JOURNAL OF COSMETIC SCIENCE 132 Sample preparation and addition-recovery tests. Three tested skin lotions (A, 5.0 mg B, 100 mg C, 200 mg) were each diluted to 50 ml with 10% methanol, derivatized, and analyzed as described above. Addition-recovery tests were carried out to assess the accuracy of the method by spiking skin lotion samples with OPP (0.005 mg for A, 0.003 mg for B, 0.003 mg for C). An aliquot of 100 μl was analyzed and the OPP concentration in each sample was determined. Recovery was calculated as follows: Total amount after spiking Spiked amount Original amount Recovery % 100 ୉ × RESULTS AND DISCUSSION DERIVATIZATION OF OPP WITH NBD-COCL For the time-course study, the reaction time was set at 1, 2, 3, 5, or 10 min at room tem- perature at various pH values. OPP (100 μl, 0.1 μg/ml), IS solution (100 μl, 0.1 μg/ml), borate buffer (100 μl, various pH values), and NBD-COCl (100 μl, 2 mg/ml) were mixed as described in Experimental. The reaction of OPP at pH 8.5 was faster than under the other pH conditions examined, and the derivatization of OPP reached a maximum at 2 min, but subsequently declined somewhat (Figure 2A). The peak area ratio of NBD-CO- OPP derivative to NBD-CO-IS derivative was most stable at pH 8.5 and 9.0 (Figure 2B). Thus, the derivatization time of 2 min at pH 8.5 was selected. CHROMATOGRAMS Figure 3 shows typical chromatograms obtained from (a) blank spiked with IS and (b) standard sample (0.1 μg/ml) spiked with IS. The retention times of NBD-CO-OPP and Figure 2A. Time courses of formation of NBD-CO-OPP derivative. Standard samples (0.1 μg/mL) were reacted with NBD-COCl in borate buffer at pH 8.0 to 10.0 at room temperature. ( ), pH 8.0 (○), pH 8.5 (□), pH 9.0 ( ), pH 9.5 (×), pH 10.0. Data are expressed as mean values of two experiments. Figure 2B. Time courses of peak area ratio for formation of NBD-CO-OPP and NBD-CO-IS derivatives. Standard sample (0.1 μg/mL) and IS solution (0.1 μg/mL) were reacted with NBD-COCl in borate buffer at pH 8.0 to 10.0 at room temperature. ( ), pH 8.0 (○), pH 8.5 (□), pH 9.0 ( ), pH 9.5 (×), pH 10.0. Data are expressed as mean values of two experiments.

DETERMINATION OF O-PHENYLPHENOL IN SKIN LOTION 133 NBD-CO-IS derivatives were 16.2 and 22.2 min, respectively. The running time was 25 min. METHOD VALIDATION Linearity. A standard curve was constructed by plotting integrated peak area ratio versus con- centration of OPP. The plot was linear (y = 6.297×–0.0217) in the range of 0.01–0.2 μg/ml with an r2 value of 0.9960. Sensitivity. The values of the lower limits of quantifi cation and detection were 10 ng/ml (absolute amount of 40 pg/20 μl injection, signal-to-noise ratio of 10:1) and 3 ng/ml (absolute amount of 12 pg/20 μl injection, signal-to-noise ratio of 3:1), respectively. As shown in Table I, the sensitivity of our method (3 ng/ml, absolute amount of 12 pg) was no less than those of various previously reported methods (range of 3.4–350 pg as absolute amount) (6,7,9,10,19–21). PRECISION AND ACCURACY Precision and accuracy for intra-day and inter-day assays of OPP are shown in Table II. In the intra-day assay, the range of standard deviation was within 4.5–6.4% of the mean, and recoveries were within the range of 91.4–96.0%. In the inter-day assay, the range of standard deviation was within 5.3–8.8% of the mean, and recoveries were within the range of 89.4–94.5%. Figure 3. Typical chromatograms of blank spiked with IS (A) and standard sample spiked with IS (B) after derivatization with NBD-COCl. Samples were reacted with NBD-COCl for 2 min at pH 8.5 at room temperature. Compounds: OPP, 0.4 ng/20 μl injection volume IS, 0.4 ng/20 μl injection volume. Retention times: 16.2 min, NBD-CO-OPP (1) 22.2 min, NBD-CO-IS (2). Chromatogram (A) shows no peak of NBD-CO-OPP (1).