Protein and peptide samples are aliquoted and mixed with Internal Standards (see Quantitation), dried in glass tubes in a vacuum concentrator and subjected to vapor phase hydrolysis by 6N HCl at 150ºC for 1.5 hours under argon atmosphere in the presence of phenol which limits the halogenation of Tyrosine residues. The samples are subsequently reconstituted in 0.4 N Borate Buffer to bring the eventual pH to 10 for optimum derivitization and transferred to the AminoQuant autosampler for automated derivatization and loading.  In cases where Cysteine data is requested, Dithiodiproprionic Acid is added to convert Cysteine to S-2-carboxyethylthio-L-cysteine (Cys-MPA) which is preserved during hydrolysis.

Insoluble samples (feeds, waste, cloth etc.) are weighed, Internal Standards added, and liquid-phase hydrolysis is used.  Enough 6N HCl is added to cover the sample and samples are exposed to 100ºC for 22 hours.

In all cases where hydrolysis is used, Asparagine and Glutamine are deamidated to their respective acids.  Results for these residues are reported as ASX and GLX to denote that these data contain the combined amounts from both the amide and the acid.  Acid hydrolysis also destroys Tryptophan.  If it is necessary to quantitate this residue, a separate assay using 4 N Methansulfonic Acid (with 0.2%w/v tryptamine) for hydrolysis.  The Tryptophan analysis has a very high %RSD (~25%) and requires ~50-100 micrograms of sample. 

Physiological samples or samples where only the free amino acids are of concern might be filtered to remove proteins or dried down and then reconstituted in 0.4 N Borate Buffer to bring the eventual pH to 10 for optimum derivitization.  No hydrolysis is involved for free amino acid determination.

The AminoQuant analyzes all samples by pre-column derivatization of the amino acids with o-phthalaldehyde (OPA) and 9-fluoromethyl-chloroformate (FMOC). OPA reacts with primary amino acids and FMOC with secondary amino acids (proline).  Both reagents react rapidly and quantitatively and give highly fluorescent and UV-absorbing isoindole derivatives.  The derivatized amino acids are separated by reverse phase HPLC and detected by UV absorbance with a diode array detector or by fluorescence using an in-line fluorescence detector.