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.