We followed the previously reported dabsyl chloride derivatization procedure for standard amino acid mixture and CSF samples and gradient HPLC analysis (18), with the exception that 1 nmol norleucine
was added as an internal calibrator before dabsylation to confirm complete derivatization.
Results were calculated with norleucine
as internal calibrator.
Pleasanton, CA, USA) using ninhydrin for postcolum derivatization and norleucine
as the internal standard.
Amino acid composition was determined with Technicon Amino Acid Analyzer (TSM-1 Technicon Instrument Basingstoke, UK) using Norleucine
as internal standard.
Amino acid derivatives were identified by their relative retention times based on the reference peaks for retention time (methionine sulfone and norleucine
, both of which were added to the internal standard solution).
Samples were hydrolyzed for 24 hours at 110[degrees]C with 6 mol/HCL containing 2 g/L reagent grade phenol and 5,000 nmol norleucine
(internal standard) in evacuated and sealed ignition tubes.
5 g sample was weighed into a 100 ml flat bottomed flask, 1 ml of Norleucine
standard solution, 5 ml of performic acid in ice bath.
For amino acid analysis, 75 nmol of norleucine
was added to 250 [micro]L of purified Lp(a).