Determination of Urea and Allantoin in Cosmetics

Urea and allantoin are added to cosmetic products for skin protection and regeneration, especially for the treatment of dry skin, and analyzed for QC purposes. As polar compounds, they are not ideal for reversed-phase HPLC separations. Neutral hydrophilic compounds like urea and allantoin are best analyzed by hydrophilic interaction chromatography (HILIC). Traditional HILIC columns use silica modified with a hydrophilic group such as diol or cyano. Analytes are adsorbed and subsequently eluted with mobile phases containing high percentages of organic solvent (>75%). These columns are hydrophilic and provide little or no retention for hydrophobic analytes. The Acclaim® Mixed-Mode HILIC-1 column contains a silica packing that is modified by a long alkyl chain with a diol group at the terminus. The diol allows the column to be operated in HILIC mode; the alkyl chain allows the column to be run in reversed-phase mode. In addition, this new column exhibits different selectivity compared to other HILIC columns. Here we separate urea and allantoin and determine their contents in a cosmetic with an Acclaim Mixed-Mode HILIC-1 column operated in the HILIC mode.

Experimental

All chromatography was conducted on an UltiMate® 3000 HPLC system consisting of an HPG 3400A pump with SRD 3400 Solvent Rack w/ degasser, a TCC-3000 thermostatted column compartment, a WPS-3000TSL autosampler, and a VWD-3400 UV-Vis detector. System control and data processing were executed with a Chromeleon® 6.80 Chromatography Workstation. All separations in the HILIC mode used an Acclaim Mixed Mode HILIC-1, 5 μm, 4.6 × 150 mm (Figure 1). Prior to analysis, 0.5 g of sample, 0.5 g diatomaceous earth, and 10 mL n-heptane were mixed in a beaker. After stirring five min, the organic phase was discarded. After extracting two more times, the residue was dried at 60°C, dissolved in 10 mL 70% acetonitrile, centrifuged, and extracted two more times with 10 mL 70% acetonitrile. All three extracts were placed in a 100-mL volumetric flask and diluted to the mark with acetonitrile. Prior to injection, extracts were filtered with a 0.45-μm filter. See Dionex Application Note 198 for more details.

Figure 1

Results and Discussion

Figure 1 shows chromatography of urea and allantoin on three columns. On a high carbon-load small-pore C18 column there is no retention of either compound (chromatogram 2). Using a polar-embedded reversed-phase column with a 100% aqueous mobile phase there is a small amount of retention for each compound and they are resolved (chromatogram 3). Chromatogram 1 shows that an Acclaim Mixed-Mode HILIC-1 column operated in the HILIC mode is the best choice for retention and resolution of urea and allantion. The same method was used to assay a cosmetic for allantion and urea. Figure 2 shows a chromatographic blank, sample analysis, and analysis of sample spiked with allantion and urea. The sample had 0.37 and 76.6 mg/g allantoin and urea, respectively, with spike recoveries of 98.4 and 97.3%. This demonstrates the method's suitability for cosmetics analysis.

Figure 2

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