Dansyl Method see Note

3.2.1. Derivatization

1. Standards were derivatized prior to column injection as follows: dansyl derivatization was performed with the addition of 100 ^L 2 N sodium hydroxide solution, 150 ^L saturated sodium bicarbonate and dansyl chloride solution.

2. The reaction mixture was incubated at 40°C for 45 min.

3. After incubation the residual dansyl chloride was removed by addition of 50 ^L ammonia.

4. After 20 min the sample was adjusted to 2.5 mL with acetonitrile.

5. The sample was filtered with a 0.22-^m filter.

6. The precolumn derivatization and the column apparatus were at 40°C.

7. Standard curves were prepared with pure compounds at different concentrations (see Note 8).

8. Solvent gradient conditions began with 50% B and ended with 90% B over 19 min (see Note 9).

3.2.2. Quantification

The internal standard (1,7-diaminoheptane) method was used (see Fig. 3 and

0 0

Post a comment