Tetracaine – selective electrodes with polymer membranes and their application in pharmaceutical formulation control

2.1 Materials and solutions

All chemicals were of analytical-reagent grade. Doubly deionized water was used to prepare all solutions. Tetracaine hydrochloride (TCH) was obtained from Aldrich, (Fig. 1). High molecular weight poly(vinyl chloride) (PVC), di-n-butyl phthalate (DBPH), tri-n-butyl phosphate (TBP), tetrahydrofuran (THF) were obtained from Fluka AG, Switzerland. Sodium tetraphenyl borate (NaTPB) was obtained from BDH, England. Phosphotungstic acid (PTA) and phosphomolybdic acid (PMA) were obtained from Merck, Germany. Injection (10 mg/mL TCH), Akorn Pharm., USA and Tetracaine HCl, eye drops (5 mg/mL TCH), Bausch & Lomb, UK were purchased from local pharmacies. The stock solution of 0.1 M TCH was prepared by dissolving the appropriate amount of TCH in 100 mL of deionized water. Standard TCH solution (1 × 10⁻²–1 × 10⁻⁶ M) was prepared by diluting the appropriate amount in deionized water. Stock solutions of 0.1 M for each of LiCl, NaCl, KCl, CaCl₂, Co(NO₃)₂, Ni(NO₃)₂, Cu(NO₃)₂, Al(NO₃)₃, CrCl₃, FeCl₃, glucose and fructose were prepared. Other diluted solutions were prepared by subsequent dilution of the stock solutions.

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Figure 1

Chemical structure of tetracaine hydrochloride (TCH).

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2.2 Apparatus

Electrochemical measurements were made using Orion digital ion analyser (Model-701A) at 25 °C in conjunction with a ceramic junction calomel electrode. A pH-meter (Orion Model 90-02) with a double junction glass electrode was used to check the pH of the solutions.

The electrochemical cell assembly used for this study was as follows: Ag/AgCl|Internal solution (0.01 M) of TCH|PVC membrane|Sample solution|Hg/Hg₂Cl₂, KCl (sat’d). A brief schematic diagram of the measuring cell is shown in Fig. 2.

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Figure 2

Schematic diagram of a conventional ion-selective membrane, measuring cell.

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2.3 Eye drops and injection samples

An appropriate amount of TCH was diluted to 100 mL with doubly deionized water in volumetric flask to obtain (1 × 10⁻³–5 × 10⁻⁵ M) of TCH.

2.4 Preparation of ion-pairs

Upon the addition of 25 mL of 1 × 10⁻² M of tetracaine hydrochloride (TCH) solutions to 25 mL of 1 × 10⁻² M sodium tetraphenyl borate (NaTPB) or 75 mL of 1 × 10⁻² M TCH to 25 mL each of phosphotungstic acid (PTA) or phosphomolybdic acid (PMA), a white or gray or green precipitate of TC-TPB or TC-PTA or TC-PMA was formed, respectively. The precipitate was filtered through Whatman filter paper No. 42, and washed with deionized water until no chloride ion was detected in the washing solution. The precipitate was dried at room temperature and ground to a fine powder in a mortar. Elemental analysis confirmed the formation of 1:1 or 3:1 or 3:1 complexes of TC-TPB or TC-PTA or TC-PMA, respectively as shown in Table 1.

2.5 Fabrication of TCH membrane electrodes

Tetracaine-selective membranes were prepared by dissolving 27.5 mg of the ion-pair complexes, 165 mg PVC and 357.5 mg of DBPH or TBP in 5 mL THF. These solutions were poured into glass Petri dishes (5 cm diameter), and were allowed to evaporate overnight at room temperature. The thickness of the obtained membrane was about 0.3 mm. Membranes (10 mm diameter) were cut out and glued to the polished end of PVC tubes by means of a PVC-THF solution. The electrode bodies consisted of a glass tube, to which the PVC tube was attached at one end and filled with an internal solution (1 × 10⁻² M of TCH), as shown in Fig. 2. The membrane was conditioned by immersing in 1 × 10⁻² M TCH solution for 3 h before measurements.

2.6 General procedure (calibration graphs)

The electrodes were calibrated by transferring 20 mL aliquots of 1 × 10⁻⁶–1 × 10⁻¹ M aqueous solutions of TCH to 50 mL beakers, followed by immersing the ion-selective membrane electrode, together with a calomel reference electrode in the solution. The potential readings were recorded after stabilization to ±0.3 mV, and the e.m.f. was plotted as a function of the logarithm of the TCH concentrations. The calibration graph was used for subsequent determinations of unknown TCH concentrations.

2.7 Selectivity of the electrodes

The selectivity coefficients, $K_{\text{TCH,}{j}^{z+}}^{\text{pot}}$ of the electrode toward different cationic species, j^Z+, were determined by the separate solution method described previously (Hassan et al., 2009) with a 1 × 10⁻³ M solution of TCH and the interferent.

3.1 Composition of the membrane

Five membrane compositions were investigated (Table 2). For each composition, the amount of polymer poly (vinyl chloride) (PVC) was kept constant (30%, w/w) while varying the percentage (w/w) of the ion-pair and plasticizer. The results reveal that the composition having the 5% ion-pair leads to exhibit a better slope (55.0 mV decade⁻¹), correlation coefficient (0.9994) and a wide concentration range (1 × 10⁻⁵–5 × 10⁻² M). In all subsequent studies electrodes made of the membrane composition No. (IV) (PVC, 30%: plasticizer, 65%: ion-pair, 5%) were used.

3.2 Sensor characteristics

The potentiometric response characteristics of the tetracaine sensors based on the use of TC-TPB or TC-PTA or TC-PMA as electroactive materials and DBPH or TBP as a plasticizer in PVC matrixes were evaluated by the IUPAC recommendations (Buck and Lindner, 1994a). Results in Table 3 show the characteristic performance of the PVC membrane sensors. As mentioned above three electroactive materials and two plasticizers were tested to evaluate their effects on the response. Better results (the better slopes) were obtained with DBPH plasticizer as shown in Table 3 and Fig. 3.

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Figure 3

The calibration curves and effect of ion-pairs and plasticizers on potentiometric response for the characteristic performance mentioned in Table 3 at 25 °C.

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3.3 Effect of plasticizer

The influence of plasticizer on the response characteristics of the tetracaine ion – selective electrode was investigated by using two plasticizers of different polarities including di-n-butyl phthalate (DBPH) and tri-n-butyl phosphate (TBP). The plasticizers dissolve the ion-pair complex and adjust both of the membrane permittivity and ion-exchanger site mobility to give the highest possible selectivity and sensitivity (Faridbod et al., 2007). The membranes incorporating DBPH showed better potential responses, i.e., better slopes and wider linearity of the calibration plots. Sensor Nos. 1–3 are preferred as it offered better life times compared to Sensor Nos. 4–6 based on the use of TPB as plasticizing solvent, Table 3. It seems that DBPH, as a lower polarity, a less viscosity (Klingender, 2008) and a relatively high mobility, with respect to TBP, provides appropriate conditions for incorporation of highly lipophilic tetracaine ion. Thus, all further studies were conducted using DBPH as plasticizer.

3.4 Effect of soaking

The performance characteristic of the tetracaine-selective electrode was investigated as a function of soaking time. The sensor No. 1 based on TC-PTA as ion-pair and DBPH as a plasticizer was soaked continuously in 1 × 10⁻² M solution of tetracaine hydrochloride (TCH) at room temperature for 1, 3, 5, 8, 24, and 48 h. The results indicate that, in the case of a conventional membrane electrode, the slope remains constant near 55 mV decade⁻¹ for up to 8 h soaking, then it slightly decreased, reaching 49.53 mV decade⁻¹ after 48 h soaking due to the leaching of the active ingredients to the bathing solution. The optimum conditioning time for the membrane sensor in 1 × 10⁻² M TCH solution was 3 h. The electrode should be kept dry in an opaque closed vessel and stored in a refrigerator while not in use.

3.5 Effect of the internal filling solution

The influence of the concentration of internal solution on the potential response of the polymeric membrane electrode for tetracaine ion was studied. The concentration was varied (from 1 × 10⁻⁴ to 1 × 10⁻¹ M) and the potential response of the electrodes has been observed as shown in Fig. 4. It was found that the best results in terms of slope (55.017 mV decade⁻¹) and working concentration range (from 1 × 10⁻⁵ to 5 × 10⁻² M) have been obtained with internal filling solution (IFS) (1 × 10⁻² M) as shown in Table 4. Thus, 1 × 10⁻² M concentration of IFS was quite appropriate for all electrodes.

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Figure 4

The effect of the internal filling (IFS) solution on the response of the tetracaine – selective electrode using sensor No. 1 based on (TC-PTA) ion-pair complex with DBPH as a plasticizer.

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3.6 The effect of pH

The influence of pH on the potential of the electrodes was investigated by measuring the e.m.f. of the cell at 1 × 10⁻⁴, 1 × 10⁻³ and 1 × 10⁻² M of TCH solutions. The pH values of the cell were adjusted by the addition of very small volumes of (0.01–0.1 M) hydrochloric acid or sodium hydroxide. The results are shown in Fig. 5; it is evident that the electrodes do not respond to pH changes in the range 2.5–6.5 for all the electrodes. At pH values higher than this range, the decrease in the potential readings is most probably attributed to the formation of the free TC base in solution, leading to a decrease in the concentration of TC cation (Hassan et al., 2011; Vaze and Srivastava, 2008). The decrease in potential readings at pH values lower than 2.5 may be due to penetration of H⁺ ion into the membrane surface or the formation of TC diprotonated species.

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Figure 5

Effect of pH on the response of proposed sensor No. 1 (pH range 2.5–6.5).

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3.7 Response time and lifetime

The time required for the proposed tetracaine-selective membrane electrodes to reach values within ±0.3 mV of the final equilibrium potentials was measured after immersion in different TCH solutions ranging from 1 × 10⁻⁵ to 1 × 10⁻² M in concentration, the measurements were from low to high concentrations. The average response time at t₉₅ varied from 10 s for TCH solutions with concentrations ⩾10⁻³ M to 22 s for solutions with concentrations ⩽10⁻⁴ M.

The average lifetime for most of reported ion-selective electrodes is in the range of 3–8 weeks. After this time the slope of the electrode will decrease, and the detection limit will increase. The tetracaine-selective electrodes were tested for 7 weeks and the calibration curve was plotted periodically with a standard solution (1 × 10⁻⁶–1 × 10⁻¹ M) of TCH. The experimental results showed that the lifetime of the sensor Nos. 1 and 2 was over 5 weeks, Table 3. During this time, the detection limit and the slope of the electrode remained almost constant, Fig. 6. While sensor No. 3 showed operative lifetime of about 3 weeks with a slight gradual decrease in slope from 54.59 to 49.56 mV decade⁻¹ and an increase in the detection limit from 5 × 10⁻⁵ to 8 × 10⁻⁵ M. It is well established that the loss of plasticizer, ionic sites from the polymeric membrane due to leaching into the sample is a primary reason for the limited lifetimes of the sensor No. 3 (El-Saharty et al., 2006).

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Figure 6

Calibration curve of sensor No. 1 as a function of time.

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3.8 Selectivity of the electrodes

The influence of some inorganic cations, such as Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Zn²⁺, Ni²⁺, Co²⁺, Cu²⁺, Al³⁺, Cr³⁺, Fe³⁺ and sugars (glucose, fructose) on the electrode response was investigated. The selectivity coefficients ( $K_{\text{TCH,}{j}^{z+}}^{\text{pot}}$ ) were used to evaluate the degree of interference. Selectivity coefficients were determined by the separate solution method (Lindner and Umezawa, 2008; Umezawa et al., 2000; Buck and Lindner, 1994b) in which the following equation was applied:

The results in Table 5 showed that tetracaine-PVC membrane electrodes are selective for tetracaine ion over many inorganic cations and sugars. It is evident from the data in Table 5 that the sensor Nos. 1 and 2 based on TC-PTA and TC-PMA complexes, respectively, have a relatively high selectivity toward tetracaine relative to interference cations and sugars, this attributed probably to the large difference in the mobilities and permeabilities of the ions in TC-PTA and TC-PMA electrodes as compared to TC-TPB electrode (sensor No. 3). The mechanism of selectivity is mainly based on the stereospecificity and electrostatic environment, and is dependent on how much similarity is present between the locations of the lipophilic sites in the two competing species in the bathing solution side and those present in the receptor of the ion-exchanger (Bakker et al., 2000). The inorganic cations do not interfere owing to the differences in ionic size, and consequently their mobilities and permeability, as compared with those of tetracaine cation. Also, the smaller the energy of hydration of the cation, the greater the response of the membrane. In the case of sugars, the high selectivity is due to the difference in polarity and lipophilic character (Kormosh et al., 2009; Ensafi and Allafchian, 2008).

3.9 Analytical applications

The prepared electrodes have been successfully used for the determination of tetracaine in aqueous solutions and in pharmaceutical preparations (eye drops and injection) by using the direct and standard addition methods and the results are summarized in Table 6. The direct method is the simplest of obtaining quantitative results using ion-selective membrane electrode (Gründler, 2007; Wang, 2006). The calibration graph was constructed and the concentration of the unknown was calculated by linear equation of the calibration curve. This method has been applied for the determination of TCH in eye drops and injection, the results are summarized in Table 6 including the repeatability data (N = 4). The content of TCH in its formulation using the proposed electrodes is with a good agreement with declared amount while the standard addition method was applied by adding a small portion (0.1 mL) of 1.0 × 10⁻² M standard TCH solution to 20 ml aliquot samples of various pure and formulation TCH concentrations (1.0 × 10⁻³–5.0 × 10⁻⁵ M). The change in potential readings (at a constant temperature of 25 °C) was recorded after each addition and used to calculate the concentration of TCH sample solutions (Buck and Lindner, 1994a) by the following equation:

In order to compare two methods for the determination of tetracaine hydrochloride (TCH) in some pharmaceutical preparations, the results obtained from ion-selective electrode using sensor No. 1 (TC-PTA-DBPH) were compared with those obtained by the standard British Pharmacopoeia (BP) method (British Pharmacopoeia on CD-ROM, 2001). The results obtained by the two different methods, (Table 7) were statistically compared, using student’s t-test and variance ratio F-test at 95% confidence level (Miller and Miller, 2005; Farrant, 1997). In all cases, the calculated t- and F-values, Table 7 did not exceed the theoretical values, which indicate that there is no significant difference between either methods in accuracy and precision in the determination of tetracaine hydrochloride (TCH) in pharmaceutical formulations.