Quantification of free, dissolved combined, particulate, and total amino acid enantiomers using simple sample preparation and more robust chromatographic procedures

The goal of this work was to develop amino acid (AA) quantification protocols using simple sample preparation steps and new chromatographic conditions optimized for a hybrid (organo-silica) C18 column resistant to alkaline samples in borate buffer. This resistance allows for larger injection volumes, and thus stronger detected signals (~3 times stronger), higher precision, and lower analysis costs compared with previous methods. Enantiomeric and non-enantiomeric separations of AA were possible in less than 0.5 mL of AA-poor water samples. Results showed that a high salt content and the sample treatment do not lead to AA loss or separation interference. The accuracy and the precision of AA quantification in a natural sample strongly depend on the blank correction. The limits of detection, measured based on the variability of these blanks, were 0.007–3.6 nM depending on the AA. The protocols developed here allow for the quantification of all forms of AA (i.e., free, dissolved combined, particulate, and total) in most natural waters, including deep ocean waters. It was even possible to quantify many AA in ultrapure water after blank correction. Despite the injection of a relatively high volume (100 µL) of alkaline borate solutions, the hybrid C18 column showed no sign of performance degradation after more than 700 injections. This durability reduces the cost of AA analysis and provides a more consistent separation. The analysis time can be greatly reduced by the use of stationary phases with a nonporous core and extended pH stability as they become available.