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Detection of the reduced forms of radical adducts on the ESR trace using HPLC-electrochemical detector-ultraviolet absorption detector-electron spin resonance-MS.

To detect and identify the electron spin resonance (ESR) silent forms of the alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) radical adducts, an electrochemical detector (ECD) was employed as a reactor in the HPLC-ECD-UV absorption detector-ESR-MS (HPLC-ECD-UV-ESR-MS). The ECD was employed to regenerate the radical forms from the reduced forms. The reduced forms of the 4-POBN/pentyl radical adducts were analyzed using the HPLC-ECD-UV-ESR-MS. On addition of the ECD applied potential of +0.3 V, a peak appeared on the ESR trace of the HPLC-ECD-UV-ESR-MS analyses, indicating that the radical forms are regenerated from the reduced forms. The HPLC-ECD-UV-ESR-MS analyses were also performed for the reaction mixtures of phenylhydrazine with CuCl(2). Two peaks (peaks I and II) were detected on the UV trace (300 nm) of the HPLC-ECD-UV-ESR-MS. The mass spectra showed that the peak I and peak II compounds are radical and reduced forms of the 4-POBN/phenyl radical adducts under the ECD applied potential of 0.0 V. Peak I was only detected on the ESR trace under the ECD applied potential of 0.0 V. In addition to peak I, peak II appeared on the ESR trace under the ECD applied potential of +0.3 V, indicating that the reduced forms are oxidized to the corresponding radical forms.