ISOLATION AND QUANTITATION OF ISOTOPICALLY LABELED AMINO-ACIDS FROM BIOLOGICAL SAMPLES

To face the problem of simultaneous isolation and quantitation of isot opically labeled amino acids in biological samples, two semi-preparati ve chromatographic methods were developed. One method was especially d esigned to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phthaldialdehyde (OPA), which i s known to be easy and reliable. Isolation of amino acids labeled with stable isotopes required another approach as we wanted to use isotope ratio mass spectroscopy (IRMS), which can only be performed on pure, non-derivatized amino acids. Because the OPA probe cannot be removed a fter isolation of the derivative, we used 9-fluorenylmethylchloroforma te (FMOC) instead. This probe is linked to an amino acid via a peptide bond which can easily be broken by gas-phase acid hydrolysis (103% re covery after 5 h at 150 degrees C: S.D=3.5%, n=14). Run time (injectio n to injection) was 60 min for the OPA method and 75 min for the FMOC method. Both fluorescence and UV absorbance detection can be employed. The coefficient of variation (C.V.) for peak area measurement was bel ow 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 mu l could be injected, representing appr oximately 200 mu l of deproteinized plasma. The methods were linear up to injection of 0.5 mu mol of all amino acids (OPA: r(2)=0.995-0.999; FMOC: r(2)=0.992-0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50-5 00 nmol), was less than 3% above 100 nmol, indicating that chromatogra phic isolation fulfils the needs of the IRMS determination. The result ing methods are suitable for the isolation and quantitation of micromo lar amounts of labeled amino acids from biological samples.