COMPARISON OF PLASMA PHENYLALANINE DETERMINATION BY DENSITOMETRY OF THIN-LAYER CHROMATOGRAMS AND BY HIGH PERFORMANCE...

To compare two chromatographic methodologies for determination of plasma phenylalanine (Phe) and their usefulness for diagnosing hyperphenylalaninemia (HPA) and phenylketonuria (PKU).

The plasma amino acids were isolated and concentrated from blood collected from infants with HPA detected by newborn screening. The plasma Phe was determined in parallel by HPLC and by image-densitometry of 2D-TLC plates.

Typical examples of 2D-TLC plates and HPLC chromatograms from infants with HPA and PKU are presented and evaluated. The Phe spot was visible on 2D - TLC plates at Phe concentrations higher than 300 µmol/L. The standard calibration curve traced after image-densitometry of the Phe spot presented high dispersion of values at each concentration of Phe, high SD values, the equation of the curve having a low R-squared value (0.862). In contrast, the standard calibration curve obtained by HPLC shows linearity on the range of concentrations from 100 - 16,000 µmol/L, extremely small SD values, the equation of the curve has a very high R-squared value (0.999).

The HPLC methodology is appropriate to confirm HPA detected by newborn or selective screening of PKU. The 2D - TLC methodology is adequate to detect patients with severe PKU.