258 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table IV (Continued) Manufacturer Perfume Estimated Bergamot Equivalent Based Upon 0.27 g/100 ml 5-MOP (ml/100 ml) Estimated 5-MOP Concentration (g/1 O0 ml) Paco Rabanne Prince Matchabelli Puig Revlon Rochas Shulton Vanda Vidal Yardley Yves Saint Laurent (Y.S.L.) Calandre Perfume 0.5 0.00 ! 35 Paco Rabanne Toilet Water 0.062 0.00017 Cachet Cologne 0 0 Wing Song Toilet Water 0 0 Aqua Brava Aftershave 0.016 0.00004 Puig Aftershave 0.0 ! 6 0.00004 Aquamarine Toilet Water 0 0 Charlie Cologne 0.0 ! 6 0.00004 Intimate Toilet Water 0.016 0.00004 Audace Perfume 2 to 2.5 0.00540 to 0.00675 Eau de Rochas 1 0.00270 Femme Cologne 0.125 0.00034 Femme Toilet Water 0. ! 25 0.00034 Femme Perfume ! to 1.5 0.00270 to 0.00405 Madame Rochas Parfum 0.031 to 0.062 0.00008 to 0.00017 de Toilet Monsieur Rochas Cologne 0.031 to 0.062 0.00008 to 0.00017 Old Spice Aftershave 0.03 ! 0.00008 Si Jolie Cologne 0.016 0.00004 Pino Sylvestre Cologne 0.75 to ! 0.00203 to 0.00270 Bond Street Cologne 0.25 to 0.5 0.00068 to 0.00135 Caprice Cologne 0 0 Cougar Aftershave 0.016 0.00004 Flair Cologne 0.125 to 0.25 0.00034 to 0.00068 Khadine Cologne 0.062 0.000 ! 7 Reverie Cologne 0.016 0.00004 Sea Jade Cologne 0 0 Shanida Cologne 0. ! 25 0.00034 Rive Gauche Cologne 0.125 0.00034 Y.S.L. After shave 0.75 to 1 0.00203 to 0.00270 Y.S.L. Toilet Water (Males) 0.75 to 1 0.00203 to 0.00270 Y.S.L. Toilet Water (Females) 0.062 to 0.125 0.00017 to 0.00034 Y.S.L. Perfume 0.25 to 0.5 0.00068 to 0.00135 bergamot oil). It was impossible to predict the presence or absence of 5-MOP from the price, kind or manufacturer of the perfume. Recovery experiments. As shown in Table IV, the concentration of 5-MOP in Paco Rabanne toilet water and Agua Brava aftershave, as determined by the C. a/bicans test, was 0.00017 and 0.00004% respectively, whereas Caprice cologne and Wind Song toilet water did not contain any 5-MOP. Following the addition of a known quantity of bergamot oil and using the same test, the concentration of 5-MOP in the resultant solu- tions was shown to be approximately 0.00135%. The recovery of bergamot oil from these perfumes was, therefore, virtually complete. Of the four resultant solutions, only Agua Brava aftershave had a TLC pattern similar to that of bergamot oil and Group A perfumes. The TLC pattern of Paco Rabanne toilet water, Caprice cologne and Wind Song toilet water was similar to that of Group B perfumes, which showed a poor separation of 5-MOP from the adjacent TLC bands. This was further confirmed by spectrophotometry where the absorption spectrum of the 5-MOP band of Agua Brava

QUANTITATIVE ANALYSIS OF BERGAPTEN 259 Table ¾ A Comparison of the Phototoxic Activity of Yves Saint Laurent Toilet Water Using Bergapten TLC'Fraction, Filter Paper Disc and Silica Gel to Which the Perfume Was Added Bergapten TLC Whole Perfume On Whole Perfume Fraction Filter Paper Disc On Silica Gel Diameter of Killing Zone (mm) 26.8 25.2 23.6 S.E.M. 0.37 0.20 0.40 aftershave was similar to that of bergamot oil and different from that of three other perfumes. The importance of TLC. The results of the phototoxic effects of YSL toilet water using the bergapten TLC fraction, 10 mm filter paper discs and silica gel containing the whole perfume, are shown in Table V. The killing zone was largest with the bergapten TLC fraction and smallest with the silica gel containing the whole perfume the filter paper disc produced an intermediate result. The values obtained for the filter paper discs and silica gel differed significantly from the result of the bergapten TLC fraction at the 1 and 0.1% levels of probability respectively, when the Student's "t" test was used. A number of perfumes produced killing of the yeast in the nonirradiated plates when the whole perfume was applied to filter paper discs. The perfumes, which on TLC were shown to contain 5-MOP (Chanel 5, Quelques Fleurs and F•te perfumes, Charlie Cologne and Cougar aftershave), produced an inhibitory zone in the control plate smaller than that obtained in the irradiated plate. Others which did not show a definite 5-MOP fraction on TLC (Bellodgia, Youth Dew, Ritz and Estee Lauder perfumes and Aquamarine toilet water) produced an inhibition zone in the control plate equal to that obtained in the irradiated plate. THE SPECTROPHOTOMETRIC TEST The results of the determination of 5-MOP concentration in some of the perfumes tested using this technique are shown in Table VI. It was impossible to perform accurate quantitative analysis of 5-MOP in Group B perfumes because the method was based on a comparison of the absorption of the 5-MOP TLC fraction of perfumes with that of bergamot oil and a significant contami- nation of this fraction was expected to yield higher results. This was indeed the case Table VI The Estimated 5-MOP Concentration in Perfumes Using the Spectrophotometric Test. Group A Represents Perfumes Which Have a Relatively Clear Bergapten TLC Fraction. In Group B Perfumes, the Bergapten TLC Fraction Shows Significant Impurities Manufacturer Perfume Group Estimated Bergamot Equivalent Based Upon 0.27 g/100 ml 5-MOP (ml/100 ml) Estimated 5-MOP Concentration (g/100 ml) Christian Dior Faberge' Rochas Vidal Eau Sauvage Toilet Water A Brut Aftershave B Eau de Rochas A Pino Sylvestre Cologne A 0.90 2.70 1.00 O.9O O.O0243 0.00729 0.00270 0.00243