High-performance liquid chromatographic analysis of physiological amino acids in human brain tumors by pre-column derivatization with phenylisothiocyanate.
A reversed-phase high-performance liquid chromatographic technique for the determination of free amino acids in five biopsies of human brain tumors (two meningiomas, one glioblastoma and two oligodendrogliomas) is described. The frozen tissues were homogenized, deproteinized with perchloric acid and neutralized with potassium hydroxide. Aliquots of the supernatant containing the physiological amino acids are used for pre-column derivatization with phenylisothiocyanate. The derivatized PTC-amino acids (phenylthiocarbamyl derivatives) are stable for a five day period if stored as a powder at -20 degrees C in an inert atmosphere and they can be analyzed on a reversed-phase column (PicoTag) using a gradient of two eluents with absorption detection at a wavelength of 254 nm. Good resolution of several amino acids (>30) is achieved within ca. 60 min. For most amino acids this method is suitable for an accurate measurement over a wide range of physiological concentrations (50-400 pmol) starting from a very small amount of sample.
Battaglia
A
A
Istituto CNR dei Composti del Carbonio Contenenti Eteroatomi I.Co.C.E.A., Bologna, Italy. battaglia@area.Bo.Cnr.it
Bertoluzza
A
A
Calbucci
F
F
Eusebi
V
V
Giorgianni
P
P
Ricci
R
R
Tosi
R
R
Tugnoli
V
V
D000596:Amino Acids / Q000378:metabolism*; D001932:Brain Neoplasms / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D017879:Isothiocyanates; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet; D013861:Thiocyanates
D000596:Amino Acids; D007202:Indicators and Reagents; D017879:Isothiocyanates; D013861:Thiocyanates; C005441:phenylisothiocyanate