JOURNAL OF COSMETIC SCIENCE 384 Next, the pH dependency (pH 7.5–10.0) was examined at the derivatization time of 10 min at 60°C. The peak area of NBD-hinokitiol was maximal at pH 9.0 (Fig. 3). Thus, the derivatization time of 10 min at pH 9.0 was selected. CHROMATOGRAMS Fig. 4 shows typical chromatograms obtained from blank (A) and standard sample (B, 1 μg/ml). The retention time of NBD-hinokitiol was 7.2 min. The running time was 15 min. Figure 2. Time course of the formation of the NBD derivative of hinokitiol. The standard sample (2 μg/ml) was reacted with NBD-F in borate buffer, pH 9.0, at 60°C. Figure 3. pH dependency of the formation of the NBD derivative of hinokitiol. The standard sample (2 μg/ml) was reacted with NBD-F for 10 min at 60°C in borate buffer at various pH values.

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).