Amino acids represent a fraction of organic matter in marine and freshwater ecosystems, and a source of carbon, nitrogen and energy. L-Amino acids are the most common enantiomers in nature because these chiral forms are used during the biosynthesis of proteins and peptide. To the contrary, the occurrence of D-amino acids is usually linked to the presence of bacteria. We investigated the distribution of L- and D-amino acids in the lacustrine environment of Terra Nova Bay, Antarctica, in order to define their natural composition in this area and to individuate a possible relationship with primary production. A simultaneous chromatographic separation of 40 L- and D-amino acids was performed using a chiral stationary phase based on teicoplainin aglycone (CHIROBIOTIC TAG). The chromatographic separation was coupled to two different mass spectrometers--an LTQ-Orbitrap XL (Thermo Fisher Scientific) and an API 4000 (ABSciex)--in order to investigate their quantitative performance. High-performance liquid chromatography coupled with mass spectrometry methods were evaluated through the estimation of their linear ranges, repeatability, accuracy and detection and quantification limits. The high-resolution mass spectrometer LTQ-Orbitrap XL presented detection limits between 0.4 and 7 μg L (-1), while the triple quadrupole mass spectrometer API 4000 achieved the best detection limits reported in the literature for the quantification of amino acids (between 4 and 200 ng L (-1)). The most sensitive method, HPLC-API 4000, was applied to lake water samples.