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Preservatives are used to inhibit the growth of microorganisms in cosmetic products. The Japanese standards for cosmetics set restrictions on the maximum amount of each preservative added to cosmetics as per the purpose of use of cosmetics. For the investigation into the actual conditions of commonly used preservatives in commercial cosmetics, we analyzed parabens, phenoxyethanol, sodium benzoate, sodium dehydroacetate, salicylic acid, and potassium sorbate by high-performance liquid chromatography (HPLC). Twenty-one samples were obtained from cosmetic product manufacturers located in 14 prefectures in Japan. Among different acid- and salt-based preservatives, sodium benzoate was observed to have been used in many products. These acid- and salt-based preservatives were used with parabens in personal washing products, such as shampoo and soap. The labels of two of the cosmetic product samples displayed inaccurate ingredient information, that is, a preservative other than the one used in the corresponding product was listed on them. The amount of preservatives used did not exceed regulatory limits in any of the analyzed samples.

Addition of medical ingredients to cosmetics is prohibited. However, last year some cases of illegal cosmetics containing steroids were successfully identified. We have already reported an analytical method to detect steroids in cosmetics [Bull. Natl. Inst. Health Sci, 126, 51-56 (2008)]. In this study, we initially examined whether this method could be applied for the detection of some new steroids as target chemicals. We then used this developed method to detect steroids in cosmetics obtained from manufacturers by spot checks. These manufacturers have been advertising the effectiveness of a steroid-free cream against atopic dermatitis. The results revealed that clobetasol propionate (CP) was present in this facial moisturizing cream, which was available in the market. The steroid was extracted with methanol. After ultrasonication and centrifugation, the resulting supernatant was injected into the high-performance liquid chromatography system equipped with an ODS column. The separation was achieved using a mixture of acetonitrile and water as the mobile phase. The retention times of the observed peaks were in accordance with those of some preservatives and CP. The presence of CP was also confirmed by thin-layer chromatography. The concentration of CP in the cream was approximately 0.039%. CP is a steroid that has the strongest effect as compared to those of other steroids. The cream was therefore recalled for safety reasons.

Strontium dichloride is one of the prohibited ingredients in cosmetics due to the Japanese Pharmaceutical Affairs Act. We established the analytical method for strontium dichloride in cosmetics by capillary electrophoresis (CE). The toothpaste was dispersed into water. After ultrasonication for 10 min, the solution was centrifuged at 3000 rpm for 10 min. The supernatant was filtrated through a membrane (0.45 microm), diluted 100-times with water, and injected into CE. The calibration curve showed linear between the concentrations of strontium dichloride (from 2 to 50 microg/ml) and the peak area of strontium ion. Detection limit of strontium dichloride is 2 microg/ml. There was no interference of the ingredients in the toothpaste.

Cadmium sulfide is one kind of the prohibited ingredients in cosmetics by the Japanese Pharmaceutical Affairs Act. We established the analytical method for cadmium sulfide in cosmetics by ICP-MS. Analytical procedures were as follows: Cosmetics of 1 g contained inorganic pigment were put into a 100 ml flask glass. After adding 100 ml of 12% nitric acid into the 100 ml flask glass, the filtrate was sonicated for 30 min. After sonicating, the mixture was centrifuged at 3000 rpm for 5 min and then the supernatant was filtrated through a millipore membrane filter (0.45 microm). After filtration, the filtrate was made up to 200 ml with 12% nitric acid. The solution was diluted 100 times with 12% of nitric acid and used as the test solution. The test solution of 100 microl was analyzed by ICP-MS (HP-4500, monitoring mass 111). The working curve from 1 to 1000 microg/l showed a linear line between the concentrations of cadmium and the peak areas. Detection limit of cadmium is 0.02 microg/l. There was no effect of the ingredients in the cosmetics on cadmium sulfide determination.

Simultaneous determination for 9 ultraviolet absorbers those set a limit to the amount in cosmetics was performed. Ultraviolet absorbers were extracted from cosmetics with tetrahydrofuran (THF) by ultrasonication. After centrifugation, the supernatant was collected, and the sample solution was injected into the HPLC. Separation was archived using an ODS column with the mixture of THF and water as the mobile phase. Detection wavelength was set at 310 nm. The linearity was obtained between the peak areas and the concentrations of each ultraviolet absorber in the range of 5 - 100 microg/ml. In 70 commercial cosmetic products, such as sunscreen, face powder, foundation, massage cream, moisture lotion, lip balm and essence, 2-ethylhexyl-p-methoxycinnamate (EMC), 2-hydroxy-4-methoxybenzophenone (HMB), 4-tert-butyl-4'-methoxydibenzoylmethane (BMB) and 2-ethylhexyl salicylate (ES) were detected.

Mercury is one kind of prohibited ingredients in cosmetics by the Japanese Pharmaceutical Affairs Act. We established the analytical method for mercury in cosmetics by ICP-MS. Analytical procedures were as followed: Ten microl of 1 g/l mercury solution and 1 g of whitening cream were put into a 50 ml plastic tube. After adding 20 ml of 12% nitric acid into the 50 ml plastic tube, the mixture was sonicated for 10 min. After sonicating, the mixture was centrifuged at 3000 rpm for 10 min and then the supernatant was filtrated through a milli-pore membrane with the pore size of 0.45 microm and 0.1 microm. After filtration, the mixture was made up to 25 ml with 7% nitric acid and used as the test solution. The test solution of 100 microl was analyzed by ICP-MS (HP-4500, monitoring mass 202). The calibration curve from 1 to 1000 microg/l showed a linear line between the concentrations of mercury and the peak areas. Detection limit of mercury was 0.1 microg/l. There was no effect of the ingredients in the whitening cream on mercury determination.

Selenium disulfide is one kind of prohibited ingredients in cosmetics by the Japanese Pharmaceutical Affairs Act. We established the analytical method for selenium disulfide in cosmetics by ICP-MS. Selenium disulfide of 20 mg was put into a teflon vessel. After adding 5 ml of concentrated nitric acid and 2 ml of the shampoo into the teflon vessel, the mixture was digested with microwave-oven. After digesting, the mixture was made up to 25 ml with milliQ water and then it was filtrated through a milli-pore membrane (0.45 micro). After filtration, the solution was diluted with 7% of nitric acid and used as the test solution. The test solution of 100 microl was analyzed by ICP-MS (HP-4500, monitoring mass 82). The working curve from 10 to 1000 microg/l showed a linear line between the concentrations of selenium and the peak areas. Detection limit of selenium disulfide is 22 microg/l. There was no effect of the ingredients in the shampoo on selenium disulfide determination.

Analytical methods for red tar colors, Helindone Pink CN (R226) and Permaton Red (R228), in cheek rouge were developed. R226 and R228 were extracted from cheek rouge with chloroform by ultrasonication. After centrifugation, the supernatant was collected for the determination of R226 and R228. Methanol was then added to the residue for the extraction of Pigment Red 57-1 (R201) and Pigment Red 57 (R202). Each R226 and R228 was separately detected by the silica-gel thin-layer chromatography using the mixture of hexane and chloroform (2:1) or (3:1), or hexane and tetrahydrofuran (THF) (2:1) as a developing solvent. For the determination of R226 and R228, the extract in chloroform was injected into the HPLC equipped with Amide colomn and UV-VIS detector (detection wavelength 535 nm and 487 nm) using the mixture of hexane and THF as mobile phase. The linearity was obtained between the peak areas and the concentrations of R226 and R228 in the range of 0.625-10 microg/ml. R201 and R202 were determined using ODS column and the mixture of acetonitrile and phosphate buffer as mobile phase. Seven cheek rouge samples were analyzed. The red tar colors listed in each cheek rouge were contained in the range of 247 to 6574 microg/g.

Disodium monofluorophosphate is one kind of the prohibited ingredients in cosmetics due to the Japanese Pharmaceutical Affairs Act. We established the analytical method for disodium monofluorophosphate in cosmetics by capillary electrophoresis (CE). The tooth paste of 1 g was put into a 50-ml plastic tube. After adding 7.0 mg of disodium monofluorophosphate and 50 ml of milliQ water into the plastic tube, the mixture was ultrasonicated for 10 min. After centrifuging, the supernatant was filtrated through a milli-pore membrane (0.45 microm). After filtration, the solutionwas put into a 100-ml volumetric flask, made up to 100 ml with milliQ water and used as the test solution. The mouthwash of 1 ml and 7.0 mg of disodium monofluorophosphate were put into a 100-ml volumetric flask, made up to 100 ml with milliQ water and used as the test solution. The testing solution was analyzed by CE. The working curve from 10 to 100 microg/ml showed a linear line between the concentrations of disodium monofluorophosphate and monofluorophosphate peak areas. Detection limit of disodium monofluorophosphate is 0.3 microg/ml. There was no interference of peak of monofluorophosphate with the ingredients in the tooth paste and mouthwash.

Cyproheptadine hydrochloride (CH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for CH was investigated by HPLC. The lotion or milky lotion of 0.5 g was put into a 10-ml volumetric flask. After adding 1.0ml of CH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol as the test solution. Creams were procedured as follows; 0.5 g of cream was put into a 10-ml volumetric flask. After adding 1.0 ml of tetrahydrofuran into the volumetric flask, the mixture was stirred for several minutes and the ingredients of the creams were dissolved. After adding 1.0 ml of CH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol. This mixture was transferred to a centrifuging tube with a cap and then the tube was centrifuged for 5 minutes at 3000 rpm. The supernatant was used as the test solution. The test solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of 1% acetic acid with 10 mmol/l sodium octanesulfonate and acetonitrile (11:9) and the detection wavelength of 286 nm. The working curve from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of CH and the peak areas. There was no interference of peak of CH with the ingredients such as methylparaben, ethylparaben in the lotions, milky lotion and creams.

Acetohexamide (AH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for AH was investigated by HPLC. The lotion or milky lotion of 0.5g was put into a 10-ml volumetric flask. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol as the testing solution. Creams were procedured as follows; 0.5 g of cream was put into a 10-ml volumetric flask. After adding 1.0ml of tetrahydrofuran into the volumetric flask, the mixture was stirred for several minutes and the ingredients of the creams were dissolved. After adding 1.0ml of AH solution at 50 microg/ml into the volumetric flask, the mixture was made up to 10ml with methanol. One milliliter of the mixture including AH at 5 microg/ml was exactly put into a test tube with a cap and then 1 ml of water and 1 ml of hexane were added. After shaking vigorously, stand for several minutes. After centrifuging, the hexane layer was eliminated and the residual mixture was used as the test solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 5.3)(3:1) and the detection wavelength of 247 nm. The working curve from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of AH and the peak areas. There was no interference of peak of AH with the ingredients such as methylparaben, ethylparaben in the lotions, milky lotion and creams.

Tetracaine hydrochloride (TH) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method for TH was investigated by HPLC. After adding 5 ml of TH solution at 10 microg/ml and 2 ml of salicylic acid solution at 75 microg/ml as the internal standard to 0.5 g of the lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1) as the testing solution. Milky lotion was procedured as follows: After adding 5 ml of TH solution at 10 microg/ml and 2 ml of internal standard solution to 0.5 g of the milky lotion, the mixture was made up to 10 ml with a mixture of water and methanol (1:1). Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. In the case of the cream, the other procedures were used: 0.5 g of cream was placed into a 10-ml volumetric flask and 1 ml of tetrahydrofuran was added. After dissolving, the mixture of methanol and water (1:1) was added to make up 10.0 ml. Two milliliter of this mixture was placed into a centrifuging tube with a cap and 2.0 ml of hexane was added. After shaking vigorously and centrifuging, the lower layer was used as the testing solution. The testing solution of 20 microl was analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of acetonitrile and 50 mmol/l phosphate buffer(pH 2.0)(7:3) and the detection wavelength of 303 nm. The working curves from 0.5 to 6.0 microg/ml showed a linear line between the concentrations of TH and the peak area ratio. There was no interference of peak of TH from the lotion, milky lotion and cream.

Estriol (EO) is nominated as the prohibited ingredients in cosmetics in Japanese Pharmaceutical Affairs Act. So the analytical method using HPLC for EO was investigated. After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the lotion into a 10-ml volumetric flask, the methanol was added to make until that volume and this solution was used as the testing solution. Milky lotion was procedured as follows: After placing 1.0 ml of EO solution at 50 microg/ml and 0.5 g of the milky lotion into a 10-ml volumetric flask, the methanol was added to make until that volume. The suspending mixture was moved to a centrifuging tube with a cap. After centrifuging at 3000 rpm for 5 minutes, the supernatant was used as the testing solution. The testing solution of 20 microl was determined by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture of water and acetonitrile (31:9) and the detection wavelength of 285 nm. The working curve from 1.0 to 6.0 microg/ml showed a linear line between the concentrations of EO and the peak area. There was no interference of peak of EO from the lotion and milky lotion.

Both phenoxyethanol and parabens are kinds of preservatives in cosmetics and are nominated as the restricted ingredients in cosmetics in Japanese Pharmaceutical Affairs Low. So the analytical methods for phenoxyethanol and parabens were investigated by HPLC. After adding methanol to phenoxyethanol and parabens in lotions, the testing solutions were analyzed by HPLC using the ODS column (CAPCELL PAK C18 column, 4.6 x 250 mm), the mixture either of 50 mmol/l phosphate(pH3.5) and acetonitrile (7:3) for phenoxyethanol or of 50 mmol/l phosphate buffer(pH3.5) and acetonitrile (3:2) for parabens and the detection wavelength of either 270 nm for pehnoxyethanol or 255 nm for parabens. The working curves from 0.5 to 5 micrograms/ml passed through the origin. There were no interference of peaks of phenoxyethanol and parabens from the cosmetics. Eleven lotions out of 42 lotions did not have phenoxyethanol. Phenoxyethanol in thirty-one cosmetics ranged from 0.009 to 1.043% and those average was 0.264%. Parabens, especially methylparaben, were detected in all lotions. The amounts of parabens ranged from 0.016 to 0.267% and their average was 0.120%. There were 7 kinds of lotions whose contents of phenoxyethanol and methylparaben were below 0.1%.