Electrochemical Behavior of Propranolol and its Major Metabolites , 4 ’ ‐ Hydroxypropranolol and 4 ’ ‐ Hydroxypropranolol Sulfate , on Glassy Carbon Electrode

Foi estudado o comportamento eletroquímico do propranolol (PRO) e seus principais metabólitos. Medidas voltamétricas foram realizadas em eletrodo de carbono vítreo (GCE). Todos os compostos examinados sofrem oxidação via eletroquímica. Em todos os casos foi observada uma separação dos picos de oxidação, sugerindo a possibilidade de eletroanálise simultânea de propranolol e seus metabólitos. A linearidade das curvas de calibração foi obtida para concentrações entre 4,22×10-1,35×10 mol L para PRO, 4,00×104,81×10 mol L para 4’-hidroxipropranolol (4’OH PH) e 3,52×10-4,22×10 mol L para o sulfato de 4’-hidroxipropranolol (4’OH PS). O método descrito é rápido e simples, e pode ser aplicado para determinar os compostos mencionados acima em amostras biológicas. Os resultados obtidos foram verificados por cromatografia líquida de ultra-alta eficiência (ultra HPLC).

PRO is consumed in sport and other stressful activities, behaving as a doping agent.The drug slows down the heart rate of animals, leading to its use in the horse racing industry.PRO is rapidly metabolized after oral administration, and even traces of the drug may be difficult to detect in biological fluids some time after administration.
The detection of the metabolites of the drug will provide proof of its consumption.
Ingested PRO is excreted mainly as the sulfate conjugate, 4'-hydroxypropranolol sulfate (4'OH PS) (Scheme 1).The determination of the presence of this metabolite could yield useful information about PRO consumption.The second interesting metabolite of PRO is 4'-hydroxypropranolol (4'OH PH), which displays β-blocking properties.
6][7] PRO and 4'OH PH exhibit natural fluorescence properties, permitting the use of HPLC methods with fluorescence detectors for both compounds.4][15] A polarographic method was used to determine the quantity of PRO in pharmaceutical formulations.PRO was combined with nitric acid to yield nitropropranolol, which was then measured in Britton-Robinson buffer on a static mercury drop electrode. 13Adsorptive stripping differential pulse voltammetry was used to quantify PRO in a tablet dosage form on a carbon paste electrode. 14Another electrochemical method used a stabilized lipid film biosensor for rapid screening of PRO in pharmaceutical pills. 15lectrochemical methods have only been applied to the determination of PRO in pharmaceutical formulations, and to the best of our knowledge, no such methods have been established to find the major metabolites of PRO.
The aim of this study was to evaluate the usefulness of cyclic voltammetry (CV) and differential pulse voltammetry (DPV) as simple and rapid methods in the determination of PRO and its metabolites.These methods have been applied to characterize the electrochemical behavior of PRO, 4'OH PS and 4'OH PH on a glassy carbon electrode (GCE).The 4'OH PH has β-adrenergic properties, while 4'OH PS is the metabolite of PRO with the highest concentration in patient urine.Therefore, a new voltammetric method for the simultaneous determination of these compounds is very important.The electrochemical behavior was also studied using multi-wall carbon nanotube (MWCNT)-modified GCE.However, those results were no better than those obtained from a bare GCE, especially for PRO and 4'OH PS, substances that could be present in urine, so the latter unmodified electrode was used.This paper also proves that selected metabolites can be determined simultaneously in the presence of PRO.PRO and its metabolites in urine samples were also determined by ultra high performance liquid chromatography (ultra HPLC) method.

Instrumentation
The voltammetric measurements were conducted using the µAUTOLAB potentiostat, type III (Eco-Chemie, Netherlands).All measurements were carried out in a threeelectrode cell with a glassy carbon electrode (GCE), or a multi-wall carbon nanotube (MWCNT)-modified GCE as the working electrode.A saturated silver/silver chloride electrode (Ag/AgCl/KCl (sat.) ) and platinum wire were used as the reference and auxiliary electrodes, respectively.Voltammetric measurements were carried out in a 3 mL electrochemical glass cell.Nitrogen was used to remove oxygen from the measured solutions.The pH of the buffer solutions was measured using a pH meter of model CP-401 (ELMETRON, Zabrze, Poland).

Preparation of the modified electrode
The MWCNT (BU-201, Bucky USA, Houston, USA) were purified by stirring in a 2 mol L -1 nitric acid solution for 24 h.Next, 2 mg of MWCNT were mixed with 1 mL of DMF.The solution was then sonicated for 3 h.A 2 µL aliquot of the solution was then placed directly onto a polished GCE and allowed to dry.It was rinsed with water before use.Stock solutions 5.00 mg (0.0141 mmol) of 4'OH PS, 5.00 mg (0.0160 mmol) of 4'OH PH and 11.23 mg of propranolol hydrochloride, correspond to 10.00 mg of PRO (0.0339 mmol) were weighed to make stock solutions of each in 10 mL volumetric flasks, by dissolving the reference substances into mixtures of methanol:water (1:1 v/v).The resulting concentrations of the stock solutions of 4'OH PS, 4'OH PH and PRO were 1.41×10 -3 mol L -1 , 1.60×10 -3 mol L -1 , and 3.39×10 -3 mol L -1 , respectively.The stock solutions were stored at 4 °C and were stable for at least three weeks.

Analytical procedure
The GCE was polished manually to a mirror finish using an alumina slurry.The particle sizes of the alumina used were 1.0, 0.3 and 0.05 µm.Before transferring the electrode to the electrolyte solutions, the GCE was cleaned with water in an ultrasonic bath.A 2 mL portion of the Britton-Robinson buffer (BR), which served as a supporting electrolyte, was transferred into the 3 mL voltammetric glass cell.Oxygen was removed by purging with pure nitrogen for 10 min.The voltammogram of the pure supporting electrolyte was recorded, and an appropriate amount of stock solution was added.The series of voltammograms were recorded at different analyte concentrations.The measurements were performed in triplicate, using fresh sample solutions.The experimental conditions for the voltammetric measurements were: initial potential -0.5 V, final potential 1.5 V, starting potential 0 V, and scan rate in the range of 0.005-0.3V s -1 .The parameters used in DPV measurements were: initial potential -0.3 V, final potential 1.5 V, scan rate 0.025 V s -1 , pulse amplitude 50 mV, and pulse width 50 ms.All the experiments were performed at ambient laboratory temperature.
The limit of detection (LOD) and the limit of quantification (LOQ) were calculated using the relationship kSD b /a, where k = 3 or k = 10, respectively, where the SD b parameter is the standard deviation of the intercept, and a is the slope of the calibration curve. 16

Determination of PRO, 4'OH PS and 4'OH PH in urine samples
Urine samples were collected 2, 4, 5 and 10 h after administration of PRO (10 mg dose of PRO was administrated), and were filtered through 0.45 µm nylon Bakerbond filters to eliminate the residues.Each 5 mL sample of urine was precipitated adding 5 mL acetonitrile and 5 mL of phosphate buffer (pH 6.88) with stirring.The samples were centrifuged (2500 rpm) for 10 min.The supernatant was filtered through a 0.45 µm nylon Bakerbond filter to remove precipitated proteins from urine samples and transferred to an Oasis ® HLB cartridge (500 mg, 6 mL).The solid phase extraction (SPE) procedure consisted of conditioning (6 mL methanol, 6 mL distilled water), sample loading, washing (4 mL distilled water), drying and elution (2×2 mL of methanol).The eluates were evaporated to dryness under a stream of nitrogen.The residues were dissolved in 2 mL BR buffer and transferred to the voltammetric cell.
The recoveries of the determined substances were established for blank human urine samples spiked (before SPE enrichment step) with known amounts of PRO and metabolites.The results obtained for real urine samples were verified by ultra HPLC method. 17

Determination of PRO, 4'OH PS and 4'OH PH by ultra HPLC method
Analyses were carried out at 20 ºC with gradient elution. 17The best gradient program evaluated for the determination using the C 18 monolithic column was defined as 0.05% trifluoroacetic acid in water (solvent A) and acetonitrile (solvent B).The gradient elution program is presented in Table 1.The PRO and its metabolites were monitored by UV detection at λ = 227 nm.
The samples were prepared similarly as reported for voltammetric determination by SPE method.The eluates were evaporated to dryness under a stream of nitrogen.The residues were dissolved in 1 mL of a methanol:water mixture (1:9, v/v) and then 1 mL was introduced to the chromatographic column.

Results and Discussion
The imino and hydroxyl groups in PRO and its metabolites suggest that the compounds may be electrochemically oxidized.The set of voltammograms shown in Figure 1 confirms this finding.As the voltammogram of PRO shows, the only one well-defined oxidation peak occurs on GCE in acidic solution.The electrochemical oxidation of the metabolites 4'OH PS and 4'OH PH results in two well-defined peaks.For each metabolite there are only three peaks appearing in the entire potential range between -0.5 and 1.5 V, two resulting from oxidation processes and one from reduction.
The first oxidation peaks for 4'OH PS and 4'OH PH, appear at 0.156 V and 0.114 V respectively and are connected with the reduction peaks at 0.098 V and 0.048 V corresponding to reversible systems, as shown by the distances between reduction and oxidation peaks, close to theoretical values 0.059 V.Those redox couples could be connected with phenol oxidation giving possibly quinone systems.The second oxidation peaks of these two metabolites are related to irreversible processes, thus making the peaks at around 0.4 V and 1.0 V analytically useful.The presence of analyzed compounds in solution does not affect the background CV, except in the region in which these molecules are being reduced or oxidized.That unchanged background suggests a lack of adsorption of the analyzed compounds on GCE surface.This observation is verified in the scan rate dependence studies, described below.

Effect of pH
The pH effect of the electrolyte was examined between pH values of 2.00-9.00for PRO and metabolites.As pH decreases, the oxidation turns more difficult and shifts to more positive potential.The position of the peak potential E p vs. pH was obtained for all examined compounds.The cyclic voltammograms of the PRO metabolites show more than one peak.Therefore, only the irreversible oxidation peaks were used to evaluate the effects of pH (for 4'OH PS peak at a potential near 1.0 V, for 4'OH PH peak at a potential near 0.4 V).The observed pH effects corroborate the use of acidic buffered solutions as supporting electrolytes.The oxidation peaks of all examined compounds were broader in neutral media, making quantification unreliable.However the highest peaks were observed at pH 3.0.Therefore this pH value was used for analytical applications.
The E p vs. pH dependence for 1.35×10 -4 mol L -1 of PRO at pH < 9 may be expressed by equation: The slope of the equation suggests that the number of electrons and protons transferred in the reaction of PRO are equal.Similar relationships were determined for metabolites of PRO.The adequate relationships are presented below for 4.22 × 10 -5 mol L -1 of 4'OH PS (equation 2) and for 4.81 × 10 -5 mol L -1 of 4'OH PH (equation 3): E p /V = 0.541 -0.053 pH In both cases, the plots have slopes close to 0.059, which is characteristic for a reaction in which the number of electrons and protons that are transferred is the same.The plots of peak potentials as a function of pH were inserted in Figures 2, 3 and 4, respectively.
The observed oxidation peak for PRO is probably connected with the oxidation of the secondary alcoholic group in the PRO molecule. 18The proposed mechanism involves 2 protons and 2 electrons according to: The irreversible oxidation peak observed for metabolites of PRO could be connected with adequate mechanism of oxidation process.

Effect of scan rate
The peak current was plotted against scan rate in order to determine whether the irreversible oxidation processes of PRO and its metabolites are diffusion-or adsorptioncontrolled. 19,20 The linear correlation between peak current and scan rate is characteristic of adsorption-controlled processes, indicating that the electroactive species are being adsorbed onto the electrode surface.The linear relationship between peak current and square root of scan rate is typical for diffusion-controlled systems, meaning that the correlation between peak current and the concentration of analyte could be used as an analytical tool.In this study, the effect of the scan rate was determined in the scan rate range of 0.005-0.25 V s -1 .The peak current for PRO solutions was found to be linear to the square root of the scan rate according to the relationship: with a correlation coefficient of R 2 = 0.9983.The dependence indicates that the process is diffusion-controlled.A similar dependence was determined for PRO metabolites.For 4'OH PS, the dependence was determined on the basis of a peak found at a potential around 1.0 V.The peak current vs. square root of scan rate is described as: I p / µA = 0.062 (v / (mV s -1 )) ½ + 0.080 (5)   with a correlation coefficient of R 2 = 0.9961.The effect of the potential scan rate on the peak current may be expressed as log(I p ) vs. log(v), which has a slope of 1.0 for the species confined to the electrode surface, or 0.5 for the diffusion controlled system.For the 4'OH PS, the dependence of log(I p ) vs. log(v) can be expressed by the equation: log (I p / µA) = 0.425 log(v / (mV s -1 )) -0.999 (6)   with a correlation coefficient of R 2 = 0.9957.The value of the slope confirms that the process is diffusion-controlled.The effect of the scan rate for 4'OH PH was determined on the basis of measurements of peak current at a potential around 0.4 V.The relationship obtained in this situation also indicates that the process is diffusion-controlled.The peak current vs. square root of scan rate may be expressed as: I p / µA = 0.035 (v / (mV s -1 )) ½ + 0.011 (7)   with a correlation coefficient of R 2 = 9976.Analogously, as for 4'OH PS, the relationship of log(I p ) vs. log(v) was examined.The dependence could be expressed by the equation: log (I p / µA) = 0.467 log (v / (mV s -1 )) -1.379 (8)   with a correlation coefficient of R 2 = 0.9981.The value of the slope confirmed that the process is diffusion-controlled.

Calibration curves of PRO, 4'OH PS and 4'OH PH
The DP voltammograms, recorded at a scan rate of 0.025 Vs -1 , were used with the calibration curves.This scan rate value was applied because, in all cases, the peaks width increases with the increase of scan rate.This effect could generate interferences, especially in simultaneous determination of PRO and metabolites, when the distance between peaks is smaller.The voltammograms were recorded in BR buffer at pH 3.00.

Determination of PRO, 4'OH PS and 4'OH PH in a mixture
The peak potentials of the compounds examined are sufficiently different to permit the simultaneous determination of PRO and its metabolites.The distances between most peak potentials are more than 0.4 V.Only the distance between the irreversible oxidation peak of 4'OH PS and the peak of PRO is less than 0.2 V, making peak separation unsatisfactory.The distinction between the two was made using the second derivative (Figure 5).
To verify the possibility of simultaneous determination, the DP voltammogram of the buffer solutions at pH 3.00 into which all the compounds were injected was recorded, as shown in Figure 5.The slopes and intercepts of the calibration curves provide the values for the LOD and LOQ, obtained for PRO and its metabolites, as shown in Table 2.

Determination of PRO, 4'OH PS and 4'OH PH on MWCNT-modified GCE
Measurements for many drugs have been more accurate on MWCNT-modified GCE than on bare electrodes, making the modification attractive for our own purposes.Figure 5 shows that the voltammograms recorded on modified electrode are practically identical to those obtained on bare GCE for 4'OH PS.The voltammograms of PRO recorded on MWCNT-modified GCE are worse than adequate voltammograms obtained on bare GCE.Therefore, only the results obtained on unmodified GCE are presented, as that method is faster, simpler and cheaper.

Determination of PRO, 4'OH PS and 4'OH PH in urine samples
The methods described here can be used to determine the presence of the above compounds in urine samples prepared as described in Experimental section.All measurements were performed in triplicate by a standard addition method.Urine samples collected from various patients were examined.The voltammograms of one set of urine sample are shown in Figure 6.The recoveries from urine were measured by spiking drug free urine with known amounts of PRO and metabolites before SPE step.The obtained results are presented in Table 3.
As expected, the level of 4'OH PH concentration was very low, and could not be observed in urine samples 2 h after administration.The peaks characteristic for PRO and 4'OH PS were observed.The matrix signals were observed at potentials lower than 1.0 V and did not have an effect on PRO and 4'OH PS determination.The peak observed at potential ca.0.65 V decreases with following scans.The additional experiments indicated that this peak is connected with oxidation reaction of uric acid.The best results were obtained in urine samples collected 2 h after administration, in which the following concentrations were determined: 3.34×10 -6 mol L -1 of PRO and 5.76×10 -6 mol L -1 of 4'OH PS.The concentrations of the examined compounds in urine collected from patients were determined to lie in the ranges of 2.15×10 -6 -4.52×10 -6 mol L -1 and 2.04×10 -6 -5.76×10 -6 mol L -1 for PRO and 4'OH PS, respectively.The results are presented in Table 4.The described method could be applied for determination of PRO and metabolites in urine sample collected from patients treated simultaneously by popular diuretic, spironolactone.The second most popular diuretic, furosemide, could interfere with PRO and it must be eliminated.
The results obtained for urine samples were verified by ultra-HPLC method.At first the ultra-HPLC chromatogram was recorded for mixture of standard solutions and presented in Figure 7.
The chromatogram recorded for urine sample collected 2, 4 and 10 h after PRO administration was presented in Figure 8.The comparison between the DPV and the ultra-HPLC method was performed through the statistical t-student test at 0.05 significance level of the mean experimental values obtained in the quantification of PRO and 4'OH PS in urine samples.The results are given in Table 5.The test shows no statistical difference between results obtained by examined methods, neither for PRO nor for 4'OH PS.
The proposed voltammetric method in comparison with chromatographic method 1-7 is easier and low cost.In the comparison with other electrochemical methods, 13,15 the described method is not time consuming, do not involve accumulation time and do not require the nitration step.4][15] To the best of our knowledge, the electroanalytical method for simultaneous determination of PRO, 4'OH PS and 4'OH PH was not yet reported.

Conclusions
A simple and rapid electroanalytical method for the determination of PRO and its metabolites has been described.The metabolites are electrochemically oxidized in two steps in a diffusion-controlled mechanism, and no adsorption effects are observed.In both cases, the first oxidation process is reversible, and the second one is irreversible.The measurements indicate that the existing difference between the peak potential positions makes simultaneous determination of PRO and its metabolites possible.The proposed method could be applied for monitoring PRO and its metabolites in biological fluids.

a
based on peak potential approximately 1.0 V; b based on peak potential approximately 0.4 V; c LOD is the limit of detection; d LOQ is the limit of quantification.Regression equation I p /µA = a C/µmol L -1 + b, where a is the slope and b the intercept of the calibration plot, C is the concentration of adequate substance.

Figure 8 .
Figure 8.The ultra-HPLC chromatogram recorded for urine sample collected from a patient 2 h (A), 4 h (B) and 10 h (C) after PRO administration

Table 1 .
Gradient elution program for ultra-HPLC equipment solvents:

Table 2 .
Linear regression equations for calibration plots obtained in the quantitative determination of PRO, 4'OH PS and 4'OH PH (n = 6)

Table 3 .
Determination of PRO, 4'OH PH and 4'OH PS in spiked urine samples