Introduction 1 2 3 4 5 6 7 8 6 2 5 9 4 10 11 12 14 15 16 17 18 19 21 22 24 25 26 27 28 29 2 2 30 31 1 2 2  L Scheme 1 Schematic representation of the principles of the biochemical assay  Experimental section Materials L  L 32 Instrumentation λ ex  λ em 27 Pro-oxidant and antioxidant assay optimization λ ex  λ em 27 Pro-oxidant and antioxidant detection system in flow-injection analysis mode 27 1 2 2 2 2 Fig. 1  AS  Liquid chromatography coupled to the pro-oxidant and antioxidant detection system 1 2 2 2 2 2 2 2 2 Results and discussion 1  L Optimization of pro-oxidant and antioxidant detection assay The optimization of the biochemical assay for the PAD system was conducted first in an off-line batch format before the optimized biochemical assay was transferred to the PAD system in FIA and HPLC modes. Paraquat was used as a model ROS-producing compound. The following parameters were optimized: enzyme concentrations (rat liver microsomes, SOD, and HRP), cofactor NADPH and substrate 4-HPAA, blocking reagents PEG3350 and PEG6000, the detergent Tween 20 (which can improve the resolution of the on-line PAD system) and organic modifiers (that are necessary when the on-line PAD system is operated on-line in gradient HPLC mode). All optimizations were performed without the presence of paraquat as the continuous ROS-producing compound in SL-B. However, paraquat (0.036 mM) was added to the SL-B in the final optimized system in order to permit measurements of both pro-oxidants and antioxidants using the on-line PAD system. 33 33 27 27 27 27 27 Optimized conditions were derived from the abovementioned experiments performed in the off-line batch assay format and subsequently translated to the on-line PAD system in FIA mode. These final conditions were: a carrier solution consisting of 10% MeOH and 100 mg/l Tween 20 at a flow rate of 100 μl/min; SL-A containing potassium phosphate buffer (50 mM; pH 7.8), rat liver microsomes (50 μg/ml), HRP (18 U/ml), and SOD (14 U/ml), and SL-B with the same buffer containing PEG6000 (1 mg/ml), NADPH (44 μM), and 4-HPAA (1.2 mM). For continuous ROS production (resulting in a stable fluorescent baseline), paraquat was present in the optimized system in SL-B at a concentration of 0.036 mM. Both superloops had a flow-rate of 100 μl/min. PAD system in flow-injection analysis mode L 2 2 Fig. 2 a b c d  2  L 2 34 35  L  L 3  L 3 Fig. 3 Injections (triplicates) of different compounds into the PAD system in FIA mode (without the continuous addition of a pro-oxidant): 1) paraquat (0.05 mM); 2) paraquat (0.05 mM) and ascorbic acid (0.1 mM); 3) menadione (0.03 mM); 4) menadione (0.03 mM) and ascorbic acid (0.1 mM)   L 1 Table 1 Initial relative increases or decreases (in fluorescence units, FU) of different ROS-producing pro-oxidant compounds and antioxidants compared to paraquat for the PAD system used in FIA mode, in gradient HPLC mode and for a traditional batch assay Pro-oxidant/Antioxidant FIA PAD system (FU/mol±SEM) Detection limit for the FIA PAD system (nmol) HPLC PAD system (FU/mol±SEM) Detection limit for the HPLC PAD system (nmol) Batch assay set-up (FU/mol±SEM) Paraquat 1.00 ± 0.08 0.07 1.00 ± 0.33 0.9 1.00 ± 0.05 Menadione 1.55 ± 0.20 0.01 0.56 ± 0.01 0.4 1.60 ± 0.33 Duroquinone 0.13 ± 0.02 0.04 0.36 ± 0.19 1.3 0.16 ± 0.07 Glutathione -2.20 ± 0.03 1.9 -0.67 ± 0.18 8.0 -1.01 ± 0.04 Ascorbic acid -4.58 ± 0.22 0.1 -1.35 ± 0.13 0.2 -1.22 ± 0.12 The detection limits of the PAD system used in the FIA and HPLC modes are also given 27 29 1 1 On-line coupling of the PAD system to gradient HPLC The PAD system in gradient HPLC mode was evaluated by analyzing the five test compounds after HPLC separation with a decreasing flow-rate gradient. The advantage of this decreasing flow-rate gradient lies with the initially high flow-rate through the column at low concentrations of organic modifier, which results in better eluting compound resolution at the start of the gradient. At higher concentrations of organic modifier, the flow rates are obviously gradually decreased (and the post-column counteracting flow rates are gradually increased) in order to obtain a continuous flow rate (1 ml/min) and concentration of organic modifier (of 10%) after mixing in the post-column counteracting gradient. This results in a constant flow rate and organic modifier concentration (after the 1:9 split) when entering the on-line PAD. The added value of this approach is that alterations in the chromatographic method can be made without much effect on the on-line PAD assay.  L 4  L  L 1 1 Fig. 4 a  bottom  top b  top  bottom  5 5 L 2 Fig. 5 a b  Conclusion This paper presents the development and validation of a HRS-based on-line post-column detection system for the detection of ROS-producing compounds as well as antioxidants in mixtures. Different parameters, such as substrate (4-HPAA) and enzyme concentrations, reaction time, temperature, additives, and organic modifier concentrations were first optimized for the PAD system used in FIA mode. Several ROS-producing compounds as well as antioxidants were successfully measured with the optimized system. The intraday and interday variabilities of the PAD system used in FIA mode were determined and found to be lower than 5%. Good sensitivities, at least comparable with similar off-line batch assay formats for individual compounds, were obtained. On-line coupling of the novel PAD system to gradient HPLC permitted the screening of individual compounds in mixtures for ROS-producing and antioxidant properties. It should be noted, however, that compounds that show fluorescence quenching or intrinsic fluorescence may interfere with the methodology. However, it may be possible to adjust the system so that another split directs some of the flow to a second on-line assay that measures these artifacts with a “negative control PAD system” in order to detect such interferences. This PAD system used in gradient HPLC mode is potentially of great value to drug discovery and toxicology and food research.