Isolation and quantitation of isotopically labeled amino acids from biological samples.

Department of Surgery, Maastricht University, Netherlands.

To face the problem of simultaneous isolation and quantitation of isotopically labeled amino acids in biological samples, two semi-preparative chromatographic methods were developed. One method was especially designed to isolate radioactively labeled amino acids for which we used derivatization with the fluorophore o-phthaldialdehyde (OPA), which is 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 after isolation of the derivative, we used 9-fluorenylmethylchloroformate (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% recovery after 5 h at 150 degrees C: S.D=3.5%, n=14). Run time (injection 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 below 2% for most OPA amino acids and below 3% for most FMOC amino acids. At maximum, a total of 1000 microl could be injected, representing approximately 200 microl of deproteinized plasma. The methods were linear up to injection of 0.5 micromol of all amino acids (OPA: r2=0.995-0.999; FMOC: r2=0.992-0.999). The C.V. of the IRMS measurement within the range which can be isolated maximally in one chromatographic run (50-500 nmol), was less than 3% above 100 nmol, indicating that chromatographic isolation fulfils the needs of the IRMS determination. The resulting methods are suitable for the isolation and quantitation of micromolar amounts of labeled amino acids from biological samples.