]. Cosmet. Sci., 57, 23-35 CTanuary/February 2006) Comparative characterization of a wool-wax extract by two complementary chromatographic techniques ERIC JOVER, CARMEN DOMINGUEZ, PILAR ERRA, and JOSEP MARIA BAYONA, Environmental Chemistry Department (BJ., J.M.B.) and Surfactant Technology Department (C.D., P.E.), I.I.Q.A.B.-C.S.I.C., Jordi Girona 18-26, E-08034 Barcelona, Spain. Accepted for publication August 17, 2005. Synopsis A detailed characterization of lipids present in a wool-wax extract, obtained by supercritical fluid extraction, is presented. Thin-layer chromatography coupled to a flame ionization detector (TLC-FID) has been used as a screening technique to identify the different chemical classes of complex mixtures. Furthermore, to confirm peak identification obtained by TLC-FID and to check its suitability as a quality control technique, preparative TLC fractionation has been carried out by reanalyzing the different fractions obtained by TLC-FID and by gas chromatography coupled to mass spectrometry in the electron impact and in the chemical ionization modes. By means of this comparative study, the TLC-FID identification has been confirmed. Also, the complementarity of the different techniques used confirmed the identification of different lipid classes, namely fatty acids, alcohols, sterols, sterane diol, stanone, steryl sulphate, hydroxy- acids, alkane diols, aliphatic and steryl esters, hydroxy steryl esters, triacylglycerol, diesters, and ceramides types II and VI. INTRODUCTION Wool wax is secreted by the sebaceous glands of sheep to protect wool fibers from external aggressions. The analysis of wool-wax fractions, obtained by means of an etha- nol-modified supercritical carbon dioxide extraction by thin-layer chromatography coupled to a flame ionization detector (TLC-FID), reveals that wool wax is formed by a complex mixture of apolar and polar lipids (1). Lanolin, which is a refined fraction of wool wax, formed mainly by apolar lipids, is an important ingredient in many phar- maceutical and cosmetic products. This interest arises from the fact that lanolin and human skin lipids have similar properties. In this way, lanolin can act as a vehicle agent to transport active products through human skin (2). It also allows stabilization of high-water-content emulsions that are applied to increase skin hydration (3 ). Recently, there has been a growing interest in several lanolin constituents such as the long-chain Address all correspondence to Eric Jover. 23

24 JOURNAL OF COSMETIC SCIENCE saturated fatty alcohols, which have shown to be anticarcinogenic ( 4) and the ceramides and cholesteryl esters, which are proposed as membrane-producer compounds (5). Lanolin is a complex lipid mixture consisting mainly of fatty acids, fatty alcohols, sterols, diols, hydroxyacids, aliphatic esters, steryl esters, diesters, and other minor constituents (2,6). Such a complexity is highlighted, for example, by the composition of the mono- ester chemical class, which is estimated to be formed roughly by 10,000 individual components (7) due to the large number of possible fatty acid and alcohol combinations. Indeed, each of them can be present in three different isomeric forms: normal lineal compounds, iso ((w-1)-monomethyl-substituted) and anteiso ((w-2)-monomethyl- substituted). In accordance with its complexity, lanolin cannot be synthetically ob- tained. Furthermore, the molecular characterization of natural wool wax and lanolin can be useful to supply additional information for further applications. Until recently, the technique for analyzing lanolin involved a hydrolysis step to cleave the ester bonds of the aliphatic and steryl esters and amides (8-10). This approach gave useful information, but was limited in determining the original mixture composition in esters, amides, fatty acids, and fatty alcohols. In order to avoid this limitation, a current trend in lipid analysis is to analyze the intact wool wax and lanolin. Accordingly, two different approaches have been developed: TLC-FID, which permits the lipid class characterization, and gas chromatography (GC) coupled to mass spectrometry (MS), which permits the sample characterization at molecular level. TLC-FID is a rapid, sensitive, and simple method for the analysis of any chemical material of low volatility that can be separated by conventional TLC. In fact, this technique has considerably improved the sensitivity of TLC. The TLC-FID technique has been commonly used for the determination of lipid classes (11), vitamins, amino acids, and a variety of other lipid compounds (12). Moreover, it has already been used specifically to characterize the lipid composition of different internal wool lipids (13,14) and wool-wax extracts (1,15), but identification confirmation with other analytical techniques is needed. Molecular characterization of complex lipid mixtures can be carried out by GC-MS in the electron impact (EI) and chemical ionization modes (CI). In the case of wool wax containing high-molecular-weight compounds such as aliphatic esters, steryl esters, or ceramides, three types of columns for GC analysis are available: short-capillary, high- temperature gas chromatography (HTGC) (16) and sub-ambient pressure gas chroma- tography (17). The highest resolution could be obtained with long HTGC columns, but for thermolabile compounds such as cholesteryl esters, sub-ambient pressure GC is more convenient, because it can elute compounds at lower temperatures. Several families of intact wool-wax lipids have been previously characterized at the molecular level, such as fatty acids, alcohols, hydroxy acids, and diols by means of GC-EIMS (18) aliphatic esters by means of HTGC-EIMS (19) and steryl esters by means of sub-ambient pressure GC-EIMS/CIMS (20). However, molecular characterization is complex, tedious, and expensive therefore, in most cases, these techniques will not be suited for routine quality control analysis. In this work, TLC-FID identification of a chosen wool-wax extract was confirmed by a combination of chromatographic techniques. Preparative thin-layer chromatography (p- TLC) was used to perform a wool-wax extract fractionation with a similar resolution and selectivity as those of the ones obtained by TLC-FID. The various fractions obtained were