398 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS -v 1• NaHCO 3 •rashing 0 5 (min) 0 5 (min) Retention time Retention time Figure 4. Effect of NaHCO 3 washing on cleanup of acidic compounds in bergamot oil. I: Bergapten, II: Citropten, IS: Chrysene, Column: 5% SE-30/Chromosorb W 60-80, 3 mm in diameter and 150 cm in length, Glass, Column temperature: 230øC, Carrier gas: N 2 30 ml/min. solutions, but the adoption of strong alkali might lead to a recleavage of bergapten in its lactone ring. For this reason, 1% sodium bicarbonate was favored for the removal of such acids without affecting the bergapten (see Figure 4-2). STEP 3 Brown et al. (6) used 14% EGS as the liquid phase for the gas chromatographic column, while Mizuishi et al. (3) adopted 2% PEG-20M. In this investigation, 5% SE-30 was employed because it was preferable to 2% OV-17, 5% NPGS and 3% DEGS column o Q o• o 0 200 400 (pE/3ml) Figure 5. Calibration curve of bergapten.

DETECTION OF BERGAPTEN 399 packings, with regard-to the separation of bergapten from citropten and chrysene, as well as with regard to the symmetry of the peaks. A typical calibration curve obtained by the internal standard method of Step 3 is illustrated in Figure 5. The plot of 15-1,500 ng of bergapten per tube gives a straight line passing through the origin. A typical gas chromatogram of bergamot oil which had passed through the procedures of Steps 1 and 2 is illustrated in Figure 3-2. Peak I (retention time 5.2 rain) and Peak II (retention time 3.2 rain) were analyzed by GC-MS the results are listed below. (Peak I) m/e (%): 216 (M +, C•2H804, 100), 201 (M+--CH3, 34), 188 (M+-CO, 16), 173 (48), 145 (27), 89 (19), 63 (13), 51 (22). (Peak II)m/e (%): 206 (M +, CnH•004, 100), 178 (M + --CO, 77), 163 (45), 135 (28), 77 (13), 69 (20), 63 02). From tR values, mass spectra and melting points, the substances corresponding to Peaks I and II were identified as bergapten and citropten, respectively. Results of bergapten determination on several samples of bergamot oil are shown in Table I. Table I Bergapten Contents in Several Bergamot Oils Sample weighed Content Oil (g) (3tg/g) 1 1 ND • 10 1 2 1 1050 3 1 990 4 1 1150 5 1 ND 6 1 ND 7 1 ND •Not detected. The maximal content amounted to about 1,000 ktg/g. It is presumed that the samples had been subjected to removal procedure of bergapten since their bergapten contents were quite small compared with those of raw oils. Mean recoveries of five trials spiked at the level of 10 or 100 ktg/g to the sample were 85.0 and 92.6%, respectively. By Table II Recoveries of Bergapten From Several Kinds of Cosmetics Content Bergapten Added Recovery Cosmetics (10 g) (•tg/g) (•tg/g) (%) Eau de Cologne ND • Skin lotion ND Hair liquid ND Hair tonic ND Pomade ND 10 65 100 93 100 92 100 90 100 92 100 88 •Not detected.