Do you search for compounds that react and generate specific responses to each oxidative agents you mentioned separately? Or a global response (cumulative signal to sum of hydroxyl radicals (•OH), singlet oxygen (¹O₂), and superoxide radicals (O₂•⁻)) to the presence of oxidative stress?

If the second case is the question, then any phenolic compound (antioxidant) will raise the signal if the oxidative stress is increased in the reaction pot. Most of the phenolic substances (quercetin, gallic acid, naringenin, kaempferol, chrysin, galangin, cinnamic acids, and so many others etc) can react with the given agents and easily detected by LC-MS.

The broad method to assay free radical may be told as electron spin resonance spectroscopy. However, the radical you have asked, may be assayed by keeping all those as substrate. Superoxide as substrate in dismutation reaction, hydroxyl radical as in Fenton reaction. Singlet oxygen lying between oxygen molecule and superoxide, is an exited oxygen molecule. It can be converted to superoxide and then assayed by dismutation reaction.

Thank you, Dr. Dipak Kumar Chattopadhyay, for your response. I am currently working with a Fenton-like system that generates hydroxyl radicals (•OH), singlet oxygen (¹O₂), and superoxide radicals (O₂•⁻). To estimate their steady-state concentrations, we rely on reaction kinetics using known second-order rate constants between the radicals and specific probe compounds.

At the moment, we are using compounds known to react selectively with these radicals, such as nitrobenzene (•OH), furfuryl alcohol (¹O₂), and p-benzoquinone (O₂•⁻). However, we are facing challenges in detecting these probes and their degradation products using LC-MS/MS. Issues include low ionization efficiency and inconsistent peak profiles.

I would greatly appreciate any literature recommendations on method development strategies for improving LC-MS/MS detection of these probe compounds. Specifically:

• Optimal ionization mode (positive/negative).
• Suitable mobile phase composition.
• Effective fragmentor voltage, collision energy, and precursor/product ion transitions for these compounds

If anyone has successfully quantified these or similar probe compounds using LC-MS/MS, your insights would be very helpful.

Thank you, Dr. İsmail Emir Akyildiz. I appreciate your suggestion regarding the use of phenolic antioxidants to evaluate oxidative stress. Indeed, compounds like quercetin, gallic acid, and cinnamic acids are useful for general oxidative stress studies, especially for a cumulative or global signal.

However, in my case, I am specifically targeting individual reactive oxygen species (ROS) - hydroxyl radicals (•OH), singlet oxygen (¹O₂), and superoxide radicals (O₂•⁻) - to determine their steady-state concentrations in a Fenton-like reaction system.

To achieve this, we rely on probe compounds with well-established second-order rate constants for specific ROS, allowing us to apply kinetic modeling. We currently use nitrobenzene for •OH, furfuryl alcohol for ¹O₂, and p-benzoquinone for O₂•⁻. The challenge is not in reactivity but in method development for detecting these probes using LC-MS/MS.

Any insights or literature references on optimizing LC-MS/MS conditions for these or similar small, semi-volatile, or poorly ionizing compounds would be greatly appreciated.

I may follow the reaction products in a non-targeted fashion (full scan first to see who is there and in increase) instead of these more GC-amenable compounds if the LC-MS is the only choice.

If you cannot monitor these molecules precisely (most probably), I would prefer to scan the products of these probes at different time scales or conditions to extrapolate the reaction and to quantify the growing signal rather than consumption.

If you do not have an APCI config, your reaction setups may produce some more LC-ESI-compatible analytes during reaction to plot the kinetics...There will possibly be a couple of transient and end products in the reaction pot, one of which may raise its hand (detectable by LC-MS) to be a target. MS/MS is optional in this case.

Once you select your target compound in the preview runs, you can perform absolute quantification and/or validation by integrating MSMS.

Thank you for the suggestion, Dr. İsmail Emir Akyildiz. I tried running a full scan, but didn’t get any meaningful peaks- mostly just scattered low-mass ions. However, I run in SIM mode and observe a reasonable area, so I plan to proceed with that for now to monitor the reaction. By this, I am confirming my compounds with three parameters: 1) Mass, 2) Retention time, and 3) UV. Did you have any thoughts on this.