American Journal of Analytical Chemistry, 2016, 7, 179191

Published Online February 2016 in SciRes.

http://www.scirp.org/journal/ajac
http://dx.doi.org/10.4236/ajac.2016.72015

Spectrophotometric Study for the

Reaction of Pentoxifylline
Hydrochloride with
1,2-Naphthoquinone-4Sulphonate: Kinetics, Mechanism
and Application for Development
of High-Throughput Kinetic
Microwell
Ashraf M. Mahmoud Assay
, Saad A. for Pentoxifylline
AlQahtani
Department
of Pharmaceutical
Analytical Chemistry,
Faculty of Pharmacy, Assiut
in
Quality
Control
Laboratory
University, Assiut, Egypt Department of Pharmaceutical Chemistry, College of
1,2*

Pharmacy, Najran University, Najran, Kingdom of Saudi Arabia

3

Department of Clinical Pharmacy, College of Pharmacy, Najran University, Najran,

Kingdom of Saudi Arabia
Received 17 December 2015; accepted 30 January 2016; published
2 February 2016
Copyright 2016 by authors and Scientific Research Publishing Inc.
This work is licensed under the Creative Commons Attribution
International License (CC BY).
http://creativecommons.org/licenses/by/4.0/

Abstra
ct
Spectrophotometric study was carried out, for the first time, to investigate
the reaction between the vasodilator pentoxifylline hydrochloride (POX) and
1,2-naphthoquinone-4-sulphonate (NQS) reagent. The reaction occurs in
alkaline medium to activate the nucleophilic substitution reaction producing
an orange-colored product measured spectrophometrically at max 472 nm.
The va-riables affecting the reaction were carefully studied and the
conditions were optimized. The ki-netics of the reaction was investigated and
its activation energy was found to be 0.262 cal/mol. Owing to its low
activation energy, the reaction proceeded easily and was successfully used
for simple and rapid assay of POX. The stoichiometry of the reaction was
determined (1:1), and the reaction mechanism was suggested. To develop a
high-throughput methodology used in quality control laboratory, a
*
Corresponding author. study of the reaction using the conventional spectrophotome-tric
comparative
versus
microwell
assay
was applied.
theS.A.
optimum
reaction conditions,
How
to cite
this paper:
Mahmoud,
A.M. andUnder
AlQahtani,
(2016) Spectrophotometric
Study
thethe
initial
rateof Pentox-ifylline Hydrochloride with 1,2-Naphthoquinone-4-Sulphonate: Kinetics,
for
Reaction

Mechanism and Application for Development of High-Throughput Kinetic Microwell Assay for
Pentoxifylline in Quality Control Laboratory. American Journal of Analytical Chemistry, 7, 179-191.
http://dx.doi.org/10.4236/ajac.2016.72015

A. M. Mahmoud, S. A.
AlQahtani

and fxed time methods were utilized for constructing the calibration graphs
for determination of POX concentrations. The linear range was 10 - 120
g/ml with good correlation coefficients (0.9987 - 0.9998). The LOD was 2.5
and 3.4 g/ml for initial rate and fxed time methods, respec-tively. The intraand inter-day accuracy and precision of the developed methods were
satisfactory, where RSD was 3.94%. The present methods have been
successfully applied to the determination of POX in its pharmaceutical tablets,
and the percentage recovery values were 97.9% - 101.9%. Therefore, we
strongly recommend the proposed methods for determination of POX in
quality
control laboratories.
Keywor

ds
Pentoxifylline HCl, 1,2-Naphthoquinone-4-Sulphonate, Kinetic

Spectrophotometry, Pharmaceutical Analysis, Micro-Well Plate

Reader

1.
Introduction
Pentoxifylline hydrochloride (POX); 3,7-dihydro-3,7-dimethyl-1-(5-oxohexyl)-1H-purine-2,6-dione is the
first clinically effective drug approved for the treatment of intermittent claudication secondary to chronic
occlusive vascular disease [1]. It was used for treatment of Reynoulds syndromes and transient ischemic
attacks, too. Moreover, it can also be used as a vasodilator to lower the blood pressure in treatment of
peripheral vascular disorders [1] [2]. Recently, POX has wide clinical applications in dermatology because it
has a variety of physi-ological effects at the cellular level, which may be very important in treating a diverse
group of diseases as pso-riasis. In most cutaneous diseases, POX was regarded as a valuable therapeutic
adjuvant. POX may best serve as a corticosteroid-sparing agent in dermatology [3]. Because of the therapeutic
importance and clinical success of POX, it has been widely used throughout many countries worldwide [3].
Numerous analytical methods have been developed for the quantitative determination of POX in pure,
phar-maceutical dosage forms and/or biological fluids. The methods published prior to 1998 have been
reviewed by Indrayanto G et al. [2]. The reported methods after 1998 include spectrophotometry [4] [5],
fluorimetry [6], high-performance liquid chromatography [7]-[13], thin layer chromatography [13], Gas
chromatography [14], and electrochemistry [15]. Despite of the higher sensitivity and selectivity of the
separation-based methods, the instrumentation and high analysis cost limited their use in quality control
laboratories. Spectrophotometry is the most convenient and widely available analytical technique due to its
inherent simplicity and low cost. Kinetic spectrophotometry has been becoming of great interest in
pharmaceutical analysis of drug content [16] owing to its improved selectivity. The application of microwell
plates as reaction vessels instead of the calibrated flasks and classical cuvettes in the pharmaceutical analysis
of drug content is becoming of great interest and broadened to the dissolution studies where higher limits of
quantification are not required [17] [18]. This application can increase the automation and high-throughput
property as well as decreasing the cost of the analysis to become more suitable for the determination of drug
substance in quality control laboratories [19] [20]. 1,2-naphthoqui-none-4-sulphonate (NQS) has been used for
the determination of many pharmaceuticals [21]-[24]. The substitu-tion reaction between NQS and POX has
not been investigated, yet. For these reasons, the present work was devoted to explore NQS in the
development of a high-throughput microwell assay, for the first time, for the de-termination of POX in its
pharmaceutical tablets. To develop an automated high-throughput methodology used in quality control
laboratory, a comparative study for the reaction using the conventional spectrophotometry versus microwell
assay
2. was made.

Experimental
2.1.
Multi-Detection Microplate Reader (BioTek Synergy 2, BioTek Instruments Inc., USA) was used for all
mea-surements in 96-microwell plates. A Shimadzu model UV-1601 PC (Kyoto, Japan) UV-VIS double beam
Instrumentation
spec-trophotometer with matched 1-cm quartz cells was used for recording the absorption spectrum.
Polypropylene microplates, 96 well with U bottom were obtained from Santa Cruz Biotechnology, Inc.
(USA). Finn pipette
18
0

A. M. Mahmoud, S. A.
AlQahtani

adjustable 8 channel-pipettes were purchased from Sigma Chemical Co.,

USA.

2.2. Chemicals, Reagents and Pharmaceutical

Formulations

Pentoxifylline hydrochloride (POX), (Servier Egypt Industries Limited, Egypt). 1,2-Naphthoquinone-4-sulpho2

nic acid (NQS) sodium salt (Aldrich Chemical Co., Milwaukee, USA) was 5 10 M aqueous solution.

Sodium hydroxide solution was 0.25 M aqueous solution. Trental (Servier Egypt Industries Ltd., Egypt),

Vasotal (T3A, Egypt), and Pexal (Mepha, Egypt) tablets are labeled to contain 400 mg of pentoxifylline
hydrochloride per tablet. All solvents and other chemicals used throughout this study were of analytical grade.

2.3. Preparation of Standard

Solutions

An accurately weighed amount (60 mg) of POX was quantitatively transferred into a 50-ml calibrated
flask, dissolved in 30 ml distilled water, completed to volume with distilled water to produce a stock solution
of 1.2 mg/ml. This stock solution was then diluted with distilled water to obtain working standard solutions
of 100 -1200 g/ml.

2.4. Preparation of Sample Solution of

Pharmaceutical Tablets

Twenty tablets of each formulation were weighed and finely powdered. A quantity of the mixed powder
equiva-lent to 40 mg of POX was transferred into a 50-ml calibrated flask and dissolved in 25 ml distilled
water. The contents of the flask were swirled, sonicated for 5 min, and then completed to the mark with
distilled water. The contents were mixed well, filtered, and the first portion of the filtrate was rejected. The
filtrate solution yield working solutions of 800 g/ml for analysis.

2.5. General Recommended

Procedures
2.5.1. Using the Conventional Spectrophotometry
Accurately measured aliquots of POX solution containing 100 - 1200 g/ml were transferred into separate 10ml calibrated flasks. Two milliliters of sodium hydroxide solution (0.25 M) was added followed by 2 ml of
2
NQS solution (5 10 M). The solution was diluted to volume with distilled water and mixed well. After
mixing, the reaction mixture was immediately transferred to a spectrophotometric cell and the absorbance was
recorded as a function of time at room temperature (25C 2C) for 10 min at 472 nm against reagent blank
treated
2.5.2.similarly.
Using the Microwell Plate Format
Hundred microliters of the standard or sample POX solution of OMZ (100 - 1200 g/ml) was transferred
into the wells of 96-microwell plate. Fifty microliters of sodium hydroxide solution (0.25 M) followed by 50
2
l of NQS solution (5 10 M) were added to each well. The absorbance intensities of the resulting
solutions were recorded as a function of time at room temperature (25C 2C) for 10 min. The measurements
were made us-ing optical filter 475 nm and multi-detection microplate reader. Blank wells were treated
similarly using 100 l of distilled water instead of POX. The absorbance intensities of the blank wells were
subtracted from those of standard or sample wells.

2.6. Determination of the Reaction

Stoichiometry

The Jobs method of continuous variation [25] was employed for determination of the stoichiometry of the
4
reac-tion between POX and NQS. Master equimolar (3.6 10 M) aqueous solutions of POX and NQS
were pre-pared. Series of 5ml portions of the master solutions of POX and NQS were made up comprising
different com-plementary proportions (0:10, 1:9 9:1, 10:0, inclusive) in 10.0 ml-calibrated flasks. Two
milliliters of 0.25 M NaOH was added to each flask, and the solutions were diluted to volume with distilled
water and mixed well. The absorbance of the solutions was measured after 10 min at 472 nm against reagent
blank treated similarly.

2.7. Data Acquisition and

Processing

The kinetic data recorded for the designed methods was transformed to the Microsoft Office Excel
software,

18
1

A. M. Mahmoud, S. A.
AlQahtani

version 2010, for curve fitting, regression analysis, and statistical calculations. In the initial rate method of analysis, the initial rate (V) of the reaction was obtained from the slope of the absorbance-time curve at definite
concentration. The calibration curve was constructed by plotting (log V) at different concentrations versus (log
C) of POX. Alternatively, the fixed time method was used for constructing the calibration curve by plotting the
ab-sorbance measured after fixed time of 10 min vs. the corresponding POX concentrations. The least squares
me-thod was used for getting the regression equation.

2.8. Validation of the Proposed Kinetic

Methods

The following validation parameters were assessed and evaluated according to ICH guidelines [26]. The standard curve for determination of POX was constructed applying the general assay procedure using either the initial rate or fixed time method. After getting the regression equation, the limit of detection (LOD) and limit of
quantitation (LOQ) were determined [26] using the formula: LOD or LOQ = SDa/b, where = 3.3 for LOD
and 10 for LOQ, SDa is the standard deviation of the intercept, and b is the slope (sensitivity) of the proposed
method. Intra- and inter-day accuracy and precision of the proposed methods were assessed by recovery studies
at three (low, medium and high) concentration levels. Recovery was determined by the standard addition
method. Known amounts of POX were added to the pre-determined drug-containing dosage forms, and then
determined by the proposed methods. The mean recovery was determined by dividing the measured
concentrations to the concentrations taken for analysis, expressed in percentages. The intra-day precision and
accuracy were deter-mined by carrying out the assay of six replicate samples of each concentration as one batch
in a single run, while the inter-day precision and accuracy were determined by repeating the assays for the same
samples at three dif-ferent days. The selectivity of the proposed method was studied by testing the
interferences liability from the excipients of the dosage forms. Common excipients such as starch, glucose,
lactose, acacia, and magnesium stearate were used in this study. The recovery was calculated each time.
Ruggedness was also tested by two dif-ferent analysts at two different elapsed times. Moreover, robustness was
evaluated at small variation in the rea-gents concentrations, reaction temperature and reaction time

3. Results and
Discussion
3.1. Involved Reaction, Design and Strategy of the
Assay Development

1,2-Naphthoquinone-4-sulphonate (Folins reagent) is frequently used as an analytical reagent for the

determi-nation of nucleophilic analytes in weak alkaline medium at normal or slightly elevated temperature. It
has been used for the determination of primary amines [21]-[23], secondary amines [24], thiols [27] and active
methylene containing compounds [28] [29] by spectrophotometry [21], [23] [24] and spectrofluorometry [22],
[27] after the reduction of the condensation product to the fluorescent 1,2-dihydroxynaphthalene-4-derivative.
In the present work, POX was found to react with NQS producing an orange-colored product measured at max
472 nm. Figure 1 shows the absorption spectrum of the reaction product of POX with alkaline NQS. The
preliminary investigation of the reaction showed that the absorbance of the colored product increased with time
to the maximum, then ra-pidly decreased without a stable period for the measurements. This finding attracted
our notice to develop an automated kinetic spectrophotometric assay for the determination of POX to
overcome this disadvantage. This can be done by carrying out the reaction in 96-microwell plate as a reaction
vessel and measuring the signal with a microplate reader depending on our previous experience [19] [20]. In
order to come to this conclusion, the reaction was investigated under various conditions of reagent
concentration and alkalinity as well as reaction time and temperature. The following sections describe the
conditions under which the reaction of POX with al-kaline NQS fulfills the requirements necessary for its
spectrophotometric analysis.

3.2. Optimization of the Reaction

Conditions

In order to investigate the effect of NQS concentration on the reaction product, different concentrations (1.0 - 10
2
10 M) of NQS working solution were tested. The results revealed that the absorbance and the initial reaction
rate increased with increasing the concentration of NQS and maximum values were attained when the NQS
3
2
concentration reached 5 10 M in the final solution (5 10 M working solution) as shown in Figure 2. Also,
2
different volumes (0.5 - 4.0 ml) from NQS working solution (5 10 M) were tested. The results indicated that
2
2 ml of 5 10 M NQS working solution yielded the highest values. Therefore further testing was performed

18
2

A. M. Mahmoud, S. A.
AlQahtani

Figure 1. Absorption spectrum of the reaction product of POX (80 g/ml)

with NQS reagent (0.005 M) in alkaline medium (0.05 M NaOH) at room
temperature.

Figure 2. Effect of molar concentrations of both NQS reagent () and

NaOH () on the absorption intensity of the reaction product of POX (80
g/ml) with NQS.
2

using 2 ml of 5 10 M solution of NQS using the conventional spectrophotometry. Using the microwell
2
plate format, the results indicated that 50 l of 5 10 M NQS solution in total volume of 200 l (plate well
volume) resulted in the highest absorbance values.
To investigate the effect of sodium hydroxide concentration on the initial reaction rate, different
concentra-tions (0.01 - 1.0 M) of NaOH solution were tested. The results showed that maximum absorbance
was obtained when NaOH concentration of 0.025M in the final solution was used (0.25 M working solution)
(Figure 2). Fur-thermore, different volumes (1.0 - 5.0 ml) from 0.25 M working solution were investigated
and indicated that 2 ml of 0.25 M NaOH solution in total volume of 10 ml was the ideal concentration.
Using the microwell plate format, the results indicated that 50 l of 0.25 M NaOH solution in total volume of
200 l gave the highest ab-sorbance values.

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A. M. Mahmoud, S. A.
AlQahtani

Because the formation of the colored product increases with time, we had to optimize the time of the
reaction between POX and NQS by monitoring the reaction at different times (0.2, 2.5, 5, 7.5, 10, 12.5, 15,
17.5, 20, 25, 30 min). Figure 3 showed that the highest absorbance values were obtained after 10 min
followed by gradual decrease in the absorbance after 12 min. This is a strong disadvantage if we use the
conventional spectrophoto-metry. However, we can overcome this disadvantage by using either the microwell
plate format or the kinetic initial rate method for the analysis.
In spite of heating the reaction mixture in the range of 25C - 50C had slight positive effect on the
reaction, however to simplify the procedure, further testing was performed at room temperature (25C
2C). The sol-vents used for NQS reagent as well as for dilution of the reaction mixture were carefully studied
using different solvents: water, methanol, ethanol, isopropanol, and acetonitrile. Water was found to be the
optimum solvent as the highest absorbance values were obtained. Table 1 shows the optimum conditions for
the reaction between POX and NQS using both the conventional and 96-microwell spectrophotometric
methods.
3.3.
Stoichiometry and Reaction

Mechanism

The stoichiometry of the reaction of POX with NQS was determined by the Jobs method of continuous
varia-tion [25]. The POX: NQS ratio was found to be 1:1. The POX molecule contains four tertiary amino
groups and only one active methylene group. The tertiary amino groups cannot react with NQS, however, the
active methy-lene group can react with alkaline NQS. Therefore, the mechanism of POX-NQS reaction was
suggested to pro-ceed according to the pathway given in Figure 4.

Figure 3. Effect of time on the absorption intensity of the reaction product of

POX (90 g/ml) with NQS (0.005 M) in alkaline medium (0.05 M NaOH) at
room temperature.

Figure 4. The proposed pathway for the reaction of POX with alkaline NQS.

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4

A. M. Mahmoud, S. A.
AlQahtani
Table 1. Optimum conditions for the substitution reaction of POX with NQS.
Conditions

Conventional spectrophotometric method

Microplate method

NQS working conc. (M)

0.05

0.05

NQS volume (l)

2000

50

NaOH working conc. (M)

0.25

0.25

NaOH volume (l)

2000

50

Reaction time (min)

10

10

Temperature (C)

25 2

25 2

Solvent

Dist. water

Dist. water

Total volume of final solution l)

10,000

200

Measuring instrument

UV-VIS Spectrophotometer

Microplate reader

Wavelength of measurements (nm)

472

475

3.4. Kinetics of the Investigated

Reaction
3.4.1. Order of the Reaction
Under the described optimum conditions (Table 1), the absorbance-time plots for the reaction of POX with alkaline NQS reagent was constructed (Figure 5). The initial rates of the reaction were determined from the
slopes tangents of the absorption-time curves. The order of the reaction with respect to NQS was determined
by investigating the reaction at different NQS concentrations keeping the concentration of POX constant. The
plot of the initial rate, against NQS concentrations was linear with a slope value of 1.007 indicating that the
reaction is a first order with respect to NQS. The plot of the initial rate, against POX concentrations (keeping the
reagents concentrations constant) was linear with a slope value of 0.953 describing a first order rate with
respect to POX, too. However under the optimized experimental conditions, the concentration of POX was
determined using rel-ative excess amounts of the analytical and other conditional reagents. Therefore,
pseudo-first order conditions were obtained with respect to POX and the reaction rate was found to obey the
logV log A t log K n log C
(1)
Equation (1) [30]:
where: V is the initial reaction rate, A is the absorbance intensity, t is the measuring time, K is the pseudofirst order rate constant, C is the molar concentration of POX, and n (slope) is the order of the reaction. The
results showed a linear plot with good correlation coefficient (r) equal 0.9977 and slope (n) equal 0.95
(1) which proved that the order of the investigated reaction was first order.
3.4.2. Activation Energy and the Standard Free Energy Change of the Product
The activation energy of the reaction under investigation can be determined from the logarithmic form of
Arrhe-nius equation [31]:
where: k is the rate, A is a constant known
as Arrhenius
frequency
factor, Ea is the activation energy, T is (2)
log k log
A Ea 2.303
RT
the absolute temperature (273+C), and R is the gas constant (1.987 cal/ molC).
The activation energy was determined by measuring the absorbance of the reaction product at different
tem-peratures; 25C, 30C, 35C, 40C, and 50C keeping the concentrations of POX, NQS and NaOH
constant. The absorbance-time plot at these temperatures were constructed to determine the rates, then plotting
log rates (k) vs 1/T to determine the slope (Ea/2.303R) of the line as shown in Figure 6. The value of the
slope was 0.0573 and the activation energy of the investigated product was 0.262 cal/mol. The positive
sign of the activation energy indicated that the product formation increased with increasing the temperature,
however, its very small value indicated the slight positive effect of heating the reaction mixture.
The standard free energy change of the reaction product was determined using the equation III [31], and
its value was 2.903 Kcal/ mol. The Negative sign of G indicates endothermic condensation reaction.
18
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A. M. Mahmoud, S. A.
AlQahtani

Figure 5. Absorbance-time curve for the reaction of POX with NQS (0.005 M)
in alkaline medium (0.05M NaOH) at room temperature. The molar concentrations of POX were 0.54
104 (), 1.08
104 (), 2.16
104 (), 3.24
4
4

10 (), and 3.6

10 M ().

Figure 6. Arrhenius plot for the activation energy of the reaction of POX (90
g/ml) with NQS reagent (0.005 M) in alkaline medium (0.05 M NaOH) at
different reaction temperatures (25C, 30C, 35C, 40C, and 50C). 1 was
added to the values of log K to eliminate the negative sign of the values.
0

G 2.303RT log K

3.5. Validation of the Proposed

Methods

Using the general assay procedures (Table 1), the following validation parameters were assessed and
evaluated according to ICH guidelines [26].
3.5.1. Linearity, Limits of Detection and
Quantitation 1) Initial Rate Method
The quantitation depending on initial rates of the POX reaction would follow a pseudo-first order
Equation
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6

(3)

A. M. Mahmoud, S. A.
AlQahtani

(1). Regression analysis using the method of least square resulted in the equation (log V = 2.129 + 0.953 log
4
4
C) with good correlation coefficient (0.998). The linear range was 0.4
10 - 4.31
10 M (10 - 120 g/ml)
with a limit of detection (LOD) and limit of quantitation of 2.51 and 7.59 g/ml, respectively. These values
con-firmed the suitability of the method for determination of POX in quality control laboratory.
2) Fixed Time Method
In this method, the absorbance of the reaction product for each POX concentration was measured at a
prese-lected fixed time. Standard curve of absorbance vs. corresponding POX concentrations was established
after 10 min. Regression equation (A = 0.0085 X 0.0124) with linear range (10 - 120 g/ml) and good
correlation
coefficients
(0.9997)
was obtained. The LOD and LOQ were 3.4 and 10.2 g/ml, respectively
3.5.2. Accuracy
and
Precision
(Table 2).
Intra- and inter-day accuracy and precision of the designed methodology were assessed [32] by recovery
studies at three concentration levels (15, 60 and 100 g/ml). Table 3 shows the values of the intra- and interday preci-sion (determined as % RSD) and accuracy (determined as % recovery) of the proposed
conventional and mi-crowell spectrophotometric methods using both initial rate and fixed time. This high
level of precision and ac-curacy indicated the suitability of the designed methodology for the analysis of POX
in quality control labora-tory. For comparative evaluation and/or recommendations of these methods
depending on the precision level, the designed microwell assay might be highly recommended for routine
analysis of POX in quality control la-boratories.
3.5.3. Selectivity of the Proposed Methodology
The designed methods have the advantage of that all measurements are performed in the visible region at
472 nm, away from the near UV absorbing interfering excipients that might be co-extracted from POXcontaining dosage forms. The potential interferences from the excipients were investigated. Samples were
prepared by mixing known amount (20 mg) of POX with various amounts of the common excipients such as
starch, glucose, lactose, acacia, and magnesium stearate. The results (Table 4) revealed that no interference
was observed from any of these excipients with the designed methods, where the average recovery results was
101.41%
1.69%.
3.5.4. Ruggedness
and Robustness
The ruggedness was tested by applying the designed kinetic methods to the assay of POX using the same
opti-mum conditions but using two different analysts and different elapsed time. Results obtained were
reproducible as RSD did not exceed 3%.
Robustness
of theand
designed
procedure for
wastheexamined
evaluating
influencemicrowell
of small variation in
Table
2. Quantitative
kinetic parameters
analytical by
performance
of the
the designed
the
spectrophotometric method after fixed time of 10 min.
Parameter

Value
1

Linear range (gml )

10 - 120

Intercept

0.0124

Standard deviation of the intercept

0.0086

Slope

0.0085

Correlation coefficient (r)

0.9997

Limit of detection, LOD (gml1)

1

3.4

Limit of quantification, LOQ (gml )

10.2

Order of the reaction (n)

0.96 (~1.0)
1

The activation energy, Ea (calmol )

0.262

The standard free energy change, G (Kcalmol1)

2.903

Molar absorptivity, (l mol cm )

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2.37 103

A. M. Mahmoud, S. A.
AlQahtani
Table 3. Evaluation of the intra- and inter-day accuracy and precision of the initial rate and fixed time methods of the
de-signed methodology for determination of POX.
Recovery (% RSD)a

Spectrophotometric method

Amount taken (gml )

Initial rate method

Conventional

96-Microwell plate

Intra-day

Fixed time method

15

97.56 2.97

98.22 3.86

60

101.30 2.12

99.03 1.88

100

100.85 1.46

101.24 1.90

15

98.01 2.15

100.31 1.91

60

101.72 0.98

100.24 1.08

100

100.10 0.71

100.36 0.84
Inter-day

Conventional

96-Microwell plate

15

97.25 3.83

60

98.35 3.94

101.10 2.14

99.15 2.31

100

101.31 1.60

100.17 1.80

15

97.86 1.87

100.71 2.08

60

101.10 1.16

100.34 1.26

100

100.81 0.85

100.15 0.91

Recovery was calculated as the amount found/amount taken

100. Values are mean RSD for six
determinations.

Table 4. Determination of POX in presence of common excipients by the designed microwell spectrophotometric
method after fixed time of 10 min.

Ingredient

Recovery ( % SD)a

Starch (50)b

100.51 2.84

Glucose (10)

103.22 2.65

Lactose (10)

102.44 2.71

Acacia (10)

101.92 2.64

Magnesium stearate (10)

98.94 2.96

Average SD

101.41 1.69

Pool SD

2.76
b

Values are mean of three determinations; Figures in parenthesis are the amounts in mg added per 20 mg of POX.
2

concentration of NQS (4.5, 5.0 and 5.5 10 M) and NaOH (0.23, 0.25 and 0.27 M) reagents, in the reaction
temperature (23C, 25C, and 27C) or in the reaction time (9, 10, 11 min) on the analytical performance of
the designed methods. In these experiments, one experimental variable was changed, whereas the others were
kept unchanged, and the recovery percentage was calculated each time. It was found that the small change in
temper-ature, reaction time, or the reagents concentrations did not significantly affected the results; where the
recovery percentages were 98% - 102% 0.82% - 2.67%. This provided an indication for the reliability of the
proposed methods during routine work.

3.6. Application of the Designed Methodology to the Analysis

of Dosage Forms

Depending on the obtained validation results, the designed procedures (initial rate or fixed time) were found
to

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A. M. Mahmoud, S. A.
AlQahtani
Table 5. Analysis of POX-containing tablets by the reported and the designed methodology for determination of
a
POX .
Spectrophotometric
method

Conventional

96-Microwell plate

Initial rate method

Tablet

Fixed time method

Label claim
(% RSD)a

t-value

F-value

0.72

101.60 2.35

0.04

0.35

0.13

0.75

100.36 2.91

0.59

0.31

97.91 3.24

0.90

0.21

99.46 2.42

0.22

0.51

Trental

99.09 0.67

0.75

0.10

100.21 1.36

0.04

0.82

Pexal

101.92 1.95

0.08

0.85

101.51 1.96

0.10

0.59

Vasotal

98.62 1.06

0.59

0.28

98.40 1.24

0.70

0.46

Label claim
(% RSD)a

t-valueb

F-value

Trental

100.96 1.28

0.09

Pexal

100.81 2.08

Vasotal

The label claim % for Trental, Pexal and Vasotal tablets determined by the reported method [5] were 98.81 1.51, 99.46 1.79, and 98.1 1.77,
b
re-spectively. Values are mean RSD of six determinations. The tabulated values of t and F at 95% confidence limit are 1.81 and 5.05, respectively.

be suitable for the routine quality control analysis of POX. The concentration of POX was calculated from its
corresponding regression equations. The proposed and the reference methods [5] have been tested on
commer-cial tablets. The results obtained by the present methods were statistically compared with those
obtained by the reference method with respect to the accuracy and precision. The recovery of the labeled
amount was 97.91% -101.92% 0.67% - 3.24% (Table 5). The results of t- and F-tests showed that there are
no significant differenc-es between the present and reference methods at 95% confidence level. This proved
the applicability of the de-signed methods for analysis of POX in its pharmaceutical tablets with
comparable analytical performance. However, the comparative evaluation and/or recommendations of these
methods might be based on the high-throughput value, automation and the precision level of these methods.
According to the above consideration, the designed microwell assay might be highly recommended for
routine analysis of POX in quality control la-boratories.

4.
Conclusion
The present investigation described the successful use of NQS reagent in the development of simple and
rapid spectrophotometric methods for the determination of POX in its tablets. The present methodology was
based on the substitution reaction of POX with NQS in alkaline medium. The kinetics of the reaction was
determined and both the initial rate and fixed time methods were successfully applied to the determination of
POX. The present methodology was carried out in 96-microwell plate as a reaction vessel and compared to the
conventional spec-trophotometry. The designed method is superior to all the reported conventional
spectrophotometric methods for determination of POX in terms of automation and high-throughput.
Furthermore, all the analytical reagents are inexpensive, have excellent shelf life, and are available in any
analytical laboratory. Therefore, the designed methodology is practical and valuable for routine use as
alternative for the existing methods for analysis of POX in quality control laboratories.

Acknowledgem
ents
The authors thank the Deanship of Scientific Research, Najran University for funding the work; Project
Code [nu/mid/14/062].

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