HPLC DETERMINATION OF KOJIC ACID 207 methanol (50 ml) was prepared and stocked at 4°C. Working standard solutions (0, 0.25, 0.5, 0.875, 1.25, 2.5, and 5 μg/ml) were prepared by dilution with 10% methanol. Borate buffer (0.1 M) was adjusted to pH 9.0 by the addition of NaOH. Borate buffer (50 μl) was added to a diluted standard sample (50 μl) then NBD-F solution in acetonitrile (50 μl, 2 mg/ml) was added. The mixture was vortexed and allowed to react for 7 min at 40°C then an aliquot (20 μl) was injected into the HPLC system. Sample preparation and addition-recovery tests. A skin-whitening cosmetic (150 μl) was di- luted to 100 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 a skin-whitening cosmetic (150 μl) with KA (30.0 or 50.0 μg) and diluting it in the same manner. An aliquot of 50 μl was analyzed, and KA concentration in the sample was determined. Recovery was calculated as follows: u100 Total amount after spiking – Spiked amount Recovery % Original amount RESULTS AND DISCUSSION DERIVATIZATION OF KA WITH NBD-F For the time course study, the reaction time was set at 2, 4, 7, 10, 13, or 16 min at 40°C. KA (50 μl, 2.5 μg/ml), borate buffer (50 μl, pH 9.0), and NBD-F (50 μl, 2 mg/ml) were mixed as described in the Experimental section. The derivatization of KA reached a max- imum at 7 min (Figure 2). Next, pH dependency (pH 8.0 to 10.0) was examined at the derivatization time of 7 min at 40°C. The peak area of NBD-KA was maximal at pH 9.0 (Figure 3). Thus, the de- rivatization time of 7 min at pH 9.0 was selected. Figure 2. Time course of formation of the NBD-KA derivative. The standard sample (2.5 μg/ml) was re- acted with NBD-F in borate buffer, pH 9.0, at 40°C.

JOURNAL OF COSMETIC SCIENCE 208 CHROMATOGRAMS Figure 4 shows typical chromatograms obtained from blank (A) and the standard sam- ple (B, 2.5 μg/ml). The retention time of NBD-KA was 7.8 min. The running time was 12.5 min. Figure 3. pH dependency of the formation of the NBD-KA derivative. The standard sample (2.5 μg/ml) was reacted with NBD-F for 7 min at 40°C in borate buffer at various pH values. Figure 4. Typical chromatograms of blank (A) and the standard sample (B, 2.5 μg/ml) after derivatization with NBD-F. Samples were reacted with NBD-F for 7 min in borate buffer, pH 9.0, at 40°C. Retention time of NBD-KA: 7.8 min (arrowed peak).