Autocatalytic nature of permanganate oxidations exploited for highly sensitive chemiluminescence detection

Manganese(II) salts catalyze the chemiluminescent oxidation of organic compounds with acidic potassium permanganate. The formation of insoluble manganese(IV) species from the reaction between manganese(II) and permanganate can be prevented with sodium polyphosphate, and therefore, relatively high concentrations of the catalyst can be added to the reagent before the lightproducing reaction is initiated. The rapid and intense emissions from these manganese(II) catalyzed chemiluminescence reactions provide highly sensitive detection and greater compatibility with liquid chromatography.

Mechanism of permanganate chemiluminescence

Spectroscopic and synthetic methods have been exploited to deduce the mechanism for acidic potassium permanganate chemiluminescence. We have employed electron paramagnetic resonance (EPR) spectroscopy with a continuous flow assembly to monitor the formation of radical intermediates in real time generated from substrate oxidation by manganese(VII). These transient species react with manganese(III) in solution to produce the  previously characterized manganese(II)* emission source. Using UV-vis, EPR, attenuated total reflection (ATR)-FTIR, and chemiluminescence spectroscopies, we have established that there are two distinct enhancement mechanisms that in combination afford a 50-fold increase in emission intensity when the reaction is conducted in the presence of phosphate oligomers. In addition to preventing disproportionation of the manganese(III) precursor, the phosphate oligomers form protective "cagelike” structures around the manganese(II)* emitter, thus preventing nonradiative relaxation pathways.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

Extending the analytical utility of acidic potassium permanganate chemiluminescence

The purpose of this work was to improve the analytical usefulness of acidic potassium permanganate chemiluminescence for the determination of various analytical compounds. This thesis also examined the fundamental chemistry involved with these types of reactions.

Chemiluminescence evidence supporting the selective role of ligands in the permanganate oxidation of micropollutants

The selective increase in the oxidation rate of certain organic compounds with permanganate in the presence of environmental "ligands" and reduced species has been ascribed to the different reactivity of the target compounds toward Mn(III), which bears striking similarities to recent independent investigations into the use of permanganate as a chemiluminescence reagent. In spite of the importance of Mn(III) in the light-producing pathway, the dependence of the oxidation mechanism for any given compound on this intermediate could not be determined solely through the emission intensity. However, target compounds susceptible to single-electron oxidation by Mn(III) (such as bisphenol A and triclosan) can be easily distinguished by the dramatic increase in chemiluminescence intensity when a permanganate reagent containing high, stable concentrations of Mn(III) is used. The differences are accentuated under the low pH conditions that favor the chemiluminescence emission due to the greater reactivity of Mn(III) and the greater influence of complexing agents. This study supports the previously postulated selective role of ligands and reducing agents in permanganate oxidations and demonstrates a new approach to explore the chemistry of environmental manganese redox processes.

Determination of neurotransmitters and their metabolites using one- and two-dimensional liquid chromatography with acidic potassium permanganate chemiluminescence detection

High-performance liquid chromatography with chemiluminescence detection based on the reaction with acidic potassium permanganate and formaldehyde was explored for the determination of neurotransmitters and their metabolites. The neurotransmitters norepinephrine and dopamine were quantified in the left and right hemispheres of rat hippocampus, nucleus accumbens and prefrontal cortex, and the metabolites vanillylmandelic acid, 3,4-dihydrophenylacetic acid, 5-hydroxyindole-3-acetic acid and homovanillic acid were identified in human urine. Under optimised chemiluminescence reagent conditions, the limits of detection for these analytes ranged from 2.5 × 10−8 to 2.5 × 10−7 M. For the determination of neurotransmitter metabolites in urine, a two-dimensional high-performance liquid chromatography (2D-HPLC) separation operated in heart-cutting mode was developed to overcome the peak capacity limitations of the one-dimensional separation. This approach provided the greater separation power of 2D-HPLC with analysis times comparable to conventional one-dimensional separations.

Enhancing permanganate chemiluminescence detection for the determination of glutathione and glutathione disulfide in biological matrices

Acidic potassium permanganate chemiluminescence enables direct post-column detection of glutathione, but its application to assess the redox state of a wider range of biological fluids and tissues is limited by its sensitivity. Herein we show that the simple on-line addition of an aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two orders of magnitude, but also provides a remarkable improvement in the selectivity of the reagent towards thiols such as glutathione (compared to phenols and amino acids that do not possess a thiol group). This enhanced mode of detection was applied to the determination of glutathione and its corresponding disulfide species in homogenised striatum samples taken from both wild type mice and the R6/1 transgenic mouse model of Huntington's disease, at both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease progression had not significantly altered the intracellular redox environment.