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Voltammetric determination of lidocaine and its toxic metabolite using C18 silica modified carbon paste electrode: Application to pharmaceutical dosage form and milk
Ahmed S. Saad, Amal M. Abou Al-Alamein, Maha M. Galal*, and Hala E. Zaazaa
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El Aini st., 11562-Cairo, Egypt
Corresponding author’s e-mail: [email protected]
Tel. +201117486474, Fax.+20223635140
Voltammetric assay of lidocaine and its milk-secreted toxic metabolite
A new voltammetric method was developed for the simultaneous determination of lidocaine hydrochloride (LH) and its toxic metabolite 2, 6-dimethylaniline (DMA) based on their oxidation and adsorption on C18 silica modified carbon paste electrode (SMCPE). Several experimental factors including effect of pH of Britton Robinson buffer solution (BRB), scan rate, percentage of silica, accumulation potential and time were studied and the optimum conditions were found to be: pH 11±0.10 (BRB), scan rate 100 mV/s, accumulation potential -0.6V, accumulation time 150 s and 5 % C18 silica. Under the optimized conditions, square wave voltammetry (SWV) showed two signals for DMA and LH at 0.56 and 0.87 V, respectively. The method revealed satisfactory results in terms of linearity (7.94 x10-6 to 1.07 x 10-4 M) for LH and (1.20 x 10-6 to 1.07 x 10-5 M) for DMA, accuracy, and precision. The limit of detection was found to be 2.19 x 10-6 and 2.15 x 10-7 M for LH and DMA in pure forms, respectively. The method was further applied for the determination of LH in its pharmaceutical dosage form and for the assay of DMA in spiked milk samples.
Keywords: Lidocaine hydrochloride; 2,6-Dimethylaniline; toxic metabolite; C18 silica modified carbon paste electrode; square wave voltammetry
1. Introduction
Lidocaine hydrochloride monohydrate (LH) , chemically known as 2-(Diethylamino)-N-(2,6-dimethylphenyl) acetamide hydrochloride monohydrate (1),(2) (See Supplementary data Fig.1) is used as a local anesthetic.
It exerts its action by preventing the generation and conduction of nerve impulse as its main site of action is the cell membrane where it interferes with the permeability of the membrane to sodium ions (3). Lidocaine hydrochloride is an official drug in both the British and United States pharmacopeias. 2,6-Dimethylaniline (DMA) is the specified official impurity of LH (1) as well as its degradation product (4). DMA is a toxic metabolite of LH (5), may cause cancer of the urinary bladder (6) and reported to be nasal carcinogen in rats where its estimated cancer potency was found to be 0.0063 mg/kg body weight/day from dose–response data for nasal cavity tumors in female and male rats (7).
The literature review revealed that different analytical methods were reported for the determination of LH either alone or in mixtures including: spectrophotometry (4,8), high performance liquid chromatography (9–12), thin layer liquid chromatography (4), gas chromatography (13–16), capillary zone electrophoresis (17–20), voltammetry (25–27) and ion selective electrode-potentiometry (21–24). Simultaneous or selective determination of LH and DMA was reported using HPLC (29–31), ion selective electrode (28) and TLC (4) methods. As far as to our Knowledge no voltammetric method is reported for the simultaneous determination of LH and DMA. Therefore the aim of this work was to develop a simple, accurate and sensitive voltammetric method for the simultaneous determination of LH and DMA in pure form and in pharmaceutical dosage. The developed method was further applied for the determination of DMA in spiked milk samples, where it has been reported that DMA is secreted in human and bovine milk as inactive toxic metabolite for LH (7).
Carbon paste electrodes (CPE) have found their place in modern electroanalysis owing to their versatility, ease of preparation, low ingredients cost, reasonable reproducibility, high sensitivity and broad potential range. Several modifications have been carried out to CPE inorder to improve the analytical performance through increasing the selectivity and sensitivity of the determination (32). Silica gel is one of the reported modifiers that has been used for the determination of some drugs owing to its attractive properties as high adsorption capacity, thermal stability and insolubility in most solvents (33–35). The developed method is based upon using C18 silica modified carbon paste electrode (CPE) for the simultaneous determination of LH and DMA.
2. Experimental
2.1. Materials and reagents
LH was kindly supplied by Egyptian International Pharmaceuticals Industries Co (EIPICO) Cairo- Egypt with 100.95% ± 1.210 purity (1). DMA, graphite (; 20 ?m), paraffin oil were purchased from Sigma-Aldrich (Germany). Bondesil C18 Silica (230-400 mesh, 40 ?m) was purchased from Agilent (USA). Britton–Robinson buffer (BRB) was prepared by mixing different volumes of 0.04 mol/L each phosphoric acid, acetic acid and boric acid (Adwic Co., Egypt). The pH was adjusted to the range of (2.0–12.0) using appropriate volumes of 0.2 mol/L NaOH (Adwic Co., Egypt). Milli-Q deionized water was used.
2.2. Standard solutions
2.2.1. Lidocaine Hydrochloride stock standard solution (10-2 M)
The solution was prepared by accurately transferring and dissolving 0.288 g of LH into a 100-mL volumetric flask containing 50 mL deionized water and completing to the mark with the same solvent. Working standard solution of 10-3 M LH was prepared by suitable dilution from the stock standard solution.
2.2.2. 2,6-Dimethylaniline stock standard solution (10-2 M)
The solution was prepared by transferring 0.121 g of DMA into a 100-mL volumetric flask containing 50 mL water and completing to the mark with the same solvent. Working standard solutions of 10-3 M DMA was prepared by suitable dilution from the stock standard solution.
2.3. Pharmaceutical formulation
Vials of Lidocaine HCl 1% batch number (140573) were purchased from the local market. Each vial is claimed to contain 35 mg LH in 3.5 mL injection and is manufactured by Egyptian International Pharmaceuticals Industries Co (EIPICO).
2.4. Preparation of electrode
Graphite powder and paraffin oil were mixed together in a glassy morter in a ratio of 7: 3 in order to prepare the bare carbon paste. This paste was then filled into the hole of the electrode body and smoothed using a filter paper until giving a glossy appearance. The electrode is a cylinderical teflon body (6 cm in length) with an orifice of 6 mm diameter and 5 mm length followed directly by a cylindrical copper rod (6 mm in diameter) to perform as an electric contact.
Modified C18 silica carbon paste electrode was prepared by mixing graphite powder with 5% of its weight silica gel followed by the addition of paraffin oil. The homogenized paste was then packed into the hole of the electrode body and smoothed on a filter paper.
2.5. Instrumental and experimental set up
For voltammetric measurements, a Metrohm (AUTOLAB, model AUT204FRA32) electrochemical device was employed. A three electrode arrangement was applied throughout. Ag/AgCl reference electrode was used together with a platinum electrode serving as the counter electrode and finally C18 silica modified carbon paste electrode as a working electrode. Adjustment of pH was carried out using a pH-meter (JENWAY model 3505 – UK).
2.6. Construction of calibration curves
Aliquots equivalent to 200 ?L LH (10-3 M) were successively added to 25 mL BRB buffer (pH 11±0.1) in the electrolytic cell. Square wave voltammetry (SWV) was applied at deposition potential -0.6 V, deposition time 150 seconds and scan rate 100 mV/s. A calibration curve was constructed relating the different concentrations to the peak current. The same procedure was applied for DMA but by adding successive increments of 30 ?L (10-3 M) solution.
2.7. Application to pharmaceutical dosage form and milk
Lidocaine hydrochloride vials labeled to contain 10 mg/mL LH. A suitable dilution was made to obtain a concentration of 1×10-5M then SWV was applied and the recovery was calculated. The accuracy of the method was also checked by applying the standard addition technique.
Skimmed milk was purchased from the local market and without any treatment it was diluted with the buffer (pH 11±0.1) in a ratio (1:1, v/v). A volume of 25 mLwas transferred to the electrolytic cell then successive aliquots of DMA were added and SWV was recorded. A calibration graph was then constructed relating the concentration to the peak current.
3. Results and discussion
Primary analysis using cyclic voltammetry of 4 x 10-3 mol/L of LH in BRB pH 10 at scan rate 100 mV/s showed an irreversible oxidation peak at +0.85 V which is attributed to the oxidation of the tertiary amino group as reported in the literature (36). The applied potential range was +0.02 to +1.5 V at room temperature at bare carbon paste electrode (Fig.1). Optimization of cyclic voltammetric conditions included studying the effect of different modifiers, effect of pH, effect of silica concentration, deposition potential, accumulation time and effect of scan rate. After the conditions were optimized, square wave voltammetry was applied for the simultaneous determination of LH and DMA.
3.1. Effect of different modifiers
Two types of silica were tested, polar silica and C18 silica. It was found that C18 silica gave higher current intensity than bare carbon paste and polar silica modified electrodes (Fig.2). Morphological study of the three electrodes using scanning electron microscope (SEM) (Fig.3), revealed that C18 silica electrode had the most homogeneous porosity and particles distribution with an almost flat morphology producing the best oxidation in addition to highest current density.
3.2. Effect of pH
Cyclic voltammograms (CV) of LH were recorded at C18 silica carbon paste electrode in BRB over pH range (2.0-12.0) at a scan rate 100 mV/s. By increasing the pH of the solution, it was observed that the peak potential shifted to lower values indicating that oxidation at the modified electrode is pH dependent and that protons are involved in the reaction (37) (See Supplementary data Fig.2). By plotting the pH against the potential a straight line was obtained which fitted to the following equation: E=1.3609-0.0465 pH (r2= 0.9867) with a slope near 59 mV indicating that the number of electrons and protons involved in the reaction is equal (See Supplementary data Fig.3). The peak current showed its maximum value at pH 11 and thus it was used in further investigation and in quantitation.
3.3. Effect of different concentrations of silica
Different electrodes containing different concentrations of C18 silica (2.5, 5 and 7.5 %) were tried. The CVs were recorded and it was found that 5 % silica gave maximum current intensity (Fig. 4).
3.4. Effect of scan rate
Different scan rates were recorded for LH (4 x 10-3 M) starting from 20 to 200 mV/s inorder to study the reaction mechanism and determine whether it is adsorption or diffusion controlled. A linear relationship was obtained by plotting the logarithm of scan rate against the logarithm of current (See Supplementary data Fig. 4). The regression equation was found to be: log Ip (?A) =0.8418 log (v)-7.6908 (r2=0.9979) showing a value of slope between 0.5 and 1, implying that LH is transported by both adsorption and diffuion processes (38). The evidence of diffusion is also supported by the linear relationship obtained by plotting peak current against the square root of scan rate (See Supplementary data Fig.5). A scan rate of 100 mV/s was chosen for the quantitative determination of LH as it showed well-shaped peak with relatively narrow peak width.
The number of electrons involved in the oxidation reaction was calculated from Laviron equation (39) using the slope obtained by plotting oxidation peak potential against the logarithm of scan rate. The value of the slope was found to be 0.132 indicating the transfer of one electron which is in accordance with the reported literature on electro-oxidation of tertiary amines (40).
3.5. Effect of deposition potential and accumulation time
Deposition potential and accumulation time may have a great impact on the sensitivity of the determination by controlling the amount of drug adsorbed at the electrode surface. Different deposition potentials were scanned in the range of -1.2 to -0.2 V and the cyclic wave voltammograms were recorded. Results revealed that the current intensity of LH was greatly enhanced upon applying a potential of -0.6 V. Upon applying this potential for different time intervals, maximum peak current of LH was obtained after accumulation time of 150 seconds as shown in (See Supplementary data Fig.6).
Cyclic voltammetry is primarily used as a tool for fundamental and diagnostic studies which provides qualitative information only about electrochemical processes. More sensitive methods such as SWV are used for the quantitative determination of electroactive substances. Thus a mixture of DMA and LH was analyzed using the optimized conditions of 5% C18 silica in BRB (pH 11±0.10) applying deposition potential of -0.6 V for 150 seconds at scan rate of 100 mV/s using SWV where two peaks were observed at 0.56 V and 0.87 V for DMA and LH respectively (Fig.5).
3.6. Method validation
Linearity: using the optimized conditions, good linearity was achieved by plotting the concentration of LH and DMA against the peak current showing good correlation coefficients, in ranges of (7.94 x10-6 to 1.07 x 10-4 M) and (1.20 x 10-6 to 1.07 x 10-5 M) for LH and DMA, respectively (See Supplementary data Fig.7 and Fig.8). The parameters of the regression equations are shown in Table 1.
Accuracy: three different samples of LH and DMA were analyzed using the same procedure under linearity and the concentrations were calculated from the regression equations showing reasonable recoveries as shown in Table 1.
Precision: intraday and interday precision were assessed by analyzing three different samples in triplicates on the same day and on three successive days respectively. RSD% was calculated and was less than 2% as shown in Table 1.
Specificity and selectivity: the proposed method was successfully applied for the determination of LH and DMA in their laboratory prepared mixtures as shown in Table 2. The method was also applied for the determination of LH in dosage form without any interference from excipients or additives. Validation of the method was further checked by applying the standard addition technique as shown in Table 3.
Limit of detection (LOD) and limit of quantitation (LOQ): LOD was determined using the following equation: LOD= 3.3 x (SD of the response/slope) while LOQ was calculated using this equation LOQ = 10 x (SD of the response/slope) where SD is the standard deviation. Results are shown in Table 1.
3.7. Application to spiked milk
Being a toxic metabolite of LH and reported to be secreted in human and bovine milk, DMA was determined in spiked milk samples by the developed SWV method where good linearity was acheived in the range of 2.39 x 10-6 – 8.33 x 10-6 M with detection limit of 2.42 x 10-7 M as shown in Table 4.
3.8. Comparison with reported method
The results of the proposed method were statistically compared to those obtained from the reported HPLC method and no significant difference was found between them as shown in Table 5.
4. Conclusion
In this work, a new sensor was developed based on carbon paste electrode modified with C18 silica for the simultaneous determination of LH and its toxic metabolite DMA. Different conditions were studied and under the optimized conditions square wave voltammetry was used for the quantitation of both LH and DMA in pure form and pharmaceutical dosage form. DMA was also determined in spiked milk samples with good recoveries. The proposed method is sensitive, accurate and precise. Complete validation was done regarding linearity, accuracy, precision, specificity and selectivity. The proposed method can be used in the quality control laboratories giving satisfactory results compared to other sophisticated, time consuming and expensive methods.

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