PHTHALATE ESTERS IN COSMETIC PRODUCTS 129 EXPERIMENT AL REAGENTS AND MATERIALS The following reagents and materials were used: Hexane was purchased from Burdick & Jackson (Muskegon, Michigan). Acetonitrile and methanol were purchased from T. J. Baker (Phillipsburg, New Jersey). 2-Propanol was purchased from Fisher Scientific (Fairlawn, New Jersey). All solvents were HPLC grade. Phthalate esters DMP (99%), DEP (99.5%), and BBP (98%) were purchased from Sigma Aldrich (Milwaukee, Wis- consin). DBP (2:::98%) and DEHP (99.5%) were purchased from Sigma Aldrich (St. Louis, Missouri). De-ionized water was prepared with a Milli-Q purification system from Millipore (Billerica, Massachusetts). Celite 545 was purchased from Fisher Scientific (Fairlawn, New Jersey). The extraction tubes and filter disks were obtained from Supelco (Bellefonte, Pennsylvania). PHTHALATE ESTER CALIBRATION STANDARDS A primary standard solution of a mixture of the five phthalate esters (- mg/ml each) was prepared by adding approximately 100 mg of each to a 100-ml amber volumetric flask and diluting to the mark with hexane. Because of the wide range of possible concen- trations in cosmetic products, three sets of working standards were prepared. One set was prepared at approximately 0.001, 0.003, 0.006, and 0.01 mg/ml by appropriate serial dilution of the stock solution. Similarly, a second set was prepared at approximately 0.01, 0.03, 0.06, and 0.1 mg/ml, and a third set for BBP, DBP, and DEHP only was prepared at 0.10, 0.30, 0.60, and 1.00 mg/ml. HPLC peak areas were determined based on duplicate injections of 20 µl, and a calibration curve was obtained by plotting peak area versus standard concentration. SAMPLE EXTRACTION To avoid contamination by environmental sources of phthalate esters, all glassware was thoroughly cleaned and rinsed with water and ethanol before use, and phthalate- containing plastics were avoided. Approximately 1 g of each cosmetic sample was weighed into a 40-ml beaker, mixed thoroughly with about 3 g of Celite, and then transferred to a 15-ml extraction tube containing a filter disk. The sample/Celite mix- ture was covered with a second filter disk and compacted firmly with a stirring rod. The prepared column was eluted with sufficient hexane to obtain 10 ml of extract rn a volumetric flask. The extraction flask was mixed well prior to HPLC analysis. HPLC ANALYSES HPLC analyses were carried out on an Agilent ll00 series HPLC, equipped with a quaternary pumping system, an in-line vacuum degasser, a variable wavelength diode array UV-visible absorbance detector, a 20-µl injection loop, and a personal computer with HP Chemstation software. Chromatographic separation was achieved using a Whatman Partisil ODS-3 5-µm guard column (7 .5 mm by 4.6 mm ID) and a Whatman Partisil ODS-3 5-µm analytical

130 JOURNAL OF COSMETIC SCIENCE column (250 mm by 4.6 mm ID), both obtained from Altech Chromatography (Deer- field, Illinois). The analytical column was housed in a thermostatted column compart- ment at room temperature. Chromatographic separation was achieved using a solvent program starting initially at 50% water, 34% acetonitrile, 13% 2-propanol, and 3% methanol that was changed linearly over 35 minutes to 15% water, 55% acetonitrile, 25% 2-propanol, and 5% methanol, and held at the final composition for an additional ten minutes. The mobile phase flow rate was 1.0 ml/min. The mobile phase was gradually returned to the initial mobile phase composition over a period of ten minutes. Phthalate esters were detected at 230 nm. QUANTITATION Four-point calibration curves were prepared for each phthalate ester. Phthalates were identified in sample extracts by comparing HPLC retention times with standards, and quantitated using the standard calibration curve for each phthalate ester. Sample extracts were diluted as necessary to confirm that the concentrations were in the linear range of the calibration curve. Calculated phthalate ester concentrations of 10 ppm or less were recorded as not detected. RECOVERY The recovery of phthalate esters from cosmetic products was determined by fortifying products with 100 and 1,000 ppm of each ester followed by extraction and HPLC analysis as described above. RESULTS AND DISCUSSION In this study, a rapid method for the determination of five phthalate esters was developed and validated. For most cosmetic sample extracts, each phthalate ester was completely separated from other components by HPLC and could be quantitated unambiguously. To further confirm that observed peaks were due to phthalate esters and not impurities, UV spectra of chromatographic peaks were evaluated to determine peak purity. For DMP it was generally necessary to use peak UV spectra to distinguish DMP from other compounds eluting near the retention time of DMP. HPLC calibration curves obtained for BBP, DBP, and DEHP were found to be linear over the concentration range of 0.001 mg/ml to 1 mg/ml. The calibration curve for DMP was linear from 0.001 mg/ml to 0.3 mg/ml, while DEP was linear from 0.001 mg/ml to 0.6 mg/ml. All regression correlation coefficients were better than 0.995. The limit of quantitation (LOQ) ranged from 1 to 10 ppm at ten times baseline noise. Figure 1 shows the chromatographic separation of the five phthalate esters and typical chromato- grams of cosmetic extracts. The presence of phthalate esters in solvents, laboratory equipment, and plastic materials has been reported by other investigators. To assure accurate quantitation of phthalate esters in the cosmetic products examined, laboratory equipment and glassware were carefully washed and thoroughly rinsed with water and ethanol before use. The HPLC