New analysis of clopidogrel bisulfate in plavix tablet and human biological fluids utilizing chemically modified carbon paste sensor

2.1 Chemicals and materials

All chemicals used for preparation of solutions were of analytical grade. Doubly distilled water was used throughout all the experiments. Clopidogrel bisulfate and its dosage form (Plavix, 75 mg /tablet) were provided by Sanofi Aventis Company Cairo-A.R.E. Tributyl phthalate (TBP), tricresyl phthalate (TCP), graphite powder, dibutyl phthalate (DBP), dioctyl phthalate (DOP), 2-nitrophenyl phenyl ether (2-NPPE), ammonium reineckate (AmmRein), sodium tetraphenylborate (NaTPB), silicotungstic acid (STA), silicomolybdic acid (SMA), phosphomolybdic acid (PMA), and phosphotungstic acid (PTA), were selective products from Aldrich.

0.5 M chloride solutions of each of the following cations: NH₄⁺, K⁺, Na⁺, Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Mg²⁺, Ba²⁺, Mn²⁺, Cr³⁺, and Fe³⁺solutions (1000 μg ml⁻¹) were obtained from Merck. Glucose anhydrous, lactose monohydrate, maltose, urea, ascorbic acid, aspirin, l-threonine, l-lysine, l-cystine, and l-glycine were obtained from Aldrich. Serum was used within 24 h as provided by VACSERA (Giza, Egypt) while urine samples were obtained from healthy volunteers. Corn oil, sodium hydroxide and hydrochloric acid are from NODCAR.

2.2 The Electrochemical system

The potentiometric measurements were carried out with a Jenway 3515 digital pH/mV meter. A WTW-packed saturated calomel sensor (SCE) was used as an external reference sensor. The electrochemical system was as follows: CMCPS/test solution//SCE. The dissolution profile was studied using the USP XXXII (United States Pharmacopeia, 2009) method with the paddle apparatus II (Liddell et al., 2007). The apparatuses used for this purpose is model “SR8Plus”, CA USA Hanson Research, with number ”73–100-116”) and the spectrophotometer double beam instrument UV-1800–2011 Shimadzu (Japan).

2.3 Sensor preparation

Chemically modified carbon paste sensors were prepared as previously described (Khorshid and Issa, 2014). The sensor was used directly for potentiometric measurements without preconditioning requirements. A fresh surface of the paste was obtained by squeezing more out. The surplus paste was wiped out and the freshly exposed surface was polished on a paper until the surface showed shiny appearance.

2.4 Construction of the calibration graphs

Different compositions covering the ranges of 0.5–5% of CLO were prepared. The measured potential was recorded using the present sensor. Data were plotted as potential versus logarithm of the drug concentration CLO⁺ activity and the calibration graph was used for subsequent determination of unknown drug concentration.

2.5 Effect of pH

The effect of pH of the test solution on the sensor was investigated by measuring its potential in solutions prepared in the concentration range of 1.0 × 10⁻⁵–1.0 × 10⁻³ M by serial dilution. The sample solution pH was monitored simultaneously with a conventional glass pH sensor. The emf readings were taken after the potential reached a constant value. The mV-readings were plotted against the pH-values for the different concentrations.

2.6 Effect of temperature on the sensor potential

The thermal stability of the sensors with calibration graphs was studied covering the range of 25–50 °C of different test solution-temperatures. The values of E° were plotted versus (t-25) in the estimation of the thermal coefficients of the sensors. The isothermal coefficient (dE°/dt) of the cell represents the slope of this line obtained by plotting of E°_cell versus (t-25) and was calculated for each sensor by the following equation (Antropov’s equation): $$\text{E}°\text{cell}=\text{E}{°}_{25}°\text{C}+(\text{dE}°/\text{dt})(t-25)$$

By subtraction of the standard sensor potential of the calomel sensor (241.2, 234.4, 230.88 and 223.57 mV) at different temperatures (25, 35, 40 and 50 °C) the values of the standard potentials of sensors (E°_elec.) were calculated.

2.7 Sensor selectivity

The matched potential method (MPM) (Umezawa et al., 2000, 1995) was applied as the previously reported method (Khorshid and Issa, 2014).

The following equation is used to calculate the selectivity values of $\text{log}{\text{K}}_{\text{CLO,}{\text{J}}^{\text{z}+}}^{\text{pot}}$ : $${\text{K}}_{\text{CLO}.{\text{J}}^{\text{z}+}}^{\text{pot}}=\frac{({\text{a}}_{\text{A}}^{-}-{\text{a}}_{\text{drug}})}{{\text{a}}_{\text{j}}}$$ where: ${\text{a}}_{\text{A}}^{-}$ is the initial concentration of drug, a_drug is the activity of the added drug and a_j is the activity of the added interfering ion producing the same increase in potential.

The separate solutions method (SSM) (Guilbault et al., 1976) was applied, to confirm (MPM):

Two potential values were measured for the same concentration of the drug and the interferents. The selectivity values of $\text{log}{\text{K}}_{\text{CLO,}{\text{J}}^{\text{z}+}}^{\text{pot}}$ are calculated using the following equation: $$\text{log}{\text{K}}_{\text{CLO,}{\text{J}}^{\text{z}+}}^{\text{pot}}=\frac{{\text{E}}_2-{\text{E}}_1}{\text{S}}+\text{log}[\text{Drug}]-\text{log}{[{\text{J}}^{\text{z}+}]}^{1/2}$$ where: E₁ and E₂ are the sensor potentials of 10⁻³ M solution of each of the CLO drug and interfering cation, J^z+, respectively and S is the slope of the calibration graph.

2.8 Potentiometric determination of CLO

Small portions (0.1 ml) of standard 10⁻² M CLO solution were added to 50 ml water-containing different concentrations of drug ranging from 10⁻⁶ to 10⁻⁴ M or its pharmaceutical dosage form using the following equation: $$C_{\text{x}}=C_{\text{s}}\left(\frac{V_{\text{s}}}{V_{\text{x}}+V_{\text{s}}}\right){\left[{10}^{n(\mathrm{\Delta }\text{E}/\text{S})}-\frac{V_{\text{x}}}{V_{\text{s}}+V_{\text{x}}}\right]}^{-1}$$ where C_x is the concentration to be determined, V_x is the volume of the original sample solution, V_s and C_s are the volume and concentration of the standard solution added to the sample to be analyzed, respectively, ΔE is the change in potential after addition of certain volume of standard solution, and S is the slope of the calibration graph. Unknown drug concentration was determined from the graph produced by plotting the logarithm of CLO⁺ activity versus the potential.

In the potentiometric titrations different concentrations that ranged from 2.09 to 41.99 mg of drug were dissolved in 50 ml bi-distilled water. Different volumes of this solution (1.0–5.0 mL) were taken and subjected against 0.0025 M STA, 0.0025 SMA, 0.0033 PTA, 0.0033 PMA, 0.01 M (NaTPB and AmmRein) using the sensor(s). Conventional S-shaped curves with first and second plots were used to determine the end points.

2.9 Determination of CLO in plavix

For sampling of tablets, five plavix tablets (75 mg/tablet) were powdered together to fine powder. An accurately weighed portion was taken from this powder, was added to 50 ml bidistilled water and the solution was completed to the mark with bidistilled water then shaken. The standard addition technique was applied for the potentiometric determination.

2.10 Content uniformity assay of plavix tablets<905> (Liddell et al., 2007)

One tablet of Plavix (75 mg/tablet) was immersed in the measuring flask and adjusted to pH 1; for measuring each sensor it was immediately immersed in the sample solution three times and then washed between each individual measurement with distilled water to reach steady potential. The content uniformity was evaluated from the calibration graph by using the mean potential. For the spectrophotometric measurements by employing UV absorbance λ max 240 nm with the standard solution.

2.11 Determination of CLO in biological fluids

2.12 In spiked urine

For urine analysis, different quantities of the drug and 5 ml urine were transferred to a 100 ml volumetric flask and left stirred for 5 min, completed to the mark with doubly bidistilled water and a small volume of (0.1–2.0 ml) 0.01 M HCl was added to give solutions of pH ranging from 4 to 5 and concentrations from 1.0 × 10⁻⁶ to 5.0 × 10⁻⁴ M drug. These solutions were subjected to the standard addition method for drug determination.

2.13 Dissolution <711> (Liddell et al., 2007)

One tablet of Plavix (75 mg/tablet) was placed in the vessel of instrument apparatus II. In vitro release study the dissolution medium (900 ml of 0.01 M HCl) pH 1.2 was maintained at 37 ± 0.5 °C for 2 h. The clopidogrel was kept in a hard gelatin capsule so the vessel was rotated at 50 rpm. At appropriate time intervals, the potential values were recorded using the clopidogrel sensor in conjunction with saturated calomel sensor (SCE) reference sensor and the amount of clopidogrel released was calculated from the calibration graph. For the spectrophotometric measurements, 5.0 ml aliquots of the dissolution solution were withdrawn, filtered, diluted with 0.01 M HCl and the concentration of samples was analyzed using a UV spectrophotometer (1800, Shimadzu, Japan) and the absorbencies were measured at λ max 240 nm. A calibration graph was used for drug release calculation.

3.1 Composition and performance characteristics of CLO sensors

The paste with no exchangers displayed no measurable response toward clopidogrel (CLO⁺) ion. For this purpose, the ion-associates of CLO₄ST, CLO₄SM, CLO₃PT, CLO₃PM, CLO-TPB and CLO-Rein were prepared. The chemical composition of the precipitates was identified and confirmed by elemental analysis (C, H, and N) at the Microanalysis Center, Cairo University, Egypt. The results are shown in Table 1.

While the investigated as modifiers for carbon paste sensors selective to clopidogrel as shown in Table 2. The influence of the binder type and concentration on the characteristics of the studied sensors was investigated by using six binders with different polarities including 2-NPPE, TCP, DOP DBP, TBP and Corn Oil.

Different binder/graphite (w/w) ratios were studied. The sensor with 2-NPPE as a solvent mediator produced the best response, as shown in Fig. 2, likely due to better dielectric characteristics of 2-NPPE compared to other solvents, and the ability of 2-NPPE to extract clopidogrel ions from the aqueous solution to the organic paste phase.

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Figure 2

Effect of different plasticizers on the response of 5% CLO-ST.

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Among the different compositions studied, a paste containing ion-exchanger complex 5.0 wt% CLO₄ST, 54.0 wt% graphite, and 41.0 wt% 2-NPPE exhibited the best response characteristics and also the lowest detection limit. Therefore, this composition was used to study various operation parameters of the sensor and the optimum composition for the best sensor is given in Table 3. This sensor was chosen in this study and its electrochemical performance characteristics were systematically evaluated according to IUPAC recommendation (Guilbault et al., 1976; Linder and Umezawa, 2008).

3.2 Reproducibility of the sensor

The repeatability of the potential reading of the CLO-ST/CMCP sensor was examined by subsequent measurements in 1.0 × 10⁻³ M CLO-H₂SO₄ solution immediately after measuring the first set of solutions at 1.0 × 10⁻⁴ M CLO-H₂SO₄. The standard deviation for 5 replicate measurements of emf was found to be 0.473 in 1 × 10⁻⁴ M solution and 0.816 in 1 × 10⁻³ solution.

It was noticed that the slope of the calibration graph obtained by CLO-ST/CMCPS was nearly constant by polishing for any time taken days and then starts to decrease gradually without polishing so it consider as a new sensor with every one polishing to the sensor. After any period of time a new section from the master paste was found to function properly.

3.3 Dynamic response time

The dynamic response time (Skoog et al., 1997) of sensor was tested by measuring the time required to achieve a steady-state potential (within ± 1 mV) after successive immersions of the sensor in a series of drug solutions, each having a 10-fold increase in concentration from 1.0 × 10⁻⁷ to 1.0 × 10⁻² M. In this study, practical response time was recorded by increasing CLO concentration by up to 10-fold. When the sensor was transferred from one concentration solution to another one, it was stabilized to a value higher than its value (8 s), which may be due to its memory effect. The sensor yielded steady potential within 10–12 s. This is most probably due to the fast exchange kinetics of association–dissociation of clopidogrel ion with the ionophores at the solution–paste interface. The potential–time plot for the response of the sensor CLO-ST is shown in Fig. 3.

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Figure 3

Potential-time plot for response of the CLO-ST/CMCPS.

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3.4 Effect of pH

The potential pH profile obtained indicates that the responses of the sensors are fairly constant over the pH range of 1.2–4.8; in this range the sensor can be safely used for CLO determination. For quantitative measurements with ion selective sensors, studies were carried out to reach the optimum experimental conditions. Therefore, the pH range from 1.2 to 4.8 was assumed to be the working pH range of the sensors. It can be seen from Fig. 4 that at pH values lower than the previously mentioned pH ranges, the potential readings increase which can be related to interference of hydronium ion while at pH values higher than pH 4.8, the potential readings decrease gradually due to the formation of free base of the drug and decrease of the protonated species in the test solutions as shown in Fig. 4.

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Figure 4

Effect of pH on the response of CLO-ST/CMCPS.

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3.5 Effect of temperature

3.5.1

3.5.1 Thermal stability of the sensors

To study the thermal stability of the sensors, calibration graphs (emf, mV vs. pDrug) were constructed at different test solution temperatures covering the range of 25–50 °C. The results indicate that the slopes of the calibration graphs are still in the Nernstian range in spite of the increase of the temperature of the test solutions up to 50 °C as shown in Fig. 5a.

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Figure 5a

Calibration graphs for CLO-ST/CMCPS at the test solution temperatures 25 °C (a), 35 °C (b), 40 °C (c) and 50 °C (d).

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3.5.2

3.5.2 Determination of the thermal coefficient of the sensors

The potential of ion-selective sensors is usually affected by the temperature of the test solution. A thermally stable sensor is characterized by low thermal temperature coefficient. This means the successful applicability of the sensor over a wide range of temperatures. To calculate the thermal coefficient of the cell (dE°/dt)_cell, the standard cell potentials, E°_cell, were determined at different temperatures from the respective calibration plots as the intercept of these plots at p Drug = 0. Knowing that E°_cell is related to (dE°/dt) by (Antropov’s equation): $$\text{E}{°}_{\text{cell}}=\text{E}{°}_{25}°\text{C}+(\text{dE}°/\text{dt})(t-25)°\text{C}$$

Plot of E°_cell versus (t-25) °C produced a straight line; the slope of this line is taken as the thermal coefficient of the cell, as shown in Fig. 5b. The value of the standard potentials of sensor (E°elec.) was calculated after the subtraction of the standard sensor potential of the calomel sensor at different temperatures (the values are 241.2, 234.4, 230.88 and 223.57 mV at 25, 35, 40 and 50 °C, respectively). Plots of (E°_elec.) versus (t-25) °C gave a straight line. The slope of the line was taken as the thermal coefficient of the sensor.

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Figure 5b

Variation of standard electrode potential and standard potential of the cell containing CLO-ST/CMCPS with changes of test solution temperatures.

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The isothermal coefficient (dE_elec./dt) of the sensor was calculated and found to be ∼0.0001 V °C⁻¹ and (dE_cell/dt) equals ∼0.0005 V °C⁻¹. These values indicate a fairly high thermal stability of the sensor within the temperature range investigated and show no large deviation from the theoretical Nernstian behavior.

3.6 Selectivity of the sensor

The selectivity coefficients presented in Table 4 indicate that, CLO-ST sensor is highly selective to clopidogrel cation. Most inorganic cations do not interfere because of the difference in their mobility and permeability as compared to clopidogrel cation. In the case of sugars and amino acids the high selectivity is related to the difference in polarity and lipophilic nature of their molecules relative to clopidogrel cation.

3.7 Quantification of CLO

In order to assess the validity of the proposed sensor, the analytical applications involve determination of the drug in its bulk powder, pharmaceutical preparation (Plavix 75 mg) and biological fluids serum & urine was applied. Applying the standard addition method (Guilbault et al., 1976), the percentage recovery for determinations of CLO in pure solution, Plavix Tablets and in spiked urine & human serum ranged from 98.2% to 99.3%, 98.0–99.0, 98.3–99.0 and 97.6–98.3 respectively, (Table 5).

While in the Calibration curve method the percentage recovery for determinations of CLO in pure solution and Plavix Tablets ranged from 98.0 to 99.4 and 98.3 to 98.7, respectively. The potentiometric titration technique usually offers the advantage of high accuracy and precision, a further advantage is that the potential break at the titration end-point must be well defined. The titration process was carried out manually in aqueous solution containing 4.20–42.0 mg CLO with average recoveries of 98.2–99.6% using NaTPB as titrant, 97.3–98.5% in Plavix tablets.

On using PTA as titrant, 97.6–99.3%, 97.0–99.0%, on using SMA as titrant, 98.0–99.7%, 97.0–99.4, and on using PMA as titrant, 98.0–99.8%, 97.8–99.5% for pure solution and Plavix tablets respectively, Fig. 6. The results applying the potentiometric titration method Table 5. The results obtained were compared with those of the official method (United States Pharmacopeia, 2009). No significant difference between two methods was observed with respect to accuracy and precision (Table 6).

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Figure 6

Potentiometric titrations of 21.0 mg CLO-H₂SO₄ with PMA (a), PTA (b), SMA (c), and STA (d) As titrants using CLO-ST/CMCPS.

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3.8 Validation of the proposed method

3.9 Analytical applications

The standard addition method was proved to be successful for the determination of clopidogrel in its bulk solutions, Plavix tablet (75 mg/tablet) and biological fluids such as human serum/urine using its prepared chemically modified carbon paste sensor.

3.9.3

3.9.3 Potentiometric monitoring of Plavix tablet dissolution Liddell et al., 2007; United States Pharmacopeia, 2009

The dissolution test was operated at 50 rpm in 900 ml 1.0x10⁻² M hydrochloric acid (simulated duodenum fluid), and potentiometric clopidogrel sensor was used. The simulated duodenum fluid was kept at 37.0 ± 0.5 °C. There are no degradation products in the vitro test. The compression recipients do not interfere. Taking into account the S-shape of the dissolution curve obtained Fig. 6. It shows that clopidogrel releases immediately after capsule was ruptured. More than 75% drug was released within 15 min and complete dissolution was achieved in 120 min.

In the potentiometric method, the potential values were continuously recorded at 1-min time intervals and compared with a calibration graph. For the UV spectrophotometric assay, fixed volumes of the dissolution medium were withdrawn, diluted with 0.01 M HCl, measured at λ max 240 nm and compared with a calibration graph. Fig. 7 shows the dissolution profiles of clopidogrel tablet using both measurement techniques. The results obtained by spectrophotometry and potentiometry are almost identical. The use of the potentiometric method sensor, however, has the advantage of in situ monitoring.

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Figure 7

Dissolution profiles of 75 mg clopidogrel bisulfate tablet obtained by potentiometric: 5.0% CLO-CMCPS, and spectrophotometric measurements at 240 nm.

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