JOURNAL OF COSMETIC SCIENCE 382 of the tropolone ring, its instability to heat, and its adsorption on the stationary phase. Quantitative determinations of hinokitiol by GC and capillary GC have been performed after derivatization with trimethylsilyl chloride and with diazomethane, respectively (1,4). Hanafusa et al. (5) presented an HPLC determination of hinokitiol in cosmetics by HPLC with ultraviolet (UV) detection by adding copper (II) to the mobile phase to form the hinokitiol–copper (II) complex. However, the sensitivity of these methods was not discussed. Also, it is diffi cult and time-consuming to treat waste containing copper (II). Endo et al. (6) developed a sensitive HPLC determination of hinokitiol based on formation of the difl uoroborane derivative, with the detection limit of 40 pg. Dyrskov et al. (7) determined hinokitiol by using a capillary zone electrophoresis–UV method, which provided the detection limit of 0.21 μM. Recently, we established an HPLC-dual UV (240 and 345 nm) method of hinokitiol determination in personal care products by using a reversed-phase C4 column with detection limits of 0.005 μg/ml (absolute amount of 1 ng) and 0.01 μg/ml (absolute amount of 2 ng) at 240 and 345 nm, respectively (8). Nevertheless, a simple, sensitive, and convenient assay that would be suitable for routine quality control of personal care products containing hinokitiol is still needed. As a fl uorescent labeling agent of primary and secondary amino groups for HPLC-fl uorescence detection (9–13), 4-fl uoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) has been used and it has also been used as a UV-labeling reagent reactive with the phenolic hydroxyl group of N-acetyltyrosine, chlorophenols, and eugenol (14–16), and the phenol-like hydroxyl group of kojic acid (17). Hinokitiol contains a reactive hydroxyl group (vinyl alcohol) derived from the tropolone structure. In this article, we present a simple HPLC–UV method for the determination of hinokitiol in skin lotion after precolumn derivatization with NBD-F. The derivatization scheme is illustrated in Fig. 1. Our data and validation results indicate that this method will be suitable for routine quality control purposes. EXPERIMENTAL APPARATUS The HPLC system consisted of a model LC-10ATvp pump (Shimadzu, Kyoto, Japan), a Rheodyne injection valve (Cotati, CA) with a 20-μl loop, and a model SPD-10Avp UV detector (Shimadzu) operating at 380 nm. The HPLC column (C18-MS-II, Nacalai Tesque, Kyoto, Japan) was 150 × 3.0 mm i.d., containing 5 μm particles of C18 packing material. The quantifi cation of peaks was performed using a Chromatopac Model C-R8A Figure 1. Scheme of hinokitiol derivatization with NBD-F.

HPLC DETERMINATION OF HINOKITIOL 383 integrator (Shimadzu). The mobile phase was prepared by the addition of acetonitrile (500 ml) to 500 ml of Milli-Q water containing trifl uoroacetic acid (0.1 v/v%). The samples were eluted from the column at room temperature at a fl ow rate of 0.40 ml/min. REAGENTS Hinokitiol, NBD-F, and seven parabens (methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, isopropyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, and benzyl 4-hydroxybenzoate) were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). General reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). Skin lotion was purchased from a market in Kanazawa city, Ishikawa Prefecture, Japan. PROCEDURES Preparation of standard solutions. A stock solution of hinokitiol (40 μg/ml) in 1% ethanol was prepared in a dark glass bottle and stored at 4°C. It was diluted appropriately with water to prepare several working solutions. The working concentration of hinokitiol was set at 0, 0.2, 0.4, 0.7, 1, 2, and 4 μg/ml. Derivatization. Ultrapure water was from a Milli-Q water purifi cation system (Simplicity® UV, Millipore Corporation, Bedford, MA). Borate buffer (0.1 M) was adjusted to pH 9.0 by the addition of NaOH. Borate buffer (50 μl) was added to each working standard solution (50 μl) then NBD-F solution in acetonitrile (2 mg/ml, 50 μl) was added. The mixture was vortexed and allowed to react for 10 min at 60°C then an aliquot (20 μl) was injected into the HPLC system. Sample preparation and addition-recovery tests. Tested skin lotion contains hinokitiol, para- ben, allantoin, perfume, and so on. Concentration of the composition was not expressed. An aliquot of skin lotion (500 μl) was diluted to 100 ml with water, and analyzed after derivatization as described earlier. Addition-recovery tests were carried out to assess the accuracy of the method by spiking skin lotion (500 μl) with hinokitiol (50 or 100 μg) and diluting it in the same manner. An aliquot of 50 μl was analyzed, and the hinokitiol concentration in the sample was determined. Recovery was calculated as follows: 100 Total amount after spiking – Spiked amount Recovery (%) Original amount = × RESULTS AND DISCUSSION DERIVATIZATION OF HINOKITIOL WITH NBD-F For the time course study, the reaction time was set at 5, 7.5, 10, 15, 20, or 30 min at 60°C. Hinokitiol (50 μl, 2 μg/ml), borate buffer (50 μl, pH 9.0), and NBD-F (50 μl, 2 mg/ml) were mixed as described in the Experimental section. The derivatization of hinokitiol reached a plateau at 10 min (Fig. 2).