! a ,u 28.00 29.00 ANALYSIS OF WOOL-WAX EXTRACT 1 3 30.00 31.00 32.00 3300 34.00 15:30 35.00 ;-- 36.00 m/z368 mlz393 jsl C � t 16:30 ·� /\. � T0 1s:3o �j.,___l ��=r��=;c.......,����UG��i��-----'J'---'r--/, --"- iL2� =,7=,���==�=:3=0==�c-;-�-= 28.00 29.00 30.00 31.00 32.00 33.00 34.00 35.00 36.00 mlz395 d m,30� � o � o �o ·��.,.......,.., -q,-�=4=,-,�JlJU�--.=.+M----}_/\--,,--A�=-\. -,.0 ���-----�....,_...,__.., 33 28.00 29.00 30.00 31.00 32.00 33.00 Time (min) Figure 6. Low-polarity compounds. (a) TLC-FID chromatogram of the p-TLC fraction K. Compound identification: 1, monoesters 2, unknown 3, diesters. The chromatogram has been scaled in order to see the minor compounds. Pattern of the different steryl esters ident:ified in fraction K. (b) GC-MS fragmentogram of cholesteryl esters (m/z 368). (c) GC-MS fragmentogram of lanosteryl esters (m/z 393). (d) GC-MS fragmentogram of dihydrolanosteryl esters (m/z 395). 34.00 35.00 36.00 of a homologue series from 33 to 54 carbon atoms having molecular weights ranging from 550 to 788, with a maximum of abundance centered in the neighborhood of the C4 7 homologue. A summary of the different lipids identified in each p-TLC spot of the wool-wax extract, according to TLC-FID and GC-MS analysis, is shown in Table II. CONCLUSIONS Comparison between p-TLC combined with TLC-FID and p-TLC combined with GC-MS was able to confirm the tentative chemical class identification carried out by TLC-FID. Furthermore, the complementary aspects of using chromatographic tech- niques of different resolution and selectivity were highlighted. On one hand, low- resolution techniques such as TLC-FID were useful as screening and quality control

34 JOURNAL OF COSMETIC SCIENCE Table II Summary of the Different Lipids Identified in an SFE Wool-Wax Extract with Their Retention-Behavior Characteristics in Preparative TLC, TLC-FID, and GC-MS Diagnostic Ions Using Silylated Derivatives Lipids Cholesteryl sulfate Cholestanediols Hydroxyacids Diols Ceramide type II: sphyngosine Ceramide type II: dihydrosphyngosine Ceramides type VI Cholestanones Sterols Fatty alcohols Fatty acids Hydroxycholesteryl esters Triacylglycerol Diesters Aliphatic esters Cholesteryl esters Lanosteryl esters Dihydrolanosteryl esters * Non- polar elution. ** Polar elution. Preparative TLC Rf Fraction 0.000-0.072 A,B,C 0.000-0.033 A,B 0.000-0.072 A,B,C 0.000-0.072 A,B,C 0. 000-0.033 A,B 0.000-0.033 A,B 0.000-0.072 A,B,C 0.072 C 0.072-0.191 C,D,E 0.132-0.283 D, E, F, G 0.191-0.368 E, F, G, H 0.237-0.283 F, G 0.283-0.467 G, H, I 0.553-0.730 J, K 0.730 K 0.730 K 0.730 K 0.730 K 1 Relative abundance between brackets. TLC-FID GC-MS Rf MW Diagnostic ions I 0.04** 0.68** 546 456 (100), 546 (1) 388-528 147 (28) 374-514 147 (28) 0.76** 681-793 311(100) 0.76** 683-795 313 (100) 0.32** 0.72-0.88** 382,472 382 (53), 472 (30-67) 0.28* 456,458 366 (100), 368 (80) 0.36* 258-566 103 (35) 0.46* 216-566 117 (81) 0.46-0.54* 456 0.54* 638 [M"}, [M-CnCOO}' 0.66* 524 [M+NH4+} 0.76* 550-788 85 (10-25) 0.76* 554-722 368 (100) 0.76* 580-762 393 (100) 0.76* 582-764 395 (100) methods for the different chemical classes, and on the other hand, due to sample complexity and to the lack of available standards, a confirmation step based on GC-MS was necessary. In this work, a wool-wax extract has been analyzed, and the presented methodology can be useful not only for wool-wax or lanolin characterization, but also for any other complex naturally occurring lipidic mixture. For the wool-wax extract analyzed, more than 15 different chemical classes have been identified, highlighting its complexity. Finally, for the first time, type II ceramides were completely characterized in wool-wax extracts. ACKNOWLEDGMENTS The authors thank Mrs. Roser Chaler and Mrs. Dori Fanjul for their technical assistance, and Dr. Roshan Paul for his English review. This research was supported by CICYT Project 2FD97-0509. The authors are grateful to Peinaje del Rfo Llobregat S.A. and Praxair Espana S.A. for supplying the raw wool and the CO2 (SFC quality), respectively. REFERENCES (1) C. Domfoguez, E. Jover, J.M. Bayona, P. Erra, Characterization of supercritical fluid extracts from raw wool by TLC-FID and GC-MS,.J. Am. Oil Chem. Soc., 80, 717-724 (2003).