Flow injection potentiometric determination of pipazethate hydrochloride

Abstract

New plastic membrane electrodes for pipazethate hydrochloride based on pipazethatium phosphotungstate, pipazethatium phosphomolybdate and a mixture of the two were prepared. The electrodes were fully characterized in terms of composition, life span, pH and temperature and were then applied to the potentiometric determination of the pipazethate ion in its pure state and pharmaceutical preparations under batch and flow injection conditions. The selectivity of the electrodes towards many inorganic cations, sugars and amino acids was also tested.

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Introduction

Pipazethate hydrochloride (PiCl), 10H-pyrido[3,2-b][1,4]benzothiadiazine-10-carboxylic acid 2-(2-piperidinoethoxy)ethyl ester,¹ is a bronchodilator that suppresses irritative and spasmodic cough by inhibiting the excitability of the cough center and the peripheral neural receptors in the respiratory passage. The response to the drug takes about 10–20 min and lasts for 4–6 h.

Pipazethate has been determined using a limited number of techniques including spectrophotometry,^2,3 TLC⁴ and HPLC.⁵ Although there is an increased demand for chemical surveillance in the environmental, medical and many other industries which has created the need for highly sensitive, selective, precise and inexpensive techniques, no ion-selective electrode (ISE) has been constructed for the determination of pipazethate. In this study, plastic membrane electrodes for Pi cation were constructed based on the incorporation of pipazethatium phosphotungstate (Pi-PTA), pipazethatium phosphomolybdate (Pi-PMA) or a mixture of the two (Pi-PTA/PMA) ion exchanger(s) in poly(vinyl chloride) (PVC) membranes plasticized with dioctylphthalate (DOP). The electrodes were fully characterized under batch conditions and then used to determine the drug both in batch and with a flow injection technique, which is considered a very efficient way of improving the performance characteristics of ISEs for various reasons, including the following: (i) the permanent liquid stream has a conditioning effect on the sensor membrane, leading to better sensitivity and stability and increased reproducibility of the e.m.f. readings; (ii) the liquid junction and streaming potentials are stable; (iii) the way in which the sample is presented to the ISE is more closely defined and reproducible under flow-through conditions than in static (batch) measurements; (iv) the streaming of solution is beneficial in that it reduces the diffusion layer thickness and hence shortens the response time; and (v) the sample is not influenced by the electrode itself as any release from the membrane (e.g., dissolution) is transported downstream.⁶ The selectivity of the electrodes was also tested in batch and FIA procedures and compared with each other.

Experimental

Reagents

All reagents used for the preparation of solutions were of analytical-reagent grade. Doubly distilled water was used for preparing solutions and as a flow stream in FIA measurements. The carrier and reagent solutions were de-gassed by means of vacuum suction. Sample solutions used for injections were freshly prepared prior to measurements. Pure grade pipazethate hydrochloride and its pharmaceutical preparations (Selgon, tablets 20 mg per tablet and drops 40 mg ml⁻¹) were provided by the Egyptian International Pharmaceutical Industries Company (EIPICO), (10th of Ramadan City, Egypt).

Apparatus

Potentiometric measurements in the batch mode were carried out with a Schott-Gerate (Hofheim, Germany) CG 820 pH-meter and pMX microprocessor pH/ion meter (WTW, Weilheim, Germany). A Techne (Cambridge, UK) Model C-100 circulator thermostat was used to control the temperature of the test solutions. A WTW packed saturated calomel electrode (SCE) was used as an external reference electrode. The electrochemical system may be represented as follows:

Ag|AgCl|filling solution|membrane|testsolution∥KCl salt bridge∥SCE  (1)

The flow injection set-up was composed of a four channel peristaltic pump (ISM 827) (Ismatec, Zurich, Switzerland) and a Model 5020 injection valve with exchangeable sample loop from Rheodyne (Cotati, CA, USA). The electrodes were connected to a WTW pMX 2000 microprocessor pH/ion meter and interfaced to a Model BD111 strip chart recorder from Kipp and Zonn (Deflt, The Netherlands).

A wall-jet cell, providing a low dead volume, fast response, good wash characteristics, ease of construction and compatibility with electrodes of various shapes and sizes, was used in flow measurements, where a Perspex cup with axially positioned inlet polypropylene tubing was mounted at the sensing surface of the electrode body. The optimized distance between the nozzle and the sensing surface of the electrode was 5 mm; this provides the minimum thickness of the diffusion layer and consequently a fast response.⁷ The ISE with flow cup, reference electrode (SCE) and the outlet tube were placed in a beaker, where the level of solution was kept 1 cm above the electrode surface.

Preparation of the ion exchangers

The ion exchangers, pipazethatium phosphotungstate (Pi₃-PTA) (buff powder) and pipazethatium phosphomolybdate (Pi₃-PMA) (yellow powder), were prepared by the addition of 150 ml of 10⁻² mol l⁻¹ PiCl solution to 50 ml of 10⁻² mol l⁻¹ of each of phosphotungstic acid or phosphomolybdic acid, respectively. The precipitates were filtered, washed thoroughly with distilled water until chloride free and air-dried. The chemical composition of the precipitates was identified and confirmed by elemental analysis (C, H, N, S, P and metal); the results are given in Table 1.

Table 1 Elemental analysis (%) of the pipazethate ion associates

Pi-PTA		Pi-PMA
Element	Found	Calculated	Found	Calculated
C	18.49	18.53	26.67	26.70
H	1.98	1.91	2.79	2.75
N	3.11	3.09	4.46	4.45
S	2.37	2.35	3.41	3.39
P	0.72	0.76	1.12	1.09
Metal	54.21	54.13	40.71	40.69

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Preparation of the electrodes

The electrodes were constructed as described previously.⁸ The membrane composition was studied by varying the percentages (w/w) of the ion exchanger(s), PVC and DOP until optimum composition that exhibits the best performance characteristics is reached. The membranes were prepared by dissolving the required amounts of PVC, ion-exchanger(s) and the plasticizer (DOP) in a 5.0 cm diameter Petri dish containing 10.0 ml of tetrahydrofuran (THF) (the total weight of ingredients was 0.35 g), the components being well mixed to ensure homogeneity of the membranes. The Petri dish was then covered with a filter-paper and left to dry in air. To obtain uniform membrane thickness, the amount of THF was kept constant and its evaporation was fixed for 24 h.

An 8 mm diameter disk was cut out from the prepared membrane and glued using PVC–THF paste to the polished end of a plastic cap attached to a glass tube. The electrode body was filled with a solution that was 10⁻² mol l⁻¹ with respect to both NaCl and PiCl and preconditioned by soaking in 10⁻³ mol l⁻¹ PiCl solution.

Results and discussion

Optimization of the ISE response in batch conditions

(i) Composition of the membranes.. Several membrane compositions were investigated in which the content of ion exchanger ranged from 3.0 to 10.0% of Pi-PTA and Pi-PMA and from 2.0 to 10.0% of each ion exchanger for Pi-PTA/PMA mixed membrane electrodes. For each composition, the electrodes were repeatedly prepared four times. The preparation process was highly reproducible, as revealed from the low relative standard deviation (RSD) values of the slopes obtained employing the prepared membranes (the mean RSD was about 0.25%).

The best performances were obtained by using compositions containing 5.0% Pi-PTA, 47.5% PVC and 47.5% DOP, 7.0% Pi-PMA, 46.5% PVC and 46.5% DOP or 3.0% Pi-PTA, 3.0% Pi-PMA, 47.0% PVC and 47.0% DOP, resulting in slopes of 59.0, 57.5 and 58.7 mV per concentration decade after minimum pre-soak times of 0.5, 1 and 0.5 h for Pi-PTA, Pi-PMA and Pi-PTA/PMA, respectively. The usable concentration range for the three electrodes was 6.3 × 10⁻⁵–0.1 mol l⁻¹. The above optimum compositions were used to prepare membrane electrodes for all further investigations.

(ii) Effect of soaking.. The continuous soaking of the electrodes in 10⁻³ mol l⁻¹ PiCl solution affects negatively their response to the pipazethatium cation, which is attributed to leaching of the active ingredients [ion-exchanger(s) and solvent mediator] to the bathing solution.⁹ It was observed that the slopes of the calibration graphs obtained using the preconditioned electrodes were nearly constant for 2 weeks in the case of Pi-PTA and Pi-PTA/PMA electrodes and for 7 d of continuous soaking in the case of the Pi-PMA electrode and then started to decrease gradually to about 50.0 mV per concentration decade after 35, 21 and 56 d and reaching about 45.0 mV per decade after 37, 28 and 63 d for Pi-PTA, Pi-PMA and Pi-PTA/PMA, respectively.

It is noteworthy that the life span of the membrane containing mixed ion exchangers is much longer than those containing an individual ion exchanger. This is most probably due to some sort of physical interaction of the two ion exchangers within the PVC network. It may also be correlated with the diffusion and partition coefficients of both the ion exchangers and the plasticizer.^10,11 Naturally, it was noted that in all cases, electrodes which had been kept dry in a closed vessel and stored in a refrigerator showed nearly constant slope values and the same response properties extending to several months. Hence it is recommended that unused electrodes be kept dry in closed vessels in a refrigerator in order to extend their life spans substantially.

(iii) Regeneration of the electrodes.. The above discussion reveals that soaking of an electrodes in the drug solution for a long time has a negative effect on the response of the membrane towards its ion. The same effect appears after working with the electrode for a long time.

The regeneration of the electrodes was tried simply by reforming the ion exchanger(s) on the external gel layer of the membrane,¹² and this was successfully achieved by soaking the exhausted electrode for 24 h in a solution that was 10⁻² mol l⁻¹ in PTA, PMA or in both PTA and PMA followed by soaking for 1, 2 and 2 h in 10⁻² mol l⁻¹ PiCl solution for Pi-PTA, Pi-PMA and Pi-PTA/PMA, respectively. The slopes of the exhausted electrodes increased from 46.5, 46.0 and 45.0 to 55.0, 53.3 and 55.0 for Pi-PTA, Pi-PMA and Pi-PTA/PMA electrodes, respectively, as a result of the regeneration process.

It was found that the life span of the regenerated electrode was limited to 2 h of continuous soaking owing to the ease of leaching of the lipophilic salts from the gel layer at the electrode surface compared with those which are attached homogeneously to the PVC network through the solvent mediator.

(iv) Effect of temperature of the test solution.. Calibration graphs [electrode potential (E_elec) versus pPi] were constructed at different test solution temperatures (25, 30, 40, 50, 60 and 70 °C) for all electrodes. The slopes and the standard electrode potentials (E°) of the electrodes at each temperature are given in Table 2.

Table 2 Performance characteristics of Pi electrodes at different temperatures

Electrode	Temperature/°C	Slope/mV per decade	E°/mV^a
a E° is the standard electrode potential versus the normal hydrogen electrode (NHE).
Pi-PTA	25	59.0	53
30	59.5	58
40	60.8	64
50	62.5	85
60	64.8	100
70	67.5	114
Pi-PMA	25	57.5	37
30	59.0	43
40	61.5	55
50	62.8	60
60	64.8	70
70	67.0	84
Pi-PTA/PMA	25	58.7	88
30	60.5	92
40	62.5	96
50	64.5	105
60	66.3	124
70	68.3	138

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For the determination of the isothermal coefficients (dE°/dt) of the electrodes, the standard electrode potentials (E°) versus the normal hydrogen electrode (NHE) at the different temperatures were obtained from the calibration graphs as the intercepts at pPi = 0 (after subtracting the values of the standard electrode potential of the SCE at these temperatures) and plotted versust − 25, where t is the temperature of the test solution in °C. A straight line plot is obtained according to the following equation:¹³

E° = E°₍₂₅₎ + (dE°/dt) (t − 25) (2)

The slopes of the straight lines obtained represent the isothermal coefficients of the electrodes (0.001355, 0.001044 and 0.001244 V °C⁻¹) for Pi-PTA, Pi-PMA and Pi-PTA/PMA, respectively. These slopes were compared with the theoretical values at the given temperature and found to be in good agreement, revealing fairly high thermal stability of the electrodes within the temperature ranges investigated. The investigated electrodes were found to be usable at temperatures up to 70 °C without any deviation from the theoretical Nernstian behaviour.

Optimization of FIA response

The flow injection measurements were carried out in a two-line configuration. The sample was injected into a distilled water stream, which was then merged with another stream of distilled water. In both lines the same tubing size was used, offering the same flow rate. The connector of the two streams was connected to the detector by a 50 cm tube of 0.4 mm id. Fig. 1 shows the configuration of the system used in measurements. The dispersion coefficient was found to be 1.7, i.e., limited dispersion that aids optimum sensitivity and fast response of the electrodes.¹⁴

Fig. 1 Schematic diagram of the flow injection system used in measurements.

(i) Injection volume.. Samples of different volumes (20–500 μl) were injected. In general, the higher the sample volume, the higher are the peak heights and residence time of the sample at the electrode surface, requiring a longer time to reach a steady state and greater consumption of sample.¹⁵ A sample loop of size 150 μl was used throughout this work, giving about 99% of the maximum peak height obtained by a 500 μl loop but with a shorter time to reach the baseline and less consumption of reagents.

(ii) Effect of flow rate.. The dependence of flow rate on the peak heights and time to recover the baseline was studied, the response of the three electrodes to a solution that is 10⁻² mol l⁻¹ being studied at different flow rates (4.15, 5.35, 7.50, 9.70, 12.50, 17.85, 23.25, 25.00, 27.00 and 30.00 ml min⁻¹). With a constant injection volume, the residence time of the sample was inversely proportional to the flow rate¹⁶ and the recovery time increased linearly with residence time of the sample at the active membrane surface. It was found that, as the flow rate increased, the peaks became higher and narrower until a flow rate of 25.00 ml min⁻¹ was reached, after which the peaks obtained at higher flow rates were nearly the same. A flow rate of 9.7 ml min⁻¹ was adopted, providing about 99% of the maximum peak height obtained by higher flow rates, a short time to reach the baseline and less consumption of the carrier. Fig. 2 shows typical recordings obtained from the Pi-PTA/PMA electrode under optimum FIA conditions.

Fig. 2 Recordings (a) and calibration graph (b) for Pi-PTA/PMA electrode under optimum FIA conditions.

Electrode response in FIA

In potentiometric detection, the electrode potential depends on the activity of the main ion sensed. This is considered to be a principle advantage of this detection method; also in flow measurements the dependence is semi-logarithmic over a wide analyte activity range according to the Nickolsky–Eisenman equation. However, the main unfavorable feature of this detection method is the slow response of electrode potential to concentration change and this is pronounced when low concentrations are measured and depends on the state of the membrane surface at the interface with the measured solution.^17,18 This slow response is a fairly good reason for the super-Nernstian sensitivities obtained in FIA measurements using the investigated electrodes at different flow rates. An increase in the slope of the calibration plots in FIA was observed compared with batch measurements, where potential is measured under conditions very close to the equilibrium at the membrane–solution interface.¹⁹

The slopes of the calibration graphs ranged from 64.5 to 72.5, from 64.0 to 71.5 and from 65.5 to 73.7 compared with 59.0, 57.5 and 58.7 mV per concentration decade for Pi-PTA, Pi-PMA and Pi-PTA/PMA, respectively. The variation of the calibration graph slope with flow rate is shown in Fig. 3.

Fig. 3 Variation of calibration graph slope for Pi-PTA (a), Pi-PMA (b) and Pi-PTA/PMA (c) electrodes with flow rate.

The usable concentration range of the electrodes in FIA is 6.3 × 10⁻⁴–1.0 × 10⁻² mol l⁻¹, which is a slightly lower than that in the batch mode, mainly owing to the difference in the dispersion coefficients in the two techniques.

Effect of pH

The effect of the pH of the test solution on the electrode potentials was studied in batch and FIA measurements. In batch measurements, the variation in potential with pH change was followed by the addition of small volumes of HCl and NaOH (0.1–1.0 mol l⁻¹), whereas in FIA, a series of solutions of 10⁻² mol l⁻¹ PiCl concentration and with pH ranging from 1.0 to 12.0 were injected into the flow stream and the peak heights, representing variation of potential response with pH, were measured.

It is evident that the electrodes do not respond to pH changes in the range 2.0–9.0 in both batch and FIA conditions. Nevertheless, at pH values lower than these values, the potential decreases gradually, which can be related to interference of hydronium ion, while the decrease that takes place at pH >9.0 can most probably be attributed to the formation of the free pipazethate base in the solution or the ionization of the hydroxyl group in such high alkaline pH media, leading to a decrease in the concentration of pipazethatium cation.

Selectivity of the electrodes

It was shown earlier for solid state membrane electrodes that the apparent selectivity coefficient measured under transient flow injection conditions may differ significantly from that measured under batch conditions.^20–23 This is interpreted by the difference in the time of interaction of interferents with the membrane surface. This difference increases with increase in the interaction of the interferent with the membrane in comparison with the main sensed ion, and also the interference process is highly dependent on the rate of diffusion and the exchange reaction of the interfering ion.²⁴ Therefore, in FIA measurements, where the sample remains in contact with the electrode for a short period of time , the apparent selectivity should be different from that found in batch conditions.

The influence of some inorganic cations, sugars and amino acids on the Pi electrodes was investigated. Under FIA conditions, the values of selectivity coefficients were calculated based on potential values measured at the top of peaks for the same concentrations of the drug and the interferent, whereas under batch conditions the separate solution method was applied and a high concentration of the interferent ion was used (10⁻² mol l⁻¹) to ensure that there is no interference if lower concentrations than this are present. Although there are many precautions that must be taken into consideration when using this method in the determination of the selectivity coefficients, especially in the case of solutions of differently charged ions, it is still the simplest way to evaluate the degree of interference that might be taking place and is used to perform measurements in important biological samples such as blood²⁵ as it is simple and easy to perform. In this work, the matched potential and the mixed solution methods, which are time consuming owing to the need to prepare many solutions and to perform many steps, were used only for determining the degree of tolerance towards sugars and amino acids and as a confirmation when −log was <3.5.

The selectivity coefficients of the electrodes (Table 3), reflect a very high selectivity of the investigated electrodes for the pipazethatium cation. The mechanism of selectivity is mainly based on the stereospecificity and electrostatic environment and it is dependent on how much fitting is present between the locations of the lipophilicity sites in the two competing species in the bathing solution side and those present in the receptor of the ion exchanger.²⁶ The inorganic cations do not interfere because of differences in ionic size, mobility and permeability. Also, the smaller the energy of hydration of the cation, the greater is the response of the membrane. The electrodes exhibit good tolerance towards sugars, amino acids and urea.

Table 3 Selectivity coefficients and tolerance values for the Pi-responsive electrodes

−log
Pi-PTA			Pi-PMA			Pi-PTA/PMA
Batch		Batch		Batch
Interferent	SSM^a	MSM^b	FIA	SSM^a	MSM^b	FIA	SSM^a	MSM^b	FIA
a SSM: separate solution method.  b MSM: mixed (matched) solution method.
Na^+	2.87	3.01	2.92	3.12	3.24	3.18	3.25	3.28	3.37
K^+	3.10	3.37	0.98	3.26	3.42	1.10	3.42	3.51	1.21
Li^+	3.19	3.34	3.23	3.31	3.45	3.39	3.38	3.48	3.41
NH•	3.25	3.37	3.34	3.33	3.54	3.43	3.42	3.57	3.46
Mg^2+	3.78	—	3.89	3.86	—	4.08	3.74	—	3.83
Ca^2+	3.91	—	4.17	3.76	—	3.98	3.89	—	4.16
Sr^2+	4.23	—	4.35	4.37	—	4.52	4.34	—	4.49
Ba^2+	4.56	—	4.64	4.47	—	4.58	4.65	—-	4.76
Mn^2+	4.63	—	4.79	4.74	—	4.94	4.85	—	4.98
Co^2+	4.55	—	4.71	4.58	—	4.65	4.87	—	4.97
Ni^2+	4.46	—	4.54	4.59	—	4.67	4.64	—	4.73
Cu^2+	4.87	—	4.95	4.76	—	4.87	4.88	—	5.00
Zn^2+	5.12	—	5.30	5.18	—	5.27	5.29	—	5.36
Pb^2+	5.43	—	5.52	5.37	—	5.45	5.49	—	5.59
Cd^2+	5.23	—	5.35	5.29	—	5.34	5.33	—	5.41
Cr^3+	5.48	—	5.58	5.52	—	5.67	5.61	—	5.70
Fe^3+	5.55	—	5.69	5.37	—	5.50	5.49	—	5.56
Al^3+	5.64	—	5.75	5.58	—	5.68	5.67	—	5.75
Glycine	—	4.52	4.57	—	4.43	4.58	—	4.52	4.59
L-Threonine	—	4.38	4.46	—	4.27	4.51	—	4.38	4.52
Alanine	—	4.76	4.98	—	4.65	4.82	—	4.73	4.86
Arginine	—	5.24	5.39	—	5.01	5.28	—	5.18	5.31
Glucose	—	5.57	5.71	—	5.25	5.41	—	5.34	5.50
Maltose	—	5.62	5.80	—	5.32	5.52	—	5.29	5.47
Lactose	—	4.97	5.21	—	5.10	5.34	—	5.14	5.35
Urea	—	4.13	4.42	—	4.20	4.37	—	4.31	4.53

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Comparing the selectivity coefficients obtained for the investigated electrodes under both batch and FIA conditions (Table 3), it is clear that in most cases, the electrodes were more selective in FIA than in batch measurement with the exception of K⁺, where the selectivity was significantly lower. This may be due to its smaller atomic size, so that it is more easily adsorbed on the electrode surface and it is more difficult to remove it by the flowing stream. In the case of other monovalent ions, the selectivity coefficients obtained were smaller to some extent (less than 0.5 decade) than the values obtained under batch conditions.

Analytical applications

PiCl was determined potentiometrically using the investigated electrodes under batch conditions by both the interpolation and standard additions methods.²⁷ In the latter, small portions (0.1 ml) of standard 0.1 mol l⁻¹ PiCl solution were added to 50 ml of water containing 1, 2, 10, 20, 110 or 220 mg of pure compound or their equivalents from pharmaceutical preparations. The change in millivolt readings was recorded after each addition and used to calculate the concentration of the PiCl sample solution.

For sampling of tablets (Selgon, 20 mg per tablet), 20 tablets were ground together and appropriate weights of each were taken as samples. The required amount (1.0–200.0 mg) was dissolved in 0.01 mol l⁻¹ HCl (about 0.1 ml mg⁻¹ of pipazethate tablets) and diluted to 50 ml with distilled water. For drops (Selgon, 40 mg ml⁻¹), the contents of three bottles were mixed and volumes equivalent to the required weights were taken and then used for the determination of the drug content by the standard additions method.

The results of the standard additions method given in Table 4 are in good agreement with those obtained from the official method¹ (the latter is based on measuring the absorbance of the drug solution in 0.1 mol l⁻¹ HCl at 252 nm). The mean recovery of the amounts taken (1.0–220.0 mg) ranged from 97.0 to 101.1% with RSD = 0.17–0.42%.

Table 4 Determination of pipazethate hydrochloride employing the different Pi electrodes applying the standard additions method under batch conditions

Pi-PTA		Pi-PMA		Pi-PTA/PMA
Sample	Taken^a/mg	Recovery (%)	RSD^b (%)	Recovery (%)	RSD^b (%)	Recovery (%)	RSD^b (%)
a Taken mg per 50 ml.  b Five determinations.
Pure solutions	1.0	98.3	0.26	97.9	0.16	98.4	0.29
2.00	98.7	0.22	98.3	0.34	98.9	0.24
10.0	99.5	0.19	99.0	0.27	99.8	0.31
20.0	100.5	0.17	99.8	0.35	100.1	0.20
110.0	100.3	0.32	99.8	0.27	100.5	0.26
220.0	100.8	0.21	100.4	0.24	100.3	0.33
Tablets (20 mg per tablet)	1.0	97.0	0.31	97.4	0.30	97.8	0.25
2.00	97.9	0.24	98.6	0.32	98.3	0.34
10.0	98.5	0.19	99.5	0.35	98.9	0.28
20.0	99.6	0.24	100.3	0.23	99.7	0.26
110.0	100.2	0.27	100.5	0.26	99.9	0.37
220.0	100.5	0.18	100.6	0.20	100.5	0.24
Drops (40 mg ml^−1)	1.0	98.5	0.18	98.9	0.34	99.0	0.25
2.00	99.2	0.25	99.5	0.25	99.8	0.34
10.0	100.5	0.31	100.4	0.31	100.2	0.25
20.0	100.8	0.42	100.3	0.40	100.6	0.31
110.0	101.0	0.35	100.5	0.36	100.5	0.20
220.0	100.6	0.38	101.1	0.28	100.9	0.37

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Under FIA conditions, a series of solutions of different concentrations were prepared from the tablets and drops and the peak heights were measured at three flow rates (4.15, 9.70 and 23.25 ml min⁻¹), then compared with those obtained from injecting a standard solution of the same concentration prepared from pure pipazethate hydrochloride. From the results obtained (Table 5), it clear that the flow rates did not affect the recovery values except for a rate of 4.15 ml min⁻¹, where the electrodes needed a longer time to attain the baseline, and the recoveries were lower than those obtained under batch conditions. The mean recoveries for the amounts taken (1.0–220 mg) ranged from 95.3 to 99.0, from 97.9 to 101.3 and from 97.5 to 101.6% with RSDs of 0.21–0.52, 0.19–0.50 and 0.19–0.56% for flow rates of 4.15, 98.7 and 23.25 ml min⁻¹, respectively.

Table 5 Determination of pipazethate hydrochloride in tablets and drops using Pi electrodes under FIA conditions

Pi-PTA		Pi-PMA		Pi-PTA/PMA
Flow rate/ ml min^−1	Taken^a/mg	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)
a (a) Tablets, (b) drops.
4.15	1.0 (a)	95.5	0.24	95.3	0.21	95.6	0.26
(b)	95.8	0.35	95.4	0.23	95.4	0.29
2.0 (a)	96.0	0.27	95.8	0.25	96.1	0.24
(b)	95.9	0.28	96.2	0.27	96.5	0.28
10.0 (a)	96.4	0.34	96.2	0.34	96.7	0.31
(b)	96.3	0.41	96.0	0.35	96.5	0.36
20.0 (a)	96.2	0.42	96.7	0.41	97.0	0.37
(b)	96.5	0.36	96.4	0.37	96.8	0.38
110.0 (a)	97.8	0.48	97.3	0.52	97.5	0.39
(b)	98.2	0.52	97.8	0.49	98.1	0.43
220.0 (a)	98.6	0.21	98.6	0.28	98.3	0.33
(b)	99.0	0.28	98.5	0.34	98.7	0.39
9.7	1.0 (a)	98.9	0.20	97.9	0.21	98.7	0.41
(b)	98.7	0.25	98.4	0.27	98.5	0.47
2.0 (a)	99.6	0.24	98.6	0.24	98.9	0.37
(b)	99.8	0.27	99.0	0.26	99.2	0.34
10.0 (a)	100.1	0.19	99.2	0.31	99.6	0.35
(b)	99.8	0.23	99.7	0.37	99.7	0.42
20.0 (a)	100.2	0.31	100.0	0.41	100.1	0.43
(b)	100.5	0.35	100.4	0.43	99.8	0.46
110.0 (a)	100.1	0.23	100.2	0.33	100.1	0.28
(b)	100.3	0.25	100.8	0.35	100.5	0.27
220.0 (a)	100.6	0.30	100.7	0.27	100.9	0.50
(b)	100.9	0.33	101.2	0.34	101.3	0.48
23.25	1.0 (a)	98.2	0.28	97.5	0.41	97.9	0.34
(b)	98.8	0.32	97.9	0.39	98.5	0.37
2.0 (a)	99.1	0.41	98.2	0.26	98.6	0.28
(b)	99.3	0.46	98.6	0.24	98.3	0.27
10.0 (a)	99.8	0.52	99.4	0.35	99.6	0.19
(b)	99.6	0.54	99.7	0.37	99.9	0.23
20.0 (a)	100.0	0.41	100.5	0.46	100.4	0.43
(b)	100.2	0.46	100.1	0.50	100.0	0.39
110.0 (a)	100.6	0.28	100.8	0.33	100.2	0.56
(b)	100.8	0.32	101.0	0.38	100.6	0.53
220.0 (a)	100.9	0.25	101.3	0.43	100.8	0.27
(b)	101.5	0.27	101.6	0.49	101.3	0.30

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The results were subjected to linear regression analysis (found values versus taken), using the computer program Excel-5, in order to establish whether the investigated electrodes exhibit any fixed or proportional bias. The slopes and intercepts of the regression lines did not differ significantly from the ideal values, revealing the absence of a systematic error during the measurements within the investigated concentration range. Also, they were compared with the results obtained from the official method,¹ which is based on measuring the absorbance of the drug solution in 0.1 mol l⁻¹ HCl at 252 nm, by applying F- and t-tests.²⁸ The values obtained (Table 6), show that the present methods are of comparable precision to the official method and there is no significant difference between the mean values obtained by the two methods.

Table 6 Statistical treatment of data obtained for the determination of pipazethate using Pi electrodes in comparison with the official method

Proposed method
Pi-PTA		Pi-PMA		Pi-PTA/PMA
Official method	Batch	FIA	Batch	FIA	Batch	FIA
a One-tailed critical F value.
Pure solutions—
x ± SE	100.08 ± 0.120	99.98 ± 0.141	100.1 ± 0.141	99.95 ± 0.524	100.12 ± 0.211	100.03 ± 0.871	100.07 ± 0.621
Probability	>0.01	>0.01	>0.01	>0.01	>0.01	>0.01
Relative error (%)	0.02	0.10	0.05	0.12	0.03	0.07
F^3,3 value (9.27)^a	4.14	5.28	3.54	4.62	3.17	3.97
Selgon tablets (20 mg per tablet)—
x ± SE	98.70 ± 0.524	98.95 ± 0.075	98.80 ± 0.411	99.48 ± 0.055	98.65 ± 0.072	99.46 ± 0.053	98.96 ± 0.097
Probability	<0.05	>0.01	>0.01	>0.01	>0.01	>0.01
Relative error (%)	1.05	1.2	0.52	1.35	0.54	1.04
F^3,3 value (9.27)^a	5.34	4.63	6.42	5.86	6.34	5  .47
Selgon drops (40 mg per ml^−1)—
x ± SE	100.5 ± 0.613	100.10 ± 0.084	100.38 ± 0.653	100.11 ± 0.125	100.28 ± 0.634	100.16 ± 0.726	100.28 ± 0.682
Probability	<0.05	>0.01	<0.05	>0.01	>0.01	>0.01
Relative error (%)	0.10	0.38	0.11	0.28	0.16	0.28
F^3,3 value (9.27)^a	3.87	5.52	5.48	5.40	5.38	5.617

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Conclusion

The application of the proposed method to the determination of pipazethate hydrochloride in its pure solutions and pharmaceutical preparations is characterized by a high degree of precision and accuracy when compared with the official method. Comparing the results obtained from Pi-PTA, Pi-PMA and Pi-PTA/PMA electrodes under batch and FIA conditions, it clear that the application of the electrodes in FIA increased considerably the selectivity of the electrodes towards the pipazethate ion and shortened the time needed for the determination of the drug in its pure state or in its pharmaceutical preparations. It is noteworthy also that the life span of the electrode containing the mixed ion exchanger is much longer than that of an electrode containing a single ion exchanger.

Acknowledgement

The authors express their thanks to Professor Dr. Marek Trojanowicz, Head of the Laboratory of Flow Analysis and Chromatography, Chemistry Department, Warsaw University, Poland, for his help in the construction of the FIA system and for his valuable guiding comments.

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