JOURNAL OF COSMETIC SCIENCE 522 METHOD VALIDATION Linearity. As shown in Figure 3, standard curves of hinokitiol on the RP-4 column, de- tected at 240 and 345 nm, were constructed by plotting integrated peak area vs hinokitiol concentration (0.05–5 μg/ml). Linear relationships were obtained: y = 667x + 15.3 at 240 nm and y = 265x + 17.5 at 345 nm. The squared regression coeffi cients (r2) at 240 and 345 nm were 0.9974 and 0.9933, respectively. Sensitivity. The detection limits at 240 and 345 nm were established to be 0.005 (1 ng, 0.03 μM) and 0.01 (2 ng, 0.06 μM) μg/ml, respectively (S/N = 3). Since previous meth- ods provided detection limits of 40 pg and 0.21 μM of hinokitiol (6,7), our method is intermediate in terms of sensitivity. Figure 2. Typical chromatograms of hinokitiol in a standard sample (1 μg/ml), obtained with RP-18, RP-8, and RP-4 columns. (A) 240 nm (B) 345 nm. (1) RP-18 (2) RP-8 (3) RP-4. Arrowed peak: hinokitiol. Figure 3. Standard curves of hinokitiol. (○) at 240 nm. ( ) at 345 nm.

MEASUREMENT OF HINOKITIOL BY HPLC 523 PRECISION AND ACCURACY Precision and accuracy for intra- and interday assays are shown in Tables I and II. In the intraday assay (Table I), the ranges of standard deviation (S.D.) from the average (C.V.) of hinokitiol at 240 and 345 nm were within the ranges of 4.1–5.8% and 3.8–6.8%, respec- tively. The recoveries were in the range of 99.4–103.0%. In the interday assay (Table II), the corresponding values were within the ranges of 4.4–8.2% and 4.7–8.7%, respec- tively, and the recoveries were in the range of 96.6–104.4%. APPLICATION TO THREE PERSONAL CARE PRODUCTS The hinokitiol levels in the skin lotion and body soap used in this study were not speci- fi ed, while the label concentration for the hair restorer was 0.5 mg/ml. Each product was examined six times with our newly developed method. Typical chromatograms are shown in Figure 4. For the hair restorer, the average values of hinokitiol level with detection at 240 nm and 345 nm were 0.536 mg/ml (C.V. = 4.1%, recovery = 107.2%) and 0.502 mg/ml (C.V. = 3.9%, recovery = 100.4%), respectively. Recovery was satisfactory. The corresponding results were 7.81 μg/ml (C.V. = 6.7%) and 8.02 μg/ml (C.V. = 6.8%) for the skin lotion and 0.182 mg/g (C.V. = 7.2%) and 0.178 mg/g (C.V. = 7.4%) for the body soap, respectively. In addition, recovery values of diluted samples spiked with a known amount of hinokitiol were examined at 240 and 345 nm. The known amount of hinokitiol (0.250 mg, 80.0 μg, Table I Intraday Assay Reproducibility for Determination of Hinokitiol Concentration (μg/ml) Measured (μg/ml) (mean ± S.D., n = 5) C.V. (%) Recovery (%) 240 nm 0.05 0.0497 ± 0.0029 5.8 99.4 0.5 0.509 ± 0.027 5.3 101.8 5 5.12 ± 0.21 4.1 102.4 345 nm 0.05 0.0515 ± 0.0035 6.8 103.0 0.5 0.508 ± 0.024 4.7 101.6 5 5.06 ± 0.19 3.8 101.2 Table II Interday Assay Reproducibility for Determination of Hinokitiol Concentration (μg/ml) Measured (μg/ml) (mean ± S.D., n = 5) C.V. (%) Recovery (%) 240 nm 0.05 0.0494 ± 0.0033 6.7 98.8 0.5 0.514 ± 0.042 8.2 102.8 5 5.22 ± 0.23 4.4 104.4 345 nm 0.05 0.0520 ± 0.0045 8.7 104.0 0.5 0.483 ± 0.037 7.7 96.6 5 5.14 ± 0.24 4.7 102.8