Amino Acid ¹³C Analysis

Amino acids are analyzed for ¹³C using a Surveyor HPLC coupled to a Delta Plus isotope ratio mass spectrometer through a Finnigan LC Isolink interface (Thermo Electron Corp.). Amino acids are separated on a Primesep A column (3.2 x 250 mm; SIELC Technologies, Prospect Heights, IL, USA) using a linear gradient program. The mobile phase for the initial 10 min of separation is 100% water, followed by a 20 min linear gradient to 100% sulfuric acid solution (0.2% v/v).  From 30-110 minutes, the mobile phase is 100% sulfuric acid solution (0.2% v/v). Flow rate is a constant 0.5 mL/min at 25ºC. After separation, the column eluent is mixed with sodium peroxidisulfate (0.8 M) and phosphoric acid (1.7 M), separately pumped at 30 µL/min. Individual amino acid peaks are then quantitatively oxidized to CO₂ as the mobile phase flows through an oxidation reactor at 99.9ºC. Each CO₂ peak is transferred from aqueous phase to He carrier gas (2 mL/min) in a separation unit. Following water removal using a two-stage Nafion™ drier, the CO₂ peaks enter the IRMS through an open split.

References

McCullagh, J., Gaye-Siessegger, J., D., and Focken, U. 2008. Determination of underivatized amino acid δ¹³C by liquid chromatography/isotope ratio mass spectrometry for nutritional studies: the effect of dietary non-essential amino acid profile on the isotopic signature of individual amino acids in fish. Rapid Communications in Mass Spectrometry. 20:2761-2768.