# Simple Spectrophotometric Method for Determination of Paroxetine in Tablets Using 1,2-Naphthoquinone-4-Sulphonate  as a Chromogenic Reagent

Authors: Ibrahim A. Darwish; Heba H. Abdine; Sawsan M. Amer; Lama I. Al-Rayes
Journal: International Journal of Analytical Chemistry
 (2009)

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## Abstract

Simple and rapid spectrophotometric method has been developed and validated for the determination of paroxetine (PRX) in tablets. The proposed method was based on nucleophilic substitution reaction of PRX with 1,2-naphthoquinone-4-sulphonate (NQS) in an alkaline medium to form an orange-colored product of maximum absorption peak (λmax) at 488 nm. The stoichiometry and kinetics of the reaction were studied, and the reaction mechanism was postulated. Under the optimized reaction conditions, Beer's law correlating the absorbance (A) with PRX concentration (C) was obeyed in the
range of 1–8 μg mL−1. The regression equation for the calibration data was: A = 0.0031 + 0.1609 C, with good correlation coefficients (0.9992).
The molar absorptivity (ε) was 5.9×105 L mol−1 1 cm−1. The limits of detection and quantitation were 0.3 and 0.8 μg mL−1, respectively. The precision of the method was satisfactory; the values of relative standard deviations did not exceed 2%. The proposed method was successfully applied to the determination of PRX in its pharmaceutical tablets with good accuracy and precisions; the label claim percentage was 97.17±1.06
%. The results obtained by the proposed method were comparable with those obtained by the official method.


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## Body

## 1. Introduction

Paroxetine; (3S,4R)-3-[(1,3-benzodioxol-5-vloxy)methyl]-4-(4-flurophenyl) piperidine (PRX) is a new generation
antidepressant drug. It exerts its antidepressant effect through a selective
inhibition for the reuptake of the neurotransmitter serotonin by the
presynaptic receptors. PRX is comparable to the tricyclic antidepressants in
their clinical efficacy,  however, PRX is safer and has greater acceptance by
the patients [1]. It is also prescribed in the treatment of related disorders,
such as obsessive-compulsive disorder, panic fits, social phobia, and posttraumatic
stress [2]. PRX is devoid of sedative effect and remarkably safe in overdose.
PRX takes 5.2 hours to reach the peak, with extended half-life (21 hours) that
allowed the introduction of formulations for once-daily dosing [3]. These
combined qualities made PRX the most widely prescribed antidepressants [4].The methods reported
for quantitative determination of PRX in tablets and/or biological fluids include voltammetry [5, 6],
densitometry [7, 8], high-performance liquid chromatography [9–14], gas
chromatography [15–17], and capillary
electrophoresis [18]. These methods offered the required sensitivity and
selectivity for the analysis of PRX in biological fluids; however, their
sophisticated instrumentation and high analysis cost limited their routine use
in quality control laboratories for analysis of PRX in its pharmaceutical tablets.Spectrophotometry is considered the most convenient analytical technique because
of its inherent simplicity, low cost, and wide availability in most quality
control laboratories. However, few spectrophotometric methods have been
reported for its determination in tablets [19–21]. These
methods were based on formation of ion-pair associates with bromophenol blue, bromothymol
blue, and bromocresol green [19], formation of charge-transfer complexes with
7,7,8,8-tetracyanoquinodimethane and chloranilic acid [20], formation of vinylamino-substituted
haloquinone derivatives with haloquinone reagents in presence of acetaldehyde [21],
and formation of condensation product with 7-chloro-4-nitrobenzofurazon [19]. These methods were associated with some major
drawbacks such as laborious multiple extraction steps in the analysis by ion
pair formation-based methods [19], and in preparation of the samples for the analysis
by the methods relied on PRX base, rather than the hydrochloride salt [20, 21].
Furthermore, the analytical reactions were long and thus the procedures were time-consuming
[19, 21]. For these reasons, the development of new alternative simple and
rapid spectrophotometric method for the determination of PRX in its tablets was
very essential.1,2-naphthoquinone-4-sulphonic
sulphonate (NQS) has been used as a chromogenic reagent for the spectrophotometric
determination of many pharmaceutical amines [22–26]. However, the
reaction between NQS and PRX has not been investigated so far. The present
study describes the evaluation of NQS as a chromogenic reagent in the
development of simple and rapid spectrophotometric method for the determination
of PRX in its tablets.


## 2. Experimental

### 2.1. Apparatus

Double beam V-530 (JASCO Co. Ltd., Kyoto, Japan)
ultraviolet-visible spectrophotometer with matched 1 cm quartz cells was used
for all the spectrophotometric measurements. pH meter, Model 350 (Bibby
Scientific Ltd., T/As Jenway, Essex, UK).


### 2.2. Reagents and Materials

Paroxetine hydrochloride (PRX; SmithKline Beecham Pharmaceuticals,
Bentford, England) was obtained and used as received; its purity was99.8±1.45%.
A solution of 0.5% (w/v) of 1,2-naphthoquinone-4-sulphonate (NQS; Aldrich
Chemical Co., St. Louis, Mo, USA) was prepared by dissolving 250 mg in 50 mL distilled
water. The solution was freshly prepared and protected from light during use. Clark
and Lubs buffer solution of pH 9 was prepared by mixing 50 mL of 0.2 M aqueous
solution of boric acid and potassium chloride (1 liter contains 12.368 g of
boric acid and 14.90 g of potassium chloride) with 21.3 mL of 0.2 M sodium hydroxide
in 200 mL standard flask [27] and adjusted by pH meter. Seroxate tablets
(SmithKline Beecham Pharmaceuticals, Brentford,  UK) are labeled to contain 20 mg paroxetine HCl
per tablet. Double distilled water was obtained through WSC-85 water
purification system (Hamilton Laboratory Glass Ltd., Ky, USA) and used
throughout the work. All solvents and materials used throughout this study were
of analytical grade.


### 2.3. Preparation of Standard and Sample Solutions

#### 2.3.1. Paroxetine Hydrochloride (PRX) Standard Solution

An accurately weighed amount (50 mg) of PRX was
quantitatively transferred into a 25 mL calibrated
flask, dissolved in 20 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mg mL−1. This stock solution was
further diluted with water to obtain working
solutions in the range of 10–80 μg mL−1.


#### 2.3.2. Tablets Sample Solution

Twenty tablets were weighed and finely
powdered. An accurately weighed quantity of the powdered tablets equivalent to
100 mg of PRX was transferred into a 100 mL calibrated flask and dissolved in about 40 mL of distilled
water. The contents of the flask were swirled, sonicated for 5 minutes, and
then completed to volume with water. The contents were mixed well and filtered rejecting
the first portion of the filtrate. The prepared solution was diluted
quantitatively with distilled water to obtain a suitable concentration for the
analysis.


### 2.4. General Recommended Procedure

Accurately measured aliquots of PRX solution
containing 10–50μg mL−1 were
transferred into separate 10 mL calibrated flasks. One milliliter of Clark
and Lubs buffer solution (pH 9) was added followed by
1 mL of NQS solution (0.5%, w/v). The reaction solution was allowed to proceed
at room temperature (25  ±  5C°) for 10 minutes. The reaction mixture was
completed to volume with methanol, and the resulting solution was measured at
488 nm against reagent blank treated similarly.


### 2.5. Determination of the Stoichiometric Ratio of the Reaction

#### 2.5.1. Job’s Method

Job’s method
of continuous variation [28] was employed. Master equimolar (5 × 10−3 M) aqueous solutions of PRX and
NQS were prepared. Series of 10 mL portions of
the master solutions of PRX and NQS were made up comprising different
complementary proportions (0 : 10,  1 : 9,…,9 : 1,  10 : 0, inclusive) in 10 mL calibrated
flasks containing 1 mL of buffer solution (pH 9). The  solution was manipulated as
described under the general recommended procedures, Section 2.4.


#### 2.5.2. Limiting Logarithmic Method

In the limiting logarithmic method [29], two sets of experiments were
carried out employing the general recommended procedures described above.  The first set of experiments was
carried out  using increasing NQS concentrations (1.9 × 10−3  − 9.6 × 10−3 M) at
fixed PRX concentration (1.37 × 10−5 M). The
second set of experiments was
carried out using increasing PRX concentrations (0.3 × 10−5  − 2.04 × 10−5 M) at
fixed NQS concentration (1.92 × 10−2 M). The
logarithms of the obtained absorbances were plotted as function of the
logarithms of the NQS and PRX concentration in the first and second sets of
experiments, respectively. The slopes of the fitting lines in both sets of
experiments were calculated.


## 2.1. Apparatus

Double beam V-530 (JASCO Co. Ltd., Kyoto, Japan)
ultraviolet-visible spectrophotometer with matched 1 cm quartz cells was used
for all the spectrophotometric measurements. pH meter, Model 350 (Bibby
Scientific Ltd., T/As Jenway, Essex, UK).


## 2.2. Reagents and Materials

Paroxetine hydrochloride (PRX; SmithKline Beecham Pharmaceuticals,
Bentford, England) was obtained and used as received; its purity was99.8±1.45%.
A solution of 0.5% (w/v) of 1,2-naphthoquinone-4-sulphonate (NQS; Aldrich
Chemical Co., St. Louis, Mo, USA) was prepared by dissolving 250 mg in 50 mL distilled
water. The solution was freshly prepared and protected from light during use. Clark
and Lubs buffer solution of pH 9 was prepared by mixing 50 mL of 0.2 M aqueous
solution of boric acid and potassium chloride (1 liter contains 12.368 g of
boric acid and 14.90 g of potassium chloride) with 21.3 mL of 0.2 M sodium hydroxide
in 200 mL standard flask [27] and adjusted by pH meter. Seroxate tablets
(SmithKline Beecham Pharmaceuticals, Brentford,  UK) are labeled to contain 20 mg paroxetine HCl
per tablet. Double distilled water was obtained through WSC-85 water
purification system (Hamilton Laboratory Glass Ltd., Ky, USA) and used
throughout the work. All solvents and materials used throughout this study were
of analytical grade.


## 2.3. Preparation of Standard and Sample Solutions

### 2.3.1. Paroxetine Hydrochloride (PRX) Standard Solution

An accurately weighed amount (50 mg) of PRX was
quantitatively transferred into a 25 mL calibrated
flask, dissolved in 20 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mg mL−1. This stock solution was
further diluted with water to obtain working
solutions in the range of 10–80 μg mL−1.


### 2.3.2. Tablets Sample Solution

Twenty tablets were weighed and finely
powdered. An accurately weighed quantity of the powdered tablets equivalent to
100 mg of PRX was transferred into a 100 mL calibrated flask and dissolved in about 40 mL of distilled
water. The contents of the flask were swirled, sonicated for 5 minutes, and
then completed to volume with water. The contents were mixed well and filtered rejecting
the first portion of the filtrate. The prepared solution was diluted
quantitatively with distilled water to obtain a suitable concentration for the
analysis.


## 2.3.1. Paroxetine Hydrochloride (PRX) Standard Solution

An accurately weighed amount (50 mg) of PRX was
quantitatively transferred into a 25 mL calibrated
flask, dissolved in 20 mL distilled water, completed to volume with the
same solvent to obtain a stock solution of 2 mg mL−1. This stock solution was
further diluted with water to obtain working
solutions in the range of 10–80 μg mL−1.


## 2.3.2. Tablets Sample Solution

Twenty tablets were weighed and finely
powdered. An accurately weighed quantity of the powdered tablets equivalent to
100 mg of PRX was transferred into a 100 mL calibrated flask and dissolved in about 40 mL of distilled
water. The contents of the flask were swirled, sonicated for 5 minutes, and
then completed to volume with water. The contents were mixed well and filtered rejecting
the first portion of the filtrate. The prepared solution was diluted
quantitatively with distilled water to obtain a suitable concentration for the
analysis.


## 2.4. General Recommended Procedure

Accurately measured aliquots of PRX solution
containing 10–50μg mL−1 were
transferred into separate 10 mL calibrated flasks. One milliliter of Clark
and Lubs buffer solution (pH 9) was added followed by
1 mL of NQS solution (0.5%, w/v). The reaction solution was allowed to proceed
at room temperature (25  ±  5C°) for 10 minutes. The reaction mixture was
completed to volume with methanol, and the resulting solution was measured at
488 nm against reagent blank treated similarly.


## 2.5. Determination of the Stoichiometric Ratio of the Reaction

### 2.5.1. Job’s Method

Job’s method
of continuous variation [28] was employed. Master equimolar (5 × 10−3 M) aqueous solutions of PRX and
NQS were prepared. Series of 10 mL portions of
the master solutions of PRX and NQS were made up comprising different
complementary proportions (0 : 10,  1 : 9,…,9 : 1,  10 : 0, inclusive) in 10 mL calibrated
flasks containing 1 mL of buffer solution (pH 9). The  solution was manipulated as
described under the general recommended procedures, Section 2.4.


### 2.5.2. Limiting Logarithmic Method

In the limiting logarithmic method [29], two sets of experiments were
carried out employing the general recommended procedures described above.  The first set of experiments was
carried out  using increasing NQS concentrations (1.9 × 10−3  − 9.6 × 10−3 M) at
fixed PRX concentration (1.37 × 10−5 M). The
second set of experiments was
carried out using increasing PRX concentrations (0.3 × 10−5  − 2.04 × 10−5 M) at
fixed NQS concentration (1.92 × 10−2 M). The
logarithms of the obtained absorbances were plotted as function of the
logarithms of the NQS and PRX concentration in the first and second sets of
experiments, respectively. The slopes of the fitting lines in both sets of
experiments were calculated.


## 2.5.1. Job’s Method

Job’s method
of continuous variation [28] was employed. Master equimolar (5 × 10−3 M) aqueous solutions of PRX and
NQS were prepared. Series of 10 mL portions of
the master solutions of PRX and NQS were made up comprising different
complementary proportions (0 : 10,  1 : 9,…,9 : 1,  10 : 0, inclusive) in 10 mL calibrated
flasks containing 1 mL of buffer solution (pH 9). The  solution was manipulated as
described under the general recommended procedures, Section 2.4.


## 2.5.2. Limiting Logarithmic Method

In the limiting logarithmic method [29], two sets of experiments were
carried out employing the general recommended procedures described above.  The first set of experiments was
carried out  using increasing NQS concentrations (1.9 × 10−3  − 9.6 × 10−3 M) at
fixed PRX concentration (1.37 × 10−5 M). The
second set of experiments was
carried out using increasing PRX concentrations (0.3 × 10−5  − 2.04 × 10−5 M) at
fixed NQS concentration (1.92 × 10−2 M). The
logarithms of the obtained absorbances were plotted as function of the
logarithms of the NQS and PRX concentration in the first and second sets of
experiments, respectively. The slopes of the fitting lines in both sets of
experiments were calculated.


## 3. Results and Discussion

### 3.1. Absorption Spectra

According to the procedure, the absorption spectrum of the product
produced by the reaction between PRX and NQS was recorded (Figure1). The
product was orange-colored exhibiting a maximum absorption peak (λmax) at 488 nm, and the λmax of NQS was 360 nm. The λmax of the product was red-shifted
by 248 nm from the λmax of RRX (240 nm). In order to eliminate
the interference, the measurements were carried out at 488 nm against the
reagent blank.Figure 1
(1) Absorption spectra of PRX against water, (2) NQS against
water, and (3) their reaction product against reagent blank.


### 3.2. Optimization of Reaction Variables

#### 3.2.1. Effect of NQS Concentration

The  studying of NQS concentrations revealed that the reaction was
dependent on NQS reagent (Figure2). The absorbance of the reaction solution
increased as the NQS concentration increased, and the highest absorption intensity
was attained at NQS concentration of 0.25% (w/v). Higher NQS concentrations up
to 1.25% had no effect on the absorption values. Further experiments were
carried out using 0.5%.Figure 2
Effect of NQS(•)
and NaOH (∘) concentrations
on the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL;
NaOH: 1 mL; NQS: 1 mL; temperature: 25  ±  5C°; reaction time: 10 minutes.


#### 3.2.2. Effect of Alkalinity and pH

To generate the nucleophile from PRX and activate the nucleophilic
substitution reaction, alkaline medium was necessary. Different inorganic bases
were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium
bicarbonate, all prepared as aqueous solution of a concentration range of 1–25  × 10−3 M. Best
results were obtained in case of sodium hydroxide where with other bases either
precipitation of white colloid occurred upon diluting the reaction solution
with organic solvent, high blank readings, nonreproducible results, and/or weak sensitivity were
observed. Studies for optimization of sodium hydroxide concentration revealed
that the optimum concentration was 2–25  × 10−3 M (Figure 2). As well, it was found that the use of alkaline buffer solution gives more
precise readings over the use of NaOH. In a separate series of experiments, the
influence of pH on the absorbance of PRX-NQS product was investigated. The
results revealed that the absorbances at pH < 6 were close to 0, indicating
that under acidity, PRX has difficulty to react with NQS (Figure 3). This was possibly
due to the fact that the amino group (piperazinyl-NH) of PRX exists in the
form of hydrochloride amine salt, thus, it loses the nucleophilic substitution
capability. At pH > 6, the absorbance increased rapidly with the increase in
the pH, as the amino group of PRX turns into the free-NH, rather than the HCl
salt, facilitating the nucleophilic substitution reaction. The maximum
absorption values were attained in the range of pH at 8–10. At pH > 10,
the absorbance of solution obviously decreased. This was attributed probably to
the increase in the amount of hydroxide ion that  holds back the condensation reaction between PRX
and NQS. In order to keep the high sensibility for determination of PRX, the
experiment was carried out at pH 9.Figure 3
Effect of pH on the reaction of PRX
with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution: 1 mL; NQS (0.5%, w/v): 1 mL; temperature: 25 ± 5C°; reaction
time: 10 minutes.


#### 3.2.3. Effect of Temperature and Time

The effect of temperature on the reaction was studied by carrying out the
reaction at different temperatures (25–90C°). The
results (Figure 4) revealed that increasing the temperature had negative effect
on the absorption values of the reaction solution. This was probably attributed
to the instability of the PRX-NQS derivative. For this reason, further
experiments were carried out at room temperature (25 ± 5C°).  The effect of time on the formation of the
reaction product was investigated by carrying out the reaction for different
times. The maximum absorbance intensity was attained after 5 minutes, and longer
reaction time up to 25 minutes did not affect the absorbance intensity (Figure 4).
For more precise results, further experiments were carried out at 10 minutes.Figure 4
Effect of temperature(∘) and time (•) on
the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution (pH 9): 1 mL; NQS (0.5%, w/v): 1 mL.


#### 3.2.4. Effect of Organic Solvents

It was found that the PRX-NQS product is insoluble in the aqueous
reaction medium. For spectrophotometric measurements, the reaction product might
be either dissolved in a miscible organic solvent of lower polarity than water or
extracted with an immiscible extractive solvent. Different solvents were tested
for dilution: methanol, ethanol, isopropanol, acetone, acetonitrile,
dimethylsulphoxide, and 1,4-dioxane. The highest readings were obtained when methanol
was used for dilution (Table1). In a separate series of experiments, different
nonmiscible solvents were tested for extraction of the PRX-NQS product: carbon
tetrachloride, chloroform, dichloromethane,  ethyl acetate,  toluene, and benzene. The highest
readings were obtained when chloroform was used for extraction. The performance
of both extractive and nonextractive procedures (in terms of sensitivity and background
readings) was comparable. In order to simplify the analytical procedures, the
simple nonextractive procedure (dilution with methanol) was chosen as optimum
condition for the further experiments.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of PRX with NQS.
Diluting solventAbsorbance(a)Extracting
solventAbsorbance(a)Methanol0.802Carbon tetrachloride0.587Ethanol0.781Chloroform0.808Isopropanol0.778Dichloromethane0.716Acetone0.245Ethyl
acetate0.362Acetonitrile0.686Toluene0.267Dimethylformamide0.346Benzene0.2391,4-dioxane0.617(a)Values for all solvents are mean of three
determinations; the  RSDs
for the readings were <3.


#### 3.2.5. Stability of the Chromogen

Under the aforementioned optimum conditions, the reaction between PRX and
NQS was completed within 5 minutes at room temperature, and the absorbance no
longer changed after standing for up to 25 minutes. The effect of time on the
stability of the chromogen was studied by following the absorption intensity of
the reaction solution (after dilution) at different time intervals. It was
found that the absorbance of the chromogen remains stable for at least 4 hours.
This allowed the processing of large batches of samples and their comfortable
measurements with convenience. This increased the convenience of the methods as
well as made it applicable for large number of samples.


### 3.3. Stoichiometry and Kinetics of the Reaction

Under the optimum conditions, the stoichiometry of the reaction between PRX
and NQS was investigated by Job [28] and limiting logarithmic [29] methods. The
symmetrical bell shape of Job’s plot (Figure 5) indicates that the NQS:PRX
ratio was 1 : 1. In the limiting logarithmic method, two straight lines were
obtained (Figure 6). The values of the slopes of these lines were 1.0201 and 0.9248,
confirming the 1 : 1 ratio for the reaction. Based on this ratio, and the
presence of only one center (piperazinyl N–H group) in PRX
molecule that is available for the substitution reaction, the reaction pathway
was postulated to be proceeded as shown in Figure 7.Figure 5
Job’s plot for determination of stoichiometry of the reaction between
PRX and NQS. [PRX]:5×10−3 M; [NQS]:
5×10−3 M;
[PRX]+[NQS]: 1 mL; Clark and Lubs buffer solution (pH 9): 1 mL; temperature: 25 ±  5C°; reaction
time: 10 minutes.Figure 6
Limiting logarithmic plot for molar reactivity of PRX with NQS.C and A
are the concentration and absorbance, respectively. For generating the first
line (⧫), [NQS]: 1.9×10−2; [PRX]:
0.3 × 10−5  −  2.04 × 10−5 M; Clark
and Lubs buffer solution (pH 9): 1 mL; temperature: 25   ±  5C°; reaction time: 10 minutes.
For generating the second line (•), [NQS]: 1.9 × 10−3  −  1.9 × 10−2 M; [PRX]:
1.37 × 10−5 M. The
other conditions are the same as those employed in generating the first line.Figure 7
Scheme for the reaction pathway of PRX with NQS.Under the optimum conditions, the absorbance-time curves for the reaction
of PRX at several concentrations (0.3 × 10−5  − 2.04 × 10−5 M) with a
fixed concentration of NQS (1.9 × 10−2 M) were
generated, and the initial reaction rates (K) were determined from the slopes
of the curves. The logarithms of the reaction rates (Log K) were plotted as a function
of logarithms of PRX concentration (log C). As seen in Figure 8, a straight
line passing through the origin with a slope value of 0.9888 was obtained by
fitting the data to the following equation:(1)LogK=logK′+nlogC, where K is reaction rate, K′ is the rate constant, C is the molar
concentration of PRX, and n (slope of regression line) is the order of
the reaction. The value of the slope (≈1) confirmed
that the reaction was first order. However, under the optimized reaction
conditions, the concentration of NQS was in much more excess than that of PRX
in the reaction solution. Therefore, the reaction was regarded as a pseudo-first-order reaction.Figure 8
Linear plot for LogC versus Log K for the kinetic reaction of PRX with
NQS. C is the PRX concentration (0.3 × 10−5  − 2.04 × 10−5 M) and K is the reaction rate (second−1). Clark
and Lubs buffer solution (pH 9): 1 mL; NQS (1.9 × 10−2): 1 mL; temperature:
25  ± 5C°.


### 3.4. The Apparent Rate Constant and Activation Energy

The  absorbance-time curves at three different temperatures (25, 40, and
60C°)
were generated using fixed concentrations of PRX (1.7 × 10−5 M) and
NQS (1.9 × 10−2 M). From
these curves, the apparent rate constants were calculated. These rates were found
to be 6.92 × 10−4, 6.7 × 10−4, and
6.25 × 10−4 second−1 at 25,
40, and 60C°,
respectively. The activation energy, defined as the minimum kinetic energy that
a molecule possess in order to undergo a reaction, was determined using
Arrhenius equation [30]:(2)Logk=logA−Ea/2.303RT, where k is the apparent
rate constant, A is the frequency factor, Ea is the activation energy, T
is the absolute temperature, and R is the gas constant. By plotting log k′ as a
function of 1/T, a straight line with a slope value of 1.775 = −Ea /2.303 R. From this data, the activation
energy was found to be 8.12 kcal mole−1. Because
of this low activation energy, the nucleophilic substitution reaction between
PRX and NQS could be
easily taken place, and NQS could be used for determination of  PRX.


### 3.5. Validation of the Method

#### 3.5.1. Calibration and Sensitivity

Calibration curve for the determination of PRX by its reaction with NQS
was constructed by plotting the absorbances as a function of the corresponding
concentrations. The regression equation for the results wasA = 0.0031+0.1609C  (r  =  0.9992), where A is the absorbance at 488 nm, C is the concentration of
PRX in μg mL−1 in the range of 1–8 μg mL−1, and r is the correlation coefficient.
The molar absorptivity (ε) was 5.9 × 105 L mol−1 cm−1. The
limit of detection (LOD) and limit of quantitation (LOQ) were determined using
the formula: LOD or LOQ  =  κSDa/b, where κ = 3 for
LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is
the slope. The LOD and LOQ were 0.3 and 0.8 μg mL−1, respectively. The precision of the proposed method
was determined by analyzing 5 replicate samples of standard PRX solution at one
concentration level. The assay gave satisfactory results; the relative standard
deviation (RSD) was less than 2%.


#### 3.5.2. Reproducibility

The reproducibility of the proposed method was determined by replicate
analysis of five separate solutions of the working standard at three
concentration levels of each drug (1.5, 3, and 6μg mL−1). The method gave satisfactory
results; RSD did not exceed 2% indicating the good reproducibility of the
proposed method. This precision level is adequate for the precision and routine
analysis of the investigated drugs in quality control laboratories.


#### 3.5.3. Accuracy and Interference Liabilities

The accuracy of the proposed method was evaluated by the standard
addition method. The recovery values of the added concentrations were 97.6  −101.3±0.84  − 1.85% (Table 2), indicating the accuracy of the proposed method. Before
proceeding with the analysis of PRX in its tablets, interference liabilities
were carried out to explore the effect of common excipients that might be added
during tablets formulation. Samples were prepared by mixing known amount (20 mg) of PRX with various amounts of the common excipients: starch, glucose, lactose,
acacia, talc, and magnesium stearate. These laboratory-prepared samples were
analyzed by the proposed method applying the general recommended procedure. The
recovery values were 97.97 −  101.53±0.39  − 1.26%, with an average recovery of
99.63±1.23% (Table 3). These data confirmed the absence of interference from any
of the common excipients with the determination of PRX by the proposed method.Table 2
Recovery studies for determination of PRX by
the proposed method.
Sample numberPRXRecovery (%  ± SD)(a)Added(μgmL−1)Found(μgmL−1)(a)12.01.9999.5±1.8523.02.9798.9±1.2034.04.05101.3±0.9245.04.8897.6±0.8456.05.9899.7±1.50(a)Values are mean of three determinations.Table 3
Analysis of PRX in presence of common excipients by the proposed method.
ExcipientRecovery (%  ± SD)(a)Starch (50)(b)99.46±0.46Glucose (10)99.58±1.23Lactose (10)100.37±0.87Acacia (10)98.84±0.39Talc (5)101.53±1.02MS(c)(10)97.97±1.26Average ± SD99.63±1.23(a)Values are mean of three determinations.(b)Figures in parenthesis are the amounts in mg  added per 20 mg of PRX.(c)MS: Magnesium stearate.


#### 3.5.4. Robustness and Ruggedness

Robustness was examined by evaluating the influence of small variation of
method variables including concentration of analytical reagent and reaction
time on the performance of the proposed methods. In these experiments, one
parameter was changed whereas the others were kept unchanged, and the recovery
percentage was calculated each time. It was found that small variation of
method variables did not significantly affect the procedures; recovery values
were 98.8 −101.5±0.85  − 1.87% (Table 4). This provided an indication for the
reliability of the proposed method during its routine application for the analysis
of PRX. Ruggedness was also tested by applying the proposed methods to the assay
of PRX using the same operational conditions but using two different
instruments at two different laboratories and different elapsed time. Results
obtained from lab-to-lab and day-to-day variations were reproducible, as the
relative standard deviations (RSDs)
did not exceed 2.54%.Table 4
Influence of small variations in
the assay conditions on the analytical performance of the proposed
spectrophotometric method for determination of PRX using NQS reagent.
ParametersRecovery (%  ± SD)(a)Recommended conditions(b)100.5±1.35NQS concentration (%, w/v)0.2598.8±0.850.75100.4±1.25Buffer solution (pH)8.898.9±1.879.2101.5±1.67Reaction time (min)599.5±1.8715100.2±0.85(a)Values
are mean of 3 determinations.(b)The recommended
conditions are given in Section 2.


#### 3.6. Application of the Proposed Method to Analysis of PRX in Tablets

It is evident from the above-mentioned results that the proposed method
gave satisfactory results with PRX in bulk. Thus, its tablets were subjected to
the analysis of their PRX contents by the proposed and the official [14] methods.
The label-claim percentage was 99.17±1.06% (Table 5). This
result was compared with that obtained from the official method by statistical analysis with respect to the accuracy (by
t-test) and precision (by
F-test). No significant differences were found between the calculated and
theoretical values of
t- and F-tests at 95%
confidence level proving similar accuracy and precision in the determination of
PRX by both methods.Table 5
Analysis of PRX-containing tablets by the proposed and the official methods.
TabletRecovery
(%  ±
RSD)(a)t-value(c)F-value(c)ProposedOfficial(b)Seroxate tablets99.17±1.06101.31±0.482.204.88(a)Values are mean of 6 determinations.(b)Reference [14].(c)The
tabulated values of
t- and F-values at 95%
confidence limit are 2.78 and 6.39, respectively.


### 4. Conclusions

The present study described the successful
evaluation of NQS reagent in the development of simple and rapid spectrophotometric
method for the accurate determination of PRX in bulk and tablets. In contrast
with the previously reported methods for analysis of PRX, the method described
herein has many advantages: it does not need expensive sophisticated apparatus,
it is simple and rapid, and it has high sensitivity. Furthermore, all the
analytical reagents are inexpensive, have excellent shelf life, and are
available in any analytical laboratory. Therefore, this method is practical and
valuable for its routine application in the analysis of PRX in quality control
laboratories.


## 3.1. Absorption Spectra

According to the procedure, the absorption spectrum of the product
produced by the reaction between PRX and NQS was recorded (Figure1). The
product was orange-colored exhibiting a maximum absorption peak (λmax) at 488 nm, and the λmax of NQS was 360 nm. The λmax of the product was red-shifted
by 248 nm from the λmax of RRX (240 nm). In order to eliminate
the interference, the measurements were carried out at 488 nm against the
reagent blank.Figure 1
(1) Absorption spectra of PRX against water, (2) NQS against
water, and (3) their reaction product against reagent blank.


## 3.2. Optimization of Reaction Variables

### 3.2.1. Effect of NQS Concentration

The  studying of NQS concentrations revealed that the reaction was
dependent on NQS reagent (Figure2). The absorbance of the reaction solution
increased as the NQS concentration increased, and the highest absorption intensity
was attained at NQS concentration of 0.25% (w/v). Higher NQS concentrations up
to 1.25% had no effect on the absorption values. Further experiments were
carried out using 0.5%.Figure 2
Effect of NQS(•)
and NaOH (∘) concentrations
on the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL;
NaOH: 1 mL; NQS: 1 mL; temperature: 25  ±  5C°; reaction time: 10 minutes.


### 3.2.2. Effect of Alkalinity and pH

To generate the nucleophile from PRX and activate the nucleophilic
substitution reaction, alkaline medium was necessary. Different inorganic bases
were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium
bicarbonate, all prepared as aqueous solution of a concentration range of 1–25  × 10−3 M. Best
results were obtained in case of sodium hydroxide where with other bases either
precipitation of white colloid occurred upon diluting the reaction solution
with organic solvent, high blank readings, nonreproducible results, and/or weak sensitivity were
observed. Studies for optimization of sodium hydroxide concentration revealed
that the optimum concentration was 2–25  × 10−3 M (Figure 2). As well, it was found that the use of alkaline buffer solution gives more
precise readings over the use of NaOH. In a separate series of experiments, the
influence of pH on the absorbance of PRX-NQS product was investigated. The
results revealed that the absorbances at pH < 6 were close to 0, indicating
that under acidity, PRX has difficulty to react with NQS (Figure 3). This was possibly
due to the fact that the amino group (piperazinyl-NH) of PRX exists in the
form of hydrochloride amine salt, thus, it loses the nucleophilic substitution
capability. At pH > 6, the absorbance increased rapidly with the increase in
the pH, as the amino group of PRX turns into the free-NH, rather than the HCl
salt, facilitating the nucleophilic substitution reaction. The maximum
absorption values were attained in the range of pH at 8–10. At pH > 10,
the absorbance of solution obviously decreased. This was attributed probably to
the increase in the amount of hydroxide ion that  holds back the condensation reaction between PRX
and NQS. In order to keep the high sensibility for determination of PRX, the
experiment was carried out at pH 9.Figure 3
Effect of pH on the reaction of PRX
with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution: 1 mL; NQS (0.5%, w/v): 1 mL; temperature: 25 ± 5C°; reaction
time: 10 minutes.


### 3.2.3. Effect of Temperature and Time

The effect of temperature on the reaction was studied by carrying out the
reaction at different temperatures (25–90C°). The
results (Figure 4) revealed that increasing the temperature had negative effect
on the absorption values of the reaction solution. This was probably attributed
to the instability of the PRX-NQS derivative. For this reason, further
experiments were carried out at room temperature (25 ± 5C°).  The effect of time on the formation of the
reaction product was investigated by carrying out the reaction for different
times. The maximum absorbance intensity was attained after 5 minutes, and longer
reaction time up to 25 minutes did not affect the absorbance intensity (Figure 4).
For more precise results, further experiments were carried out at 10 minutes.Figure 4
Effect of temperature(∘) and time (•) on
the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution (pH 9): 1 mL; NQS (0.5%, w/v): 1 mL.


### 3.2.4. Effect of Organic Solvents

It was found that the PRX-NQS product is insoluble in the aqueous
reaction medium. For spectrophotometric measurements, the reaction product might
be either dissolved in a miscible organic solvent of lower polarity than water or
extracted with an immiscible extractive solvent. Different solvents were tested
for dilution: methanol, ethanol, isopropanol, acetone, acetonitrile,
dimethylsulphoxide, and 1,4-dioxane. The highest readings were obtained when methanol
was used for dilution (Table1). In a separate series of experiments, different
nonmiscible solvents were tested for extraction of the PRX-NQS product: carbon
tetrachloride, chloroform, dichloromethane,  ethyl acetate,  toluene, and benzene. The highest
readings were obtained when chloroform was used for extraction. The performance
of both extractive and nonextractive procedures (in terms of sensitivity and background
readings) was comparable. In order to simplify the analytical procedures, the
simple nonextractive procedure (dilution with methanol) was chosen as optimum
condition for the further experiments.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of PRX with NQS.
Diluting solventAbsorbance(a)Extracting
solventAbsorbance(a)Methanol0.802Carbon tetrachloride0.587Ethanol0.781Chloroform0.808Isopropanol0.778Dichloromethane0.716Acetone0.245Ethyl
acetate0.362Acetonitrile0.686Toluene0.267Dimethylformamide0.346Benzene0.2391,4-dioxane0.617(a)Values for all solvents are mean of three
determinations; the  RSDs
for the readings were <3.


### 3.2.5. Stability of the Chromogen

Under the aforementioned optimum conditions, the reaction between PRX and
NQS was completed within 5 minutes at room temperature, and the absorbance no
longer changed after standing for up to 25 minutes. The effect of time on the
stability of the chromogen was studied by following the absorption intensity of
the reaction solution (after dilution) at different time intervals. It was
found that the absorbance of the chromogen remains stable for at least 4 hours.
This allowed the processing of large batches of samples and their comfortable
measurements with convenience. This increased the convenience of the methods as
well as made it applicable for large number of samples.


## 3.2.1. Effect of NQS Concentration

The  studying of NQS concentrations revealed that the reaction was
dependent on NQS reagent (Figure2). The absorbance of the reaction solution
increased as the NQS concentration increased, and the highest absorption intensity
was attained at NQS concentration of 0.25% (w/v). Higher NQS concentrations up
to 1.25% had no effect on the absorption values. Further experiments were
carried out using 0.5%.Figure 2
Effect of NQS(•)
and NaOH (∘) concentrations
on the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL;
NaOH: 1 mL; NQS: 1 mL; temperature: 25  ±  5C°; reaction time: 10 minutes.


## 3.2.2. Effect of Alkalinity and pH

To generate the nucleophile from PRX and activate the nucleophilic
substitution reaction, alkaline medium was necessary. Different inorganic bases
were tested: sodium hydroxide, disodium hydrogen phosphate, and sodium
bicarbonate, all prepared as aqueous solution of a concentration range of 1–25  × 10−3 M. Best
results were obtained in case of sodium hydroxide where with other bases either
precipitation of white colloid occurred upon diluting the reaction solution
with organic solvent, high blank readings, nonreproducible results, and/or weak sensitivity were
observed. Studies for optimization of sodium hydroxide concentration revealed
that the optimum concentration was 2–25  × 10−3 M (Figure 2). As well, it was found that the use of alkaline buffer solution gives more
precise readings over the use of NaOH. In a separate series of experiments, the
influence of pH on the absorbance of PRX-NQS product was investigated. The
results revealed that the absorbances at pH < 6 were close to 0, indicating
that under acidity, PRX has difficulty to react with NQS (Figure 3). This was possibly
due to the fact that the amino group (piperazinyl-NH) of PRX exists in the
form of hydrochloride amine salt, thus, it loses the nucleophilic substitution
capability. At pH > 6, the absorbance increased rapidly with the increase in
the pH, as the amino group of PRX turns into the free-NH, rather than the HCl
salt, facilitating the nucleophilic substitution reaction. The maximum
absorption values were attained in the range of pH at 8–10. At pH > 10,
the absorbance of solution obviously decreased. This was attributed probably to
the increase in the amount of hydroxide ion that  holds back the condensation reaction between PRX
and NQS. In order to keep the high sensibility for determination of PRX, the
experiment was carried out at pH 9.Figure 3
Effect of pH on the reaction of PRX
with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution: 1 mL; NQS (0.5%, w/v): 1 mL; temperature: 25 ± 5C°; reaction
time: 10 minutes.


## 3.2.3. Effect of Temperature and Time

The effect of temperature on the reaction was studied by carrying out the
reaction at different temperatures (25–90C°). The
results (Figure 4) revealed that increasing the temperature had negative effect
on the absorption values of the reaction solution. This was probably attributed
to the instability of the PRX-NQS derivative. For this reason, further
experiments were carried out at room temperature (25 ± 5C°).  The effect of time on the formation of the
reaction product was investigated by carrying out the reaction for different
times. The maximum absorbance intensity was attained after 5 minutes, and longer
reaction time up to 25 minutes did not affect the absorbance intensity (Figure 4).
For more precise results, further experiments were carried out at 10 minutes.Figure 4
Effect of temperature(∘) and time (•) on
the reaction of PRX with NQS. PRX (40μgmL−1): 1 mL; Clark
and Lubs buffer solution (pH 9): 1 mL; NQS (0.5%, w/v): 1 mL.


## 3.2.4. Effect of Organic Solvents

It was found that the PRX-NQS product is insoluble in the aqueous
reaction medium. For spectrophotometric measurements, the reaction product might
be either dissolved in a miscible organic solvent of lower polarity than water or
extracted with an immiscible extractive solvent. Different solvents were tested
for dilution: methanol, ethanol, isopropanol, acetone, acetonitrile,
dimethylsulphoxide, and 1,4-dioxane. The highest readings were obtained when methanol
was used for dilution (Table1). In a separate series of experiments, different
nonmiscible solvents were tested for extraction of the PRX-NQS product: carbon
tetrachloride, chloroform, dichloromethane,  ethyl acetate,  toluene, and benzene. The highest
readings were obtained when chloroform was used for extraction. The performance
of both extractive and nonextractive procedures (in terms of sensitivity and background
readings) was comparable. In order to simplify the analytical procedures, the
simple nonextractive procedure (dilution with methanol) was chosen as optimum
condition for the further experiments.Table 1
Effect of diluting and extracting solvents on
the intensity of the reaction product of PRX with NQS.
Diluting solventAbsorbance(a)Extracting
solventAbsorbance(a)Methanol0.802Carbon tetrachloride0.587Ethanol0.781Chloroform0.808Isopropanol0.778Dichloromethane0.716Acetone0.245Ethyl
acetate0.362Acetonitrile0.686Toluene0.267Dimethylformamide0.346Benzene0.2391,4-dioxane0.617(a)Values for all solvents are mean of three
determinations; the  RSDs
for the readings were <3.


## 3.2.5. Stability of the Chromogen

Under the aforementioned optimum conditions, the reaction between PRX and
NQS was completed within 5 minutes at room temperature, and the absorbance no
longer changed after standing for up to 25 minutes. The effect of time on the
stability of the chromogen was studied by following the absorption intensity of
the reaction solution (after dilution) at different time intervals. It was
found that the absorbance of the chromogen remains stable for at least 4 hours.
This allowed the processing of large batches of samples and their comfortable
measurements with convenience. This increased the convenience of the methods as
well as made it applicable for large number of samples.


## 3.3. Stoichiometry and Kinetics of the Reaction

Under the optimum conditions, the stoichiometry of the reaction between PRX
and NQS was investigated by Job [28] and limiting logarithmic [29] methods. The
symmetrical bell shape of Job’s plot (Figure 5) indicates that the NQS:PRX
ratio was 1 : 1. In the limiting logarithmic method, two straight lines were
obtained (Figure 6). The values of the slopes of these lines were 1.0201 and 0.9248,
confirming the 1 : 1 ratio for the reaction. Based on this ratio, and the
presence of only one center (piperazinyl N–H group) in PRX
molecule that is available for the substitution reaction, the reaction pathway
was postulated to be proceeded as shown in Figure 7.Figure 5
Job’s plot for determination of stoichiometry of the reaction between
PRX and NQS. [PRX]:5×10−3 M; [NQS]:
5×10−3 M;
[PRX]+[NQS]: 1 mL; Clark and Lubs buffer solution (pH 9): 1 mL; temperature: 25 ±  5C°; reaction
time: 10 minutes.Figure 6
Limiting logarithmic plot for molar reactivity of PRX with NQS.C and A
are the concentration and absorbance, respectively. For generating the first
line (⧫), [NQS]: 1.9×10−2; [PRX]:
0.3 × 10−5  −  2.04 × 10−5 M; Clark
and Lubs buffer solution (pH 9): 1 mL; temperature: 25   ±  5C°; reaction time: 10 minutes.
For generating the second line (•), [NQS]: 1.9 × 10−3  −  1.9 × 10−2 M; [PRX]:
1.37 × 10−5 M. The
other conditions are the same as those employed in generating the first line.Figure 7
Scheme for the reaction pathway of PRX with NQS.Under the optimum conditions, the absorbance-time curves for the reaction
of PRX at several concentrations (0.3 × 10−5  − 2.04 × 10−5 M) with a
fixed concentration of NQS (1.9 × 10−2 M) were
generated, and the initial reaction rates (K) were determined from the slopes
of the curves. The logarithms of the reaction rates (Log K) were plotted as a function
of logarithms of PRX concentration (log C). As seen in Figure 8, a straight
line passing through the origin with a slope value of 0.9888 was obtained by
fitting the data to the following equation:(1)LogK=logK′+nlogC, where K is reaction rate, K′ is the rate constant, C is the molar
concentration of PRX, and n (slope of regression line) is the order of
the reaction. The value of the slope (≈1) confirmed
that the reaction was first order. However, under the optimized reaction
conditions, the concentration of NQS was in much more excess than that of PRX
in the reaction solution. Therefore, the reaction was regarded as a pseudo-first-order reaction.Figure 8
Linear plot for LogC versus Log K for the kinetic reaction of PRX with
NQS. C is the PRX concentration (0.3 × 10−5  − 2.04 × 10−5 M) and K is the reaction rate (second−1). Clark
and Lubs buffer solution (pH 9): 1 mL; NQS (1.9 × 10−2): 1 mL; temperature:
25  ± 5C°.


## 3.4. The Apparent Rate Constant and Activation Energy

The  absorbance-time curves at three different temperatures (25, 40, and
60C°)
were generated using fixed concentrations of PRX (1.7 × 10−5 M) and
NQS (1.9 × 10−2 M). From
these curves, the apparent rate constants were calculated. These rates were found
to be 6.92 × 10−4, 6.7 × 10−4, and
6.25 × 10−4 second−1 at 25,
40, and 60C°,
respectively. The activation energy, defined as the minimum kinetic energy that
a molecule possess in order to undergo a reaction, was determined using
Arrhenius equation [30]:(2)Logk=logA−Ea/2.303RT, where k is the apparent
rate constant, A is the frequency factor, Ea is the activation energy, T
is the absolute temperature, and R is the gas constant. By plotting log k′ as a
function of 1/T, a straight line with a slope value of 1.775 = −Ea /2.303 R. From this data, the activation
energy was found to be 8.12 kcal mole−1. Because
of this low activation energy, the nucleophilic substitution reaction between
PRX and NQS could be
easily taken place, and NQS could be used for determination of  PRX.


## 3.5. Validation of the Method

### 3.5.1. Calibration and Sensitivity

Calibration curve for the determination of PRX by its reaction with NQS
was constructed by plotting the absorbances as a function of the corresponding
concentrations. The regression equation for the results wasA = 0.0031+0.1609C  (r  =  0.9992), where A is the absorbance at 488 nm, C is the concentration of
PRX in μg mL−1 in the range of 1–8 μg mL−1, and r is the correlation coefficient.
The molar absorptivity (ε) was 5.9 × 105 L mol−1 cm−1. The
limit of detection (LOD) and limit of quantitation (LOQ) were determined using
the formula: LOD or LOQ  =  κSDa/b, where κ = 3 for
LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is
the slope. The LOD and LOQ were 0.3 and 0.8 μg mL−1, respectively. The precision of the proposed method
was determined by analyzing 5 replicate samples of standard PRX solution at one
concentration level. The assay gave satisfactory results; the relative standard
deviation (RSD) was less than 2%.


### 3.5.2. Reproducibility

The reproducibility of the proposed method was determined by replicate
analysis of five separate solutions of the working standard at three
concentration levels of each drug (1.5, 3, and 6μg mL−1). The method gave satisfactory
results; RSD did not exceed 2% indicating the good reproducibility of the
proposed method. This precision level is adequate for the precision and routine
analysis of the investigated drugs in quality control laboratories.


### 3.5.3. Accuracy and Interference Liabilities

The accuracy of the proposed method was evaluated by the standard
addition method. The recovery values of the added concentrations were 97.6  −101.3±0.84  − 1.85% (Table 2), indicating the accuracy of the proposed method. Before
proceeding with the analysis of PRX in its tablets, interference liabilities
were carried out to explore the effect of common excipients that might be added
during tablets formulation. Samples were prepared by mixing known amount (20 mg) of PRX with various amounts of the common excipients: starch, glucose, lactose,
acacia, talc, and magnesium stearate. These laboratory-prepared samples were
analyzed by the proposed method applying the general recommended procedure. The
recovery values were 97.97 −  101.53±0.39  − 1.26%, with an average recovery of
99.63±1.23% (Table 3). These data confirmed the absence of interference from any
of the common excipients with the determination of PRX by the proposed method.Table 2
Recovery studies for determination of PRX by
the proposed method.
Sample numberPRXRecovery (%  ± SD)(a)Added(μgmL−1)Found(μgmL−1)(a)12.01.9999.5±1.8523.02.9798.9±1.2034.04.05101.3±0.9245.04.8897.6±0.8456.05.9899.7±1.50(a)Values are mean of three determinations.Table 3
Analysis of PRX in presence of common excipients by the proposed method.
ExcipientRecovery (%  ± SD)(a)Starch (50)(b)99.46±0.46Glucose (10)99.58±1.23Lactose (10)100.37±0.87Acacia (10)98.84±0.39Talc (5)101.53±1.02MS(c)(10)97.97±1.26Average ± SD99.63±1.23(a)Values are mean of three determinations.(b)Figures in parenthesis are the amounts in mg  added per 20 mg of PRX.(c)MS: Magnesium stearate.


### 3.5.4. Robustness and Ruggedness

Robustness was examined by evaluating the influence of small variation of
method variables including concentration of analytical reagent and reaction
time on the performance of the proposed methods. In these experiments, one
parameter was changed whereas the others were kept unchanged, and the recovery
percentage was calculated each time. It was found that small variation of
method variables did not significantly affect the procedures; recovery values
were 98.8 −101.5±0.85  − 1.87% (Table 4). This provided an indication for the
reliability of the proposed method during its routine application for the analysis
of PRX. Ruggedness was also tested by applying the proposed methods to the assay
of PRX using the same operational conditions but using two different
instruments at two different laboratories and different elapsed time. Results
obtained from lab-to-lab and day-to-day variations were reproducible, as the
relative standard deviations (RSDs)
did not exceed 2.54%.Table 4
Influence of small variations in
the assay conditions on the analytical performance of the proposed
spectrophotometric method for determination of PRX using NQS reagent.
ParametersRecovery (%  ± SD)(a)Recommended conditions(b)100.5±1.35NQS concentration (%, w/v)0.2598.8±0.850.75100.4±1.25Buffer solution (pH)8.898.9±1.879.2101.5±1.67Reaction time (min)599.5±1.8715100.2±0.85(a)Values
are mean of 3 determinations.(b)The recommended
conditions are given in Section 2.


### 3.6. Application of the Proposed Method to Analysis of PRX in Tablets

It is evident from the above-mentioned results that the proposed method
gave satisfactory results with PRX in bulk. Thus, its tablets were subjected to
the analysis of their PRX contents by the proposed and the official [14] methods.
The label-claim percentage was 99.17±1.06% (Table 5). This
result was compared with that obtained from the official method by statistical analysis with respect to the accuracy (by
t-test) and precision (by
F-test). No significant differences were found between the calculated and
theoretical values of
t- and F-tests at 95%
confidence level proving similar accuracy and precision in the determination of
PRX by both methods.Table 5
Analysis of PRX-containing tablets by the proposed and the official methods.
TabletRecovery
(%  ±
RSD)(a)t-value(c)F-value(c)ProposedOfficial(b)Seroxate tablets99.17±1.06101.31±0.482.204.88(a)Values are mean of 6 determinations.(b)Reference [14].(c)The
tabulated values of
t- and F-values at 95%
confidence limit are 2.78 and 6.39, respectively.


## 3.5.1. Calibration and Sensitivity

Calibration curve for the determination of PRX by its reaction with NQS
was constructed by plotting the absorbances as a function of the corresponding
concentrations. The regression equation for the results wasA = 0.0031+0.1609C  (r  =  0.9992), where A is the absorbance at 488 nm, C is the concentration of
PRX in μg mL−1 in the range of 1–8 μg mL−1, and r is the correlation coefficient.
The molar absorptivity (ε) was 5.9 × 105 L mol−1 cm−1. The
limit of detection (LOD) and limit of quantitation (LOQ) were determined using
the formula: LOD or LOQ  =  κSDa/b, where κ = 3 for
LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is
the slope. The LOD and LOQ were 0.3 and 0.8 μg mL−1, respectively. The precision of the proposed method
was determined by analyzing 5 replicate samples of standard PRX solution at one
concentration level. The assay gave satisfactory results; the relative standard
deviation (RSD) was less than 2%.


## 3.5.2. Reproducibility

The reproducibility of the proposed method was determined by replicate
analysis of five separate solutions of the working standard at three
concentration levels of each drug (1.5, 3, and 6μg mL−1). The method gave satisfactory
results; RSD did not exceed 2% indicating the good reproducibility of the
proposed method. This precision level is adequate for the precision and routine
analysis of the investigated drugs in quality control laboratories.


## 3.5.3. Accuracy and Interference Liabilities

The accuracy of the proposed method was evaluated by the standard
addition method. The recovery values of the added concentrations were 97.6  −101.3±0.84  − 1.85% (Table 2), indicating the accuracy of the proposed method. Before
proceeding with the analysis of PRX in its tablets, interference liabilities
were carried out to explore the effect of common excipients that might be added
during tablets formulation. Samples were prepared by mixing known amount (20 mg) of PRX with various amounts of the common excipients: starch, glucose, lactose,
acacia, talc, and magnesium stearate. These laboratory-prepared samples were
analyzed by the proposed method applying the general recommended procedure. The
recovery values were 97.97 −  101.53±0.39  − 1.26%, with an average recovery of
99.63±1.23% (Table 3). These data confirmed the absence of interference from any
of the common excipients with the determination of PRX by the proposed method.Table 2
Recovery studies for determination of PRX by
the proposed method.
Sample numberPRXRecovery (%  ± SD)(a)Added(μgmL−1)Found(μgmL−1)(a)12.01.9999.5±1.8523.02.9798.9±1.2034.04.05101.3±0.9245.04.8897.6±0.8456.05.9899.7±1.50(a)Values are mean of three determinations.Table 3
Analysis of PRX in presence of common excipients by the proposed method.
ExcipientRecovery (%  ± SD)(a)Starch (50)(b)99.46±0.46Glucose (10)99.58±1.23Lactose (10)100.37±0.87Acacia (10)98.84±0.39Talc (5)101.53±1.02MS(c)(10)97.97±1.26Average ± SD99.63±1.23(a)Values are mean of three determinations.(b)Figures in parenthesis are the amounts in mg  added per 20 mg of PRX.(c)MS: Magnesium stearate.


## 3.5.4. Robustness and Ruggedness

Robustness was examined by evaluating the influence of small variation of
method variables including concentration of analytical reagent and reaction
time on the performance of the proposed methods. In these experiments, one
parameter was changed whereas the others were kept unchanged, and the recovery
percentage was calculated each time. It was found that small variation of
method variables did not significantly affect the procedures; recovery values
were 98.8 −101.5±0.85  − 1.87% (Table 4). This provided an indication for the
reliability of the proposed method during its routine application for the analysis
of PRX. Ruggedness was also tested by applying the proposed methods to the assay
of PRX using the same operational conditions but using two different
instruments at two different laboratories and different elapsed time. Results
obtained from lab-to-lab and day-to-day variations were reproducible, as the
relative standard deviations (RSDs)
did not exceed 2.54%.Table 4
Influence of small variations in
the assay conditions on the analytical performance of the proposed
spectrophotometric method for determination of PRX using NQS reagent.
ParametersRecovery (%  ± SD)(a)Recommended conditions(b)100.5±1.35NQS concentration (%, w/v)0.2598.8±0.850.75100.4±1.25Buffer solution (pH)8.898.9±1.879.2101.5±1.67Reaction time (min)599.5±1.8715100.2±0.85(a)Values
are mean of 3 determinations.(b)The recommended
conditions are given in Section 2.


## 3.6. Application of the Proposed Method to Analysis of PRX in Tablets

It is evident from the above-mentioned results that the proposed method
gave satisfactory results with PRX in bulk. Thus, its tablets were subjected to
the analysis of their PRX contents by the proposed and the official [14] methods.
The label-claim percentage was 99.17±1.06% (Table 5). This
result was compared with that obtained from the official method by statistical analysis with respect to the accuracy (by
t-test) and precision (by
F-test). No significant differences were found between the calculated and
theoretical values of
t- and F-tests at 95%
confidence level proving similar accuracy and precision in the determination of
PRX by both methods.Table 5
Analysis of PRX-containing tablets by the proposed and the official methods.
TabletRecovery
(%  ±
RSD)(a)t-value(c)F-value(c)ProposedOfficial(b)Seroxate tablets99.17±1.06101.31±0.482.204.88(a)Values are mean of 6 determinations.(b)Reference [14].(c)The
tabulated values of
t- and F-values at 95%
confidence limit are 2.78 and 6.39, respectively.


## 4. Conclusions

The present study described the successful
evaluation of NQS reagent in the development of simple and rapid spectrophotometric
method for the accurate determination of PRX in bulk and tablets. In contrast
with the previously reported methods for analysis of PRX, the method described
herein has many advantages: it does not need expensive sophisticated apparatus,
it is simple and rapid, and it has high sensitivity. Furthermore, all the
analytical reagents are inexpensive, have excellent shelf life, and are
available in any analytical laboratory. Therefore, this method is practical and
valuable for its routine application in the analysis of PRX in quality control
laboratories.



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*Source: 237601-2009-04-22.xml*