J. Cosmet. Sci., 63, 205–212 (May/June 2012) 205 Determination of kojic acid in a skin-whitening cosmetic by high-performance liquid chromatography coupled with ultraviolet detection after pre-column derivatization with 4-fl uoro-7-nitro-2,1,3-benzoxadiazole YASUHIKO HIGASHI and YOUICHI FUJII, Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Ho-3, Kanagawa-machi, Kanazawa 920-1181, Japan. Accepted for publication November 8, 2011. Synopsis Various methods for the determination of kojic acid (KA), a skin-whitening agent, have been reported by high-performance liquid chromatography (HPLC). In this study, the concentration of KA in a skin-whitening cosmetic was analyzed by HPLC with ultraviolet detection (380 nm) after pre-column derivatization with 4-fl uoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) in order to improve the sensitivity. The HPLC column was 150 mm × 3.0 mm i.d., containing 5 μm particles of C18 packing material. The mobile phase was prepared by the addition of acetonitrile (550 ml) to 450 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 0.35 ml/min. The retention time of NBD-KA was 7.8 min. A standard curve was obtained after derivatization with NBD-F in borate buffer (pH 9.0) at 40°C for 7 min. The calibration plot was linear, in the range of 0.25–5 μg/ml with an r2 value of 0.9982, and the lower limit of detection was 0.06 μg/ml (at a signal-to-noise ratio of 3:1 ab- solute amount of 0.4 ng/20 μl injection). The coeffi cient of variation was less than 9.6%. It was found that the amount of KA in a skin-whitening cosmetic was 237 ± 14 μg/ml (range: 219–255 μg/ml). Recovery in addition-recovery tests was within the range of 83.4% to 98.1%. INTRODUCTION Various methods for the determination of kojic acid (KA, 5-hydroxy-2-hydroxymethyl- 4-pyrone, a skin-whitening agent) have been reported, including high-performance liquid chromatography (HPLC) (1–3), ion-pair liquid chromatography (4), and electrochemis- try (5,6). In addition, Liu et al. (7) recently reported a method of determination of KA by using a carbon nanotubes/alizarin red S composite fi lm-modifi ed electrode this method has a lower limit of detection of KA of 0.014 μg/ml, and was applied for the determination of KA levels in vinegar and sauce. However, this system is expensive and complicated. Address all correspondence to Yasuhiko Higashi at y-higashi@hokuriku-u.ac.jp.

JOURNAL OF COSMETIC SCIENCE 206 Derivatization with an ultraviolet (UV)-absorbing agent generally offers good selectivity and sensitivity, and an HPLC–UV method for the assay of KA would be desirable for routine quality control, since the equipment is inexpensive, widely available, and easy to use. There has been no previous report of such an HPLC method for the detection of KA after derivatization. As a fl uorescent labeling agent of primary and secondary amino groups for HPLC-fl uores- cence detection (8–12), 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 (13–15). KA contains a phenol- like hydroxyl group. In this paper, we present a simple HPLC–UV method for the deter- mination of KA in a skin-whitening cosmetic after pre-column derivatization with NBD-F. The derivatization scheme is illustrated in Figure 1. 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) was 150 mm × 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 integra- tor (Shimadzu). The mobile phase was prepared by the addition of acetonitrile (550 ml) to 450 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 0.35 ml/min. REAGENTS KA (99%) and NBD-F were obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). A skin-whitening cosmetic was purchased from a market in Kanazawa city, Ishi- kawa Prefecture, Japan. Other general reagents were obtained from Wako Pure Chemical Industries (Osaka, Japan). PROCEDURES Derivatization. Ultrapure water was from a Milli-Q water purifi cation system (Simplic- ity® UV, Millipore Corporation, Bedford, MA). A standard solution of KA (100 mg) in Figure 1. Scheme of KA derivatization with NBD-F.