Battaglia A, Bertoluzza A, Calbucci F, Eusebi V, Giorgianni P, Ricci R, Tosi R, Tugnoli V
Istituto CNR dei Composti del Carbonio Contenenti Eteroatomi I.Co.C.E.A., Bologna, Italy.
J Chromatogr B Biomed Sci Appl. 1999 Jun 25;730(1):81-93. doi: 10.1016/s0378-4347(99)00188-7.
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.
描述了一种反相高效液相色谱技术，用于测定5例人脑肿瘤活检组织（2例脑膜瘤、1例胶质母细胞瘤和2例少突胶质细胞瘤）中的游离氨基酸。将冷冻组织匀浆，用高氯酸脱蛋白，并用氢氧化钾中和。含有生理性氨基酸的上清液等分试样用于用异硫氰酸苯酯进行柱前衍生化。衍生化的PTC-氨基酸（苯硫代甲酰基衍生物）如果在惰性气氛中于-20℃以粉末形式储存，可稳定保存5天，并且可以在反相柱（PicoTag）上使用两种洗脱液的梯度进行分析，在254nm波长处进行吸收检测。在约60分钟内可实现几种氨基酸（>30种）的良好分离。对于大多数氨基酸，该方法适用于从极少量样品开始，在广泛的生理浓度范围（50-400pmol）内进行准确测量。
