Direct determination of amino acids by hydrophilic interaction liquid chromatography with charged aerosol detection

• A broad review of amino acid analysis by HPLC.
• Novel 18-min separation and quantitation of amino acids without derivatization.
• Comparison of quantitative data from UV absorbance and charged aerosol detection.
• Quantitation of amino acid content in protein hydrolysates of selected nutritional supplements.

A chromatographic analytical method for the direct determination of amino acids by hydrophilic interaction liquid chromatography (HILIC) was developed. A dual gradient simultaneously varying the pH 3.2 ammonium formate buffer concentration and level of acetonitrile (ACN) in the mobile phase was employed. Using a charged aerosol detector (CAD) and a 2nd order regression analysis, the fit of the calibration curve showed R2 values between 0.9997 and 0.9985 from 1.5 mg/mL to 50 μg/mL (600 ng to 20 ng on column). Analyte chromatographic parameters such as the sensitivity of retention to the water fraction in the mobile phase values (mHILIC) were determined as part of method development. A degradation product of glutamine (5-pyrrolidone-2-carboxylic acid; pGlu) was observed and resolved chromatographically with no method modifications. The separation was used to quantitate amino acid content in acid hydrolysates of various protein samples.