Table II.
Relative quantification of each POBN adduct in all three redox forms formed from LOX-catalysed EPA peroxidation.
| Reaction Time (min) |
POBN/•C5H9 |
POBN/•C2H5 |
POBN/•C4H7O2 |
POBN/•C7H11O2 |
POBN/•C10H15O2 |
ξ POBN/•L(OH)2 |
||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Ox★ m/z 263 |
Rad† m/z 264 |
Red‡ m/z 265 |
Ox★ m/z 223 |
Rad† m/z 224 |
Red‡ m/z 225 |
Ox★ m/z 281 |
Rad† m/z 282 |
Red‡ m/z 283 |
Ox★ m/z 321 |
Rad† m/z 322 |
Red‡ m/z 323 |
Ox★ m/z 361 |
Rad† m/z 362 |
Red‡ m/z 363 |
Ox★ m/z 529 |
|
| 0.5 | 0.26± 0.03 | 1.75± 0.13 | 0.12± 0.01 | 0.00± 0.00 | 0.09± 0.00 | 0.09± 0.0 | 0.00± 0.00 | 0.04± 0.00 | 0.03± 0.01 | 0.12± 0.00 | 0.04± 0.00 | 0.00± 0.00 | 0.00± 0.00 | 0.02± 0.00 | 0.09± 0.05 | 0.00±0.00 |
| 1 | 0.33± 0.02 | 2.33± 0.28 | 0.14± 0.02 | 0.18± 0.05 | 0.20± 0.03 | 0.11± 0.01 | 0.00± 0.00 | 0.09± 0.01 | 0.05± 0.00 | 0.18± 0.01 | 0.06± 0.01 | 0.02± 0.00 | 0.00± 0.00 | 0.04± 0.00 | 0.18± 0.02 | 0.01±0.00 |
| 2 | 0.48± 0.05 | 3.18± 0.16 | 0.22± 0.03 | 0.30± 0.04 | 0.24± 0.01 | 0.11± 0.00 | 0.04± 0.00 | 0.11± 0.00 | 0.05± 0.01 | 0.32± 0.04 | 0.08± 0.01 | 0.03± 0.00 | 0.00± 0.00 | 0.07± 0.01 | 0.21± 0.07 | 0.03±0.00 |
| 5 | 1.03± 0.15 | 6.54± 1.43 | 0.66± 0.11 | 1.07± 0.13 | 1.13± 0.19 | 0.38± 0.05 | 0.13± 0.02 | 0.37± 0.07 | 0.15± 0.03 | 0.76± 0.12 | 0.07± 0.01 | 0.06± 0.01 | 0. 07± 0.03 | 0. 01± 0.00 | 0.36± 0.20 | 0.04±0.00 |
| 15 | 1.28± 0.22 | 7.79± 1.06 | 1.60± 0.15 | 1.06± 0.20 | 0.82± 0.15 | 0.24± 0.04 | 0.17± 0.05 | 0.37± 0.06 | 0.10± 0.02 | 0.87± 0.13 | 0.19± 0.03 | 0.14± 0.03 | 0.06± 0.02 | 0.15± 0.02 | 0.31± 0.10 | 0.12±0.02 |
| 30 | 0.90± 0.03 | 5.51± 1.31 | 0.51± 0.08 | 1.43± 0.10 | 1.68± 0.05 | 0.38± 0.07 | 0.17± 0.01 | 0.54± 0.03 | 0.10± 0.00 | 0.54± 0.03 | 0.07± 0.01 | 0.05± 0.00 | 0.03± 0.01 | 0.01± 0.00 | 0.51± 0.02 | 0.04±0.01 |
| 45 | 0.87± 0.14 | 5.04± 0.67 | 0.46± 0.14 | 1.65± 0.37 | 1.92± 0.28 | 0.36± 0.08 | 0.21± 0.04 | 0.59± 0.10 | 0.10± 0.03 | 0.60± 0.09 | 0.09± 0.02 | 0.08± 0.01 | 0.10± 0.03 | 0.01± 0.00 | 0.57± 0.32 | 0.06±0.03 |
| 60 | 1.10± 0.03 | 9.18± 1.04 | 0.80± 0.09 | 2.74± 0.27 | 3.15± 0.22 | 0.55± 0.05 | 0.40± 0.06 | 1.05± 0.11 | 0.15± 0.02 | 0.69± 0.03 | 0.14± 0.03 | 0.17± 0.01 | 0.08± 0.03 | 0.01± 0.00 | 0.59± 0.23 | 0.24±0.07 |
Oxidized forms of POBN radical adducts, e.g. nitrone adducts, are structural analogues of nitrone compounds (spin trap POBN as well as its isotope-substitute D9-POBN). Thus, oxidized forms of POBN radical adducts are directly quantified (1:1) in the absolute concentration unit when D9-POBN (2.0 μg/ml) was used as internal standard.
The different ionization efficiencies among the redox forms of POBN adduct are measured and calculated using an LOX-catalysed linoleic acid peroxidation as a model system where two main POBN adducts (POBN/•C8H15O2 and POBN/•C5H11) are formed and their reduced forms can also be obtained following the reaction with a large amount of ascorbic acid. Under our experiment condition, by comparing MS efficiencies for the above two POBN radical adducts and their reduced forms toward to D9-POBN (2.0 μg/mL, internal standard), we defined response factors of D9-POBN (standard) vs POBN radical adduct vs reduced adduct forms as 1:2.05:2.87. Thus, those values were used to normalize original MS peak area, e.g. peaks in Figure 6, for data in the Table.
spin adduct as well as the reduced form failed to produce reliable statistical data, both due to overlap with the necessary overdose of POBN.
Note, the ESR-active peak 2, m/z 240 for POBN/•C2H4OH, was not listed since it was not an EPA-related radical product and has been characterized in many previous studies. Quantanalysis version 1.8 for Agilent 6300 Series Ion trap LC/MS was used to process the integration and calculation. Data are expressed as means ± SD from n≥3.