and also by GC-PCIMS using the pseudomolecular ions [M+NH 4 +} (Figure 66). Molecular weights ranged from 554 to 722 for the cholesteryl esters, from 580 to 762 for the lanosteryl esters, and from 582 to 7 64 for the dihydrolanosteryl esters. Lanosteryl and dihydrolanosteryl esters presented similar patterns, with acid chains ranging from C 10 to C 23 , with a maximum at C 1 6 . Lanosteryl distribution was monomodal, and dihydrolanosteryl presented a bimodal distribution with a minor maximum at C 1 4 . Cholesteryl esters showed acid chains ranging from C 11 to C 23 , with a maximum at C 19 . In Figure 6a, a TLC-FID chromatogram of fraction K is shown, and in Figure 66, the GC-MS patterns of the different steryl esters found in this fraction are displayed. A huge amount of aliphatic esters are present in wool wax, as has already been described (7,20). They were identified using GC-MS ions, m/z 85, which are characteristic of an alkyl chain [CH 3 -(CH 2 ) n -COOH2+} and the molecular ion [M+}. They are composed