J. Cosmet. Sci., 66, 129–137 (March/April 2015) 129 Simple determination of o-phenylphenol in skin lotion by high-performance liquid chromatography coupled with fl uorescence detection after pre-column derivatization with 4-(N-chloroformylmethyl-N-methylamino)-7-nitro- 2,1,3-benzoxadiazole YASUHIKO HIGASHI and KAZUNORI KONNO, Department of Analytical Chemistry, Faculty of Pharmaceutical Sciences, Hokuriku University, Kanagawa-machi, Kanazawa 920-1181, Japan. Accepted for publication February 15, 2015. Synopsis o-Phenylphenol (OPP) in skin lotion was quantitated by high-performance liquid chromatography coupled with fl uorescence detection after pre-column derivatization with 4-(N-chloroformylmethyl-N-methylamino)- 7-nitro-2,1,3-benzoxadiazole (NBD-COCl) in borate buffer (pH 8.5) at room temperature for 2 min. The col- umn [150 mm × 3.0 mm internal diameter (i.d.)], which contained 5 μm particles of C18 packing material, was eluted at room temperature (fl ow rate: 0.5 ml/min) with mobile phase prepared by addition of acetonitrile (550 ml) to 450 ml of Milli-Q water containing trifl uoroacetic acid (0.1 v/v%). 2-Hydroxyfl uorene was used as an internal standard. The retention times of NBD-CO-OPP and NBD-CO-IS derivatives were 16.2 and 22.2 min, respectively. The calibration plot was linear in the range of 0.01–0.2 μg/ml with an r2 value of 0.9960, and the lower limit of detection was 0.003 μg/ml (at a signal-to-noise ratio of 3:1 absolute amount of 12 pg/20 μl injection). The coeffi cient of variation was less than 8.8%. Contents of OPP in three skin lotions were deter- mined with the present system, and the recovery from spiked samples was satisfactory. INTRODUCTION o-Phenylphenol (OPP) has antibacterial and antiviral activities, and is widely used in house- holds, industry, and hospitals to disinfect surfaces, and as a preservative in cosmetics, plas- tics, etc. (1,2). Although OPP is an irritant for skin and mucous membranes, it exhibited low acute toxicity in animal experiments (3). Dermal administration of OPP promoted skin carcinogenesis in CD-1 female mice initiated with 7,12-dimethylbenz[a]anthracene (4). The Ministry of Health, Labour and Welfare in Japan recommends that the upper limit level of OPP in cosmetics should be 0.30 g/100 g for mucous membranes and for skin areas that are not washed after application, although the level has not been decided for the Address all correspondence to Yasuhiko Higashi at y-higashi@hokuriku-u.ac.jp.

JOURNAL OF COSMETIC SCIENCE 130 case of cosmetics where the skin is washed after application (5). On the other hand, the Japanese government approved the use of OPP as a food additive for citrus fruits in 1977 with the permitted maximum residue level of 10 ppm in whole fruits (6,7). The World Health Organization’s view on the toxicity of OPP is as follows (8): “A health-based value of 1 mg/l can be calculated for OPP on the basis of an ADI of 0.4 mg/kg of body weight, based on a NOAEL of 39 mg/kg of body weight per day in a 2-year tox icity study for decreased body weight gain and hyperplasia of the urinary bladder and carcinogenicity of the urinary bladder in male rats, using an uncertainty factor of 100. Because of its low toxicity, however, the health-based value derived for OPP is much higher than OPP con- centrations likely to be found in drinking-water. Under usual conditions, therefore, the presence of OPP in drinking-water is unlikely to represent a hazard to human health.” Analysis of OPP in grapefruit juice has been performed by high-performance liquid chromatography with ultraviolet absorption detection (HPLC—UV) after pre-column labeling with 4-fl uoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) (9). While this system is simple, it shows poor sensitivity. Yang et al. (7) developed a highly sensitive method of OPP determination by HPLC with electrochemical detection, using a microbore column this afforded a detection limit of 3.4 pg. Gas chromatography—mass spec- trometric (GC—MS) methods for determination of OPP after derivatization with pen- tafl uorobenzoyl bromide and ferrocenecarboxylic acid chloride have been applied to beer and citrus fruit samples, respectively (1,2). Blasco et al. (10) used liquid chroma- tography (LC)—atmospheric pressure chemical ionization MS for OPP determination in fruits and vegetables. However, MS or electrochemical detection requires expensive equipment. Instead, a simple and inexpensive method is desirable for routine OPP analysis. NBD-F has been used as a fl uorescence labeling agent of primary and secondary amino groups for HPLC—fl uorescence detection (11—15). The NBD heterocyclic ring is strongly fl uorescent, but NBD-labeling at the phenolic hydroxyl group of N-acetyltyrosine, chlorophenols, eugenol or OPP does not afford a fl uorescent derivative, so NBD-F has been used for labeling of these compounds in combination with UV detection (9,16— 19). Here, we set out to develop a simple, more sensitive HPLC-fl uorescence analysis for determination of OPP in skin lotion by means of pre-column derivatization with 4-(N- chloroformylmethyl-N-methylamino)-7-nitro-2,1,3-benzoxadiazole (NBD-COCl), which is expected to be available as a fl uorescence labeling agent for the phenolic hy- droxyl group of OPP. The derivatization scheme is shown in Figure 1. EXPERIMENTAL APPARATUS The HPLC system comprised a model L-6200 pump (Hitachi, Tokyo, Japan), a Rheo- dyne injection valve (Cotati, CA) with a 20-μl loop and a model RF-10A fl uorometer (Shimadzu, Kyoto, Japan) operating at an excitation wavelength of 470 nm and an emission wavelength of 540 nm. The HPLC column (ODS-4 GL Science, Tokyo, Ja- pan) was 150 mm × 3.0 mm i.d. in size, and contained 5 μm particles of C18 packing material. Quantifi cation of peaks was performed using a Chromatopac Model C-R3A integrator (Shimadzu, Kyoto, Japan). The mobile phase was prepared by the addition of acetonitrile (550 ml) to 450 ml of Milli-Q water containing trifl uoroacetic acid