# Flow Injection Potentiometric Assay of  Hexoprenaline in Its Pure State, Pharmaceutical Preparations,  and Biological Samples

Authors: Rasha M. El-Nashar
Journal: Journal of Automated Methods and Management in Chemistry
 (2008)

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## Abstract

Different hexoprenaline (Hx2SO4)  conventional and coated wire electrodes were constructed and evaluated. Membranes were based on hexoprenalinium phosphotungstate (Hx-PTA) and hexporenalinium phosphomolybdate (Hx-PMA). The electrodes were fully characterized in terms of their composition, response time, life span, pH, and temperature and then were applied to the potentiometric determination of the hexoprenalinium ion in its pure state, pharmaceutical preparations, and biological samples, urine and plasma, under batch and flow injection conditions. The selectivity of the electrodes towards many inorganic cations, sugars, amino acids, and some other brochodilatures of close chemical composition was also tested.


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## Body

## 1. Introduction

Hexoprenaline sulphate, N,N′-Hexamethylene
bis [2-amino-1-(3,4-dihydroxy-phenyl)ethanol] sulphate [1], [CAS-32266-10-7], is
a selective β-sympathomimetic agent which has a double
action. It can be used as  a bronchospasmatic agent that can reduce bronchial
secretion and promotes the  efficiency of the bronchial epithelium besides being
used as a sympathomimetic agent that relaxes the uterus by decreasing or
arresting both the frequency and intensity of the uterine contraction thus
inhibits both the spontaneous and oxytocin-induced labour [2].A limited number of methods are available in literature for the determination and assay of
hexoprenaline in its pure state or pharmaceutical preparations including
colorimetric determination usingNaNO2 in alkaline medium [3] and
HPLC [4].Ion-selective electrodes have been increasingly used for quantitative
measurement of drugs. Potentiometric methods based on this technique are
simple, rapid, and offer enough selectivity towards the drugs in the presence
of various pharmaceutical excipients [5, 6]. Yet, there is no literature data
about the use of ion-selective electrodes for the determination of
hexoprenaline.In this study, plastic membrane electrodes (conventional) for Hx-cation
have been constructed based on the incorporation of Hx-PTA or Hx-PMA ion exchanger
in polyvinyl chloride (PVC) membranes plasticized with dioctylphthalate (DOP).
The electrodes were fully characterized in batch conditions and then used for
the determination of Hx-cation in its pure state, pharmaceutical preparation, and
biological samples both in batch and flow injection (FIA) techniques, which are considered  a very efficient way of
automation and improving the performance of characteristics of ion-selective
electrodes. In flow analysis, a high-sampling rate can be attained within a
short period of time besides being able to handle micro volumes of different
concentrations with equivalent accuracy and precision to batch conditions.
Also, coated graphite, silver, copper, and platinum wire electrodes were also
constructed and applied to assay the above-mentioned samples under both
conditions (batch and FIA).


## 2. Experimental

## 2.1. Reagents and Materials

All chemicals used for preparation of solutions were of analytical
grade. Doubly distilled water was used for preparing solutions and as a flow
stream in FIA measurements. The carrier and reagent solutions were degassed by
means of vacuum-suction pump. Sample solutions used for injections were freshly
prepared prior to measurements. Pure grade hexoprenaline sulphate and its
pharmaceutical preparations (Asmadol, tablets 0.5 mg/tablet and Gynipral, 0.5 mg/tablet) were provided by the Arab Drug Company, ADCo, Egypt.


## 2.2. Apparatus

The potentiometric measurements in
batch mode were carried out with a Schott-Gerate CG 820 pH-meter (Hofheim,
Germany) and WTW microprocessor pH/ion meter pMX 2000 (Weilheim, Germany). A Techne
circulator thermostat Model C-100 (Cambridge,
England) was
used to control the temperature of the test solutions. A WTW packed saturated Calomel
(SCE) was used as an external reference electrode. The electrochemical system
may be represented as follows.Ag/AgCl/filling solution/membrane/test solution//KCl salt bridge//SCE.The flow injection setup was composed of a 4-channel
peristaltic pump (Ismatec, ISM 827), (Zurich, Switzerland), injection valve model 5020 with
exchangeable sample loop from Rheodyne (Cotati,California, USA). The working electrodes
were connected to WTW microprocessor pH/ion meter pMX 2000 (Weilheim, Germany) and  interfaced to a
strip chart recorder Model BD111 from Kipp and Zonn (Deflt, Netherlands).A wall-jet cell, providing 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 is mounted at the sensing
surface of the electrode body. The optimized distance between 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 ion-selective
electrode 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. This arrangement shields the membrane from the solution in
the beaker, and the electrode response is, therefore, determined solely by the
carrier stream. Any disturbance in the flow pattern, for example, a short
interruption of the liquid flow or a strong convection current within the
beaker immediately results in erratic performance owing to backmixing [8]. Figure 1 represents a schematic diagram of the flow injection system used in
measurements.Figure 1
Schematic diagram of the flow injection system used in measurements.


## 2.3. Preparation of the Ion Exchangers

The
ion exchangers, hexoprenalinium-phosphotungstate[Hx3‐PMA], (buff powder), and hexoprenalinium-phospho-molybdate [Hx3‐PMA],
(yellowish green powder), were
prepared by the addition of 150cm3 of 10−2M hexoprenaline sulphate (Hx2SO4)
solution to  100cm3 of 10−2M of each of phosphotungstic acid or
phosphomolybdic  acid, respectively. The precipitates  were filtered, washed
thoroughly with distilled water until sulphate free and then air dried. The
chemical composition of the precipitates was identified and confirmed by
elemental analysis (C, H, N, S) at  the microanalysis unit, Cairo University, Egypt.


## 2.4. Preparation of Conventional and Coated Wire Electrodes

The conventional-type electrodes
were constructed as previously described [9]. 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, and Nernestian behavior is reached. A 5 mm diameter disc was
cut out from the prepared membrane and  attached to a Phillip's body filled with a
solution that is 10−2M with respect to
both NaCl and Hx2SO4 and preconditioned by soaking in 10−3MHx2SO4 solution.The coated wire electrodes were prepared using a graphite rode and platinum,
pure silver and pure copper wires, 15 cm length and 5 mm diameter each. One of
the two ends of the rode or wire is used for connection while the other, about
one cm length, is dipped in a solution of the same optimum membrane composition
used for the conventional electrode, which was previously mixed and the solvent
was evaporated slowly until an oily concentrated mixture is formed. Two drops
of this mixture are then introduced and spread on the surface of the solid
electrode and then is kept to dry at room temperature for about 24 hours.


## 2.5. Potentiometric Determination ofHx2SO4

In batch measurements,Hx2SO4 has been determined potentiometrically using the investigated electrodes by
both the interpolation and standard additions methods. In the latter, small
portions (0.1cm3) of standard 10−2MHx2SO4  solution were
added to  50cm3 water-containing
concentrations (10−6–10−2MHx2SO4 covering the range (5.186–518.600 mg) of pure compound or their equivalents
from pharmaceutical preparations. The change in mV readings was recorded after
each addition and  used to calculate the concentration of Hx2SO4 sample
solution using the following [10]:(1)Cx=Cs(VsVx+Vs)(10n(ΔE/s)−VxVx+V)−1, where Cx and Vx are the concentration and the volume of the unknown,
respectively, Cs and Vs  the concentration and the volume of the standard,
respectively, S  the
slope of the calibration graph, and ΔE  is the change in millivolt due to the
addition of the standard.For sampling of tablets, (Asmadol,tablets 0.5 mg/tablet, and Gynipral, 0.5 mg/tablet), 20 tablets
were ground together and appropriate weights of each were taken as samples. The
required amount (5.186–518.600 mg) was dissolved in 0.1 M HCl, (about 0.1cm3/mg tablets) and  completed to 50cm3 with distilled water, and
then the standard additions technique was applied as described above.For preparation of biological samples
(plasma and urine), fresh whole blood was centrifuged at 3000 rpm for 10 minutes.
The supernatant was transferred to a test tube and used as blank plasma.
Different amounts ofHx2SO4 (5.186–518.600 mg) and 1 mL plasma or 5 mL urine were transferred to a 100 mL volumetric flask
and completed to the mark with 0.01 M HCl; these solutions were measured using
the standard additions method.In FIA, the peak heights obtained by
a series of solutions of tablets or biological fluids were compared to those
obtained from a standard series of solutions prepared using the pure drug and then  used for
calculating the recovery percentages ofHx2SO4 in tablets
and biological samples.


## 3. Results and Discussion

### 3.1. Optimization of the ISE Response in Batch Conditions

#### 3.1.1. Composition of the Membranes

Several membrane compositions were investigated in which the content of
ion exchanger ranged from 1.0 to 20.0% of Hx-PTA and Hx-PMA. 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 0.98%).The best performances were obtained by using compositions containing 10.0%
Hx-PTA, 45.0% PVC, and 45.0% DOP; or 5.0% Hx-PMA, 47.5% PVC, and 47.5% DOP for
Hx-PTA and Hx-PMA electrodes, respectively. These compositions were also used
to prepare the coated wire electrodes. The usable concentration range for the
prepared electrodes was5.5×10−6–1.0×10−2M.


#### 3.1.2. Effect of Soaking

Any plastic membrane electrode needs
a preconditioning by soaking in the respective ion solution before use. This
process activates the formation of a thin gel layer at the membrane surface at
which the exchange process can take place. The time of preconditioning varies
according to the physical properties of the membranes and depends on diffusion
and equilibrium
at the interface of the membrane and the soaking solution. Fast establishment
of equilibrium is certainly a sufficient condition for fast response [11]. In
this work, the
presoak time ranged from 1/2 up to 2 hours depending on the membrane nature.
For the coated wire electrodes, the electrodes were left to dry in air for 24 hours
before use to stabilize the formed membrane layer.Nevertheless, the continuous soaking
of the electrodes in10−3MHx2SO4 solution affects negatively their response to the
hexoprenalinium cation, which can be attributed to leaching of the active
ingredients [ion exchanger(s) and solvent mediator] to the bathing solution [12].For the conventional-type
electrodes, it was noticed that the slopes of the calibration graphs obtained
using the preconditioned electrodes remained almost constant for 10 days and then to decrease gradually to 50.0 mV per
concentration decade after 6 weeks and reaching about 45.0 mV per concentration
decade after 8 and 9 weeks of continuous soaking for Hx-PTA and Hx-PMA
electrodes, respectively.For the coated graphite electrodes,
the slopes of the calibration graphs were constant for the first 5 days and then decrease to about 50.0 mV per  concentration decade after 7 and 6 days and reaching 45.0 mV per
concentration decade after 15 and 12 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated copper electrodes, the slopes were constant for
the first 5 days and decreased
gradually reaching 50.0 mV per concentration decade after 7 and 9 days reaching
45.0 mV per concentration decade after 10 and 11 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated silver electrodes, the slopes were constant for
the first 2 days and decrease
gradually reaching 50.0 mV per concentration decade after 4 and 6 days reaching
45.0 mV per concentration decade after 7 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated platinum electrodes, the life span was limited to
only 48 hours of continuous soaking for the two electrodes.This variation in properties is
highly related to the nature of the membranes and their adherence and
interaction with the different supporting electrodes. Also, it can be
correlated with the diffusion and partition coefficients of the ion exchangers
and the plasticizer [13–16].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. Table1 shows the response characteristics of the
prepared electrodes.Table 1
Response
characteristics of the electrodes under investigation.
ElectrodeMin Presoak time (h)Slope mV/concentration decadeResponse time (s)LifespanHx-PTA conventional (I)1/258.7308 weeksHx-PTA graphite (II)1.057.3252 weeksHx-PTA Copper (III)1.057.02010 daysHx-PTA Silver (IV)1.556.4207 daysHx-PTA Platinum (V)1.555.02048 hoursHx-PMA Conventional (I)1.059.3309 weeksHx-PMA graphite (II)2.058.52512 daysHx-PMA Copper (III)1.558.02011 daysHx-PMA Silver (IV)1.555.4207 daysHx-PMA Platinum (V)1.555.02048 hrs


#### 3.1.4. Effect of Temperature of the Test Solution

Calibration graphs [electrode
potential(Eelec) versus pHx] were constructed at different test
solution temperatures (25, 30, 40, 50, 60, and 70C°) for all the electrodes.
For the determination of the isothermal coefficients (dE°/dt) of the electrodes,
the standard electrode potentials (E°) against normal hydrogen electrode, at the different
temperatures, were obtained from the calibration graphs as the intercepts at
pHx = 0 (after subtracting the corresponding values of the standard electrode
potential of the Calomel at these temperatures) and  plotted versus (t−25), where t is the
temperature of the test solution in C° (Figure 2). A straight line plot is obtained according
to the following [17]:
(2)E°=E(25)°+(dE°dt)(t−25).Figure 2
Variation ofE° of Hx-PTA (a) and Hx-PMA (b) graphite electrodes with temperature.The slopes of the straight lines obtained  represent the isothermal
coefficients of the electrodes which were found to be7.6×10−4and1.1×10−4⁢V/C° for Hx-PTA and Hx-PMA, respectively. These slopes were
compared with the theoretical values at the given temperature and  found to be in a
good agreement to them revealing a fairly high thermal stability of the
electrodes within the investigated temperature ranges. The investigated
electrodes were found to be usable up to temperatures reaching 70C° without any
deviation from the theoretical Nernstian behavior at these temperatures.


### 3.2. Optimization of FIA Response

There are many important variables
that affect the response of an ion-selective electrode on operation in FIA
conditions. These factors should be studied and taken into consideration on
designing the flow system, the most important of which are dispersion
coefficient, sample volume, flow rate, and carrier composition.


#### 3.2.1. Dispersion Coefficient

Dispersion coefficient(D) is one of
the most important factors to be taken into consideration on constructing a FIA system because it
shows how much the original sample solution is diluted on its way towards the
sensor, and how much time has  elapsed between the sample injection and readout.In case of potentiometric detection
using ISE, limited dispersion,(D=1–3) is preferable because the original
composition of the sample  should be measured and the sample should only be
diluted to the detection limit of the ISE [18, 19]. The dispersion coefficient,
determined by measuring the ratio between the peak height obtained at steady
state conditions (where the sample acts as carrier stream) and at the state of
maximum peak height, maximum dispersion, (where the sample is injected in the
carrier stream), was found to be 1.2 and this value is affected by many
parameters  as
sample volume, flow rate, and channel geometry.


#### 3.2.2. Sample Volume

Samples of different volumes (20.0,
37.5, 75.0, 150.0, 340.0, and 500.0μl) were injected; changing the injected sample
volume is a powerful way to affect the dispersion process. An increase in the
peak height, sensitivity of measurement, and longer residence time of the
sample at the electrode surface can be achieved by increasing the volume of the
injected sample solution, but this requires longer time to reach a steady state
after each injection and higher sample consumption [20]. A sample loop of size
75.0 μl was used through out this work giving about
95% of the maximum peak height obtained by a 500.0 μl loop but with a shorter time to reach the
baseline and of course less consumption of reagents.


#### 3.2.3. Flow Rate

The dependence of the peak heights
and time required to recover the baseline on flow rate was studied where the
response of the electrodes under investigation to a solution, that is,1.0×10−2MHx2SO4, was studied at different flow rates (4.15,
5.35, 7.50, 9.70, 12.50, 17.85, 23.25, 25.00, 27.00, and 30.0 mL/min).With constant injection volume, the
residence time of the sample is inversely proportional to the flow rate [21].
Therefore, low flow rate would seem most likely to produce a steady state
signal but will also lead to increased response time due to increased residence
time of the sample at the active membrane surface (thicker boundary layers).It was found that, as the flow rate
increased, the peaks become higher and narrower until a flow rate of 23.25 mL/min,
where the peaks obtained at higher flow rates are nearly the same. A flow rate
of 7.50 mL/min, which led to 95% of the maximum peak height obtained by higher
flow rates, was used for Hx-conventional-type electrodes, while a flow rate of
5.35 mL/min was used for the Hx-coated wire electrodes offering a higher
residence time at the surface of the metallic electrodes.


#### 3.2.4. Carrier Composition

The composition of the carrier
should be as similar as possible to that of samples; this is highly
advantageous for baseline stability, response time, and characteristics [22].
The baseline attainment for the studied electrodes takes a very short time.A two-line configuration FIA system was used to study the effect of pH
and addition of main ion carrier in case of any need for baseline
stabilization. It was found that the addition of a small concentration of the
studied drugs(1.0×10−3–1.0×10−5M) as a carrier stream in
the second channel of the flow system did not affect this stabilization time
but only led to a small increase in the peak heights and higher consumption of
reagents, that is, why only water was used as a carrier in a single-channel
manifold FIA system throughout the present work.


#### 3.2.5. Electrode Response in FIA

In potentiometric detection, the
electrode potential depends on the activity of the main sensed ion. This can be
considered as a principle advantage of this method; also in flow measurements,
the dependence is semilogarithmic over a wide analyte activity range according
to the Nickolsky-Eisenman equation, but the main unfavorable feature of this
detection is the slow response of the electrode potential to concentration
change, and this is pronounced when low concentrations are measured which in
turn depends on the state of the membrane surface  at the interface with the
measured solution [23]. This slow response is a quite 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 to batch measurements, where potential
is measured in conditions very close to the equilibrium at membrane solution
interface [24]. The slopes of the calibration graphs ranged from 59.7 to 77.0 mV/concentration
decade for the studied electrodes. The variation of calibration graph slope with
flow rate is shown in Table 2. Figure 3 is a representative for the recording
obtained on measuring solutions having pHx of 5.5–2.0 using Hx-PTA conventional
electrode at optimum FIA conditions.Table 2
Variation of the slopes of the calibration graphs (mV/concentration decade) with flow rate.
Flow rate4.155.357.509.7012.5017.8523.2525.0027.0030.00ElectrodeSlope
(mV/ concentration decade)Hx-PTA (I)63.164.565.4*68.269.772.475.475.976.377.0Hx-PTA (II)61.061.9*62.764.566.868.269.670.471.272.6Hx-PTA (III)62.564.1*64.965.866.367.268.469.370.371.2Hx-PTA (IV)61.562.7*63.564.966.767.368.269.069.270.3Hx-PTA (V)63.364.5*66.267.568.369.770.471.271.972.3Hx-PTA (I)59.760.461.8*63.064.766.267.167.868.069.3Hx-PTA (II)61.463.2*64.565.767.168.469.569.870.270.7Hx-PTA (III)62.563.7*64.665.967.268.369.870.370.970.6Hx-PTA (IV)61.362.4*63.565.066.267.868.970.471.573.0Hx-PTA (V)62.063.1*63.965.766.868.169.270.370.971.6Slop* at flow rate selected as optimum working condition.Figure 3
Recordings obtained for hexoprenaline solution having pHx = 5.5–2.0 using Hx-PTA conventional electrode at optimum FIA conditions.


### 3.3. Effect of pH

For batch measurements, the effect
of pH on the potential readings of the electrodes was studied by varying the pH
of the test solution of different concentrations(1.0×10−4,1.0×10−3,and1.0×10−2M) of the pharmaceutical compounds gradually and  measuring the
respective potential readings. For FIA measurements, the effect of pH of the
test solution on the electrode potentials was studied by preparing a series of
solutions of concentration, that is, 1.0×10−4,1.0×10−3,and1.0×10−2M of the drug and pH ranging from 1.0–10.0 which were
then injected in the flow stream and the peak heights, representing variation
of potential response with pH  were recorded.In both conditions, the change in pH
does not affect the potential readings or peak heights, in FIA conditions,
within the pH range 2.5–8.0. In this
range, the electrodes can be used safely for the respective determination. At
pH values lower than this range, the potential readings and the peak heights
decrease gradually with pH which may be due to the penetration of the hydronium
ion into the membrane gel layer. While at pH higher than the given ranges, the
potential readings and the peak heights decrease gradually which can be
attributed to the formation of the free base of the drug and disappearance of
the protonated species [25]. Figure 4 is a representative graph for the effect
of pH on the recordings of a solution, that is, 1.0×10−3M
hexoprenaline measured using the five Hx-PTA electrodes under optimum FIA
conditions.Figure 4
Effect of pH of the test solution of
concentration1×10−3M hexoprenaline on the potential response of a-Hx-PTA(I), b-Hx-PTA(II), c-Hx-PTA(III), d-HX-PTA(IV), and e-Hx-PTA(V) electrodes at optimum FIA conditions.


### 3.4. Selectivity of the Electrodes

The selectivity coefficientKDrug,JZ+pot is the main source of information concerning
interferences on the electrode response. In analytical applications, the
coefficients must be very small so that the electrode exhibits a Nernstian
dependence on the primary ion over a wide concentration range.The response of the electrodes towards different substances and ionic
species such as inorganic cations, amino acids, sugars, and bronchodilators pharmaceutical
compounds of close chemical structure to hexoprenaline such as salbutamol,
terbutaline, and orciprenaline sulphates was checked in both batch and FIA
conditions, and the values of the selectivity coefficients, shown in Table3, were
used to evaluate their degree
of interference.Table 3
Selectivity coefficients and tolerance values for the Hx conventional-type electrodes.
Hx-PTAHx-PMABatchFIABatchFIAInterferentSSM*MSM**SSM*MSM**Na+3.424.625.233.184.354.98K+2.983.685.753.344.825.32Mg2+4.75—6.215.45—6.74Ca2+5.32—5.985.87—6.48Cr3+3.89—4.964.62—6.08Fe3+4.21—5.664.51—6.14Al3+6.83—7.555.29—6.59Theronine—5.958.20—5.746.79Leucine—5.458.97—6.327.86Valine—7.629.32—5.877.36Glycine—5.966.87—6.359.42Alanine—8.6210.75—7.449.87Salbutamol—7.6510.94—5.9810.24Orciprenaline—6.8510.44—6.4710.53Terbutiline—6.779.87—6.8510.42Urea—6.508.09—7.1210.88Lactose—4.986.42—4.687.82Glucose—4.637.01—4.796.89Maltose—5.016.85—5.138.44Fructose—5.237.35—5.587.96Saccharose—4.997.12—5.628.90Gum xanthine—6.327.93—5.787.38SSM*: separate solution method.MSM**: mixed solution method.For many
years, the method of determination of selectivity of membrane electrodes in
potentiometric measurements was a subject of discussion in the analytical
literature [26–28]. In ideal case, this should reflect the physicochemistry of
the processes involved in the formation of membrane potential in the presence
of the main sensed ion and interferent.In batch measurements, the separate
solution method [29] was used mainly to determine the selectivity coefficient
value. Although there are many restrictions that must be taken into
consideration on using this method in the determination of the selectivity
coefficients specially in case of solutions of differently charged ions, it is
still the simplest way to show whether interference takes place or not and is
used to perform measurements in important biological samples as blood [17] as
it is simple and easy to perform. A considerably high concentration of the
interferent ion is used 1.0×10−2M to ensure that there is no
interference if lower concentrations than this are present. The mixed solution
method [30] was used only as a confirmation when the KDrug,JZ+pot is less than 3.5 as it is time-consuming due
to the need of preparation of many solutions and performing many steps.The matched potential method [31] was used to determine the tolerance
values of neutral interferents
such as amino acids, sugars and to determine the selectivity coefficient values
for all the studied ions in FIA conditions. It was shown earlier for solid
state membrane electrodes that the apparent (or conditional) selectivity
coefficient measured in transient flow injection conditions may differ
significantly from that measured in batch conditions [32–34]. This is
interpreted by a difference in time of interaction of interferent with the
membrane surface and the rate at which the exchange process at the membrane
surface takes place which increases with  increase of interaction of interferent with
membrane in comparison to the main sensed ion. This is in good agreement with
earlier results that interferences caused by ions that have exchange reaction
constants greater than unity are governed to a most significant degree by
diffusion processes [35]. Therefore, in FIA measurements, where sample remains
in contact with the electrode for a short period of time, the apparent
selectivity of the membrane should be different from that found in batch
conditions. The electrodes exhibit good tolerance towards sugars and amino
acids as the presence of these species up to 4–11 folds did not affect
significantly the potential reading. This indicates that the presence of these
species is tolerated to a very high extent. This high tolerance is mainly
attributed to the differences in polarity and lipophilic nature of their
molecules relative to those of the drug under investigation.The selectivity coefficients of the
electrodes (shown in Table3) reflect a very high selectivity of the
investigated electrodes for their respective drug even in the presence of some
bronchodilators of nearly very close structure to that of hexoprenaline.


### 3.5. Analytical Applications

Several methods are applied for
quantitative analysis using ion-selective electrodes in batch and FIA
conditions: (i) in batch conditions, direct calculation of the concentration
applying Nernst equation, (ii) in FIA, peak height comparison, and (iii) in
both conditions, standard additions method, which is frequently applied in FIA
conditions as the large consumption of reagents and time will be the mean
trouble as we need to prepare many series of solutions to make just one
measurement. So, it was only applied for the assay of biological samples (urine
and plasma) to overcome the matrix effect of these samples.The results of the standard additions method under batch conditions were
found to be in good agreement with those obtained from the official method
(which involves the spectrometric measurement in 0.1 M HCl at 250 nm) [1]. The
mean recovery of the amounts taken using the conventional and coated wire
electrodes is comparable
and ranged from 97.5 to 102.4% with RSD = 0.12–0.65%. Representative results of application of the standard additions
method in batch conditions using Hx-PTA electrodes are shown in Table 4.Table 4
Determination of hexoprenaline employing the different Hx-PTA electrodes applying the standard additions method under batch conditions.
Hx-PTA (I)Hx-PTA (II)Hx-PTA (III)Hx-PTA (IV)Hx-PTA (V)SampleTakenRecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)/(a)mg(%)(%)(%)(%)(%)(%)(%)(%)(%)(%)Pure solutions5.18698.70.1999.50.1599.70.2599.20.3998.70.2451.86099.50.1599.70.2199.40.2998.60.2498.30.35518.6099.80.19100.20.3599.90.3199.50.3599.00.18Asmadol®5.18698.80.0998.50.3097.80.2499.60.28100.00.3151.860100.20.3699.30.1998.40.19100.00.44100.60.46518.60101.40.2499.90.2899.30.35100.50.37101.60.52Gynipral®5.18698.50.1899.20.3798.60.41100.10.1999.60.2251.860100.50.16100.10.1999.80.3399.80.2598.80.36518.6099.90.21100.90.26100.40.2799.40.42100.40.48Urine5.18697.50.15100.00.37100.20.2299.60.1798.90.2151.86098.60.19100.60.42100.10.34100.20.2697.60.38518.6099.80.23101.20.36101.60.16101.50.3799.40.29Plasma5.18698.00.4199.30.2998.60.3399.40.3697.50.3651.86098.60.2598.70.1799.20.2498.50.4598.30.45518.6098.80.2299.60.23100.40.4497.30.3898.60.40Taken(a)
mg per 50 mL.Five(b)
determinations.As for FIA conditions, the results obtained from peak heights comparison
method at optimum flow rates for pure solutions and the pharmaceutical
preparations were in agreement to those obtained under batch conditions and
that of the official method and the recovery values ranged from 97.0 to 101.5%
with RSD = 0.12–0.74%.As for urine and plasma samples, the results obtained at optimum flow
rates were much lower than under batch conditions. On using higher flow rates
reaching 23.5 mL/min, the recovery values were getting worse and this proves
that the sample needs a much longer time in contact with the electrode surface
besides a longer time of washing to remove matrix components from the electrode
surface.Results comparable to batch conditions were only obtained when the flow
was lowered to 2.5 mL/min, although this decreased the sampling rate, but the
recovery values were improved from 83.2 to 97.4% with RSD 0.18–0.78%, at optimum
flow rate, to reach 98.0–102.6% with RSD 0.24–0.65%, at flow rate 2.5 mL/min. Results
of the recovery values obtained for urine and plasma samples on using the
different Hx-electrodes (conventional and wire coated) at optimum flow-rate
value and after lowering the flow rate to 2.5 mL/min are given in Table5.Table 5
Determination of hexoprenaline in plasma and urine employing the different Hx-PTA and Hx-PMA electrodes under FIA conditions.
UrinePlasmaTaken5.186 mg51.860 mg518.600 mg5.186 mg51.860 mg518.600 mgRecovery*ABABABABABABHx-PTA (I)85.398.288.699.590.5100.484.699.787.6100.295.4100.8Hx-PTA (II)84.198.389.1100.392.3100.785.298.586.9101.496.899.7Hx-PTA (III)83.798.788.298.994.3101.283.2100.888.9100.597.0102.6Hx-PTA (IV)84.299.289.4100.595.499.983.9100.090.1100.995.5101.5Hx-PTA (V)85.498.492.3101.293.898.984.7101.295.4100.492.8100.7Hx-PMA (I)86.399.990.1100.396.2100.283.9100.793.199.997.499.6Hx-PMA (II)83.7100.289.998.694.799.686.199.689.8100.196.198.9Hx-PMA (III)84.6100.593.2100.893.8101.388.698.590.799.895.4100.0Hx-PMA (IV)88.098.691.6102.196.5100.585.9100.492.3101.393.8101.4Hx-PMA (V)86.999.390.4101.397.0100.286.2100.994.3100.295.3100.6A: recovery at optimum flow rate (7.50 mL/min for conventional electrodes, 5.35 mL/min for coated wire electrodes).B: recovery at flow rate 2.50 mL/min.Average* of three determinations.


### 3.6. Statistical Validation of the Obtained Results

The linearity of the presented electrodes was tested by measuring a
series of different concentrations of pHx in the range 5.5–2.0 using the
different Hx electrodes for 3 consecutive days, and these results were
subjected to linear regression analysis (found versus taken), using sigma plot
10.0, in order to establish whether the investigated electrodes exhibit any
fixed 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. The accuracy
of the results, recovery values of 97.5–102.4% with RSD = 0.12–0.65%,  tested using Studentst-test [36], at confidence limit 99%, and the calculated values were
much lower than the tabulated ones at that confidence limit. The repeatability
(intraday) precision was tested by measuring a solution of concentration 1.0×10−2M
three times in the same day using the same electrode, while the intermediate
(interday) precision was studied measuring a series of solution of pHx 5.5–2.0
for three consecutive days and in both cases F-test was applied to
compare the obtained with those of the official method.Table6 represents some of the studied statistical parameters of data
obtained for the determination of Hexoprenaline using Hx conventional
electrodes in comparison with the official method. It is clear that presented electrodes
are of a 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 Hexoprenaline using Hx conventional electrodes in comparison with the official method.
Hx-PTAHx-PMABatchFIABatchFIAPure solutionsX±S.E.*100.23±0.8599.85±0.7499.80±0.93101.27±0.21Relative error (%)0.130.150.070.22F3,3 value (9.27)**2.562.983.454.21Slope of regression line0.9980.9860.9950.978Intercept of regression line−0.087−0.0730.045−0.062Asmadol®X±S.E.*100.54±0.23100.75±0.7499.63±0.44100.82±0.50Relative error (%)0.050.130.090.24F3,3 value
(9.27)**3.784.122.682.49Slope of regression line0.9990.9880.9860.993Intercept of reg. line0.012−0.023−0.0310.009Gynipral®X±S.E.*100.81±0.34100.25±0.62100.26±0.48101.2±0.21Relative error (%)0.140.520.180.31F3,3 value
(9.27)**4.573.512.883.69Slope of regression line0.9920.9890.9960.985Intercept of regression line−0.0910.0360.0180.042UrineX±S.E.*100.54±0.4199.48±0.56100.56±0.45100.12±0.34Relative error (%)0.090.120.240.06F3,3 value
(9.27)**3.743.924.535.26Slope of regression line0.9910.9840.9750.994Intercept of regression line0.029−0.015−0.026−0.035PlasmaX±S.E.*100.2±0.8599.8±0.7499.8±0.93100.36±0.54Relative error (%)0.180.140.250.32F3,3 value
(9.27)**2.873.193.572.96Slope of regression line0.9990.9780.9820.990Intercept of regression line−0.014−0.0260.0340.091X±S.E.*: average ± standard error.One** tailed critical F-value.
N.B.: for the official method (X ± S.E.) =100.8±0.65.The limit of detection
of the studied electrodes (LOD), defined as the Hx concentration corresponding to the
intersection of the extrapolation of the linear part of the calibration curve, ranged
from6.3×10−6to5.8×10−6M, while the limit of quantification (LOQ), defined as the last point
corresponding to the intersection of the liner part of the calibration curve,
was found to be 5.0×10−6M for the studied electrodes.


## 3.1. Optimization of the ISE Response in Batch Conditions

### 3.1.1. Composition of the Membranes

Several membrane compositions were investigated in which the content of
ion exchanger ranged from 1.0 to 20.0% of Hx-PTA and Hx-PMA. 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 0.98%).The best performances were obtained by using compositions containing 10.0%
Hx-PTA, 45.0% PVC, and 45.0% DOP; or 5.0% Hx-PMA, 47.5% PVC, and 47.5% DOP for
Hx-PTA and Hx-PMA electrodes, respectively. These compositions were also used
to prepare the coated wire electrodes. The usable concentration range for the
prepared electrodes was5.5×10−6–1.0×10−2M.


### 3.1.2. Effect of Soaking

Any plastic membrane electrode needs
a preconditioning by soaking in the respective ion solution before use. This
process activates the formation of a thin gel layer at the membrane surface at
which the exchange process can take place. The time of preconditioning varies
according to the physical properties of the membranes and depends on diffusion
and equilibrium
at the interface of the membrane and the soaking solution. Fast establishment
of equilibrium is certainly a sufficient condition for fast response [11]. In
this work, the
presoak time ranged from 1/2 up to 2 hours depending on the membrane nature.
For the coated wire electrodes, the electrodes were left to dry in air for 24 hours
before use to stabilize the formed membrane layer.Nevertheless, the continuous soaking
of the electrodes in10−3MHx2SO4 solution affects negatively their response to the
hexoprenalinium cation, which can be attributed to leaching of the active
ingredients [ion exchanger(s) and solvent mediator] to the bathing solution [12].For the conventional-type
electrodes, it was noticed that the slopes of the calibration graphs obtained
using the preconditioned electrodes remained almost constant for 10 days and then to decrease gradually to 50.0 mV per
concentration decade after 6 weeks and reaching about 45.0 mV per concentration
decade after 8 and 9 weeks of continuous soaking for Hx-PTA and Hx-PMA
electrodes, respectively.For the coated graphite electrodes,
the slopes of the calibration graphs were constant for the first 5 days and then decrease to about 50.0 mV per  concentration decade after 7 and 6 days and reaching 45.0 mV per
concentration decade after 15 and 12 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated copper electrodes, the slopes were constant for
the first 5 days and decreased
gradually reaching 50.0 mV per concentration decade after 7 and 9 days reaching
45.0 mV per concentration decade after 10 and 11 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated silver electrodes, the slopes were constant for
the first 2 days and decrease
gradually reaching 50.0 mV per concentration decade after 4 and 6 days reaching
45.0 mV per concentration decade after 7 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated platinum electrodes, the life span was limited to
only 48 hours of continuous soaking for the two electrodes.This variation in properties is
highly related to the nature of the membranes and their adherence and
interaction with the different supporting electrodes. Also, it can be
correlated with the diffusion and partition coefficients of the ion exchangers
and the plasticizer [13–16].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. Table1 shows the response characteristics of the
prepared electrodes.Table 1
Response
characteristics of the electrodes under investigation.
ElectrodeMin Presoak time (h)Slope mV/concentration decadeResponse time (s)LifespanHx-PTA conventional (I)1/258.7308 weeksHx-PTA graphite (II)1.057.3252 weeksHx-PTA Copper (III)1.057.02010 daysHx-PTA Silver (IV)1.556.4207 daysHx-PTA Platinum (V)1.555.02048 hoursHx-PMA Conventional (I)1.059.3309 weeksHx-PMA graphite (II)2.058.52512 daysHx-PMA Copper (III)1.558.02011 daysHx-PMA Silver (IV)1.555.4207 daysHx-PMA Platinum (V)1.555.02048 hrs


### 3.1.4. Effect of Temperature of the Test Solution

Calibration graphs [electrode
potential(Eelec) versus pHx] were constructed at different test
solution temperatures (25, 30, 40, 50, 60, and 70C°) for all the electrodes.
For the determination of the isothermal coefficients (dE°/dt) of the electrodes,
the standard electrode potentials (E°) against normal hydrogen electrode, at the different
temperatures, were obtained from the calibration graphs as the intercepts at
pHx = 0 (after subtracting the corresponding values of the standard electrode
potential of the Calomel at these temperatures) and  plotted versus (t−25), where t is the
temperature of the test solution in C° (Figure 2). A straight line plot is obtained according
to the following [17]:
(2)E°=E(25)°+(dE°dt)(t−25).Figure 2
Variation ofE° of Hx-PTA (a) and Hx-PMA (b) graphite electrodes with temperature.The slopes of the straight lines obtained  represent the isothermal
coefficients of the electrodes which were found to be7.6×10−4and1.1×10−4⁢V/C° for Hx-PTA and Hx-PMA, respectively. These slopes were
compared with the theoretical values at the given temperature and  found to be in a
good agreement to them revealing a fairly high thermal stability of the
electrodes within the investigated temperature ranges. The investigated
electrodes were found to be usable up to temperatures reaching 70C° without any
deviation from the theoretical Nernstian behavior at these temperatures.


## 3.1.1. Composition of the Membranes

Several membrane compositions were investigated in which the content of
ion exchanger ranged from 1.0 to 20.0% of Hx-PTA and Hx-PMA. 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 0.98%).The best performances were obtained by using compositions containing 10.0%
Hx-PTA, 45.0% PVC, and 45.0% DOP; or 5.0% Hx-PMA, 47.5% PVC, and 47.5% DOP for
Hx-PTA and Hx-PMA electrodes, respectively. These compositions were also used
to prepare the coated wire electrodes. The usable concentration range for the
prepared electrodes was5.5×10−6–1.0×10−2M.


## 3.1.2. Effect of Soaking

Any plastic membrane electrode needs
a preconditioning by soaking in the respective ion solution before use. This
process activates the formation of a thin gel layer at the membrane surface at
which the exchange process can take place. The time of preconditioning varies
according to the physical properties of the membranes and depends on diffusion
and equilibrium
at the interface of the membrane and the soaking solution. Fast establishment
of equilibrium is certainly a sufficient condition for fast response [11]. In
this work, the
presoak time ranged from 1/2 up to 2 hours depending on the membrane nature.
For the coated wire electrodes, the electrodes were left to dry in air for 24 hours
before use to stabilize the formed membrane layer.Nevertheless, the continuous soaking
of the electrodes in10−3MHx2SO4 solution affects negatively their response to the
hexoprenalinium cation, which can be attributed to leaching of the active
ingredients [ion exchanger(s) and solvent mediator] to the bathing solution [12].For the conventional-type
electrodes, it was noticed that the slopes of the calibration graphs obtained
using the preconditioned electrodes remained almost constant for 10 days and then to decrease gradually to 50.0 mV per
concentration decade after 6 weeks and reaching about 45.0 mV per concentration
decade after 8 and 9 weeks of continuous soaking for Hx-PTA and Hx-PMA
electrodes, respectively.For the coated graphite electrodes,
the slopes of the calibration graphs were constant for the first 5 days and then decrease to about 50.0 mV per  concentration decade after 7 and 6 days and reaching 45.0 mV per
concentration decade after 15 and 12 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated copper electrodes, the slopes were constant for
the first 5 days and decreased
gradually reaching 50.0 mV per concentration decade after 7 and 9 days reaching
45.0 mV per concentration decade after 10 and 11 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated silver electrodes, the slopes were constant for
the first 2 days and decrease
gradually reaching 50.0 mV per concentration decade after 4 and 6 days reaching
45.0 mV per concentration decade after 7 days for Hx-PTA and Hx-PMA electrodes,
respectively. For the coated platinum electrodes, the life span was limited to
only 48 hours of continuous soaking for the two electrodes.This variation in properties is
highly related to the nature of the membranes and their adherence and
interaction with the different supporting electrodes. Also, it can be
correlated with the diffusion and partition coefficients of the ion exchangers
and the plasticizer [13–16].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. Table1 shows the response characteristics of the
prepared electrodes.Table 1
Response
characteristics of the electrodes under investigation.
ElectrodeMin Presoak time (h)Slope mV/concentration decadeResponse time (s)LifespanHx-PTA conventional (I)1/258.7308 weeksHx-PTA graphite (II)1.057.3252 weeksHx-PTA Copper (III)1.057.02010 daysHx-PTA Silver (IV)1.556.4207 daysHx-PTA Platinum (V)1.555.02048 hoursHx-PMA Conventional (I)1.059.3309 weeksHx-PMA graphite (II)2.058.52512 daysHx-PMA Copper (III)1.558.02011 daysHx-PMA Silver (IV)1.555.4207 daysHx-PMA Platinum (V)1.555.02048 hrs


## 3.1.4. Effect of Temperature of the Test Solution

Calibration graphs [electrode
potential(Eelec) versus pHx] were constructed at different test
solution temperatures (25, 30, 40, 50, 60, and 70C°) for all the electrodes.
For the determination of the isothermal coefficients (dE°/dt) of the electrodes,
the standard electrode potentials (E°) against normal hydrogen electrode, at the different
temperatures, were obtained from the calibration graphs as the intercepts at
pHx = 0 (after subtracting the corresponding values of the standard electrode
potential of the Calomel at these temperatures) and  plotted versus (t−25), where t is the
temperature of the test solution in C° (Figure 2). A straight line plot is obtained according
to the following [17]:
(2)E°=E(25)°+(dE°dt)(t−25).Figure 2
Variation ofE° of Hx-PTA (a) and Hx-PMA (b) graphite electrodes with temperature.The slopes of the straight lines obtained  represent the isothermal
coefficients of the electrodes which were found to be7.6×10−4and1.1×10−4⁢V/C° for Hx-PTA and Hx-PMA, respectively. These slopes were
compared with the theoretical values at the given temperature and  found to be in a
good agreement to them revealing a fairly high thermal stability of the
electrodes within the investigated temperature ranges. The investigated
electrodes were found to be usable up to temperatures reaching 70C° without any
deviation from the theoretical Nernstian behavior at these temperatures.


## 3.2. Optimization of FIA Response

There are many important variables
that affect the response of an ion-selective electrode on operation in FIA
conditions. These factors should be studied and taken into consideration on
designing the flow system, the most important of which are dispersion
coefficient, sample volume, flow rate, and carrier composition.


### 3.2.1. Dispersion Coefficient

Dispersion coefficient(D) is one of
the most important factors to be taken into consideration on constructing a FIA system because it
shows how much the original sample solution is diluted on its way towards the
sensor, and how much time has  elapsed between the sample injection and readout.In case of potentiometric detection
using ISE, limited dispersion,(D=1–3) is preferable because the original
composition of the sample  should be measured and the sample should only be
diluted to the detection limit of the ISE [18, 19]. The dispersion coefficient,
determined by measuring the ratio between the peak height obtained at steady
state conditions (where the sample acts as carrier stream) and at the state of
maximum peak height, maximum dispersion, (where the sample is injected in the
carrier stream), was found to be 1.2 and this value is affected by many
parameters  as
sample volume, flow rate, and channel geometry.


### 3.2.2. Sample Volume

Samples of different volumes (20.0,
37.5, 75.0, 150.0, 340.0, and 500.0μl) were injected; changing the injected sample
volume is a powerful way to affect the dispersion process. An increase in the
peak height, sensitivity of measurement, and longer residence time of the
sample at the electrode surface can be achieved by increasing the volume of the
injected sample solution, but this requires longer time to reach a steady state
after each injection and higher sample consumption [20]. A sample loop of size
75.0 μl was used through out this work giving about
95% of the maximum peak height obtained by a 500.0 μl loop but with a shorter time to reach the
baseline and of course less consumption of reagents.


### 3.2.3. Flow Rate

The dependence of the peak heights
and time required to recover the baseline on flow rate was studied where the
response of the electrodes under investigation to a solution, that is,1.0×10−2MHx2SO4, was studied at different flow rates (4.15,
5.35, 7.50, 9.70, 12.50, 17.85, 23.25, 25.00, 27.00, and 30.0 mL/min).With constant injection volume, the
residence time of the sample is inversely proportional to the flow rate [21].
Therefore, low flow rate would seem most likely to produce a steady state
signal but will also lead to increased response time due to increased residence
time of the sample at the active membrane surface (thicker boundary layers).It was found that, as the flow rate
increased, the peaks become higher and narrower until a flow rate of 23.25 mL/min,
where the peaks obtained at higher flow rates are nearly the same. A flow rate
of 7.50 mL/min, which led to 95% of the maximum peak height obtained by higher
flow rates, was used for Hx-conventional-type electrodes, while a flow rate of
5.35 mL/min was used for the Hx-coated wire electrodes offering a higher
residence time at the surface of the metallic electrodes.


### 3.2.4. Carrier Composition

The composition of the carrier
should be as similar as possible to that of samples; this is highly
advantageous for baseline stability, response time, and characteristics [22].
The baseline attainment for the studied electrodes takes a very short time.A two-line configuration FIA system was used to study the effect of pH
and addition of main ion carrier in case of any need for baseline
stabilization. It was found that the addition of a small concentration of the
studied drugs(1.0×10−3–1.0×10−5M) as a carrier stream in
the second channel of the flow system did not affect this stabilization time
but only led to a small increase in the peak heights and higher consumption of
reagents, that is, why only water was used as a carrier in a single-channel
manifold FIA system throughout the present work.


### 3.2.5. Electrode Response in FIA

In potentiometric detection, the
electrode potential depends on the activity of the main sensed ion. This can be
considered as a principle advantage of this method; also in flow measurements,
the dependence is semilogarithmic over a wide analyte activity range according
to the Nickolsky-Eisenman equation, but the main unfavorable feature of this
detection is the slow response of the electrode potential to concentration
change, and this is pronounced when low concentrations are measured which in
turn depends on the state of the membrane surface  at the interface with the
measured solution [23]. This slow response is a quite 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 to batch measurements, where potential
is measured in conditions very close to the equilibrium at membrane solution
interface [24]. The slopes of the calibration graphs ranged from 59.7 to 77.0 mV/concentration
decade for the studied electrodes. The variation of calibration graph slope with
flow rate is shown in Table 2. Figure 3 is a representative for the recording
obtained on measuring solutions having pHx of 5.5–2.0 using Hx-PTA conventional
electrode at optimum FIA conditions.Table 2
Variation of the slopes of the calibration graphs (mV/concentration decade) with flow rate.
Flow rate4.155.357.509.7012.5017.8523.2525.0027.0030.00ElectrodeSlope
(mV/ concentration decade)Hx-PTA (I)63.164.565.4*68.269.772.475.475.976.377.0Hx-PTA (II)61.061.9*62.764.566.868.269.670.471.272.6Hx-PTA (III)62.564.1*64.965.866.367.268.469.370.371.2Hx-PTA (IV)61.562.7*63.564.966.767.368.269.069.270.3Hx-PTA (V)63.364.5*66.267.568.369.770.471.271.972.3Hx-PTA (I)59.760.461.8*63.064.766.267.167.868.069.3Hx-PTA (II)61.463.2*64.565.767.168.469.569.870.270.7Hx-PTA (III)62.563.7*64.665.967.268.369.870.370.970.6Hx-PTA (IV)61.362.4*63.565.066.267.868.970.471.573.0Hx-PTA (V)62.063.1*63.965.766.868.169.270.370.971.6Slop* at flow rate selected as optimum working condition.Figure 3
Recordings obtained for hexoprenaline solution having pHx = 5.5–2.0 using Hx-PTA conventional electrode at optimum FIA conditions.


## 3.2.1. Dispersion Coefficient

Dispersion coefficient(D) is one of
the most important factors to be taken into consideration on constructing a FIA system because it
shows how much the original sample solution is diluted on its way towards the
sensor, and how much time has  elapsed between the sample injection and readout.In case of potentiometric detection
using ISE, limited dispersion,(D=1–3) is preferable because the original
composition of the sample  should be measured and the sample should only be
diluted to the detection limit of the ISE [18, 19]. The dispersion coefficient,
determined by measuring the ratio between the peak height obtained at steady
state conditions (where the sample acts as carrier stream) and at the state of
maximum peak height, maximum dispersion, (where the sample is injected in the
carrier stream), was found to be 1.2 and this value is affected by many
parameters  as
sample volume, flow rate, and channel geometry.


## 3.2.2. Sample Volume

Samples of different volumes (20.0,
37.5, 75.0, 150.0, 340.0, and 500.0μl) were injected; changing the injected sample
volume is a powerful way to affect the dispersion process. An increase in the
peak height, sensitivity of measurement, and longer residence time of the
sample at the electrode surface can be achieved by increasing the volume of the
injected sample solution, but this requires longer time to reach a steady state
after each injection and higher sample consumption [20]. A sample loop of size
75.0 μl was used through out this work giving about
95% of the maximum peak height obtained by a 500.0 μl loop but with a shorter time to reach the
baseline and of course less consumption of reagents.


## 3.2.3. Flow Rate

The dependence of the peak heights
and time required to recover the baseline on flow rate was studied where the
response of the electrodes under investigation to a solution, that is,1.0×10−2MHx2SO4, was studied at different flow rates (4.15,
5.35, 7.50, 9.70, 12.50, 17.85, 23.25, 25.00, 27.00, and 30.0 mL/min).With constant injection volume, the
residence time of the sample is inversely proportional to the flow rate [21].
Therefore, low flow rate would seem most likely to produce a steady state
signal but will also lead to increased response time due to increased residence
time of the sample at the active membrane surface (thicker boundary layers).It was found that, as the flow rate
increased, the peaks become higher and narrower until a flow rate of 23.25 mL/min,
where the peaks obtained at higher flow rates are nearly the same. A flow rate
of 7.50 mL/min, which led to 95% of the maximum peak height obtained by higher
flow rates, was used for Hx-conventional-type electrodes, while a flow rate of
5.35 mL/min was used for the Hx-coated wire electrodes offering a higher
residence time at the surface of the metallic electrodes.


## 3.2.4. Carrier Composition

The composition of the carrier
should be as similar as possible to that of samples; this is highly
advantageous for baseline stability, response time, and characteristics [22].
The baseline attainment for the studied electrodes takes a very short time.A two-line configuration FIA system was used to study the effect of pH
and addition of main ion carrier in case of any need for baseline
stabilization. It was found that the addition of a small concentration of the
studied drugs(1.0×10−3–1.0×10−5M) as a carrier stream in
the second channel of the flow system did not affect this stabilization time
but only led to a small increase in the peak heights and higher consumption of
reagents, that is, why only water was used as a carrier in a single-channel
manifold FIA system throughout the present work.


## 3.2.5. Electrode Response in FIA

In potentiometric detection, the
electrode potential depends on the activity of the main sensed ion. This can be
considered as a principle advantage of this method; also in flow measurements,
the dependence is semilogarithmic over a wide analyte activity range according
to the Nickolsky-Eisenman equation, but the main unfavorable feature of this
detection is the slow response of the electrode potential to concentration
change, and this is pronounced when low concentrations are measured which in
turn depends on the state of the membrane surface  at the interface with the
measured solution [23]. This slow response is a quite 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 to batch measurements, where potential
is measured in conditions very close to the equilibrium at membrane solution
interface [24]. The slopes of the calibration graphs ranged from 59.7 to 77.0 mV/concentration
decade for the studied electrodes. The variation of calibration graph slope with
flow rate is shown in Table 2. Figure 3 is a representative for the recording
obtained on measuring solutions having pHx of 5.5–2.0 using Hx-PTA conventional
electrode at optimum FIA conditions.Table 2
Variation of the slopes of the calibration graphs (mV/concentration decade) with flow rate.
Flow rate4.155.357.509.7012.5017.8523.2525.0027.0030.00ElectrodeSlope
(mV/ concentration decade)Hx-PTA (I)63.164.565.4*68.269.772.475.475.976.377.0Hx-PTA (II)61.061.9*62.764.566.868.269.670.471.272.6Hx-PTA (III)62.564.1*64.965.866.367.268.469.370.371.2Hx-PTA (IV)61.562.7*63.564.966.767.368.269.069.270.3Hx-PTA (V)63.364.5*66.267.568.369.770.471.271.972.3Hx-PTA (I)59.760.461.8*63.064.766.267.167.868.069.3Hx-PTA (II)61.463.2*64.565.767.168.469.569.870.270.7Hx-PTA (III)62.563.7*64.665.967.268.369.870.370.970.6Hx-PTA (IV)61.362.4*63.565.066.267.868.970.471.573.0Hx-PTA (V)62.063.1*63.965.766.868.169.270.370.971.6Slop* at flow rate selected as optimum working condition.Figure 3
Recordings obtained for hexoprenaline solution having pHx = 5.5–2.0 using Hx-PTA conventional electrode at optimum FIA conditions.


## 3.3. Effect of pH

For batch measurements, the effect
of pH on the potential readings of the electrodes was studied by varying the pH
of the test solution of different concentrations(1.0×10−4,1.0×10−3,and1.0×10−2M) of the pharmaceutical compounds gradually and  measuring the
respective potential readings. For FIA measurements, the effect of pH of the
test solution on the electrode potentials was studied by preparing a series of
solutions of concentration, that is, 1.0×10−4,1.0×10−3,and1.0×10−2M of the drug and pH ranging from 1.0–10.0 which were
then injected in the flow stream and the peak heights, representing variation
of potential response with pH  were recorded.In both conditions, the change in pH
does not affect the potential readings or peak heights, in FIA conditions,
within the pH range 2.5–8.0. In this
range, the electrodes can be used safely for the respective determination. At
pH values lower than this range, the potential readings and the peak heights
decrease gradually with pH which may be due to the penetration of the hydronium
ion into the membrane gel layer. While at pH higher than the given ranges, the
potential readings and the peak heights decrease gradually which can be
attributed to the formation of the free base of the drug and disappearance of
the protonated species [25]. Figure 4 is a representative graph for the effect
of pH on the recordings of a solution, that is, 1.0×10−3M
hexoprenaline measured using the five Hx-PTA electrodes under optimum FIA
conditions.Figure 4
Effect of pH of the test solution of
concentration1×10−3M hexoprenaline on the potential response of a-Hx-PTA(I), b-Hx-PTA(II), c-Hx-PTA(III), d-HX-PTA(IV), and e-Hx-PTA(V) electrodes at optimum FIA conditions.


## 3.4. Selectivity of the Electrodes

The selectivity coefficientKDrug,JZ+pot is the main source of information concerning
interferences on the electrode response. In analytical applications, the
coefficients must be very small so that the electrode exhibits a Nernstian
dependence on the primary ion over a wide concentration range.The response of the electrodes towards different substances and ionic
species such as inorganic cations, amino acids, sugars, and bronchodilators pharmaceutical
compounds of close chemical structure to hexoprenaline such as salbutamol,
terbutaline, and orciprenaline sulphates was checked in both batch and FIA
conditions, and the values of the selectivity coefficients, shown in Table3, were
used to evaluate their degree
of interference.Table 3
Selectivity coefficients and tolerance values for the Hx conventional-type electrodes.
Hx-PTAHx-PMABatchFIABatchFIAInterferentSSM*MSM**SSM*MSM**Na+3.424.625.233.184.354.98K+2.983.685.753.344.825.32Mg2+4.75—6.215.45—6.74Ca2+5.32—5.985.87—6.48Cr3+3.89—4.964.62—6.08Fe3+4.21—5.664.51—6.14Al3+6.83—7.555.29—6.59Theronine—5.958.20—5.746.79Leucine—5.458.97—6.327.86Valine—7.629.32—5.877.36Glycine—5.966.87—6.359.42Alanine—8.6210.75—7.449.87Salbutamol—7.6510.94—5.9810.24Orciprenaline—6.8510.44—6.4710.53Terbutiline—6.779.87—6.8510.42Urea—6.508.09—7.1210.88Lactose—4.986.42—4.687.82Glucose—4.637.01—4.796.89Maltose—5.016.85—5.138.44Fructose—5.237.35—5.587.96Saccharose—4.997.12—5.628.90Gum xanthine—6.327.93—5.787.38SSM*: separate solution method.MSM**: mixed solution method.For many
years, the method of determination of selectivity of membrane electrodes in
potentiometric measurements was a subject of discussion in the analytical
literature [26–28]. In ideal case, this should reflect the physicochemistry of
the processes involved in the formation of membrane potential in the presence
of the main sensed ion and interferent.In batch measurements, the separate
solution method [29] was used mainly to determine the selectivity coefficient
value. Although there are many restrictions that must be taken into
consideration on using this method in the determination of the selectivity
coefficients specially in case of solutions of differently charged ions, it is
still the simplest way to show whether interference takes place or not and is
used to perform measurements in important biological samples as blood [17] as
it is simple and easy to perform. A considerably high concentration of the
interferent ion is used 1.0×10−2M to ensure that there is no
interference if lower concentrations than this are present. The mixed solution
method [30] was used only as a confirmation when the KDrug,JZ+pot is less than 3.5 as it is time-consuming due
to the need of preparation of many solutions and performing many steps.The matched potential method [31] was used to determine the tolerance
values of neutral interferents
such as amino acids, sugars and to determine the selectivity coefficient values
for all the studied ions in FIA conditions. It was shown earlier for solid
state membrane electrodes that the apparent (or conditional) selectivity
coefficient measured in transient flow injection conditions may differ
significantly from that measured in batch conditions [32–34]. This is
interpreted by a difference in time of interaction of interferent with the
membrane surface and the rate at which the exchange process at the membrane
surface takes place which increases with  increase of interaction of interferent with
membrane in comparison to the main sensed ion. This is in good agreement with
earlier results that interferences caused by ions that have exchange reaction
constants greater than unity are governed to a most significant degree by
diffusion processes [35]. Therefore, in FIA measurements, where sample remains
in contact with the electrode for a short period of time, the apparent
selectivity of the membrane should be different from that found in batch
conditions. The electrodes exhibit good tolerance towards sugars and amino
acids as the presence of these species up to 4–11 folds did not affect
significantly the potential reading. This indicates that the presence of these
species is tolerated to a very high extent. This high tolerance is mainly
attributed to the differences in polarity and lipophilic nature of their
molecules relative to those of the drug under investigation.The selectivity coefficients of the
electrodes (shown in Table3) reflect a very high selectivity of the
investigated electrodes for their respective drug even in the presence of some
bronchodilators of nearly very close structure to that of hexoprenaline.


## 3.5. Analytical Applications

Several methods are applied for
quantitative analysis using ion-selective electrodes in batch and FIA
conditions: (i) in batch conditions, direct calculation of the concentration
applying Nernst equation, (ii) in FIA, peak height comparison, and (iii) in
both conditions, standard additions method, which is frequently applied in FIA
conditions as the large consumption of reagents and time will be the mean
trouble as we need to prepare many series of solutions to make just one
measurement. So, it was only applied for the assay of biological samples (urine
and plasma) to overcome the matrix effect of these samples.The results of the standard additions method under batch conditions were
found to be in good agreement with those obtained from the official method
(which involves the spectrometric measurement in 0.1 M HCl at 250 nm) [1]. The
mean recovery of the amounts taken using the conventional and coated wire
electrodes is comparable
and ranged from 97.5 to 102.4% with RSD = 0.12–0.65%. Representative results of application of the standard additions
method in batch conditions using Hx-PTA electrodes are shown in Table 4.Table 4
Determination of hexoprenaline employing the different Hx-PTA electrodes applying the standard additions method under batch conditions.
Hx-PTA (I)Hx-PTA (II)Hx-PTA (III)Hx-PTA (IV)Hx-PTA (V)SampleTakenRecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)RecoveryRSD(b)/(a)mg(%)(%)(%)(%)(%)(%)(%)(%)(%)(%)Pure solutions5.18698.70.1999.50.1599.70.2599.20.3998.70.2451.86099.50.1599.70.2199.40.2998.60.2498.30.35518.6099.80.19100.20.3599.90.3199.50.3599.00.18Asmadol®5.18698.80.0998.50.3097.80.2499.60.28100.00.3151.860100.20.3699.30.1998.40.19100.00.44100.60.46518.60101.40.2499.90.2899.30.35100.50.37101.60.52Gynipral®5.18698.50.1899.20.3798.60.41100.10.1999.60.2251.860100.50.16100.10.1999.80.3399.80.2598.80.36518.6099.90.21100.90.26100.40.2799.40.42100.40.48Urine5.18697.50.15100.00.37100.20.2299.60.1798.90.2151.86098.60.19100.60.42100.10.34100.20.2697.60.38518.6099.80.23101.20.36101.60.16101.50.3799.40.29Plasma5.18698.00.4199.30.2998.60.3399.40.3697.50.3651.86098.60.2598.70.1799.20.2498.50.4598.30.45518.6098.80.2299.60.23100.40.4497.30.3898.60.40Taken(a)
mg per 50 mL.Five(b)
determinations.As for FIA conditions, the results obtained from peak heights comparison
method at optimum flow rates for pure solutions and the pharmaceutical
preparations were in agreement to those obtained under batch conditions and
that of the official method and the recovery values ranged from 97.0 to 101.5%
with RSD = 0.12–0.74%.As for urine and plasma samples, the results obtained at optimum flow
rates were much lower than under batch conditions. On using higher flow rates
reaching 23.5 mL/min, the recovery values were getting worse and this proves
that the sample needs a much longer time in contact with the electrode surface
besides a longer time of washing to remove matrix components from the electrode
surface.Results comparable to batch conditions were only obtained when the flow
was lowered to 2.5 mL/min, although this decreased the sampling rate, but the
recovery values were improved from 83.2 to 97.4% with RSD 0.18–0.78%, at optimum
flow rate, to reach 98.0–102.6% with RSD 0.24–0.65%, at flow rate 2.5 mL/min. Results
of the recovery values obtained for urine and plasma samples on using the
different Hx-electrodes (conventional and wire coated) at optimum flow-rate
value and after lowering the flow rate to 2.5 mL/min are given in Table5.Table 5
Determination of hexoprenaline in plasma and urine employing the different Hx-PTA and Hx-PMA electrodes under FIA conditions.
UrinePlasmaTaken5.186 mg51.860 mg518.600 mg5.186 mg51.860 mg518.600 mgRecovery*ABABABABABABHx-PTA (I)85.398.288.699.590.5100.484.699.787.6100.295.4100.8Hx-PTA (II)84.198.389.1100.392.3100.785.298.586.9101.496.899.7Hx-PTA (III)83.798.788.298.994.3101.283.2100.888.9100.597.0102.6Hx-PTA (IV)84.299.289.4100.595.499.983.9100.090.1100.995.5101.5Hx-PTA (V)85.498.492.3101.293.898.984.7101.295.4100.492.8100.7Hx-PMA (I)86.399.990.1100.396.2100.283.9100.793.199.997.499.6Hx-PMA (II)83.7100.289.998.694.799.686.199.689.8100.196.198.9Hx-PMA (III)84.6100.593.2100.893.8101.388.698.590.799.895.4100.0Hx-PMA (IV)88.098.691.6102.196.5100.585.9100.492.3101.393.8101.4Hx-PMA (V)86.999.390.4101.397.0100.286.2100.994.3100.295.3100.6A: recovery at optimum flow rate (7.50 mL/min for conventional electrodes, 5.35 mL/min for coated wire electrodes).B: recovery at flow rate 2.50 mL/min.Average* of three determinations.


## 3.6. Statistical Validation of the Obtained Results

The linearity of the presented electrodes was tested by measuring a
series of different concentrations of pHx in the range 5.5–2.0 using the
different Hx electrodes for 3 consecutive days, and these results were
subjected to linear regression analysis (found versus taken), using sigma plot
10.0, in order to establish whether the investigated electrodes exhibit any
fixed 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. The accuracy
of the results, recovery values of 97.5–102.4% with RSD = 0.12–0.65%,  tested using Studentst-test [36], at confidence limit 99%, and the calculated values were
much lower than the tabulated ones at that confidence limit. The repeatability
(intraday) precision was tested by measuring a solution of concentration 1.0×10−2M
three times in the same day using the same electrode, while the intermediate
(interday) precision was studied measuring a series of solution of pHx 5.5–2.0
for three consecutive days and in both cases F-test was applied to
compare the obtained with those of the official method.Table6 represents some of the studied statistical parameters of data
obtained for the determination of Hexoprenaline using Hx conventional
electrodes in comparison with the official method. It is clear that presented electrodes
are of a 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 Hexoprenaline using Hx conventional electrodes in comparison with the official method.
Hx-PTAHx-PMABatchFIABatchFIAPure solutionsX±S.E.*100.23±0.8599.85±0.7499.80±0.93101.27±0.21Relative error (%)0.130.150.070.22F3,3 value (9.27)**2.562.983.454.21Slope of regression line0.9980.9860.9950.978Intercept of regression line−0.087−0.0730.045−0.062Asmadol®X±S.E.*100.54±0.23100.75±0.7499.63±0.44100.82±0.50Relative error (%)0.050.130.090.24F3,3 value
(9.27)**3.784.122.682.49Slope of regression line0.9990.9880.9860.993Intercept of reg. line0.012−0.023−0.0310.009Gynipral®X±S.E.*100.81±0.34100.25±0.62100.26±0.48101.2±0.21Relative error (%)0.140.520.180.31F3,3 value
(9.27)**4.573.512.883.69Slope of regression line0.9920.9890.9960.985Intercept of regression line−0.0910.0360.0180.042UrineX±S.E.*100.54±0.4199.48±0.56100.56±0.45100.12±0.34Relative error (%)0.090.120.240.06F3,3 value
(9.27)**3.743.924.535.26Slope of regression line0.9910.9840.9750.994Intercept of regression line0.029−0.015−0.026−0.035PlasmaX±S.E.*100.2±0.8599.8±0.7499.8±0.93100.36±0.54Relative error (%)0.180.140.250.32F3,3 value
(9.27)**2.873.193.572.96Slope of regression line0.9990.9780.9820.990Intercept of regression line−0.014−0.0260.0340.091X±S.E.*: average ± standard error.One** tailed critical F-value.
N.B.: for the official method (X ± S.E.) =100.8±0.65.The limit of detection
of the studied electrodes (LOD), defined as the Hx concentration corresponding to the
intersection of the extrapolation of the linear part of the calibration curve, ranged
from6.3×10−6to5.8×10−6M, while the limit of quantification (LOQ), defined as the last point
corresponding to the intersection of the liner part of the calibration curve,
was found to be 5.0×10−6M for the studied electrodes.


## 4. Conclusion

The present work offers two conventional
and 8 coated wire electrodes for the determination of hexoprenaline sulphate in
its pure state and pharmaceutical preparation in batch and FIA conditions. It
is clear from the obtained data that, for all the determined drugs, the
presented electrodes have the same usable concentration, temperature, and pH
range, selectivity and can be applied to the determination of their respective
drugs with nearly the same precision and accuracy, but it is noteworthy to
mention that the lifespan of the wire coated electrodes  much less than the conventional type,
although such type of electrodes is much easier in construction.The automation of the analysis by applying FIA lead to higher analysis
rates (typically from 100 to 300 samples/hr compared to 5–10 samples/hr in
batch conditions, enhanced response time) often less than 10–30 seconds
between sample injection and detector response, compared to 5–10 minutes in
case of batch conditions, much more rapid start up and shut down times besides
the ease of monitoring many sorts of errors that may take place in batch
conditions such as incorrect addition of reagents and mixing problems beside
shorting the time and decreasing the amount of sample needed for analysis. All
of the above-mentioned advantages of flow-injection automation make this application
feasible and economic to be used for routine analysis and quality control.The results obtained from the applications of the electrodes were
compared with the official method for assaying the drug under investigation,
andF-and t-tests were applied to compare the precision and mean
values obtained, respectively, and the obtained values were much smaller than
the tabulated ones.Thus, it is clear that the presented electrodes are of high accuracy,
precision, and selectivity compared to the official method beside being of low
cost, fast response which results
in high-sample measurements/hr, easy to apply without any steps of sample pretreatment
or extraction, in case of biological samples, besides there is no need of
complicated instruments; thus, the proposed electrodes can be very convenient
for routine analysis of hexoprenaline in pharmaceutical preparations and
biological samples.



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*Source: 586310-2008-04-30.xml*