Validation of HPLC Method for Quantitative Determination of N-acetylglucosamine in Skin-Lightening Microemulsion

The aim of this study was to validate a high performance liquid chromatography (HPLC) method for quantitative determination of N-acetylglucosamine (NAG) in a microemulsion (ME). Pseudoternary phase diagram was constructed to find ME formulation for preparation of NAG-loaded ME for skin-lightening propose. A NH2 column (5 mu m particle size, 250 x 4.6 mm) was used as a chromatographic column. The various ratios (65:35, 70:30, or 75:25 v/v) of acetonitrile and water mixtures were studied as mobile phase to investigate the potential for NAG determination. The flow rate was set at 0.5 mL/min. The column temperature was controlled at 30 degrees C. Each sample was injected by auto-sampler with the injection volume of 10 mu L. The UV detection wavelength was operated at 194 nm. The quantification was performed using peak area counts. NAG peak was found at a retention time of 9.8, 11.5, and 14.1 min when acetonitrile:water ratios of 65:35, 70:30, and 75:25 v/v were used, respectively. The mixture of acetonitrile and water at 70:30 v/v was chosen for quantitative NAG analysis due to symmetric NAG peak and high specificity. NAG standard solutions and NAG-spiked MEs were used for the assay validation. Percent recovery (%recovery) was within 100 +/- 2% and percent relative standard deviation (%RSD) was less than 2%, translating for accuracy and precision, respectively. The calibration curve also showed a good linear relationship. LOD and LOQ values were 035 and 0.67 mu g/mL, respectively. One g of 1% NAG-loaded ME was extracted and quantified to contain NAG of 9.94 +/- 0.05 mg or 99.40 +/- 0.53% labeled amount. It could be concluded that the investigated HPLC and extraction methods were appropriate for analysis of NAG in NAG-loaded ME, a novel formulation.