Amino acid analysis
BLOOD
Reverse Phase-High Pressure Liquid Chromatography (RP-HPLC) (Hewlett Packard series 1050) was carried out on the human samples [Amino acids are separated due to varying binding affinities between the PTC-bound amino acids and the C18-bound silicon beads within the column. The separated amino acids were eluted from the column and the eluted fraction measured by UV spectrometry at 254 nm. Measurement of the area under these peaks, using ClarityLite® software, and comparison with a standard solution containing known concentrations of amino acids, was used to calculate the amino acid concentrations in human blood. Plasma Alanine was analysed as fold change from visit 1 for Placebo and Metformin using the retention area under curve values, due to lack of alanine standard. Blood plasma was centrifuged at 4 °C for 20 min at 13,000rpm and 50 μl of the resulting supernatant was transferred to a fresh tube. Prior to loading, samples underwent a derivatisation procedure to enable binding of PITC to amino acids. Briefly, Trifluroacetic acid (TFA) was used to remove the protective 