ARTICLE pubs.acs.org/IECR
Development of Electrochemical Method for the Determination of Chlorzoxazone Drug and its Analytical Applications to Pharmaceutical Dosage Form and Human Biological Fluids Jyothi C. Abbar and Sharanappa T. Nandibewoor* P. G. Department of Studies in Chemistry, Karnatak University, Dharwad 580 003, India ABSTRACT: The electrochemical behavior of chlorzoxazone drug at glassy carbon electrode has been investigated for the first time using cyclic and square wave voltammetric techniques. The dependence of the current on pH, concentration, and scan rate was investigated to optimize the experimental conditions for determination of chlorzoxazone. The anodic peak was characterized and the process was diffusion-controlled. The number of electrons transferred in the oxidation process was calculated and a plausible oxidation mechanism was proposed. In the range of 8.0 107 to 1.0 105 M, the current measured by square wave voltammetry presents a good linear property as a function of the concentration of chlorzoxazone with a detection limit of 4.41 108 M with good selectivity and sensitivity. The proposed method was successfully applied to chlorzoxazone determination under physiological condition in pharmaceutical samples and for the detection of chlorzoxazone in human biological fluids. This method can be employed in clinical analysis, quality control, and routine determination of drugs in pharmaceutical formulations.
1. INTRODUCTION Drug analysis is one of the important tools for drug quality control. Therefore, the development of a simple, sensitive, rapid, and reliable method for the determination of drugs is of great importance. Chlorzoxazone (CHZ) is an effective muscle relaxant, 5-chloro-3H-benzooxazol-2-one, as shown in Scheme 1.1 It is a centrally acting agent for painful musculoskeletal conditions. CHZ acts primarily at the level of the spinal cord and subcortical areas of the brain where it inhibits multisynaptic reflex arcs involved in producing and maintaining skeletal muscle spasm of varied etiology. It is used to decrease muscle tone and tension and thus to relieve spasms and pain associated with musculoskeletal disorders.2,3 Various analytical methods for therapeutic monitoring have been reported in the literature for the determination of Chlorzoxazone in commercial dosage form and biological fluids such as high-performance liquid chromatography (HPLC),4 reverse phasehigh performance liquid chromatography (RP-HPLC),5 liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS),6 and spectrophotometry.7 The main problems encountered in using such methods are either the need for derivatization or the need for time-consuming extraction procedures. Voltammetric methods satisfy many of the requirements for such tasks particularly owing to their inherent selectivity, rapid response, high sensitivity, low cost, simplicity, and relatively short analysis time for the determination of organic molecules, including drugs and related molecules in pharmaceutical dosage forms and biological fluids.8 Electrochemical methods, especially differential pulse voltammetry (DPV) and square wave voltammetry (SWV), make it possible to decrease the analysis time as compared to the timeexhaustive chromatographic methods.9 The advantages of SWV over other electroanalytical techniques are greater speed of analysis, lower consumption of electroactive species in relation r 2011 American Chemical Society
Scheme 1. Chemical Structure of Chlorzoxazone
to the other electroanalytical techniques, and fewer problems with blocking of the electrode surface. To the best of our knowledge, until now there is no report in the literature on usingt a voltammetric method for the determination of CHZ. The aim of this study is to establish suitable experimental conditions, to investigate the electrochemical behavior and oxidation mechanism of CHZ at glassy carbon electrode by cyclic voltammetry, and to develop a square wave voltammetric method for the direct determination of CHZ under the physiological pH = 7.0 and physiological concentration which lies within the range studied in this work,10 i.e., 8.0 to 2.0 μM, for samples such as pharmaceuticals and human biological fluids. The proposed method has advantages such has no time-consuming sample preparation step prior to drug assay, high sensitivity, rapid response, good reproducibility, and low detection limit compared to other reported methods. Hence, we here report the voltammetric behavior of CHZ for the first time by cyclic and square wave voltammetric method at glassy carbon electrode.
2. EXPERIMENTAL SECTION 2.1. Reagents and Chemicals. CHZ was purchased from Sigma-Aldrich and used without further purification. A stock Received: September 24, 2011 Accepted: November 22, 2011 Revised: November 21, 2011 Published: November 22, 2011 111
dx.doi.org/10.1021/ie2021812 | Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
solution of CHZ (10 mM) was prepared in methanol. The phosphate buffers from pH 3.0 to 11.2 were prepared according to the method of Christian and Purdy.11 The CHZ-containing tablets (Cipla Ltd. Batch No. AA0141) were purchased from a local pharmacy. Other reagents used were of analytical or chemical grade. All solutions were prepared with doubly distilled water. 2.2. Instrumentation and Analytical Procedure. Electrochemical measurements were carried out on a CHI 630D electrochemical analyzer (CH Instruments Inc., USA). The voltammetric measurements were carried out in a 10-mL singlecompartment three-electrode glass cell with Ag/AgCl as a reference electrode, a platinum wire as counter electrode, and a 2-mm diameter glassy carbon electrode as working electrode. All the potentials are given against the Ag/AgCl (3 M KCl). pH measurements were performed with an Elico LI120 pH meter (Elico Ltd., India). All experiments were carried out at an ambient temperature of 25 ( 0.1 °C. Polishing of the glassy carbon electrode (GCE) was done on microcloths (Buehler) glued to flat mirrors. Al2O3 (0.3 μm) was used for polishing before each experiment. Before transferring the electrode to the solution, it was rinsed thoroughly with methanol and doubly distilled water. After this mechanical treatment, the GCE was placed in 0.2 M phosphate buffer solution, and various voltammograms were recorded until a steady-state baseline voltammogram was obtained. The parameters for square wave voltammetry (SWV) were initial potential: 0.7 V; final potential: 1.3; increase potential: 0.004 V; amplitude: 0.025 V; frequency: 15 Hz; quiet time: 2 s; sensitivity: 1 104 A/V. 2.3. Area of the Electrode. The area of the electrode was obtained by the cyclic voltammetric method using 1.0 mM K3Fe(CN)6 as a probe at different scan rates. For a reversible process, the following RandlesSevcik formula can be used.12 Ipa ¼ 0:4463ðF 3 =RTÞ1=2 n3=2 A0 D0 1=2 C0 υ1=2
Figure 1. Cyclic voltammograms at glassy carbon electrode in 0.2 M phosphate buffer solution (pH = 7.0): (a) 5.0 104 M CHZ (), and (b) blank run (o). Scan rate: 50 mV s1. Inset: Successive cyclic voltammograms of 5.0 104 M CHZ at glassy carbon electrode.
calibration graph or regression analysis. To study the accuracy of the proposed method and to check the interferences from excipients used in the dosage form, recovery experiments were carried out. The concentration of CHZ was calculated using standard addition method. 2.5. Plasma Sample Preparation. Human blood samples were collected in dry and evacuated tubes (which contained saline and sodium citrate solution) from a healthy volunteer. The samples were handled at room temperature and were centrifuged for 10 min at 1500 rpm for the separation of plasma within 1 h of collection. The samples were then transferred to polypropylene tubes and stored at 20 °C until analysis. The plasma samples, 0.2 mL, were deproteinized with 2 mL of methanol. After vortexing for 15 min, the mixture was then centrifuged for 15 min at 6000 rpm, and supernatants were collected. The supernatants were spiked with known amounts of CHZ. Appropriate volumes of this solution were added to phosphate buffer pH = 7.0 as supporting electrolyte and the voltammograms were then recorded.
ð1Þ
where Ipa refers to the anodic peak current, n is the number of electrons transferred, A0 is the surface area of the electrode, D0 is diffusion coefficient, υ is the scan rate, and C0 is the concentration, respectively, of K3Fe(CN)6. For 1.0 mM K3Fe(CN)6 in 0.1 M KCl electrolyte, T = 298 K, R = 8.314 J K1 mol1, F = 96 480 C mol1, n = 1, D0 = 7.6 106 cm2 s1, then from the slope of the plot of Ipa vs υ1/2, relation, the electroactive area was calculated. In our experiment the slope was 3.42 106 μA (V s1)1/2 and the area of electrode was calculated to be 0.04615 cm2. 2.4. Sample Preparation. Ten pieces of CHZ-containing tablet, i.e., Cip Zox (Cipla Ltd., Batch AA0141) were weighed and ground to a homogeneous fine powder in a mortar. A portion equivalent to a stock solution of a concentration of about 1.0 mM was accurately weighed and dissolved in methanol. The contents were sonicated for 20 min to affect complete dissolution. The excipient was separated by filtration and the residue was washed three times with methanol. The filtrate was transferred into a 100-mL calibrated flask and diluted to a final volume with the same solvent. Appropriate solutions were prepared by taking suitable aliquots from this stock solution and diluting them with the phosphate buffer solutions. Each solution was transferred to the voltammetric cell. The square wave voltammograms were subsequently recorded following the optimized conditions. The content of the drug in tablet was determined referring to the
3. RESULTS AND DISCUSSION 3.1. Cyclic Voltammetric Behavior of CHZ. The electrochemical behavior of CHZ at glassy carbon electrode was investigated using cyclic voltammetry (CV) at physiological pH = 7.0. The cyclic voltammograms obtained for 5.0 104 M CHZ solution at a scan rate of 50 mV s1 exhibit a well-defined irreversible anodic peak at about 0.9072 V at glassy carbon electrode. The results are shown in Figure 1. It was found that the oxidation peak current of CHZ showed a remarkable decrease during the successive cyclic voltammetric sweeps (Figure 1 inset). The significant decrease in the oxidation peak current was noticed with the number of successive sweeps. This phenomenon may be attributed to the fouling of the electrode surface due to adsorption of the oxidation product on the electrode surface.13 However, no peak was observed in the reverse scan, suggesting that the oxidation process is an irreversible one. This oxidized product in turn did not show any reoxidized or reduced peak at the extended ranges of potential which ensures that the oxidized product may not be electroactive 112
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
Figure 3. Influence of pH on the shape of anodic peak. pH 3.0 (a), 4.2 (b), 5.0 (c), 6.0 (d), 7.0 (e), 8.0 (f), 11.2 (g). Other conditions are as in Figure 1.
Figure 2. Variation of the cyclic voltammetric anodic peak current with accumulation time. Other conditions are as in Figure 1.
at the surface of GCE. Therefore, the voltammograms corresponding to the first cycle were generally recorded. 3.2. Influence of Accumulation Potential and Time. The influences of accumulation potential and accumulation time have been studied by cyclic voltammetric method. Usually open circuit accumulation is widely used in electroanalytical chemistry to improve the sensitivity. The influence of accumulation time ranging from 0 to 90 s on the oxidation of CHZ at GCE was as shown in Figure 2. The oxidation peak current increased greatly at the first 60 s and then slowly leveled off. Therefore, the optimal accumulation time of 60 s was employed in further experiments. With the change of accumulation potential, the peak current of CHZ varied slightly. So, the accumulation potential had no such effect on the peak current of CHZ. Therefore the accumulation was carried out at open-circuit conditions. 3.3. Influence of pH. The electrode reaction might be affected by pH of the medium. The electro-oxidation of 5.0 104 M CHZ was studied over the pH range of 3.011.2 in phosphate buffer solution by cyclic voltammetry. It was observed that the peak potential shifted to less positive values with increase in the pH of the buffer solution (Figure 3). However, with increase in the pH of solution, the peak potential was shifted to less positive values until pH = 7.0, thereafter becoming almost pH independent (Figure 4). Basically, two linear regions were obtained, one between pH 3.0 and 7.0, i.e., pH < pKa with a slope of 55 mV/pH and another between pH 7.0 and 11.2, i.e., pH > pKa with a slope of 13.2 mV/pH. The intersection of the curve was located around pH = 7.5. The increase in the slope between pH 3.0 and 7.0 indicated the presence of an antecedent acidbase equilibrium with pKa of about 7.5 which is supposed to correspond to the pKa value of CHZ.14 The linear relationship between Ep and pH can be expressed by the following equation:
Figure 4. Influence of pH on the peak potential of CHZ. Inset: Variation of peak current of CHZ with pH. Other conditions are as in Figure 1.
intensity decreases. Because the best result with respect to sensitivity accompanied with sharper response was obtained with pH = 7.0, it was selected for further experiments. 3.4. Influence of Scan Rate. Useful information involving electrochemical mechanism generally can be acquired from the relationship between peak current and scan rate. Therefore, the voltammetric behavior of CHZ at different scan rates was also studied using cyclic voltammetry. Scan rate studies were carried out to assess whether the processes on glassy carbon electrode were under diffusion or adsorption-controlled. The influence of the square root of scan rate on the peak current showed a linear relationship in the range of 5 to 400 mV s1 (Figure 5A) which is of a typical diffusion controlled process,13 and the equation can be expressed as
Ep ¼ 1:302 0:055pH; r ¼ 0:9915 ðbetween pH 3:0 and 7:0Þ The slope of this equation is found to be 55 mV/pH. This closeness of the slope to the expected theoretical value of 59 mV/pH suggests that the number of electrons transferred is equal to that of the hydrogen ions taking part in the electrode reaction. From the plot of Ipa vs pH (Figure 4 inset) it is clear that the intensity was increased to a high value at pH = 7.0, then the peak
Ip ðμAÞ ¼ 77:58υ1=2 ðV 1=2 s1=2 Þ 0:4083,
r ¼ 0:997
A plot of logarithm of anodic peak current vs logarithm of scan rate gave a straight line with a slope of 0.502 (Figure 5B), which is close to the theoretical value of 0.5 for a purely 113
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
Figure 5. (A) Dependence of peak current on the square root of scan rate. (B) Dependence of the logarithm of peak current on logarithm of scan rate. (C) Relationship between peak potential and logarithm of scan rate.
diffusion-controlled process15 which in turn further confirms that the process is diffusion controlled where the electroactive species CHZ diffuses from the bulk solution to a planar electrode surface. Data fit yields the equation log Ip ðμAÞ ¼ 0:502log υðV s1 Þ þ 1:899,
redox potential. Other symbols have their usual meanings. Thus the value of αn can be easily calculated from the slope of Ep vs log υ. In this system, the slope is 0.0583, taking T = 298 K, and substituting the values of R and F, αn was calculated to be 1.014. According to Bard and Faulkner,17 α can be given as
r ¼ 0:9953
α¼
The Ep of the oxidation peak was also dependent on scan rate. The peak potential shifted to more positive values on increasing the scan rate, which confirms the irreversibility of the oxidation process, and a linear relationship between peak potential and logarithm of scan rate (Figure 5C) can be expressed by the following equation: Ep ðVÞ ¼ 0:9789 þ 0:0583log υðV s1 Þ,
47:7 mv Ep Ep=2
ð3Þ
where Ep/2 is the potential where the current is at half the peak value. So, from this we obtained the value of α to be 0.70. Further, the number of electrons (n) transferred in the electro-oxidation of CHZ was calculated to be 1.41. The value of k0 can be0 determined from the intercept of the above plot if the value of E0 0 is known. The value of E0 in eq 2 can be obtained from the intercept of Ep vs υ curve by extrapolating to the vertical axis at system the intercept for Ep vs log υ plot was υ = 0.18 In our 0 0.8778 and E0 was obtained to be 0.8778; the k0 was calculated to be 4.3 102 cm s1. 3.5. Mechanism. In the proposed method, CHZ oxidation is a one-electron one-proton process and the possible product of oxidation is found to be 2-amino-4-chloro-phenol with liberation of carbon dioxide and the mechanism is as shown in Scheme 2. This observation was in accordance with earlier reports.19,20 3.6. Calibration Curve and Detection Limit. To develop a rapid and sensitive voltammetric method for the determination
r ¼ 0:9899
For an irreversible electrode process, according to Laviron,16 Ep is defined by the following equation: ! 2:303RT RT 0 2:303RT 00 Ep ¼ E þ log log υ þ αnF αnF αnF ð2Þ where α (alpha) is the transfer coefficient, k0 is the standard heterogeneous rate constant of the reaction, n is the number of 0 electrons transferred, υ (nu) is the scan rate, and E0 is the formal 114
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
Scheme 2. Plausible Mechanism of Electro-oxidation of CHZ at Glassy Carbon Electrode
Figure 7. Plot of peak current against the concentration of CHZ.
Table 1. Comparison of Linear Range and Detection Limits for CHZ to Different Classical Methods linear range (μg/mL)
detection limit (μg/mL)
ref
25.075.0
0.0090
4
9.020.0
0.0250
5
1.050.0
0.0194
21
0.0074 (= 4.41 108 M)
present work
0.1351.69 (= 0.810 μM)
The peak current Ip increased with the increase in square wave amplitude from 5 to 75 mV or square wave frequency in the range of 5 to 55 Hz, but the peak potential shifted to less negative values and the peak changed unshapely. So 25 mV was chosen as the optimum amplitude and 15 Hz was chosen as the optimum frequency. Square-wave voltammograms obtained with increasing amounts of CHZ showed that the peak current increased linearly with increasing concentration, as shown in Figure 6. Using the optimum conditions described above, linear calibration curves were obtained for CHZ in the range of 1 105 to 8 107 M. The linear equation (Figure 7) was Ip ðμAÞ ¼ 0:1487CðμMÞ þ 1:7703,
r ¼ 0:9873
Deviation from linearity was observed for more concentrated solutions, due to the adsorption of CHZ or its oxidation product on the electrode surface. Related statistical data of the calibration curves were obtained from the five different determinations. The limit of detection (LOD) and quantification (LOQ) were 4.41 108 M and 1.47 107 M, respectively. The LOD and LOQ were calculated using the following equations:
Figure 6. Square-wave voltammograms with increasing concentration of CHZ in pH 7.0 buffer solution on glassy carbon electrode with CHZ concentrations: 0.8 (1), 1 (2), 2 (3), 4 (4), 5 (5), 6 (6), 7 (7), 8 (8), and 10 (9) μM.
LOD ¼ 3s=m; LOQ ¼ 10s=m
of CHZ, square-wave voltammetric method was adopted as the peaks obtained are better defined at lower concentration of CHZ than those obtained by cyclic voltammetry, with a lower background current, resulting in improved resolution. According to the obtained results, it was possible to apply this technique to the quantitative determination of CHZ. The phosphate buffer solution of pH = 7.0 was selected as the supporting electrolyte for the quantification of CHZ as it gave a maximum peak current at pH = 7.0. The influence of the square wave parameters such as amplitude and frequency on the peak current was investigated.
where, s is the standard deviation of the peak currents of the blank (five runs), and m is the slope of the calibration curve. During the actual analysis, the analytical response was checked through the peak potential and its height. No change in peak potential was observed within an hour, while its height changed about (2% for five different quantitative determinations. The detection limits reported for different classical methods are tabulated in Table 1. This method was better as compared with other reported classical methods.4,5,21 115
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
Table 2. Analytical Precision and Accuracy of CHZ Determination by Square-Wave Voltammetry added (M) intraday 8.0 10
a
7
founda (M) 7.9398 10
SD
7
Table 4. Results of the Assay and the Recovery Test of CHZ in Pharmaceutical Preparations Using Square-Wave Voltammetry
accuracy bias (%) RSD (%)
SWV
HPLC method4
0.100
0.76
1.26
1.0 105 0.9828 105 0.020 interday 8.0 107 7.9183 107 0.087
1.72 1.02
2.13 1.10
labeled claim (mg)
500.00
500.00
amount found (mg)
497.8a
508.16b
1.0 105 0.9713 105 0.026
2.86
2.72
recovery %
99.56
101.63
RSD %
0.62
0.70
bias % calculated t
0.44 1.58
tthc: 2.776 Fth: 4.53
Average of six determinations.
Table 3. Influence of Potential Excipients on the Voltammetric Response of 1.0 105 M CHZ excipients (1.0 mM) + drug (1.0 10
5
M)
potential observed (V)
signal change (%)
only CHZ
0.8320
citric acid + CHZ
0.8401
+0.81
dextrose + CHZ glucose + CHZ
0.8403 0.8441
+0.83 +1.21
gum acacia + CHZ
0.8442
+1.22
lactose + CHZ
0.8404
+0.84
starch + CHZ
0.8400
+0.80
sucrose + CHZ
0.8523
+2.03
calculated F
1.32
amount of pure drug added (mg)
25.00
amount found (mg) [a]
24.91
recovery %
99.64
RSD %
1.08
a Mean value of five determinations. b Mean value of seven determinations. c th: theoretical.
0
A comparison with an official reference determination method has not been possible in any pharmacopoeias, because so far no other procedure for the quantitation of CHZ from pharmaceutical formulations has been reported. For this reason, the proposed method was compared with the literature method.4 Table 4 compares the results of the analysis of CHZ between proposed and literature method; the proposed method is sensitive, selective, and more precise than the HPLC assay. The F and Student t tests were carried out on the data and statistically examined the validity of the obtained results by HPLC and voltammetric methods. According to the Student t and variance ratio F-test, the calculated t and F values were less than the theoretical values in either test at the 95% confidence level. This indicates that there is no significant difference between the performances of the proposed and HPLC method with regard to accuracy and precision. The recovery test of CHZ ranging from 8.0 to 2.0 μM was performed using square-wave voltammetry. Recovery studies were carried out after the addition of known amounts of the drug to various preanalyzed preparations of CHZ. The recoveries in different samples were found to lie in the range from 96.52% to 101.82%, with RSD of 2.05%. 3.9. Detection of CHZ in Spiked Human Plasma Samples. The applicability of the SWV to the determination of chlorzoxazone in spiked human plasma sample was investigated. The recoveries from human plasma were measured by spiking drugfree plasma with known amounts of CHZ. The plasma samples were prepared as described in Section 2.5. A quantitative analysis can be carried out by adding the standard solution of CHZ into the detect system of plasma sample. The calibration graph was used for the determination of spiked CHZ in plasma samples. The detection results obtained for five plasma samples are listed in Table 5. The recovery determined was in the range from 97.27% to 101.14% and the RSD was 1.54%. 3.9. Detection of CHZ in Urine Samples. The developed square wave voltammetric method was also applied for the determination of chlorzoxazone in spiked urine samples. The recoveries from urine were measured by spiking drug-free urine with known amounts of CHZ. The urine samples were diluted 100 times with the phosphate buffer solution before analysis without further pretreatments. A quantitative determination can be carried out by adding the standard solution of CHZ into the
Precision of the method was investigated by intra- and interday determination of CHZ at two different concentrations (n = 6) within the linear range. Accuracy of the methods expressed as bias% and RSD% for intra and inter days are as shown in Table 2, which indicated high precision of the proposed method. To ascertain the repeatability of the analysis, 6 measurements of 1 105 M CHZ solution were carried out using glassy carbon electrode at intervals of 30 min. The RSD value of peak current was found to be 2.47%, which indicated that electrode has good repeatability. As to the reproducibility between days, it was similar to that of within a day repeatability if the temperature was kept almost unchanged. 3.7. Effect of Excipients. For the possible analytical application of the proposed method, the effect of some common excipients used in pharmaceutical preparations was examined. The tolerance limit was defined as the maximum concentration of the interfering substance that caused an error less than (5% for determination of CHZ. The effects of these excipients on the voltammetric response was carried by analyzing sample solutions containing a fixed amount of chlorzoxazone (1.0 105 M) spiked with various excess amount of each excipient under the same experimental conditions. The experimental results (Table 3) showed that hundred-fold excess of citric acid, dextrose, glucose, gum acacia, lactose, starch, and sucrose did not interfere with the voltammetric signal of CHZ. Thus, the procedures were able to assay CHZ in the presence of excipients, and hence it can be considered specific. 3.8. Determination of CHZ in Pharmaceutical Preparations and Recovery Test. The proposed method was validated for the determination of CHZ in pharmaceutical preparations in “Cipzox” tablets (500 mg per tablet) as a real sample by applying SWV using the standard addition method. The procedure for the tablet analysis was followed as described in section 2.4. The results are in good agreement with the content marked in the label (Table 4). 116
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
Table 5. Determination of CHZ in Human Plasma Samples human plasma spiked (μM) detecteda (μM) recovery (%) RSD (%)
a
sample 1
8.0
7.782
97.27
2.43
sample 2
6.0
6.053
100.88
2.80
sample 3
4.0
3.985
99.64
1.42
sample 4
2.0
2.022
101.14
2.35
sample 5
1.0
1.982
99.13
2.68
Average of six determinations.
Table 6. Determination of CHZ in Urine Samples
a
urine
spiked (μM)
detecteda (μM)
sample 1
8.0
sample 2 sample 3
6.0 4.0
sample 4
2.0
recovery (%)
RSD (%)
7.848
98.11
1.35
6.062 4.036
101.03 100.92
2.03 2.91
2.063
103.19
2.85
Average of six determinations.
detect system of urine sample. The calibration graph was used for the determination of spiked CHZ in urine samples. The detection results of four urine samples obtained are listed in Table 6. The recovery determined was in the range from 98.11% to 103.19% and the RSD was 1.87%. Thus, satisfactory recoveries of the analyte from the real samples and a good agreement between the concentration ranges studied and the real ranges encountered in the urine samples when treated with the drug make the developed method applicable in clinical analysis.
’ NOMENCLATURE CHZ = Chlorzoxazone GCE = glassy carbon electrode DPV = differential pulse voltammetry SWV = square wave voltammetry CV = cyclic voltammetry Ipa = anodic peak current (μA) n = number of electrons transferred A0 = surface area of the electrode (cm2) D0 = diffusion coefficient (cm2 s1) υ = scan rate (mV s1) C0 = concentration (mol dm3) Ep = peak potential (V) Ip = peak current (μA) α = transfer coefficient k0 = standard rate constant of the reaction (cm s1) F =0 Faraday constant (C mol1) E0 = formal redox potential (V) R = gas constant (J K1 mol1) T = temperature (K) LOD = limit of detection (mol dm3) LOQ = limit of quantification (mol dm3) s = standard deviation of the peak currents m = slope of the calibration curve RSD = relative standard deviation ’ REFERENCES (1) Bailey, L. C.; Remington. The Science and Practice of Pharmacy, Vol. II, 19th ed.; Mack Publishing Company: Pennsylvania, 1995. (2) Budavari, S.; O'Neil, M. J.; Smith, A.; Heckelman, P. E.; Kinneary, J. F. The Merck Index: An Encyclopedia of Chemicals, Drugs and Biologicals, 13th ed.; Merck: USA, 2001. (3) Lunn, G. HPLC Methods for Pharmaceutical Analysis, Vol. II; Wiley: New York, 1999. (4) Pawar, U. D.; Naik, A. V.; Sulebhavikar, A. V.; Datar, T. A.; Mangaonkar, K. V. Simultaneous determination of aceclofenac, paracetamol and chlorzoxazone by HPLC in tablet dose form. E-J. Chem. 2009, 6, 289–294. (5) Reddy, P. B. Simultaneous estimation of nimesulide and chlorzoxazone in pharmaceutical formulations by a RP-HPLC method. Int. J. Chem. Tech. Res. 2009, 1, 283–285. (6) Wanga, X.; Hub, L.; Tonga, S.; Zhengc, Y.; Yea, F.; Lina, D.; Linb, G.; Zhangb, X.; Wuc, H. Determination of chlorzoxazone in rat plasma by LC-ESI-MS/MS and its application to a pharmacokinetic study. Anal. Lett. 2010, 43, 2424–2431. (7) Sastry, C. S. P.; Chintalapati, R.; Sastry, B. S.; Lakshmi, C. S. R. Spectrophotometric determination of chlorzoxazone in pure state and formulations through oxidative coupling of its hydrolysis product. Anal. Lett. 2000, 33, 2501–2513. (8) Zima, J.; Svancara, I.; Barek, J.; Vytras, K. Recent advances in electroanalysis of organic compounds at carbon paste electrodes. Crit. Rev. Anal. Chem. 2009, 39, 204–227. (9) Erk, N. Voltammetric behavior and determination of moxifloxacin in pharmaceutical products and human plasma. Anal. Bioanal. Chem. 2004, 378, 1351–1356. (10) Ono, S.; Hatanaka, T.; Hotta, H.; Tsutsui, M.; Satoh, T.; Gonzalez, F. J. Chlorzoxazone is metabolized by human CYP1A2 as well as by human CYP2E1. Pharmacogenetics 1995, 5, 143–150. (11) Christian, G. D.; Purdy, W. C. Residual current in orthophosphate medium. J. Electroanal. Chem. 1962, 3, 363–367. (12) Rezaei, B.; Damiri, S. Voltammetric behavior of multi-walled carbon nanotubes modified electrode-hexacyanoferrate(II) electrocatalyst system as a sensor for determination of captopril. Sens. Actuators B 2008, 134, 324–331.
4. CONCLUSIONS The voltammetric oxidation of CHZ at glassy carbon electrode in phosphate buffer solution under physiological condition, i.e., pH = 7.0, has been investigated. CHZ undergoes one electronone proton change and is a diffusion-controlled process. A suitable oxidation mechanism was proposed. The peak current was linear to CHZ concentrations over a certain range, under the selected conditions. This helps in voltammetric determination of selected analyte as low as 4.41 108 M and can be used successfully to assay the drug in pharmaceutical dosage form as well as in spiked urine samples. High percentage recovery and study of excipients showed that the method is free from the interferences of the commonly used excipients and additives in the formulations of drugs. In addition, the results obtained in the analysis of CHZ in spiked urine samples demonstrated the applicability of the method in real sample clinical analysis. The proposed method is suitable for quality control laboratories as well as pharmacokinetic studies where economy and time are essential. ’ AUTHOR INFORMATION Corresponding Author
*Tel.: +91 836 2215286. Fax: +91 836 2747884. E-mail:
[email protected].
’ ACKNOWLEDGMENT J.C.A. thanks UGC, New Delhi for the award of Research Fellowship in Science for Meritorious Students (RFSMS). 117
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
Industrial & Engineering Chemistry Research
ARTICLE
(13) Prashanth, S. N.; Ramesh, K. C.; Seetharamappa, J. Electrochemical oxidation of an immunosuppressant, mycophenolatemofetil, and its assay in pharmaceutical formulations. Int. J. Electrochem 2011, 1–7. (14) Heel, R. C.; Avery, G. S. In Drug Treatment, 2nd ed.; Avery, G. S., Ed.; ADIS: Sidney, 1980; pp 12121222. (15) Gosser, D. K. Cyclic Voltammetry: Simulation and Analysis of Reaction Mechanisms; VCH: New York, 1993; p 43. (16) Laviron, E. General expression of the linear potential sweep voltammogram in the case of diffusionless electrochemical systems. J. Electroanal. Chem. 1979, 101, 19–29. (17) Bard, A. J.; Faulkner, L. R. Electrochemical Methods Fundamentals and Applications, 2nd ed.; Wiley, 2004; p 236. (18) Yunhua, W.; Xiaobo, J.; Shengshui, H. Studies on electrochemical oxidation of azithromycin and its interaction with bovine serum albumin. Bioelectrochemistry 2004, 64, 91–97. (19) Glenn, A. E.; Meredith, F. I.; Morrison, W. H.; Bacon, C. W. Identification of intermediate and branch metabolites resulting from biotransformation of 2-benzoxazolinone by fusarium verticillioides. Appl. Environ. Microbiol. 2003, 69, 3165–3169. (20) Bray, H. G.; Clowes, R. C.; Thorpe, W. V. The metabolism of aminophenols, o-formamidophenol, benzoxazole, 2-methyl- and 2-phenylbenzoxazoles and benzoxazolone in the rabbit. Biochem. J. 1952, 51, 70–78. (21) Yadav, A. H.; Kothapalli, L. P.; Barhate, A. N.; Pawar, H. I.; Pawar, C. R. RP-HPLC method for determination of aceclofenac, chlorzoxazone and paracetamol in bulk and pharmaceutical formulation. Int. J. Pharma Res. Dev. 2009, 1, 1–7.
118
dx.doi.org/10.1021/ie2021812 |Ind. Eng. Chem. Res. 2012, 51, 111–118
