An LC-MS Assay to Measure Superoxide Radicals and Hydrogen Peroxide in the Blood System

Abstract

Red blood cells are constantly exposed to reactive species under physiological or pathological conditions or during administration of xenobiotics. Regardless of the source, its accurate quantification is paramount in the area of theragnostics, which had been elusive up until now. Even if there are a lot of approaches to evaluate the oxidative stress, very sensitive methods are missing for the blood system. We therefore sought to apply a highly sensitive approach, by liquid chromatography coupled to mass spectrometry (UPLC-MS), for the quantification of reactive species such as superoxide radical and hydrogen peroxide using dihydroethidium (DHE) and coumarin boronic acid (CBA) probes respectively through the detection of 2-hydroxyethidium (2OH-E⁺) and 7-hydroxycoumarin (COH). The use of the high-resolution mass spectrometry associated to UPLC ensured a selective detection of superoxide and hydrogen peroxide in the blood system under diverse conditions such as oxidized red blood cells (RBCs), untreated and treated parasitized RBCs. Moreover, this technique allowed the determination of reactive species in human plasma. This protocol provides a huge opportunity for in-depth study of several pathological conditions vis-a-vis their treatment in modern medicine.

Keywords: Plasmodium falciparum; human plasma; hydrogen peroxide species; liquid-chromatography; mass spectrometry; microvesicles; red blood cells; superoxide radicals.

Figure 1

Formation of specific adducts from reaction of probes with targeted reactive oxygen species. CBA: Coumarin boronic acid (detected as the formiate adduct C₁₀H₈BO₆⁻; m/z 235.0419 (th)), CBE: pinacolate ester of Coumarin boronic acid, COH: 7-hydroxycoumarin (detected as the deprotonated form C₉H₅0₃⁻; m/z 161.0244 (th)), DHE: dihydroethidium (detected as the protonated form C₂₁H₂₂N₃⁺; m/z 316.1808 (th)) and 2OH-E⁺: 2-hydroxyethidium (detected as a cation C₂₁H₂₀N₃O⁺; m/z 330,1601 (th)).

Figure 2

Summary of the sample the parasitized red blood cell (pRBC) or red blood cell (RBC) preparation or the LC–MS analysis. Note: pRBCs: Plasmodium infected red blood cells, MeOH: methanol, DHE: dihydroethydium, ART: artemisinin.

Figure 3

Example of the calibration curve obtained with 2OH-E⁺ (A) and COH (B).

Figure 4

Extracted mass chromatogram based on m/z 330 and corresponding 2OH-E⁺ mass spectrum (A). Extracted mass chromatogram base on m/z 161 and corresponding COH mass spectrum (B).

Figure 5

Quantification of superoxide (A) and H₂O₂ (B) in PHZ treated RBCs comparatively with untreated ones. Note: results represent six replicates from three independent experiments, value = mean ± SD. * = Significant (p < 0.05).

Figure 6

Quantification of superoxide (A) and H₂O₂ (B) in parasitized RBCs treated with artemisinin comparatively to the untreated ones. Note: results represents six replicates from three independent experiments, value = mean ± SD. * = Significant (p < 0.05).

Figure 7

Extracted mass chromatogram base on m/z 330 (A) standard 500 nM 2OH-E⁺, (B) non diluted human plasma and (C) human plasma diluted 100 times.