Comparison of Fluorescent Techniques Using Two Enzymes Catalysed for Measurement of Atmospheric Peroxides

: Atmospheric peroxides, especially hydrogen peroxide (H 2 O 2 ), are essential oxidants. The peroxide concentration is closely related to the extent of OH radicals and the O 3 cycle in the tropospheric atmospheric chemistry. However, only a few studies have investigated their atmospheric concentrations in China because of inadequacies in the measurement techniques or higher costs of analytical instruments. Therefore, it is essential to design a suitable analysis method of peroxides with higher sensitivity, lower detection limit, and low cost. In view of that, this study investigated the optimum analysis conditions of two H 2 O 2 analytical techniques: the high-performance liquid chromatography (HPLC) with ﬂuorescence detection using two-enzyme catalysis of horseradish-peroxidase (HRP method) and Hemin (Hemin method). Furthermore, these two analysis methods were systematically compared in terms of detection limit, calibration curve, precision, accuracy, and applicability for the ﬁrst time. The ﬁndings showed that the HRP method had a lower detection limit, higher sensitivity, and better applicability for detecting H 2 O 2 and methyl hydroperoxide (MHP) than the Hemin method. Moreover, the HRP method is better suitable for H 2 O 2 and MHP detection, which requires low detection limits and high sensitivity. Besides this, the Hemin method is inexpensive and is more suitable for detecting hydroxyl alkyl peroxides (C ≥ 3). The atmospheric concentrations (average) of H 2 O 2 and MHP were 0.60 ± 0.37 ppb and 0.081 ± 0.039 ppb, respectively, as determined by the HRP method. Importantly, atmospheric peroxide concentrations were higher on sunny days than on cloudy days in Beijing in September 2016. H 2 O 2 concentrations showed a diurnal variation with the lowest value in the morning and two peaks at 13:00–17:00. In contrast, MHP concentrations were lowest in the morning and highest after 17:00. Photochemical reactions were responsible for the production of H 2 O 2 and MHP. The reactions of O 3 and oleﬁns emitted by motor vehicles also caused H 2 O 2 concentration to increase during the evening rush hour.


Introduction
In recent years, many cities have witnessed tremendous growth in motor vehicle numbers in China. This growth, in turn, has led to a substantial increase in the emissions of pollutants, such as nitrogen oxides (NOx) and volatile organic compounds (VOCs), further resulting in higher concentrations of secondary pollutants, such as ozone (O 3 ) and peroxide. Peroxides play a crucial role in tropospheric atmospheric chemistry and negatively affect human health and plant growth [1][2][3][4][5].
Atmospheric peroxides mainly include hydrogen peroxide (H 2 O 2 ) and organic peroxides, such as methyl hydroperoxide (MHP) and hydroxymethyl peroxide (HMHP). They are generated by photochemical reactions with no artificial and natural emission sources. H 2 O 2 is most prevalent, followed by MHP. Peroxides, particularly H 2 O 2 , are not only Atmosphere 2022, 13, 659 2 of 12 primary oxidants for the oxidization of SO 2 to SO 4 2− in the aqueous phase when the pH is less than 4.5 [6]. The concentration of peroxides is closely related to the extent of OH radicals present and the O 3 cycle in the tropospheric atmospheric chemistry [7,8]. Therefore, researchers have focussed on peroxides and have conducted extensive measurements in the atmosphere since 1985. However, the database is still limited, especially in China, because of the inadequacies in the measurement techniques and higher costs of analytical instruments. Hence, more cost-effective analytical methods with lower detection limits and higher sensitivity are needed to determine peroxide concentration in the atmosphere.
Since the 1980s, the concentrations of atmospheric peroxides have been mainly measured by enzyme catalyzation-fluorescence detection technology, which has a lower detection limit [9,10]. This method is based on the oxidation of a non-fluorescent substrate into a potent fluorescence substance and using an enzyme as the catalyst. Horseradish peroxidase (HRP) is often used as an enzyme to catalyze the peroxide oxidation of phydroxyphenylacetic acid (POPHA) into a dimer of POPHA with strong fluorescence properties. This provides more stability and a lower detection limit to the fluorescence method [11]. Hellpointner and Gab [12] successfully separated and quantitatively analyzed H 2 O 2 and partial organic peroxides in atmospheric samples by combining enzyme catalyzation-fluorescence detection technology and high-performance liquid chromatography (HPLC), followed by a post-column derivation reaction. However, this method can only quantify H 2 O 2 and alkyl peroxides of C ≤ 2 because the pH value restricts the specificity of HRP enzyme catalysis. Hence, peroxide with C ≥ 3 cannot be well quantified [13,14]. Furthermore, an additional pump is required to provide a solution that adjusts the pH of the detected solution because the optimum pH of HRP-catalysed reaction and fluorescence detection is different [13,15,16]. Therefore, Zhang and Dasgupta [13] used an inexpensive hematin method, instead of expensive HRP methods, to measure H 2 O 2 and other organic peroxides. They discovered that the catalytic activity of hematin was similar to that of HRP for H 2 O 2 and was 1/10th of HRP for MHP. The hydroxyl alkyl peroxides (C ≥ 3) can be detected using the H 2 O 2 standard curve [12] because the optimum pH in this reaction system was 10.5. Some hydroxyl alkyl peroxides, such as HOCH 2 OOCH 2 OH (BMHP), can rapidly decompose into H 2 O 2 . A pump-delivered pH-adjusting solution was not needed because the pH of the post-column derivatization reaction (using hematin as an enzyme) was consistent with that of fluorescence detection. Hence, the experimental facility was simplified and economical. Qi et al. [14] devised a method of using cheaper Hemin instead of expensive HRP as a catalyst for analyzing H 2 O 2 and some water-soluble organic peroxides because Hemin can be quickly converted to hematin in an alkaline solution. Xu and Chen [17] investigated the optimal analysis conditions of Qi's method. However, no attempts have been made to systematically compare these two methods regarding the detection limit, precision and other factors.
This study investigated the optimum analysis conditions of two analytical methods of peroxides using the HRP (HRP method) and Hemin (Hemin method) as catalysts and compared the two methods. After that, this study measured the concentrations of atmospheric peroxides using the more sensitive and accurate method.

Experimental Methods
An HPLC post-column derivatization system with a fluorescence detector (LC-20A, Shimadzu, Kyoto, Japan) was used to analyze the concentrations of H 2 O 2 and MHP. Peroxides were separated by the ODS-II column (5 um, 4.6 × 250 mm, GL Science, Tokyo, Japan) and then reacted with p-hydroxyphenyl acetic acid (POPHA, Sigma-Aldrich, St. Louis, MO, USA) to produce the fluorescent dimer of POPHA in the presence of an enzyme. The fluorescence detector was used to determine the fluorescence intensity of the dimer, which shared a linear relationship with the concentration of peroxides. The reaction tube and chromatographic column were placed in the column oven (GL Science, Tokyo, Japan) at 1-2 • C to maintain the stability of enzyme activity [18] and reduce the decomposition of peroxides. The chromatographic column, pipes, and joints made of Peek material were used in the HPLC system to reduce peroxides' decomposition and adsorption. The quantity of injected sample was 20 µL. Figure 1 is the schematic diagram of the HPLC system. The system using HRP as an enzyme included three high-pressure pumps to deliver the mobile phase (pH: 3.5 H 3 PO 4 , Fuchen Chemical Reagent, Tianjin, China), the HRP enzyme reagent (6.25 U/mL HRP, Sigma-Aldrich, St. Louis, MO, USA; 6.57 × 10 −5 mol/L POPHA, Sigma-Aldrich, St. Louis, MO, USA; 0.01 mol/L KH 2 PO 4 , Fuchen Chemical Reagent, Tianjin, China), and the alkali solution (pH 10.5 NH 4 Cl/NH 3 ·H 2 O, Fuchen Chemical Reagent, Tianjin, China). The system using Hemin as the enzyme system included two high-pressure pumps to deliver the mobile phase (pH: 3.5 H 3 PO 4 ) and the hemin enzyme reagent (8 × 10 −6 mol/L Hemin, Sigma-Aldrich, St. Louis, MO, USA; 8 × 10 −5 mol/L POPHA; and pH: 10.5 NH 4 Cl/NH 3 ·H 2 O, Fuchen Chemical Reagent, Tianjin, China). Every day, all reagents were prepared afresh using deionized distilled water (DDW) obtained from a DDW apparatus (RFD240NA, Advantec, Tokyo, Japan). The calibration curves of H 2 O 2 and MHP were built using their diluted standard solutions. Moreover, 1 mL of DDW as the blank sample was injected into the HPLC system and analyzed daily. Notably, single-point calibration checks were performed every two hours. The H 2 O 2 standard solution was prepared by diluting the commercially available H 2 O 2 solution (35%, Sigma-Aldrich, St. Louis, MO, USA). Its concentration was calibrated once every two months [18,19]. MHP standard solutions were synthesized and calibrated according to the relevant literature [15,20]. Similarly, H 2 O 2 and MHP were identified according to the retention times of their respective standards.
Atmosphere 2022, 13, x FOR PEER REVIEW 3 of 12 dimer, which shared a linear relationship with the concentration of peroxides. The reaction tube and chromatographic column were placed in the column oven (GL Science, Tokyo, Japan) at 1-2 °C to maintain the stability of enzyme activity [18] and reduce the decomposition of peroxides. The chromatographic column, pipes, and joints made of Peek material were used in the HPLC system to reduce peroxides' decomposition and adsorption. The quantity of injected sample was 20 μL. Figure 1 is the schematic diagram of the HPLC system. The system using HRP as an enzyme included three high-pressure pumps to deliver the mobile phase (pH: 3.5 H3PO4, Fuchen Chemical Reagent, Tianjin, China), the HRP enzyme reagent (6.25 U/mL HRP, Sigma-Aldrich, St. Louis, MO, USA; 6.57 × 10 −5 mol/L POPHA, Sigma-Aldrich, St. Louis, MO, USA; 0.01 mol/L KH2PO4, Fuchen Chemical Reagent, Tianjin, China), and the alkali solution (pH 10.5 NH4Cl/NH3·H2O, Fuchen Chemical Reagent, Tianjin, China). The system using Hemin as the enzyme system included two high-pressure pumps to deliver the mobile phase (pH: 3.5 H3PO4) and the hemin enzyme reagent (8 × 10 −6 mol/L Hemin, Sigma-Aldrich, St. Louis, MO, USA; 8 × 10 −5 mol/L POPHA; and pH: 10.5 NH4Cl/NH3·H2O, Fuchen Chemical Reagent, Tianjin, China). Every day, all reagents were prepared afresh using deionized distilled water (DDW) obtained from a DDW apparatus (RFD240NA, Advantec, Tokyo, Japan). The calibration curves of H2O2 and MHP were built using their diluted standard solutions. Moreover, 1 mL of DDW as the blank sample was injected into the HPLC system and analyzed daily. Notably, single-point calibration checks were performed every two hours. The H2O2 standard solution was prepared by diluting the commercially available H2O2 solution (35%, Sigma-Aldrich, St. Louis, MO, USA). Its concentration was calibrated once every two months [18,19]. MHP standard solutions were synthesized and calibrated according to the relevant literature [15,20]. Similarly, H2O2 and MHP were identified according to the retention times of their respective standards.

Field Observation
The atmospheric concentrations of peroxides were measured on the top of Building No. 3 of the Chinese Research Academy of Environmental Sciences (40°04' N, 116°42' E) in September 2016 ( Figure 2). A nebulization-reflux concentration technique [21,22], which turns the trapped solution into mist, was used to sample H2O2 and MHP at a velocity of 2.5 L/min for 30 min at 1-h intervals from 7:00 to 19:00 daily. The sampling system was constructed by connecting the sampler, pump, and mass flowmeter. The sampler was made by installing the mist chamber ( Figure 3) comprising 8 mL of trapping solution-DDW-on a filter holder with a 0.45-μm filter to prevent DDW from entering the flowmeter. The air was circulated through the mist chamber once the sample system was energized. The peroxide in the chamber was immediately dissolved in the DDW mist and then collected. Blank samples were collected via the same method as the peroxide samples, except that the air pump was not turned on. MHP concentrations obtained during the measurement period were corrected for their low sampling efficiency (73%, the results of the lab's research). Once the sampling was finished, the samples were immediately analyzed in the HRP method by the HPLC system at wavelengths of λex = 315 nm (excitation) and λem = 405 nm (emission). The flow rates of the mobile phase, enzyme reagent, and

Field Observation
The atmospheric concentrations of peroxides were measured on the top of Building No. 3 of the Chinese Research Academy of Environmental Sciences (40 • 04' N, 116 • 42' E) in September 2016 ( Figure 2). A nebulization-reflux concentration technique [21,22], which turns the trapped solution into mist, was used to sample H 2 O 2 and MHP at a velocity of 2.5 L/min for 30 min at 1-h intervals from 7:00 to 19:00 daily. The sampling system was constructed by connecting the sampler, pump, and mass flowmeter. The sampler was made by installing the mist chamber ( Figure 3) comprising 8 mL of trapping solution-DDW-on a filter holder with a 0.45-µm filter to prevent DDW from entering the flowmeter. The air was circulated through the mist chamber once the sample system was energized. The peroxide in the chamber was immediately dissolved in the DDW mist and then collected. Blank samples were collected via the same method as the peroxide samples, except that the air pump was not turned on. MHP concentrations obtained during the measurement period were corrected for their low sampling efficiency (73%, the results of the lab's research). Once the sampling was finished, the samples were immediately analyzed in the HRP method by the HPLC system at wavelengths of λ ex = 315 nm (excitation) and λ em = 405 nm (emission). The flow rates of the mobile phase, enzyme reagent, and alkali solution were set at 0.5, 0.2, and 0.1 mL/min, respectively. Notably, the column oven's temperature was set at 4 • C.
alkali solution were set at 0.5, 0.2, and 0.1 mL/min, respectively. Notably, the column oven's temperature was set at 4 °C.

Data Analysis
Except for precision, all experiments, such as the experiment of optimum analysis conditions and accuracy, were performed thrice to ensure precision and accuracy. The relative standard deviations (RSD) were calculated from three replicate measurements. The precision experiments were performed six times sequentially. The RSD was calculated using six parallel measurement results. The results of peroxide concentrations in field observations were the average concentrations of 30-min sampling.

Optimum Analysis Conditions of Two Methods
H2O2 (400 μg/L) was selected to examine the optimum analysis conditions of two methods, including the flow rate of reagents, reaction temperature, and others because H2O2 is the most prevalent and essential peroxide in the atmosphere. The results were expressed by the signal-to-noise (SNR) ratio (peak area).
The optimum excitation wavelength (λex) and emission wavelength (λem) were investigated when the flow rates of mobile phase, enzyme reagent, and alkali solution were set at 0.5 mL/min, 0.2 mL/min, and 0.2 mL/min, respectively, and the reaction temperature was set at 25 °C. The HRP and Hemin methods were used to analyze H2O2 solution at λex of 310 nm, 315 nm, 320 nm, and 325 nm, respectively, and λem of 400 nm, 415 nm, 420 nm, and 425 nm, respectively. The results demonstrated that the SNR of H2O2 was highest at λex = 315 nm and λem = 405 nm for both the HRP and Hemin methods ( Figure 4). Atmosphere 2022, 13, x FOR PEER REVIEW 4 of 12 alkali solution were set at 0.5, 0.2, and 0.1 mL/min, respectively. Notably, the column oven's temperature was set at 4 °C.

Data Analysis
Except for precision, all experiments, such as the experiment of optimum analysis conditions and accuracy, were performed thrice to ensure precision and accuracy. The relative standard deviations (RSD) were calculated from three replicate measurements. The precision experiments were performed six times sequentially. The RSD was calculated using six parallel measurement results. The results of peroxide concentrations in field observations were the average concentrations of 30-min sampling.

Optimum Analysis Conditions of Two Methods
H2O2 (400 μg/L) was selected to examine the optimum analysis conditions of two methods, including the flow rate of reagents, reaction temperature, and others because H2O2 is the most prevalent and essential peroxide in the atmosphere. The results were expressed by the signal-to-noise (SNR) ratio (peak area).
The optimum excitation wavelength (λex) and emission wavelength (λem) were investigated when the flow rates of mobile phase, enzyme reagent, and alkali solution were set at 0.5 mL/min, 0.2 mL/min, and 0.2 mL/min, respectively, and the reaction temperature was set at 25 °C. The HRP and Hemin methods were used to analyze H2O2 solution at λex of 310 nm, 315 nm, 320 nm, and 325 nm, respectively, and λem of 400 nm, 415 nm, 420 nm, and 425 nm, respectively. The results demonstrated that the SNR of H2O2 was highest at λex = 315 nm and λem = 405 nm for both the HRP and Hemin methods ( Figure 4).

Data Analysis
Except for precision, all experiments, such as the experiment of optimum analysis conditions and accuracy, were performed thrice to ensure precision and accuracy. The relative standard deviations (RSD) were calculated from three replicate measurements. The precision experiments were performed six times sequentially. The RSD was calculated using six parallel measurement results. The results of peroxide concentrations in field observations were the average concentrations of 30-min sampling.

Optimum Analysis Conditions of Two Methods
H 2 O 2 (400 µg/L) was selected to examine the optimum analysis conditions of two methods, including the flow rate of reagents, reaction temperature, and others because H 2 O 2 is the most prevalent and essential peroxide in the atmosphere. The results were expressed by the signal-to-noise (SNR) ratio (peak area).
The optimum excitation wavelength (λ ex ) and emission wavelength (λ em ) were investigated when the flow rates of mobile phase, enzyme reagent, and alkali solution were set at 0.5 mL/min, 0.2 mL/min, and 0.2 mL/min, respectively, and the reaction temperature was set at 25 • C. The HRP and Hemin methods were used to analyze H 2 O 2 solution at λ ex of 310 nm, 315 nm, 320 nm, and 325 nm, respectively, and λ em of 400 nm, 415 nm, 420 nm, and 425 nm, respectively. The results demonstrated that the SNR of H 2 O 2 was highest at λ ex = 315 nm and λ em = 405 nm for both the HRP and Hemin methods ( Figure 4).  The optimal flow rates of the mobile phase, enzyme reagent, and alkali solution were investigated at optimal wavelengths and reaction temperature of 25 °C. The sample solution was transported by the mobile phase through the column to the detector in the HPLC system ( Figure 1). The amount of the mobile phase can affect the shape of the chromatographic peak, the detection limit, and the analysis time. If the flow rate of the mobile phase is low, the separation of H2O2 by a column is slower, resulting in a longer retention time of H2O2 and a broader chromatographic peak. In contrast, high flow rates can decrease the concentration of enzyme reagent and fluorescence signals. Therefore, this study aimed to determine an optimum flow rate that could achieve the lowest detection limit and shorter analysis time on the premise of a sharp peak shape. H2O2 solution was analyzed at the flow rate (set) of both the enzyme reagent and the alkali solution (0.2 mL/min), and the mobile phase rates were set at 0.3, 0.4, 0.5, and 0.6 mL/min. The results showed that the mobile phase's flow rates of 0.5 mL/min and 0.3 mL/min were best suitable for the HRP and Hemin methods, respectively (Figure 5a). The enzyme reagent was crucial in this reaction system. Notably, the amount of enzyme could affect the rate of fluorescent reaction. The findings showed that the optimal flow rate of enzyme reagent for both methods was 0.2 mL/min (Figure 5b). The optimum pH of fluorescent detection for the dimer of PHPAA was about 10.5 [14]. The H2O2 solution was analyzed at flow rates of 0.1, 0.2, and 0.3 mL/min of the alkali solution to achieve the optimum pH of the detection solution. The results showed that the SNR of H2O2 reached the highest value when the flow rate of alkali solution was set at 0.1 mL/min for the HRP method ( Figure 5c). The temperature is an essential factor in chemical reactions. In general, the speed of enzymatic fluorescence reaction increases with an increase in temperature [11], whereas the quantum yield and fluorescence intensity of the fluorescent substance decrease with The optimal flow rates of the mobile phase, enzyme reagent, and alkali solution were investigated at optimal wavelengths and reaction temperature of 25 • C. The sample solution was transported by the mobile phase through the column to the detector in the HPLC system ( Figure 1). The amount of the mobile phase can affect the shape of the chromatographic peak, the detection limit, and the analysis time. If the flow rate of the mobile phase is low, the separation of H 2 O 2 by a column is slower, resulting in a longer retention time of H 2 O 2 and a broader chromatographic peak. In contrast, high flow rates can decrease the concentration of enzyme reagent and fluorescence signals. Therefore, this study aimed to determine an optimum flow rate that could achieve the lowest detection limit and shorter analysis time on the premise of a sharp peak shape. H 2 O 2 solution was analyzed at the flow rate (set) of both the enzyme reagent and the alkali solution (0.2 mL/min), and the mobile phase rates were set at 0.3, 0.4, 0.5, and 0.6 mL/min. The results showed that the mobile phase's flow rates of 0.5 mL/min and 0.3 mL/min were best suitable for the HRP and Hemin methods, respectively (Figure 5a). The enzyme reagent was crucial in this reaction system. Notably, the amount of enzyme could affect the rate of fluorescent reaction. The findings showed that the optimal flow rate of enzyme reagent for both methods was 0.2 mL/min (Figure 5b). The optimum pH of fluorescent detection for the dimer of PHPAA was about 10.5 [14]. The H 2 O 2 solution was analyzed at flow rates of 0.1, 0.2, and 0.3 mL/min of the alkali solution to achieve the optimum pH of the detection solution. The results showed that the SNR of H 2 O 2 reached the highest value when the flow rate of alkali solution was set at 0.1 mL/min for the HRP method (Figure 5c).  The optimal flow rates of the mobile phase, enzyme reagent, and alkali solution were investigated at optimal wavelengths and reaction temperature of 25 °C. The sample solution was transported by the mobile phase through the column to the detector in the HPLC system ( Figure 1). The amount of the mobile phase can affect the shape of the chromatographic peak, the detection limit, and the analysis time. If the flow rate of the mobile phase is low, the separation of H2O2 by a column is slower, resulting in a longer retention time of H2O2 and a broader chromatographic peak. In contrast, high flow rates can decrease the concentration of enzyme reagent and fluorescence signals. Therefore, this study aimed to determine an optimum flow rate that could achieve the lowest detection limit and shorter analysis time on the premise of a sharp peak shape. H2O2 solution was analyzed at the flow rate (set) of both the enzyme reagent and the alkali solution (0.2 mL/min), and the mobile phase rates were set at 0.3, 0.4, 0.5, and 0.6 mL/min. The results showed that the mobile phase's flow rates of 0.5 mL/min and 0.3 mL/min were best suitable for the HRP and Hemin methods, respectively (Figure 5a). The enzyme reagent was crucial in this reaction system. Notably, the amount of enzyme could affect the rate of fluorescent reaction. The findings showed that the optimal flow rate of enzyme reagent for both methods was 0.2 mL/min (Figure 5b). The optimum pH of fluorescent detection for the dimer of PHPAA was about 10.5 [14]. The H2O2 solution was analyzed at flow rates of 0.1, 0.2, and 0.3 mL/min of the alkali solution to achieve the optimum pH of the detection solution. The results showed that the SNR of H2O2 reached the highest value when the flow rate of alkali solution was set at 0.1 mL/min for the HRP method (Figure 5c). The temperature is an essential factor in chemical reactions. In general, the speed of enzymatic fluorescence reaction increases with an increase in temperature [11], whereas the quantum yield and fluorescence intensity of the fluorescent substance decrease with The temperature is an essential factor in chemical reactions. In general, the speed of enzymatic fluorescence reaction increases with an increase in temperature [11], whereas the quantum yield and fluorescence intensity of the fluorescent substance decrease with the rise in temperature [23]. Furthermore, peroxides are also unstable at high temperatures, and their decomposition rates will increase with increasing temperature [24]. Therefore, a suitable temperature is crucial for fluorescent reactions. H 2 O 2 solution was analyzed at the fluorescent reaction temperatures of 20, 25, 30, 35, and 40 • C. The findings showed that the SNRs at different temperatures were almost the same with 1% of RSD, implying that the reaction was unaffected by temperature within 20-40 • C in the HRP method ( Figure 6). However, the SNR increased with an increase in temperature in the Hemin method, as expected, because high temperature can enhance the activity of Hemin ( Figure 6).
Atmosphere 2022, 13, x FOR PEER REVIEW 6 of 12 the rise in temperature [23]. Furthermore, peroxides are also unstable at high temperatures, and their decomposition rates will increase with increasing temperature [24]. Therefore, a suitable temperature is crucial for fluorescent reactions. H2O2 solution was analyzed at the fluorescent reaction temperatures of 20, 25, 30, 35, and 40 °C. The findings showed that the SNRs at different temperatures were almost the same with 1% of RSD, implying that the reaction was unaffected by temperature within 20-40 °C in the HRP method ( Figure 6). However, the SNR increased with an increase in temperature in the Hemin method, as expected, because high temperature can enhance the activity of Hemin ( Figure 6).

Comparison of Two Methods
Two analytical methods were compared for detection limits, calibration curves, precision, accuracy, and applicability.

Detection Limit
The detection limit in this study is defined as the concentration of H2O2 solution that corresponds to three times the mean value of 20 noises detected under the optimum analysis conditions. The detection limits of H2O2 and MHP in the liquid phase were 0.04 μg/L and 0.64 μg/L, respectively, in the HRP method, and were 0.61 μg/L and 61 μg/L, respectively, in the Hemin method (Table 1). At the sampling flow of about 180 L, the detection limits of H2O2 and MHP in the gaseous phase were 0.4 ppt and 4.5 ppt, respectively, in the HRP method, and 4 ppt and 282 ppt, respectively, in the Hemin method (Table 1). These results indicated that the detection limits of gaseous H2O2 and MHP in the HRP method were 10 and 62 times, respectively, lower than that in the Hemin method. This study's results are different from Qi's [14]. This result means that low levels of H2O2 and MHP can be analyzed more accurately using the HRP method than the Hemin method.

Calibration Curve and Sensitivity
The value of five times the detection limit of H2O2 in the liquid phase was taken as the lowest point of calibration curves, which is 0.2 μg/L in the HRP method and 3 μg/L in the Hemin method respectively. The lowest calibration curve points of MHP were 3 μg/L and 300 μg/L in the HRP method and Hemin method, respectively. The calibration curves

Comparison of Two Methods
Two analytical methods were compared for detection limits, calibration curves, precision, accuracy, and applicability.

Detection Limit
The detection limit in this study is defined as the concentration of H 2 O 2 solution that corresponds to three times the mean value of 20 noises detected under the optimum analysis conditions. The detection limits of H 2 O 2 and MHP in the liquid phase were 0.04 µg/L and 0.64 µg/L, respectively, in the HRP method, and were 0.61 µg/L and 61 µg/L, respectively, in the Hemin method (Table 1). At the sampling flow of about 180 L, the detection limits of H 2 O 2 and MHP in the gaseous phase were 0.4 ppt and 4.5 ppt, respectively, in the HRP method, and 4 ppt and 282 ppt, respectively, in the Hemin method (Table 1). These results indicated that the detection limits of gaseous H 2 O 2 and MHP in the HRP method were 10 and 62 times, respectively, lower than that in the Hemin method. This study's results are different from Qi's [14]. This result means that low levels of H 2 O 2 and MHP can be analyzed more accurately using the HRP method than the Hemin method.

Calibration Curve and Sensitivity
The value of five times the detection limit of H 2 O 2 in the liquid phase was taken as the lowest point of calibration curves, which is 0.2 µg/L in the HRP method and 3 µg/L in the Hemin method respectively. The lowest calibration curve points of MHP were 3 µg/L and 300 µg/L in the HRP method and Hemin method, respectively. The calibration curves of H 2 O 2 and MHP in the two methods were created under their optimum analysis conditions. The results demonstrated that the calibration curves for H 2 O 2 were linear between 0.2 µg/L and 1000 µg/L in the HRP method and between 3 µg/L and 4000 µg/L in the Hemin  Figure 7). The calibration curves for MHP were linear between 3 µg/L and 3000 µg/L in the HRP method and between 300 µg/L and 4000 µg/L in the Hemin method. The correlation coefficient of the calibration curve for H 2 O 2 and MHP in the HRP method (>0.999) was higher than using the Hemin method (between 0.99 and 0.999). The slope of the calibration curve of H 2 O 2 for the HRP method (152,324) was 4.6 times higher than that of the Hemin method (27,129). The slope of MHP for the HRP method (46,232) was 171 times higher than that of the Hemin method (268). These results indicate that the HRP method was more sensitive in detecting H 2 O 2 and MHP than the Hemin method. of H2O2 and MHP in the two methods were created under their optimum analysis conditions. The results demonstrated that the calibration curves for H2O2 were linear between 0.2 μg/L and 1000 μg/L in the HRP method and between 3 μg/L and 4000 μg/L in the Hemin method (Table 2, Figure 7). The calibration curves for MHP were linear between 3 μg/L and 3000 μg/L in the HRP method and between 300 μg/L and 4000 μg/L in the Hemin method. The correlation coefficient of the calibration curve for H2O2 and MHP in the HRP method (>0.999) was higher than using the Hemin method (between 0.99 and 0.999). The slope of the calibration curve of H2O2 for the HRP method (152324) was 4.6 times higher than that of the Hemin method (27,129). The slope of MHP for the HRP method (46232) was 171 times higher than that of the Hemin method (268). These results indicate that the HRP method was more sensitive in detecting H2O2 and MHP than the Hemin method.   Table 3 shows the slopes of eight standard curves of H2O2 and eight results of H2O2 standard solutions of 100 ppb and 600 ppb measured in the HRP method on different days within a 16-day period. The RSD of the slope was 0.77%, and the RSDs of H2O2 standard solutions of 100 ppb and 600 ppb were 2.2% and 1.3%, respectively. These results indicate high repeatability on peroxide calibrations.   Table 3 shows the slopes of eight standard curves of H 2 O 2 and eight results of H 2 O 2 standard solutions of 100 ppb and 600 ppb measured in the HRP method on different days within a 16-day period. The RSD of the slope was 0.77%, and the RSDs of H 2 O 2 standard solutions of 100 ppb and 600 ppb were 2.2% and 1.3%, respectively. These results indicate high repeatability on peroxide calibrations. Standard solutions mixed with the same concentration of H 2 O 2 (150 µg/L), HMHP (400 µg/L), and different concentrations of MHP were analyzed by two methods. MHP concentration in the mixed solution of the Hemin method (4000 µg/L) was four times higher than that of the HRP method (1000 µg/L). Figure 8 is the chromatogram obtained by two methods. H 2 O 2 , HMHP, and MHP were detected at the retention times of 6, 7.7, and 12 min. The signal values of H 2 O 2 , HMHP, and especially, MHP in the HRP method were significantly higher than those in the Hemin method ( Figure 8). This result indicates that the sensitivity of the HRP method in detecting H 2 O 2 , HMHP, and MHP was higher than that of the Hemin method. Zhang and Dasgupta [13] studied the kinetic profiles of HRP and hemin catalytic systems for analyzing H 2 O 2 . Their results indicated that the fluorescence development rate of HRP and Hemin was 80% and 55% of the highest catalytic activity of hematin after 30-s reactions. The initial rate of fluorescence development with HRP was higher than that with hematin. The reaction time was about 15 s in this study, meaning that the HRP method showe d more sensitivity to H 2 O 2 and HMHP than the hemin method. The above results indicate that the relative sensitivities of the HRP and Hemin methods in detecting H 2 O 2 in this study were different from the data of Zhang and Dasgupta [13] and Qi [14]. Zhang and Dasgupta [13] mainly studied the fluorescence kinetics of HRP and hematin. They did not examine the analysis method of peroxides. They gave HRP and hematin calibration data but did not explain any analysis conditions. While analyzing peroxides, many factors, such as enzyme activity and pH value of the reaction system, can affect the fluorescence reaction that determines the magnitude of signal value. Therefore, the difference mentioned above between this study and Zhang and Dasgupta [13] was not fully understood. Qi [14] investigated the analysis method of peroxides using Hemin instead of HRP. However, the concentration (Table 4), manufacturer, date of manufacture of HRP, and the filling material and manufacturing process of the chromatographic column used in this study are different from Qi [14]. These factors may cause differences in the relative sensitivities of the HRP and Hemin methods in detecting H 2 O 2 between the two methods. As for MHP, the HRP method exhibited much higher sensitivity than the Hemin method [13].
Atmosphere 2022, 13, x FOR PEER REVIEW 8 of 12 higher than that of the HRP method (1000 μg/L). Figure 8 is the chromatogram obtained by two methods. H2O2, HMHP, and MHP were detected at the retention times of 6, 7.7, and 12 min. The signal values of H2O2, HMHP, and especially, MHP in the HRP method were significantly higher than those in the Hemin method ( Figure 8). This result indicates that the sensitivity of the HRP method in detecting H2O2, HMHP, and MHP was higher than that of the Hemin method. Zhang and Dasgupta [13] studied the kinetic profiles of HRP and hemin catalytic systems for analyzing H2O2. Their results indicated that the fluorescence development rate of HRP and Hemin was 80% and 55% of the highest catalytic activity of hematin after 30-s reactions. The initial rate of fluorescence development with HRP was higher than that with hematin. The reaction time was about 15 s in this study, meaning that the HRP method showe d more sensitivity to H2O2 and HMHP than the hemin method. The above results indicate that the relative sensitivities of the HRP and Hemin methods in detecting H2O2 in this study were different from the data of Zhang and Dasgupta [13] and Qi [14]. Zhang and Dasgupta [13] mainly studied the fluorescence kinetics of HRP and hematin. They did not examine the analysis method of peroxides. They gave HRP and hematin calibration data but did not explain any analysis conditions. While analyzing peroxides, many factors, such as enzyme activity and pH value of the reaction system, can affect the fluorescence reaction that determines the magnitude of signal value. Therefore, the difference mentioned above between this study and Zhang and Dasgupta [13] was not fully understood. Qi [14] investigated the analysis method of peroxides using Hemin instead of HRP. However, the concentration (Table 4), manufacturer, date of manufacture of HRP, and the filling material and manufacturing process of the chromatographic column used in this study are different from Qi [14]. These factors may cause differences in the relative sensitivities of the HRP and Hemin methods in detecting H2O2 between the two methods. As for MHP, the HRP method exhibited much higher sensitivity than the Hemin method [13].

Precision
The low concentration (20 µg/L), medium concentration (400 µg/L), and high concentration (800 µg/L) of the H 2 O 2 standard curve were analyzed six times to evaluate the precision of the two methods ( Table 5). The RSD of the six results measured H 2 O 2 solution Atmosphere 2022, 13, 659 9 of 12 at low, medium, and high concentrations of 0.44%, 0.49%, and 0.35% for the HRP method, respectively. Similarly, these low, medium, and high concentrations were 0.97%, 0.49%, and 0.47% for the Hemin method, displaying that the repeatability of the two methods was excellent.  (Table 6).

Applicability of Methods
The results presented in Figure 6 demonstrated that the catalytic reaction rate using the HRP method was free from temperature effects within 15-40 • C. In contrast, the Hemin method's rate increased with increased temperature ( Figure 6). This result suggests that the HRP method has broader applicability than the Hemin method.
The above results show that the HRP method had a lower detection limit, higher sensitivity, and better applicability for analyzing atmospheric H 2 O 2 and MHP than the Hemin method. The Hemin method is more economical than the HRP method.

Measurement of Atmospheric Peroxides Concentrations
Atmospheric peroxide concentrations were measured using the HRP method from 07:00 to 19:00 for eight days in September 2016. A total of 96 data sets were obtained. During the period, the measurements of blank samples, parallel samples, and spiked recovery rates in the whole monitoring process were conducted twice a day to ensure the precision and accuracy of the measurements. The relative errors of the two parallel samples ranged from 2.5% to 6.3%, and the recovery rates ranged from 96% to 105%. These results indicated that the measurement of atmospheric H 2 O 2 and MHP by the HRP methods had good repeatability and high accuracy.
During the measurement period, only H 2 O 2 and MHP were quantitatively detected. The concentrations of H 2 O 2 ranged from 0.13 ppb to 1.6 ppb, with an average value of 0.60 ± 0.37 ppb. Similarly, the MHP concentrations ranged from 0.020 ppb to 0.21 ppb with an average value of 0.081 ± 0.039 ppb, respectively (Table 7). Their concentrations were significantly higher on sunny days than cloudy days (Table 8, Figure 9) because H 2 O 2 and MHP were secondary products of photochemical reactions. Moreover, stronger solar radiation and higher temperature on sunny days promoted the production of H 2 O 2 and MHP. average value of 0.081 ± 0.039 ppb, respectively (Table 7). Their concentrations were significantly higher on sunny days than cloudy days (Table 8, Figure 9) because H2O2 and MHP were secondary products of photochemical reactions. Moreover, stronger solar radiation and higher temperature on sunny days promoted the production of H2O2 and MHP.  The concentrations of O3 and H2O2 generally showed diurnal variations similar to those of solar radiation. On most days, their concentrations started to increase with the increase of solar radiation from 9:00 and peaked at 14:00 or 15:00 when the solar radiation was high. This is primarily induced by photolysis and O3 and the photolysis and photooxidation of VOCs emitted from anthropogenic and natural sources. In addition, H2O2 concentration showed a second peak at 17:00-18:00 when the solar radiation decreased to a lower level on most days, whereas O3 did not show a second peak. This may be caused by the reaction of O3 and a large amount of olefins emitted from motor vehicles during the evening rush hour [25]. The photolysis of NO2 generates O3, and the reduction of solar radiation leads to a decrease in O3 generation. MHP concentration showed a different diurnal variation from that of H2O2. MHP concentration increased from around noon with fluctuations on most sunny days, and the highest value occurred after 17:00. This result indicates that MHP, like H2O2, was also generated by photochemical reactions; however, it also has different production and reduction pathways from H2O2 [26]. The concentrations of O 3 and H 2 O 2 generally showed diurnal variations similar to those of solar radiation. On most days, their concentrations started to increase with the increase of solar radiation from 9:00 and peaked at 14:00 or 15:00 when the solar radiation was high. This is primarily induced by photolysis and O 3 and the photolysis and photooxidation of VOCs emitted from anthropogenic and natural sources. In addition, H 2 O 2 concentration showed a second peak at 17:00-18:00 when the solar radiation decreased to a lower level on most days, whereas O 3 did not show a second peak. This may be caused by the reaction of O 3 and a large amount of olefins emitted from motor vehicles during the evening rush hour [25]. The photolysis of NO 2 generates O 3 , and the reduction of solar radiation leads to a decrease in O 3 generation. MHP concentration showed a different diurnal variation from that of H 2 O 2 . MHP concentration increased from around noon with fluctuations on most sunny days, and the highest value occurred after 17:00. This result indicates that MHP, like H 2 O 2 , was also generated by photochemical reactions; however, it also has different production and reduction pathways from H 2 O 2 [26].

Conclusions
The optimum analytical conditions for determining peroxides using the HRP and Hemin methods were investigated in this study. The results indicated that the optimum excitation and emission wavelengths are 315 nm and 405 nm, respectively, for the two methods. The optimum flow rates of the mobile phase, enzyme reagent, and alkali solution for the HRP method are 0.5, 0.2, and 0.1 mL/min, respectively. Those of the mobile phase and enzyme reagent for the Hemin method are 0.3 and 0.2 mL/min, respectively. Compared