METHYLPARABEN IN O/W EMULSIONS 49 mg MP/ml stock solution or 0.10, 0.15, 0.20, 0.26, or 0.30 ml of the 2.0-mg MP/ml stock solution to 10 ml of the emulsion. The six spiked emulsions ranged from 100.5 % to 103.0% of theory in 0.5% intervals. The spiked emulsions were mixed for 30 minutes with a magnetic stirrer and analyzed in duplicate for MP. A blank emulsion was prepared without MP. ANALYTICAL PROCEDURE The separation of MP from the emulsion was accomplished by solid-phase extraction using a 1-ml Oasis HLB 1-cc (30 mg) cartridge (Waters, Milford, MA). This cartridge was activated by passing 1 ml of methanol through the column by gravity flow to solvate the alkyl chains of the sorbent. The cartridge was then conditioned with 1 ml of 10% methanol in ddH20. A sample (0.1 to 1.0 ml) of emulsion was then added to the cartridge. The MP, as well as unwanted compounds, was retained in the sorbent. The cartridge was then washed with 1 ml of 10% acetonitrile in ddH20 to elute the unwanted compounds. It was assumed that the washing solution, which is weaker than the eluent, minimally elutes the MP from the sorbent. MP was finally eluted from the sorbent with 1 ml of acetonitrile:ddH20 (60:40) and collected in a 25-ml volumetric flask. One half milliliter of 0.1% BP in acetonitrile:ddH20 (60:40) was added as an internal standard. The final volume was adjusted to 25 ml with acetonitrile:ddH20 (60:40). The average of two replicates was reported. The concentration of MP in the eluent from the Oasis HLB cartridge was determined in triplicate by HPLC. The mobile phase was acetonitrile:ddH20 (60:40). A 3.9 x 150- mm Nova-pak C-18 reversed-phase column (Waters, Milford, MA) was used. The HPLC system consisted of a 20-pl injector (model 715, Rheodyne, Cotati, CA), pump (model 510 Waters, Milford, MA), automated gradient controller (model 680, Waters, Milford, MA), UV/visible wavelength detector (model 441, Waters, Milford, MA) and integrator (model 3390A, Hewlett-Packard, Wilmington, DE). The flow rate was 1.0 ml/min. UV detection was carried out at 254 nm at 0.05 a.u.f.s. A stock solution of 1 mg MP in the mobile phase was prepared. The stock solution was diluted with additional mobile phase to obtain solutions ranging from 10 to 40 pg MP/ml that were used to prepare the standard curve. BP (20 pg/ml) was incorporated in the standard solutions as an internal standard. The MP content of the eluent from the Oasis HLB cartridge was determined by finding, from the standard curve, the MP concentration that corresponded to the average peak area ratio of MP and BP. RESULTS AND DISCUSSION Figure 1A shows a typical chromatogram obtained when an MP and BP standard solution was analyzed. The resolution factors for MP and BP were 7.5 and 5.2, respec- tively, confirming that the selected HPLC system was appropriate for analysis of MP. The calibration curve, based on the peak area ratios of MP to BP, was constructed by performing linear least-square regression on triplicate analysis of four standard solutions (10, 20, 30, and 40 l•g/ml). The equation of the calibration curve relating the peak-area ratio (y) to the methylparaben concentration (x) in this range was y = 0.0479x + 0.2199, with R 2 = 0.995. The reproducibility of the retention time of MP (1.4 min) and BP (2.5

50 JOURNAL OF COSMETIC SCIENCE MP BP I I I I 0 1 2 3 Minutes MP BP B I I I I 0 1 2 3 Minutes Figure 1. Typical chromatograms obtained from injection of (A) MP and BP standard solution and (B) an extracted solution of MP containing BP as internal standard. min) was observed consistently during the analysis of a series of MP standard solutions. The same resolution was achieved when samples extracted by the SPE procedure were analyzed (Figure lB). The effect of the sample volume on MP recovery is shown in Figure 2. An increase in recovery was observed when the volume of the sample was increased from O. 1 to 0.5 mi. The best recovery (95.0%) was achieved when 0.5 ml of emulsion was transferred to the cartridge. Increasing the volume of the sample to more than 0.5 ml did not show an improved recovery. It is believed that the sorbent may have reached its highest efficiency when the sample volume loaded onto the cartridge was at 0.5 mi. In order to assure that the proposed SPE procedure was reproducible, five randomly selected cartridges were used to extract MP from the emulsion. A sample volume of 0.5