*2.4. Electrochemistry*

A single-line flow system was coupled with potentiostat using a flow cell provided by DropSens, for the SPCE electrodes (DRP110). The Electrochemical Flow Injection Analysis system (FIA-EC) used is composed of a four-channel Peristaltic Pump—MINIPULS® 3 (Gilson, Villiers-le-Bel, France), Injection Valve 77521 Rheodyne (with a 100 μL sample

loop), Flow Cell from DropSens (model DRP-FLWCL), and Potentiostat (Autolab PGSTAT 101). A boxed connector for screen-printed electrodes from Dropsens was used to connect the SPCEs. Each measurement was performed in triplicate.

#### *2.5. Determination of Apparent Rate Constants and Half-Lives*

The kinetics of PON decay and the scavenging effect of myoglobin on peroxynitrite at pH 9 were assessed using the calculation of half-lives and apparent rate constants of peroxynitrite decay. The static method was used for the measurements of concentrations over time, as pH 9 offers the possibility of having slower decays and changes dynamics toward specific chemical decay reactions. The terms "peroxynitrite" and "PON" are widely accepted for a mixture of ONOO− and ONOOH, depending on the pH. At pH 9, ONOO− is assumed to be in excess over the protonated form ONOOH, based on the pKa of PON 6.8. The term "peroxynitrate" refers to a mixture of O2NOO− and O2NOOH, depending on the pH. Trivial names: ONOO<sup>−</sup>, peroxynitrite; ONOOH, peroxynitrous acid; O2NOO<sup>−</sup>, peroxynitrate; O2NOOH, peroxynitric acid.

For the determination of the kinetic parameters, we took in consideration the previously proposed model by which the bimolecular decomposition of PON is dominant at pH 9, according to the following reaction [30,31]:

$$\text{HOONO} + \text{ONOO}^- \rightarrow 2\text{NO}\_2^- + \text{O}\_2 + \text{H}^+ + 2\text{e}^- \tag{1}$$

According to this model, the rate law should follow pseudo first-order kinetics at pH 9, due to the excess of ONOO<sup>−</sup>. Our aim was to validate the FIA-EC method as a tool for the determination of rate order and kinetic parameters by comparing the results with the classical UV-Vis method. In this particular case, the first purpose was to establish by both UV-Vis and FIA methods if the reaction follows indeed pseudo first-order kinetics or second/higher-order kinetics at pH 9.

Two approaches were used for the confirmation of rate order and the calculation of apparent rate constants and half-lives: the first method (namely called from now on "Method A") uses the plotting of all the integrated rate law data, according to the assumed rate order. Briefly, this was done as follows: the linearity (from the value of R2) of the graphs *ln(concentration)* (for (pseudo) first-order) or *1/concentration* (for second-order) vs. *time* and the correlation of the observed half-life (extrapolation from the graph t1/2obs) with the calculated half-life (t1/2calc). The half-lives for (pseudo) first-order and secondorder reactions were calculated with the following formulas, respectively: ln2/k and 1/k·C0PON. C0PON is the initial concentration of PON and k is the (apparent) rate constant. For the linearity, we considered the R<sup>2</sup> values. If R<sup>2</sup> approaches 1, is significantly higher than the R<sup>2</sup> of the other model, *and* there is good similarity between t1/2calc and t1/2obs, the corresponding apparent order of the reaction is attributed to the detriment of the other apparent order. Each decay rate constant determination was plotted for a total of 180 s.

The second method (namely called from now on "Method B") was the "half-life method" described by Ira Levine, in the "Physical Chemistry" book, chapter 16, "Reaction kinetics" [32]. This method can be applied when the rate law has the form r = k[A]n. Based on Equation (1) and considering the excess of ONOO<sup>−</sup>, this method would follow the equation r = k[ONOOH]n, therefore determining the rate order in ONOOH. According to this method adapted to our particular case, one first plots the *concentration* vs. *time* and then should be able to fit the equation with a single-exponential decay function if n = 1 (based on Equation (2), where parameter k (the apparent pseudo first-order rate constant in our case, kobs) is solved after compilation. Secondly, one performs the extrapolation of t1/2 for various concentrations and then plots the *logt*1/2 vs. *logC*0 [32]. The slope of the logarithmic graph (that should have a linear fit) will establish the order of the reaction in reactant A, *n*, where *n* = 1 − *slope*. The two methods, A and B, were compared at the end.

$$\mathbf{C}\_{\rm{PON}} = \mathbf{C}\_{\rm{OPON}} \times \mathbf{e}^{-\rm{kt}} \tag{2}$$

where CPON is the PON concentration at a specific moment in time.

Both A and B kinetic methods were used for the UV-Vis method and for our proposed FIA-EC method. The molar extinction coefficient of 1670 M−1cm−<sup>1</sup> was used to calculate the concentration of ONOO− at 302 nm [33,34].
