INTERACTIONS OF COSMETIC PIGMENTS WITH PRESERVATIVES 85 The numbers of surviving bacteria and yeast were determined by the plate count method. The necessary controls and blanks were carried out concurrently with the test experiments. CALCULATION OF INACTIVATION DEGREE The inactivation degree (ID) of bactericidal activity in the presence of cosmetic pig- ments was calculated with the following equation: d/c d' a ID - - A x l0 B - b/a b ß c where "a" represents the number of viable cells in the sample tube which contains the cosmetic pigments, the preservative, and microorganism "b" is the number in the blank tube which contains the cosmetic pigment and microorganism "c" is the number in the control 1 which contains the preservative and the microorganism and "d" is the number in the control 2 which contains only the microorganism. When the resulting B is about 0, the bactericidal activity is considered to be little affected by the cosmetic pigment tested. On the other hand, when B is an integral number (ID 1), this indicates inactivation by pigment. Thus the exponential number B in the equation is directly related to the degree of inactivation of the preservative by the cosmetic pigment. DETERMINATION OF ADSORBED PRESERVATIVE The experimental procedure is shown in Figure 1. The incubation mixture (without inoculum) was vigorously shaken and incubated at 37øC for 5 hr. After centrifugation, the solubilized amounts of preservatives in the supernatant solutions were determined by high performance liquid chromatography (HPLC) and compared with the amounts originally added. A model ALC/GPC 201 HPLC (Waters Associates) equipped with a Waters Model 730 data module was used in this study. The chromatographic column (4 mm X 150 mm) was stainless steel. Warm water ran through the jacket to maintain the column temperature at 40øC. The column was packed with Lichrosorb PR-18 (5Ix, Merk). Samples were injected into the HPLC column using a Waters Model 710 autosampler. All experiments were done under isocratic conditions (H20:CH3OH = 20:80). Flow rate was 1.5 ml/min. •H AND 13C-NMR ANALYSIS The experimental procedure is shown in Figure 2. One gram of ultramarine blue and 0.2 g of MP were dispersed in 100 ml of distilled water and incubated at 37øC for 5 hr. After removing the pigment fraction by centrifugation, this supernatant fraction was freeze-dried to recover the solubilized MP. The •H and •3C-NMR spectra of the recovered MP were measured in perdeuteroacetone with tetramethylsilane as internal standard, using a JEOL JMN-FX 100 NMR spectrometer.

86 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS ppt /./[ Cosmetic pigment l : .erwV:',ieVr e Incubate for 5 hr at 37øC Centrifugation I Supernatant Determinate the preservative by HPLC technique. Figure 1. Method of determining the adsorbed preservative. IR MEASUREMENTS The infrared spectra of the recovered MP (Figure 2) were measured in KBr disks using a FT-IR (Gigilabo Co.). MEASUREMENT OF THE CONCENTRATIONS OF METAL IONS Measurements of the concentrations of various metal ions found in the recovered MP (Figure 2) were performed with an inductively coupled plasma emission spectrometer JY-48P (Jobin Yvon Co.). /•/tultramarine blue MP dist. water • keep for 5 hr centrifugation I • 1 supernatant ppt free•ze-dried redissolve in (CDa)aCO determine •H-NMR and •aC-NMR Figure 2. Method of measurement of •H and 13C-NMR spectra. at 37øC