4 JOURNAL OF COSMETIC SCIENCE 8% phosphoric acid solution and was charred by heating at 180°C for 5 min. Each spot was densitometrically determined with a Shimadzu CS-900 photodensitometer (Kyoto, Japan) and was compared to a calibration reference curve. Duplicate analyses were performed for each measurement. DETERMINATION OF MEA WITH HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) This procedure was performed with slight modifications of methods previously reported (6). Thus, tricosanoic acid as the IS of MEA and ADAM were added to the integral lipid solution (CHC1 3 /CH 3 OH 1: 1) and the solution was then derivatized for 1 hr at room temperature. Aliquots of the derivatized integral lipid solution were injected into an HPLC apparatus equipped with a fluorescence detector (Hitachi 1-6000 series, Hitachi, Ibaragi, Japan) and a 12.5-cm x 4.0-mm Superspher 60 RP-Se (Merck, Darmstadt, Germany) kept at 40°C. The derivatives were detected at EX.365 nm and EM.412 nm. Two solvents of (A) acetonitrile/water 85: 15 and (B) acetonitrile were used for separa­ tion at a flow rate of 1.5 ml/min with a gradient elution program, which included first (A) 100%, second (B) 2.5%/min to (B) 100% (40 min), and finally holding of (B) 100% for 20 min. The content of MEA was calibrated based on an equivalent molar sensitivity of MEA to the IS. Duplicate analyses were performed for each measurement. DETERMINATION OF CH WITH GC This procedure was performed with slight modifications of previously reported methods (7). Thus, analyses were performed on a Hewlett-Packard 5890 series GC equipped with a flame ionization detector, an Ultra #2 25-m x 0.25-mm x 0.33-µm fused silica capillary column (Hewlett-Packard), and a Hewlett-Packard 3396 Series II integrator. Aliquots of the lipid solution were injected via split mode in a ratio of 1 :50 at 340°C. The temperature of the detector was maintained at 340°C. Helium was used at a flow rate of 1.0 ml/min. The column temperature was programmed from 200°C to 320 ° C at 20°C/min, which was held for 10 min. The content of CH was determined using a calibration curve. Duplicate analyses were performed for each measurement. DETERMINATION OF CERs WITH HPLC Procedures for alkaline hydrolysis of CERs for the isolation of sphingoids were per­ formed, based on a slight modification of a method reported for CERs of the stratum corneum (24). Thus, the lipids were saponified by heating for 18 hr at 65°C in 10 ml 1 N KOH in CH 3 OH/hexane 5:1. Ten micrograms of threo-dihydrosphongosine was then added as the IS. By means of liquid-liquid extraction, CHC1 3 extracts were ob­ tained. The sphingoids extracted were isolated by preparative TLC (silica gel 60) because hydrolyzed fatty acids interrupted the reaction between sphingoid and OPA. The sphin­ goids isolated were derivatized at room temperature for 30 min with an added solution of OPA methanol, a 2-ME/methanol solution, and a sodium tetraborate aqueous solu­ tion. Aliquots of the derivatives were injected into the same HPLC equipment used for the MEA analysis with a 15-cm x 4.6-mm 1-Column ODS (Kagakuhin-Kensa-Kyoukai, Tokyo, Japan) kept at 40°C. The derivatives were detected at EX.344 nm and EM.433 nm. Two solvents of (A) CH 3 OH/water 85:15 and (B) CH 3 OH were used for separation

HAIR LIPID COMPOSITION 5 at a flow rate of 1.0 ml/min with a gradient elution program, which included first holding (A) 100% for 15 min, second (B) 5%/min to (B) 100% (20 min), and finally holding of (B) 100% for 15 min. The content of sphingoids was calibrated based on area ratios of the peaks of interest to the IS (threo-dihydrosphingosine), resulting in CER content. Duplicate analyses were performed for each measurement. STATISTICAL ANALYSES Correlation analyses among the level of lipids and a principal component analysis (PCA) with a correlation matrix for hair lipid composition were performed using Excel Tahenryo-Kaiseki version 3.0 software (Esumi, Tokyo, Japan). RESULTS MOLECULAR SPECIES OF CERs IN HUMAN HAIR A total ion GC/MS chromatogram of silylated CERs in extractable lipids is shown in Figure 1, and those in integral lipids exhibited almost the same peak pattern. Peaks A-F in Figure 1 had distinctive fragments: (A) m/z = 313, 3 70, (B) m/z = 299, 313, 458, (C) m/z = 313,398, (D) m/z = 313,426, (E) m/z = 313,454, and (F) m/z = 313,480. It has been established that a mass spectrum of silylated CERs can be characterized by a C2-C3 fragmentation of its sphingoids amidified with its N-acyl moiety (19,25). Therefore, fragments of m/z = 311 or m/z = 313 and m/z = [M-311] or m/z = [M-313] provide sufficient information about their sphingoids such as sphingenine (sphingosine) or sphinganine (dihydrosphingosine) and about their N-acyl moieties. Accordingly, the peaks were identified as follows: (A) 2-N-palmitoylamino-octadecane-1,3-diol, (B) 2-N­ a-hydroxypalmitoylamino-octadecane-1,3-diol, ( C) 2-N-stearoylamino-octadecane-1,3- 15 B A C 20 F D E TlmMnn 2!5 A B C D E F �o � H �o �: �o � OH �OH �o � OH OH 0 � Figure 1. A GC/MS total ion chromatogram of silylated CERs from extractable lipids. A, B, C, D, E, and F show their molecular structure.