[Determination of amino acids by precolumn derivatization with o-phthaldialdehyde (OPA) and reversed-phase high...

A manual pre-column derivatization procedure has been developed for the determination of amino acids in sample by reversed-phase high performance liquid chromatography. OPA/3-mercaptopropionic acid (3-MPA) were used as derivatization reagents and separation of OPA-amino acids derivatives was performed on a 5 microm partical-size ODS column. A phosphate buffer of 10 mmol/L pH 7.2 (PB) containing 0.3% tetrahydrofuran (THF) was used as mobile phase (A) and a mixture of PB-methanol-acetonitrile (50:35:15) as mobile phase (B). A single linear gradient program was employed and the signals were detected at 340 nm with UV detector. Under the above conditions all 18 amino acids in standard solution were separated to baseline in 40 minutes. The precision of analysis was observed from the reproducibility of the retention times and peak areas of eight consecutive injections of 18 amino acids standard solution and the results showed that both retention times and peak areas were highly reproduceable with the RSD 0.09%-0.48% for retention times and 1.2%-4.9% for peak areas. The linearity of UV response was tested by injecting of derivatized amino acid with different concentrations from 25 to 250 micromol/L for each of 18 amino acids. The r values were 0.996-0.999. External standardization method was used for the quantitation. The method was applied to analyze amino acid composition of bovine serum albumin (BSA) and the results were in consistence with literature values. Free amino acids in rat plasma were also determined and good results were obtained.