2.3.2. Procedure II: Derivatised fluorescence assay
Serum (250 ml) containing the standard mixture was extracted
with 125 ml 2 M HClO4. The extract was centrifuged
for 10 min at 5000 g. The supernatant was neutralised with
2 M potassium carbonate (K2CO3) to pH 6.0e6.5 and centrifuged
at 5000 g for 10 min to remove precipitated potassium
perchlorate. The supernatant (150 ml), 10 ml 20 mM potassium
hexacyanoferrate (III), 150 ml acetonitrile and 50 ml 0.1 M
DPE were mixed and incubated at 37 C (40 min) for the
derivatisation. An aliquot (20 ml) of the mixture was injected
into the chromatograph.