HPLC DETERMINATION OF HINOKITIOL 385 METHOD VALIDATION Linearity. A standard curve was constructed by plotting the integrated peak area vs the concentration of hinokitiol. The plot was linear (y = 271.8x + 3.252) in the range of 0.2 to 4 μg/ml, with an r2 value of 0.9985. Sensitivity. The values of the lower limits of quantifi cation and detection were 0.17 (abso- lute amount of 1.1 ng/20 μl injection signal-to-noise ratio of 10:1) and 0.05 μg/ml (absolute amount of 0.33 ng/20 μl injection signal-to-noise ratio of 3:1), respectively. As shown in Table I, the sensitivity of the presented method is about 8-fold less than that of the method by Endo et al. (6) and can be classed as moderate, compared with other reported methods (6–8), although the sensitivity values of some GC methods were not described (1,4). PRECISION AND ACCURACY Precision and accuracy for intra- and interday assays of hinokitiol are shown in Table II. In the intraday assay, the range of standard deviation was within 4.6% to 7.1% of the Figure 4. Typical chromatograms of blank (A) and the standard sample (B, 1 μg/ml) after derivatization with NBD-F. Samples were reacted with NBD-F for 10 min in borate buffer, pH 9.0, at 60°C. Retention time of NBD-hinokitiol: 7.2 min (arrowed peak).

JOURNAL OF COSMETIC SCIENCE 386 mean, and recoveries were within the range of 92.0% to 102.8%. In the interday assay, the range of standard deviation was within 5.4% to 9.4% of the mean, and recoveries were within the range of 90.5% to 101.0%. INTERFERENCE Skin lotion usually contains paraben, ester possessing phenol-like hydroxyl group, as an effective preservative. Therefore, selectivity was preliminarily investigated on the detec- tion of NBD-hinokitiol peak. All retention times of methyl 4-hydroxybenzoate, ethyl 4-hydroxybenzoate, propyl 4-hydroxybenzoate, isopropyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, isobutyl 4-hydroxybenzoate, and benzyl 4-hydroxybenzoate deriva- tives (each paraben concentration 1 μg/ml) by NBD-F were 6.9 min. Peaks of tested paraben derivatives were found to be slightly separated from NBD-hinokitiol peak (re- tention time 7.2 min). One question arises about same retention time of paraben deriva- tives. Although it is considered that these parabens will be hydrolyzed to generate Table I Sensitivity of Various Methods for Determination of Hinokitiol Method Limit of detection Reference Capillary GC Not described 1 GC Not described 4 HPLC 40 pg 6 Capillary zone electrophoresis 0.21 μM 7 HPLC 1 ng (at 240 nm), 2 ng (at 345 nm) 8 HPLC 0.33 ng 0.05 μg/ml This article Table II Intra- and Interday Assay Reproducibility for Determination of Hinokitiol Hinokitiol (μg/ml) Measured (μg/ml, mean ± S.D., n = 5) C.V. (%) Recovery (%) Intraday assay 0.2 0.184 ± 0.013 7.1 92 1 0.942 ± 0.058 6.2 94.2 4 4.11 ± 0.19 4.6 102.8 Interday assay 0.2 0.181 ± 0.017 9.4 90.5 1 0.962 ± 0.081 8.4 96.2 4 4.04 ± 0.22 5.4 101 C.V.: Coeffi cient of variation.