DETERMINATION OF RETINOIDS IN COSMETICS 489 1/3 (v/v) isopropanol, 1/3 (v/v) ethyl acetate, and 0.1% BHT. Each stock solution con- tained 1 mg/ml of one of the following retinoids: 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 13-cis-retinol, all-trans-retinol, all-trans-retinaldehyde, or all-trans- retinyl palmitate. Working standard solutions were prepared from the stock solutions and were used for HPLC peak identifi cation and calibration of detector response. Each working standard contained equal concentrations of all seven retinoids appropriately di- luted in the hexane/isopropanol/ethyl acetate/BHT solvent (above). Ten working stan- dards containing the following concentrations of all seven retinoids were prepared: 0.0003, 0.001, 0.002, 0.003, 0.01, 0.02, 0.03, 0.1, 0.2, and 0.3 mg/ml. Three sets of calibration curves were used to cover the range of expected retinoid concentrations in the samples. One set of calibration curves was derived from data obtained by duplicate injec- tions of the standards having retinoid concentrations of 0.0003, 0.001, 0.002, and 0.003 mg/ml. Data from duplicate injections of the 0.003, 0.01, 0.02, and 0.03 mg/ml stan- dards were used to obtain a second calibration set of curves. The third set of calibration curves was obtained from data collected following duplicate injections of the 0.03, 0.1, 0.2, and 0.3 mg/ml standards. Each set of calibration curves was obtained using a linear regression analysis of peak areas versus standard concentrations for each of the seven reti- noids. Retinoids isolated from samples were identifi ed by comparing their retention times and UV absorbance maxima with those of standards, and then were quantifi ed by using the appropriate standard calibration curve for each retinoid. When not in use, stan- dards and sample extracts were tightly sealed, stored at 2°C, and protected from light they were stable for up to three months. SAMPLE EXTRACTION Approximately 300 mg of each sample was weighed into a 40-ml beaker, mixed thor- oughly with about 1.7 g of Celite, and then transferred to a 10-ml extraction tube. The sample/Celite mixture was covered with a fi lter disk and compacted. The extraction tube was eluted into a 10-ml volumetric fl ask with enough extraction solvent (1/3 (v/v) hexane, 1/3 (v/v) isopropanol, 1/3 (v/v) ethyl acetate, and 0.1% BHT) to fi ll the volumetric fl ask to the mark. At the end of the extraction period, the eluate was thoroughly mixed and a 20-μl aliquot was immediately injected into the HPLC. Duplicate injections were per- formed for all samples. During preparation and analysis, exposure of standards and extracted samples to air, light, and heat was minimized to prevent oxidation or decompo- sition. When not in use, standards and sample extracts were tightly sealed, stored at 2°C, and protected from light. HPLC METHOD Chromatographic analyses were carried out with an Agilent 1100 series HPLC chromato- graph, equipped with quaternary pumps, a vacuum degasser, an auto-injector, a variable- wavelength diode array UV-visible absorbance detector, and a personal computer with chromatographic and spectrographic software. The mobile phase was degassed with an in-line degasser. Chromatographic separation was achieved using a Symmetry C18 analyti- cal column (250 mm × 4.6 mm with a 5-μm particle size). The column was eluted at 1 ml/min using a gradient starting at 25% solvent A (0.4 M ammonium acetate/1.0% acetic acid pH 5.3 buffer) and 75% solvent B (methanol),
JOURNAL OF COSMETIC SCIENCE 490 changing linearly to 80% solvent B and 20% solvent C (dichloromethane) in 30 minutes, and ending at 70% solvent B and 30% solvent C (35 minutes), with a fi nal ten-minute holding period. The HPLC system was then gradually returned to the initial conditions at a fl ow rate of 1 ml/min in preparation for the next sample (approximately ten minutes). Peak areas of analytes detected at 330 nm were used for quantitation. Additionally, ab- sorption maxima for retinoic acid and retinaldehyde at 350 and 380 nm, respectively, were monitored to further confi rm identifi cation of these two analytes. HP Chemsta- tionTM software was used for the treatment of data and the generation of reports. RECOVERY STUDIES Mass recovery was determined by spiking four different retinoid-free sample matrixes (three different lotions and a cream, Table I) at 50, 500, 5000, and 50000 μg/g, with each trans-retinoid isomer followed by extraction and HPLC analysis as described above. Lev- els of retinoids in the unspiked lotions and creams used for recovery studies were well below the limits of detection (LOD). Further details on LODs for each retinoid are de- scribed in the Method Performance section (p. 495). Levels of retinoids found in analysis of consumer cosmetic products were in the range of retinoids used in the recovery studies (Table III). The average recovery at each spiking concentration was determined using the following formula (21): mass recovery (%) = (retinoid found/retinoid spiked) × 100%. QUANTIFICATION External standard calibrations were used for quantifi cation. To determine analyte concen- trations, peak areas were calculated for each retinoid, and the amount of analyte was Table I Mass Recovery of Retinoic Acid, Retinol, and Retinyl Palmitate from Consumer Cosmetic Skin Care Product Sample Matrixes Spike/sample Matrix Retinoic acid Retinol Retinyl palmitate 50 μg/g/Sample A Lotion 95.7% 100.3% 88.9% 50 μg/g/Sample B Lotion 94.7% 102.7% 94.8% 50 μg/g/Sample C Cream 98.0% 97.6% 93.7% 50 μg/g/Sample D Lotion 102.0% 100.4% 95.3% 500 μg/g/Sample A Lotion 99.6% 99.8% 94.4% 500 μg/g/Sample B Lotion 102.2% 105.9% 98.8% 500 μg/g/Sample C Cream 102.0% 104.3% 102.5% 500 μg/g/Sample D Lotion 98.9% 100.4% 95.5% 5000 μg/g/Sample A Lotion 98.2% 99.0% 96.4% 5000 μg/g/Sample B Lotion 97.8% 98.5% 96.9% 5000 μg/g/Sample C Cream 100.7% 98.7% 96.9% 5000 μg/g/Sample D Lotion 95.3% 91.6% 91.3% 50000 μg/g/Sample A Lotion 101.4% 98.8% 96.8% 50000 μg/g/Sample B Lotion 99.4% 99.4% 97.0% 50000 μg/g/Sample C Cream 103.4% 100.9% 98.2% 50000 μg/g/Sample D Lotion 103.5% 95.4% 96.2%
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