248 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS tained. The 5-MOP content of this sample of bergamot oil was previously determined with the use of a spectrophotometric technique and was found to be 0.27 g/100 ml (2). Random samples of perfumes were supplied by local stores during the years 1974 to 1976. Two methods for the quantitative analysis of 5-MOP in perfumes were used: the Candida albicans phototoxicity test (5) and the spectrophotometric test (6). THE CANDIDA ALBICANS PHOTOTOXICITY TEST The test consists of the application of test substances to Sabouraud's dextrose agar plates • (2.5 to 3 mm uniform agar thickness) streaked with Candida a/b/cans and sub- sequent exposure to UVA duplicate plates were kept in the dark as control. Phototoxic substances are then identified by the presence of a clear zone of killing of the yeast around the discs in the 'irradiated plate greater than that obtained in the con- trol nonirradiated plate. DOSE-RESPONSE RELATIONSHIP It was first of all important to find out whether the test was useful for the quantitative assay of 5-MOP and other psoralens. An attempt was made, therefore, to derive dose- response curves which relate the concentration of the psoralen used to the diameter of the killing zone. Various concentrations of the bergamont oil, pure 5-MOP and pure 8-MOP in ethanol were prepared. Aliquots of 2 /xl of each of these solutions were spotted on 20 x 20 cm aluminium-backed, thin-layer chromatography silica gel plates, thickness 0.25 mm (E. Merck). The plates were processed by the ascending method with hexane-ethyl acetate (75:25) to a distance of 8 cm from the origin, allowed to dry and viewed under ultraviolet light (Camag ultraviolet viewer, peak 366 nm). The fluorescent 5-MOP band in bergamot oil was identified by its yellowish-green fluorescence and its Rf, which was identical to that of standard 5-MOP. The 5-MOP fractions of bergamot oil spots were marked and the silica gel containing them was scraped onto clean paper and added to Sabouraud's dextrose agar plates freshly seeded with C. a/b/cans each spot occupying a circle 8 to 10 mm in diameter. Since the size of the killing zone varies with the thickness of the agar (5,7) and possibly with other fac- tors as well, it was considered important when comparing the phototoxic effects of psoralens to apply similar quantities of each compound to the same agar plate. In these experiments, therefore, concentrations were chosen so that a similar range of quan- tities of 5-MOP derived by TLC from bergamot oil, pure 5-MOP and 8-MOP were ap- plied to each agar plate (Table I). For example, 0.00025 to 0.001% solutions of pure 5-MOP and 8-MOP and the equivalent of 0.00034 to 0.00135% 5-MOP present in 0.125 to 0.5% bergamot oil (which contains 0.27% 5-MOP) were used in plate 1. Similarly, 0.0025 and 0.005% pure 5-MOP and 8-MOP and 0.0027 and 0.0075% 5-MOP in bergamot oil were applied to plate 2. Concentrations of 40% bergamot oil and 0.1% pure 5-MOP and 8-MOP were applied to two separate plates because of the large killing zones produced. One set of plates was kept in the d•rk and another set, •Myocological peptone (Oxoid L 40) 10 g, Agar No. 1 (Oxoid L 11) 15 g, Dextrose 40 g/litre pH ap- proximately 5.6.

QUANTITATIVE ANALYSIS OF BERGAPTEN 249 Table I The Concentration of Bergamot Oil (Coty), 5-MOP and 8-MOP Applied to Each Sabouraud's Dextrose Agar Plate Bergamot Oil 5-MOP 8-MOP ml/100 ml g/100 ml g/100 ml Plate No. 1 0.125 0.00025 0.00025 0.25 0.0005 0.0005 0.5 0.001 0.001 2 1 0.0025 0.0025 2.5 0.005 0.005 3 5 0.01 0.01 4 10 0.025 0.025 5 20 0.05 0.05 6 40 0.1 -- 7 -- -- 0.1 with the plate covers on, was irradiated for two days with UVA from two fluorescent tubes (Philips Blacklight TW 20 W/08, wave-length range 300-355-400nm), placed 7 cm apart and 20 cm above the plates. The experiment was repeated six times and the diameter of the killing zone was measured, the minimum measurable zone being the diameter of the silica gel spot. In comparing the efficacy of various photoactive psoralens, using this test, it was necessary to ensure that any difference in phototoxic effects based on the size of the killing zone is not due to a relative diffusion of the psoralens in the agar layer. A tur- bidimetric test using Sabouraud's liquid medium was devised. 2 In this test, 3 ml of the medium were delivered to 35 x 10 mm sterile tissue culture petri dishes (Flow Labora- tories). The inoculom consisted of a suspension of C. albicans prepared by transferring 2 to 3 dabs from a two-day slant culture on a sterile cotton swab to 6 ml sterile water, to obtain an optical density of the suspension of approximately 1.2 at 600 nm. A volume of 0.1 ml was added to each of the petri dishes and a total of 20 dishes were prepared. Fresh solutions of 0.005% 5-MOP and 8-MOP in ethanol (w/v) were made and 0. ! ml of each added separately to each culture followed by gentle swirling to mix the contents. Five petri dishes containing 5-MOP and another five containing 8-MOP were exposed for 24 hr to UVA as previously described. Duplicates were covered with light- excluding material as controls. With the use of a glass rod, the contents of each culture plate were then mixed and any yeast growth on the walls of the petri dish brought into suspension. The optical density was then recorded at 600 nm. THE 5-MOP CONTENT OF PERFUMES As the presence of 5-MOP in perfumes is a result of the addition of 5-MOP containing bergamot oil, it was considered important in the assessment of 5-MOP concentration in perfumes to use bergamot oil of known concentration of 5-MOP as reference instead of pure 5-MOP. Aliquots of 5/xl of the perfumes and 5/xl of each 0.25, 0.5 and 1% bergamot oil Coty (contains 0.27 g/100 ml 5-MOP) in ethanol were 2Pancreatic digest of casein (Oxoid L 42) 5 g, Peptic digest of fresh meat (Oxoid L 49) 5 g, Dextrose 20 g per litre pH approximately 5.7.