Reagents

Metoprolol was supplied by Sigma-Aldrich (≥98%). Organic solvents such as methanol, acetonitrile and formic acid, and other chemicals used were HPLC-grade from Aldrich and J-baker. Graphite powder was obtaind from University Kebangsaan Malaysia, Malaysia. All aqueous solutions were prepared with de-ionized water (DIW) with conductivity ≤18 S/cm at ambient temperature. NaCl was purchased from Merck with high purity ≥99.5%. Metoprolol (0.01 g) dissolve in 10 mL of methanol to prepare a stock solution of 1000 mg/L. Subsequent concentrations 2, 5 and 10 mg/L were prepared after diluting in de-ionized water.

Preparation of a Graphite-polyvinyl Chloride Composite Electrode

The graphite-PVC composite electrode was prepared by mixing together a weighed portion of graphite powder (100 mesh in size and 99.9% purity, Aldrich chemical Company) and polyvinyl chloride (PVC) in 8 mL tetrahydrofuran (THF) solvent and swirled flatly to homogeneous followed by drying in an oven at 50 ℃ for 3 h. The mixture was then placed in 2 cm diameter stainless steel mould and pressed at 10 ton/cm² using hydraulic machine (ATLAS auto touch HYDRAULIC). A typical pellet contained approximately 95% of graphite powder, and approximately 5% of PVC. The total weighed of pellet obtained is approximately 2g. Graphite-PVC pellet connected to a silver wire with silver conducting paint and sealed to a glass rode. Subsequently, epoxy gum was applied to cover the silver wire connected surface as show in Fig. 1.

Fig.1. Steps of preparation of graphite-PVC electrode 

A 100 mL of metoprolol solution was electrochemically treated at different interval times (0, 10, 20, 30, 40, 50, 60, 70 and 80 min). However, consumption energy was investigated at different values of applied voltages and sodium chloride during the electrochemical oxidation process to ensure that the present study is preferred for this purpose. Metoprolol was tested without switching on power as a control test to confirm that the removal of metoprolol is not due to the adsorption to the electrodes.

A solution of 2 mg/L of metoprolol was subjected to non-electrochemical process (i.e. switch off direct current (DC) power supply) for 60 min at a fixed concentration of 5000 mg/L NaCl. All samples were analysed and compared to the origin solution using LC-TOF/MS.

Set up of Electrochemical Process

The schematic experimental setup is as shown in Fig. 2. The reaction was carried out using 100 mL pyrex glass. The glass pyrex electrochemical cell (reactor) was placed on magnetic stirring block in order to keep its contents well mixed during the experiment. Graphite-PVC pellet and Pt plate were used paralleled as anode and cathode, respectively. The distance between the electrodes was 4 cm. The electrodes were then connected to a direct current (DC) power supply (CPX200 DUAL, 35V 10A PSU).

Fig.2. The schematic diagram of the electrochemical process use 

Solid Phase Extraction (SPE) Method

All samples after electrochemical treatment were filtered through 0.45 μm GF/F glass fiber filter before SPE experiments. The SPE cartridges were preconditioned with, 2 mL of MeOH and 2 mL of deionized water at a flow rate of 3 mL/min. After the preconditioning step, aliquots of 100 mL of sample were loaded into the cartridge. Samples were passed through the cartridges at a flow rate of 3 mL/min. The cartridges were later dried under vacuum for approximately 5 min at a flow rate of 15 mL/min to remove excess of water.

The pharmaceutical compounds were subsequently eluted to a 12 mL glass tubes by sequentially passing 5 mL of methanol, then evaporated to dryness under a gentle stream of N₂ gas. Dry extracts were then reconstituted with 1.0 mL of MeOH-DIW (10:90, v/v) and then transferred to 250 (L deactivated glass insert with polymer feet inserted in amber glass vials from Agilent Technologies (USA). The extract (30 µL) was automatically injected into LC-ESI-TOF/MS system for analysis. All SPE steps have been presented in Fig. 3.

Fig. 3 Solid phase extraction setup. 

Each by-product was identified based on mass value (m/z) and retention times. Quantification was carried out using the TOF mode by extracting the narrow window extracted ion chromatogram (nwXIC) of the molecular ion for each compound (typically extracted using a 20 mDa window) compared with previous studies that used 20 and 50 mDa windows [²⁷,²⁸].

Positive identification of the target compounds was based on (a) accurate mass measurement of the base ion with an error of ≤ ± 3.5 ppm for most compounds; and (b) LC retention time of the analyte compared with that of a standard with an error of ≤ ± 0.3% for most of compounds.

Liquid Chromatography-time of flight/Mass Spectrometry

Separation of the studied pharmaceuticals was performed on the dionex Ultimate 3000/LC 09115047 (USA) system equipped with a vacuum degasser, a quaternary pump, and an auto-sampler. Sample aliquots of 30 (L were injected to Gemini 5 (m NX 110A C18 column (2 mm x 150 mm, Phenomenex). Electrospray ionization (ESI) source was utilized as ionization source.

Metoprolol and one of its by-products were analysed in positive ionization mode ESI (+). The elution off the column was achieved with a mobile phase consisting of (A) 0.1% FA in DIW and (B) ACN-MeOH (3:1, v/v) at 0.3 mL/min. the gradient elution is as follow:

Three by-products were analysed in negative ionization mode (ESI () and they eluted off the column with a mobile phase consisting of a mixture of ACN-MeOH (2:3, v/v) as mobile phase (B) and 0.1% FA in DIW as mobile phase (A) at 0.3 mL/min. the gradient elution is as follow:

Mass spectrometry was performed on ESI-TOF instrument (Bruker/Germany). The results were obtained with the following settings: MS capillary voltages, 4000/3500 [ESI (+)/ESI (-)]; drying gas flow rate, 8.0 L/min; drying gas temperature, 190 ℃; and nebulizer pressure, 4.0 bar. Two molecular ions, [M+H] ⁺ and [M(H]^-, were observed for ESI (+) and ESI (-) analysis, respectively. All analytes were acquired using an independent reference spray via the LockSpray interference to ensure accuracy and reproducibility. A mixture of sodium hydroxide and formic acid (FA) was used as the lock mass m/z 90.9766 to 974.8132. The accurate mass was calculated using Daltonics Data Analysis software incorporated in the instrument.

Electrochemical Evaluations

Removal of metoprolol was investigated using electrochemical treatment process under different conditions such as initial concentration, applied voltage and sodium chloride. It was observed that the efficiency of electrochemical process was impacted by rate of generation the oxidizing agent ClO- which is the most effective oxidizing agent for the removal of pollutants such as metoprolol.

Effect of Initial Concentration

Fig. 4 shows the removal% of metoprolol for each initial concentration (2, 5 and 10 mg/L). Experimental results showed that all trends of removal% have different profile which means decreased as the initial concentration increased. By comparing initial concentration of 10, 5 and 2 mg/L, their removal% are > 95% after 60 min. This phenomenon has been reported previously by ^{Mussa et al. (2016)} [²⁹]. It was observed that low removal% was accompanied with high initial concentration (10 mg/L), at the beginning of the treatment, whilst high removal% was found with low initial concentration (2 mg/L). The reason may be attributed to the fact that the number of moles of MTP at high initial concentration is more than its number at low initial concentration.

Fig. 4 Impact of initial concentration of metoprolol on the removal% (5V, 5000 mg/L NaCl, 100 mL solution). 

Effect of NaCl

Several experiments were conducted to study the removal behavior under various concentrations of NaCl (1000, 3000 and 5000 mg/L). Experimental results showed that most MTP was eliminated very effectively using graphite-PVC anode using 5000 mg/L NaCl, the removal of MTP reduced sharply at low concentration of NaCl (1000 mg/L and 3000 mg/L) as shown in Fig. 5. Almost no removal was found for the lowest concentration of 1000 mg/L of NaCl. The effect of NaCl concentration on the removal of MTP from aqueous solution by electrochemical treatment process using graphite-PVC anode can be discussed as presented in equations 1-4, by the considering the formation of active oxidizing agent (ClO-).

Fig. 5 Impact of NaCl concentration on the removal% (2 mg/L of MTP, 5V, 100 mL solution). 

In electrochemical treatment process, low consumption energy is preferred for this type of treatment unless the removal% is high. From Table 1, it was observed that the highest consumption energy of 2.804 Wh/mg and low removal% of < 10% were determined using 1000 mg/L NaCl after 70 min of electrochemical treatment process. Average energy consumption values of the electrochemical process measured are mostly similar between 3000 and 5000 mg/L NaCl.

The lowest consumption energy (0.548 Wh/mg) was determined using 5000 mg/L NaCl while 0.595 Wh/mg was determined using 3000 mg/L NaCl after 10 and 20 min, respectively. From the results, 5000 mg/L NaCl was the best choice for further experiments.

Effect of Applied Voltage

The effect of applied voltage on the electrochemical treatment of MTP was also investigated through the determination of removal% and consumption energy. Fig. 6 shows that the removal% are in range of 9.8 to 60%, 31 to 100% and 22 to 93% at 3, 5 and 7 V, respectively. Normally, a greater removal of MTP is associated with a greater value of applied voltage. Comparing the removal% of 3, 5 and 7V, it is obvious that the efficiency of electrochemical treatment at 7 V was greater than that of 3 V.

Fig. 6 Impact of applied voltage on the removal% (2 mg/L of MTP, 5000 mg/L NaCl, 100 mL solution). 

It is well known that high applied voltage may mean high consumption of energy, so this concept was considered in this study. From Table 1 consumption energy was very high at 7 V comparing to 5 V. On the other hand, removal% was almost same between 7 and 5 V under same conditions. It could be concluded that the removal% at 5 V is more preferred compared to 7 V since the consumption energy is much lower 0.666 Wh/mg compared to 7V of 1.117 Wh/mg. Although energy consumption at 5 and 3 V is almost similar, 5 V was selected due to high removal% was gained 98% compared to 3 V (31.2%) after 30 min.

Electro-reaction Kinetics

The kinetic studies of the degradation metoprolol were investigated under different conditions: NaCl concentration, applied voltage and initial concentration as presented in Table 2. It is well known that most of catalytic reactions could follow a second-order reaction, as described by the equation 5 [³⁰,³¹].

where, k _app is the apparent second-order rate coefficient; [ClO^-]_t is the total concentration of ClO^- species at the reaction time of t; [MTP]_t is the total concentration of the MTP at the reaction time of t.

In the presence of a high excess of ClO^-, the concentration remains always constant throughout the reaction (i.e. [ClO^-]_t = [ClO^-]_o). Hence, k _app . [ClO^-]_t could be regarded as a constant so the pseudo-first-order kinetic constant k _obs is introduced as obtained in equation 6.

After integration equation 6 then rearranged it, we get equation 7 as presented here:

According to this equation, ln ([MTP]_t/[MTP]₀) was plotted versus time to generate straight line, correlation coefficient (R²) (0.9334, with a slope of (k _obs. Electrocatalytic-kinetics rate constants were presented in Table 2. It was observed that in the presence of NaCl and high applied voltage, the rate constant value becomes more enhanced. The reason may due to the generation of ClO^-which is contributed strongly for the elimination purpose.

However, the value of k _obs of 0.0801 min^-1 was the greatest at 5000 mg/L NaCl compared to 0.1 g and 0.3 g NaCl which was 0.0016 and 0.0136 min^-1, respectively. In case of applied voltage, high k _obs of 0.0801 min^-1 was observed at 5 V which is (8) times greater than k _obs at 3 V. For further investigation, electro-reaction kinetics was tested at different initial concentration of MTP. It was observed that the greatest rate constant was at initial concentration of 2 mg/L whilst the smallest rate constant was recorded at initial concentration of 10 mg/L. k _obs values were arranged as follow: 0.0801, 0.0564 and 0.0427 min^-1 for 2, 5 and 10 mg/L, respectively. These results are quite good compared to the previous work which is provided on electrochemical fenton on metoprol [³²,³³]. The reason why low k _obs was obtained with high initial concentration at fixed volume (100 mL) may due to the fact that the number of molecules of MTP at 10 mg/L are more than its number of molecules at 2 mg/L hence only few molecules could be removed at high concentration compared to the low concentration under same conditions.

Identification of the by-products

So far, few studies have been reported about the electrochemical degradation of metoporlol and its by-products. In order to accomplish this objective, the samples were analyzed by LC-TOF/MS after electrochemical degradation process. Table 3 presents the retention times, elemental composition, chemical structure, signal to noise ratio and ring double bond for the by-products of metoporlol as obtained by LC-ToF/MS instrument. The error was ranged between 2 and 2.9 ppm.

Table 3 Accurate mass measurements obtained by LC-TOF/MS for the by-products after electrochemical oxidation treatment of metoprolol. 

No.	tR (min)	Composition molecular ion	Chemical structure	Error ppm	m/z	RDB
MTP	5.101	C15H26NO3 [M+H]+		192	268.1009	3.5
PI-P1	9.200	C16H18Cl2NO4 [M+H]+		1892	358.0716	7.5
NI-P1	8.197	C12H13O4 [M˗H]˗		177	221.0773	6.5
NI-P2	9.067	C13H10Cl4NO2 [M˗H]˗		44	351.9329	7.5
NI-P3	11.275	C15H12Cl2NO2 [M˗H]˗		30	308.0200	9.5

One by-product PI-P1 was detected in positive ion mode and it is corresponded to compound with molecular formula C₁₆H₁₈Cl₂NO₄ (m/z358.0716). Three by-products were detected in negative ionisation mode; NI-P1, NI-P2 and NI-P3 could correspond to C₁₂H₁₃O₄ (m/z221.0773), C₁₃H₁₀Cl₄NO₂ (m/z351.9329) and C₁₅H₁₂Cl₂NO₂ (m/z308.0200), respectively. All LC chromatograms of by-products were presented in Fig. 7 combined with the mass spectra for each product.

Fig. 7 Extracted ion chromatogram vs mass spectrometry profile for one by-products detected in PI mode (PI-P1) and three by-products detected in NI mode (NI-P1 to NI-P3) of metoprolol after electrochemical treatment under 5V, 5000 mg/L NaCl, 20 min and 200 μg/L. 

Elucidation of the by-products

This study reports to elucidate clearly the formation of chlorinated by-products of metoprolol in two ionization modes positive ionization (PI) and negative ionization (NI). It is important that most of by-products had at least two chlorine atoms in their structure molecule, thus indicating that chlorination reactions can occur during the electrochemical oxidation process treatment when solution contains chlorine. Two different chlorinated by-products can be elucidated (C₁₆H₁₈Cl₂NO₄, PI-P1 and C₁₃H₁₀Cl₄NO₂, NI-P2). Fig. 8a shows the spectra of by-product with two chlorine atoms (358.0716). The most striking aspects of these spectra are the clusters of intense peaks that are each separated by 2 m/z units. However, the peaks at m/z 358, 360 and 362 in the spectrum of [C₁₆H₁₈Cl₂NO₄] has isotopic patterns that refers to M, M+2, and M+4, respectively. Careful examination of this compound shows that the peaks at m/z358, 360 and 362 in compound PI-P1 spectrum has approximate intensities 100.0%, 67% and 11%, respectively relative to each other, which confirms the presence of two Cl atoms. On the other hand, product NI-P3 (C₁₅H₁₂Cl₂NO₂ , m/z 308.0200) in negative ionization mode shows that this compound has same profile of PI-P1 product. The tetra-chlorine adduct was presented in product NI-P2 (C₁₃H₁₀Cl₄NO₂, m/z 351.9329) resulting five peaks M, M+2, M+4, M+6 and M+8 which are represented m/z 351.9, 353.9, 355.9, 358.0 and 360.0, respectively with probability percentages 75%, 100%, 50%, 11% and 1%, respectively (Fig. 8b).

Fig. 8 Elucidation profile of the chlorinated by-products during electrochemical oxidation process of metoprolol; (a) di-chlorine atoms; (b) tetra-chlorine atoms. 

The oxidation pathway shown in Scheme 1 involves cycling the open chain of metoprolol as the ring double bond increased. However, product NI-P1 (m/z 221) has eluted at tR8.197 min. The mass spectrum of NI-P1 did not indicate the presence of halogens. Furthermore, the product [M-C₃H₈N-4H+O] loss 47 Da due to losing the terminal chain (C₃H₇N).Loss of water, hydrogen abstraction and forming cycle in the ESI− product ion mass spectra of (NI-P3, m/z 308) indicated that the increasing of ring double bond from four to nine as shown in Table 3. The mass value of the product was increased by 40 Da due to two chlorine atoms have been added. In the same manner to NI-P3, two by-products PI-P1 and NI-P2 were formed by the addition of 90 and 84 Da, respectively. For the PI-P1, two chlorine atoms and one methyl group have been substituted on the benzene ring producing C₁₆H₁₈Cl₂NO₄ while NI-P2 loss two methyl groups and water molecule but four chlorine atoms have been substituted on the benzene ring forming product of C₁₃H₁₀Cl₄NO₂.

Scheme 1 Proposed pathway degradation after electrochemical degradation process of metoprolol under these conditions: 5000 mg/L NaCl, 5V, 200µg/L, 100 mL of solution. 

Monitoring of By-products After Electrochemical Oxidation Process

The monitoring of the chromatographic areas of the by-products of metoprolol during the electrochemical process is necessary to evaluate the efficiency of electrochemical process. 100 ml of metoprolol solutions were treated electrochemically using graphite-PVC electrode under 5V, 5000 mg/L NaCl and 200 μg/L. The evolution of the chromatographic areas with electrochemical oxidation time for the by-products was shown in Fig. 9. It was observed that the degradation of metoprolol is simultaneously accompanied by the appearance of the oxidation by-products, which are already formed in the first 20 minutes of the treatment. As observed in the degradation curves for PI and NI modes, the most of by-products increased steadily at 20 min of electrochemical oxidation treatment then decreased with the time of electrolysis. PI-P1 by-product was increased until 20 min then reduced with time, it was completely removed after 80 min. The by-product NI-P1 has the highest peak area compared to others while NI-P2 has the lowest peak area in NI mode. In the light of the above results, it was observed that all chlorinated and non-chlorinated by-products were completely removed after 120 min indicating that the efficiency of electrochemical process to degrade the by-products after they formed.

Fig. 9 Monitoring the by-products after electrochemical degradation of metoprolol: 100 mL of solution,5 V, 5000 mg/L NaCl, and 200μg/L metoprolol.