Analysis of citrulline and metabolic related amino acids in plasma by derivatization and RPLC. Application of the extrapolative internal standard calibration method

• The extrapolative internal standard method was used for analysis of amino acids in plasma by HPLC after derivatization
• The analytical challenge is to correct matrix effects and also to control the chemical reaction under reproducible conditions
• The comparison between EISM with standard addition method would be very informative of trouble with depletion of reagents.
• Citrulline and metabolically related amino acids in plasma were determined by EISM and figures of merit were presented.

We propose to apply the extrapolative internal standard calibration method (EISM) for quantitative analysis of citrulline and other metabolically related amino acids in plasma by pre-column derivatization, liquid chromatography and UV detection. Whenever derivatization is required and an amino acid-free matrix is nonexistent, the analytical challenge is to detect and correct matrix effects and, also to control the experimental conditions of the chemical reaction, mainly the concentration of the derivatizing reagents.Plasma samples contain free amino acids, non-peptidic amino acids and other amino compounds, such as biogenic amines, that would react with most derivatizing reagents and interfere in the analysis. In this particular work, and considering the scope of the intended application, chromatographic conditions were chosen for the analysis of citrulline, arginine, glutamine, glutamic acid, proline and ornithine by precolumn reaction with FMOC-Cl and RP-HPLC-UV analysis.Linear least square regression of analyte to internal standard (IS) signal ratio versus the amount of spiked standard amino acid were established and linear regression parameters for each curve were calculated. The information provides by an IS, which is also derivatized, can be used to detect matrix effects due to the chemical reaction. This calibration method was validated and the figures of merit were estimated.