TABLE 1.
Chromatographic properties of 5-hydroxy fatty acids and their 5-oxo metabolites Fatty acids were analyzed by automated precolumn extraction/RP-HPLC as described under Materials and Methods. All of the analogs contain a Δ6 trans-double bond and either a 5S-hydroxyl or a 5-oxo group as indicated. The tR values for the 5-oxo products were confirmed by comparison with authentic standards.
|
Analog
|
tR
|
Precolumn Solvent (%
MeOH)a
|
Gradientb
|
|
|---|---|---|---|---|
| 5-Hydroxy | 5-Oxo | |||
| min | ||||
| 12:2 | 5.8 | 7.05 | 15 | 1 |
| 14:2 | 8.3 | 9.6 | 15 | 1 |
| 16:2 | 10.8 | 11.8 | 30 | 1 |
| 18:1 | 14.1 | 15.4 | 30 | 2 |
| 18:2 | 13.3 | 14.2 | 30 | 1 |
| 20:1 | 17.6 | 18.4 | 30 | 2 |
| 20:2 | 15.7 | 16.5 | 30 | 1 |
| Δ6,8,14-20:3 | 13.9 | 14.8 | 30 | 1 |
| Δ6,8,11-20:3 | 14.3 | 15.2 | 30 | 1 |
| 20:4 | 12.8 | 13.7 | 30 | 1 |
| 20:5 | 11.5 | 12.4 | 30 | 1 |
| 20:4-Me | 11.9 | 13.1 | 30 | 3 |
| 18h-18:2 | 5.6 | 6.8 | 15 | 4 |
The solvent used to equilibrate the precolumn was MeOH/water containing 2.5 mM H3PO4
All mobile phases were linear gradients between two solvents: gradient 1 [25–92% solvent B (acetonitrile/acetic acid (100/0.02)] in solvent A [water/acetic acid (100:0.02)] over 16 min; gradient 2 (40–95% B over 20 min); gradient 3 (45–99% acetonitrile in water over 16 min); and gradient 4 (35–75% B over 15 min)