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.
Kataoka
H
H
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Nakai
K
K
Katagiri
Y
Y
Makita
M
M
D000596:Amino Acids / Q000652:urine*; D000818:Animals; D002849:Chromatography, Gas; D002852:Chromatography, Ion Exchange; D004032:Diet; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006868:Hydrolysis; D008892:Milk; D009943:Organophosphorus Compounds / Q000652:urine*; D010768:Phosphoserine / Q000652:urine; D010769:Phosphothreonine / Q000652:urine; D019000:Phosphotyrosine; D010783:Photometry; D011485:Protein Binding; D014238:Trichloroacetic Acid; D014443:Tyrosine / Q000031:analogs & derivatives / Q000652:urine
D000596:Amino Acids; D009943:Organophosphorus Compounds; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014443:Tyrosine; D014238:Trichloroacetic Acid