Analysis of free and bound O-phosphoamino acids in urine by gas chromatography with flame photometric detection.

A selective and sensitive method has been developed for the analysis of free and bound forms of O-phosphoamino acids, such as O-phosphoserine, O-phosphothreonine and O-phosphotyrosine, in urine samples by gas chromatography (GC). For free O-phosphoamino acid analysis, the urine sample was extracted with trichloroacetic acid and run through an ion exchange column. For total (free plus bound) O-phosphoamino acid analysis, the urine sample was hydrolysed in acid and base in order to release the O-phosphoamino acid from peptides or proteins. O-Phosphoamino acids in these prepared samples were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and then measured by GC with flame photometric detection (FPD-GC). The calibration curve was linear in the range of 10-500 ng for each O-phosphoamino acid, and the detection limits were 30-80 pg as injection amounts. O-Phosphoamino acids in the urine samples could be selectively determined by the FPD-GC method without any influence from coexisting substances. The recoveries of O-phosphoamino acids added to urines and urine hydrolysates were 90-98% and relative standard deviations were 1.5-8.0%. By using this method, we investigated an age dependence of O-phosphoamino acid excretion in normal subjects.