Spectrophotometric Determination of Pantoprazole Sodium in Pharmaceuticals Using N-Bromosuccinimide, Methyl Orange and Indigo Carmine as Reagents Basavaiah, K.*+, Anil kumar, U.R., Kalsang tharpa Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, INDIAVinay, K.B. Process Analytical Laboratory, Advinus Therapeutics, Peenya, II Phase, Bangalore-58, INDIAABSTRACT:Two sensitive spectrophotometric methods are presented for the assay of pantoprazole sodium sesqui hydrate (PNT) in bulk drug and in formulations using N-bromosuccinimide (NBS) and two dyes, methyl orange and indigo carmine, as reagents. The methods involve the addition of a known excess of NBS to PNT in acid medium, followed by determination of unreacted oxidant by reacting with a fixed amount of either methyl orange and measuring the absorbance at 520 nm (method A) or indigo carmine and measuring the absorbance at 610 nm (method B). In both methods, the amount of NBS reacted corresponds to the amount of PNT and the measured absorbance is found to increase linearly with the concentration of PNT which is corroborated by the correlation coefficients of 0.9959 and 0.9985 for method A and method B, respectively. The systems obey Beer’s law for 0.1 - 2.0 µg mL-1 and 0.5 - 6.0 µg mL-1 for method A and method B, respectively. The limits of detection and quantification are also reported for both methods. Intra-day and inter-day precision, and accuracy of the methods have been evaluated. The methods were successfully applied to the assay of PNT in tablet preparations and the results were statistically compared with those of the reference method by applying Student’s t-test and F-test. No interference was observed from the common tablet excipients. The accuracy of the methods was further ascertained by performing recovery studies via standard-addition method. KEY WORDS:Pantoprazole sodium, Assay, Spectrophotometry, N-bromosuccinimide, INTRODUCTION
Pantoprazole sodium sesqui hydrate (PNT) is
sesqui hydrate . Pantoprazole inhibits H+ K+ AT Pase
chemically known as sodium 5-(difluoromethoxy)-2-
pump function thereby healing the acid related
conditions. PNT is chemically more stable than omeprazole
*To whom correspondence should be addressed. +E-mail: [email protected]
and lansoprazole in neutral to mildly acidic conditions,
N-bromosuccinimide (NBS): An approximately 0.01
but under strongly acidic medium, active species is
M NBS solution was prepared by dissolving about 1.8 g
formed. PNT like omeprazole and lansoprazole also has a
of chemical (SRL Research Chemicals, India) in water
role in the eradication of Helicobacter Pylori .
with the aid of heat and diluted to one litre with water and
The literature survey reveals that only few methods
standardized . The solution was kept in an amber
are available for the determination of PNT in dosage
coloured bottle and was diluted appropriately to get 80
forms and include HPLC [3-5], HPTLC , UV spectro-
and 340 µg mL-1 NBS for use in method A and method
B, respectively. The NBS solution was stored in a
Visible spectrophotometry, because of its simplicity,
cost-effectiveness, sensitivity, selectivity , fair accuracy
Hydrochloric acid (5 M): Concentrated hydrochloric
and precision, has remained competitive in an era
acid (S.D. Fine Chem., Mumbai, India; sp. gr. 1.18) was
chromatographic techniques for pharmaceutical analysis.
diluted appropriately with water to get 5 M acid.
However, only two visible spectrophotometric methods
are found in the literature for the assay of PNT. In a
was first prepared by dissolving accurately weighed 58.8
method reported by Salamaet al.  PNT was quantified
mg of dye ( S.D. Fine Chem., Mumbai, India, 85 % dye
by stability-indicating procedure through chelation with
content) in water and diluting to 100 mL in a calibrated
iron(III) in aqueous-ethanol medium to form an orange
flask and filtered using glass wool. It was further diluted
chelate peaking at 455 nm. The method is applicable over
to obtain a working concentration of 50 µg mL-1.
Indigo carmine (200 µg mL-1 ): A 1000 µg mL-1 stock
In a report by Monstafaet al.  two methods
based on charge transfer complexation reaction using
standard solution was first prepared by dissolving
2,3-dichloro-5,6-dicyano-1,4 benzo quinine (DDQ), a π
accurately weighed 112 mg of dye (S.D. Fine Chem.,
acceptor and iodine as σ-acceptor with linearity ranges of
Mumbai, India, 90 % dye content) in water and diluting
to volume in a 100 mL calibrated flask. The solution was
The same article describes one more procedure based on
then diluted 5-fold to get the working concentration of
ternary complex formation of PNT with eosin and copper
(II) with a linear range of 4-26 µg mL-1. But all the
Methanol-ammonia 4.0 % v/v: Methanol ( S.D. Fine
methods involve the use of organic solvents and the last
Chem., Mumbai) and ammonia ( S.D. Fine Chem.,
method involves liquid-liquid extraction step.
Mumbai) were prepared as per the reference method .
The present investigation aims to develop simple,
Standard solution of pantoprazole sodium: Pharma-
sensitive and cost-effective methods for the determination
ceutical grade PNT, certified to be 99.8 % pure was
of PNT in pure form and in dosage forms using the
procured from Cipla India Ltd, Mumbai, India, and was
visible spectrophotometric technique. The methods utilize
used as received. A stock standard solution containing
NBS, methyl orange and indigo carmine as reagents. The
500 µg mL-1 PNT solution was prepared by dissolving
proposed methods have the advantages of speed and
accurately weighed 50 mg of pure drug in water and
simplicity besides being accurate and precise, and can be
diluting to 100 mL in a calibrated flask with water. The
adopted by the pharmaceutical laboratories for industrial
solution was diluted stepwise to get working
concentrations of 5 and 20 µg mL-1 PNT for method A
A Systronics model 106 digital spectrophotometer
Spectrophotometry using methyl orange (method A).
with 1-cm matched quartz cells was used for all absorbance measurements.
Different aliquots (0.2 - 4.0 mL) of a standard 5 µg mL-1
PNT solution were transferred into a series of 10 mL
Reagents and Standards
calibrated flasks by means of a micro burette and the total
All chemicals used were of analytical purity grade and
volume was adjusted to 4 mL by adding adequate quantity
all solutions were prepared in distilled water.
of water. To each flask were added 1 mL each of 5 M
Spectrophotometric Determination of …
HCl and 1 mL of NBS solution (80 µg mL-1), the last
NBS after allowing the reaction between PNT and a
being measured accurately. The flasks were stoppered,
measured amount of NBS to be complete. The residual
content mixed and let stand for 15 min with occasional
NBS was determined by reacting it with a fixed amount
shaking. Finally, 1 mL of 50 µg mL-1 methyl orange
of either methyl orange or indigo carmine dye. The
solution was added (accurately measured) and the volume
methods make use of bleaching action of NBS on the
was diluted to the mark with water and mixed well. The
dyes, the decolouration being caused by the oxidative
absorbance of each solution was measured at 520 nm
PNT when added in increasing concentrations to
Spectrophotometry using indigo carmine (method B).
a fixed concentration of NBS, consumes the latter
Varying aliquots (0.25-3.0 mL) of a standard 20 µg mL-1
proportionally and there occurs a concomitant fall in the
PNT solution were transferred into a series of 10 mL
concentration of NBS. When a fixed concentration of
calibrated flasks by means of a micro burette and the total
dye is added to decreasing concentrations of NBS, a
volume was brought to 3 mL by adding water. To each
concomitant increase in the concentration of dye results.
flask were added 1 mL each of 5 M hydrochloric acid and
Consequently, a proportional increase in the absorbance
340 µg mL-1 of NBS solution (by meansofamicro burette).
The content was mixed well and the flasks were kept
aside for 10 min with intermittent shaking. Finally, 1 mL
Preliminary experiments were performed to fix the
of 200 µg mL-1 indigo carmine solution was added to
upper concentrations of the dyes that could be determined
each flask, the volume was diluted to the mark with
spectrophotometrically, and these were found to be 5 and
water, mixed well and absorbance measured against a
20 µg mL-1 for methyl orange and indigo carmine
reagent blank at 610 nm after 10 min. In either method, a
respectively. A NBS concentration of 8.0 µg mL-1 was
standard graph was prepared by plotting the absorbance
found to bleach the red colour due to 5 µg mL-1 methyl
versus the concentration of PNT . The concentration of
orange whereas 34.0 µg mL-1 NBS was required to
the unknown was read from the calibration graph or
destroy the blue colour due to 20 µg mL-1 indigocarmine.
computed from the regression equation derived using
For both steps, i.e., the reaction between PNT and NBS,
and the determination of the latter by reacting with the
dye, HCl medium was found to be ideally suited. The
Procedure for tablets
absorbance of the dye was not affected in 0.5 to 1.5 M
A quantity of the finely ground tablet powder
HCl concentrations. However, 1 mL of 5 M HCl was
equivalent to 50 mg of PNT was accurately weighed into
selected for oxidation of drug in both methods and the
a 100 mL calibrated flask, 60 mL of water was added and
same quantity of acid was maintained for bleaching step.
shaken for 20 min; the volume was finally diluted to the
The oxidation reaction was found to be complete in 15
mark with water, mixed well and filtered using a Whatman
min for method A and 10 min for method B and contact
No. 42 filter paper.First 10 mL portion of the filtrate was
times upto 30 min had no effect on the absorbance of
discarded and a suitable aliquot of the subsequent portion
dyes. The absorbance of either dyes solution even in the
(500 µg mL-1 PNT) was diluted appropriately to get 5 and
presence of reaction product was found to be stable for
20 µg mL-1 concentrations for analysis by method A and
method B, respectively. For comparison  the same
tablet powder was extracted with 0.1 M NaOH, and after appropriate dilution to 10 µg mL-1 with 0.1 M
NaOH, the absorbance was measured at 295 nm.
A linear correlation was found between absorbance at
λmax and concentration of PNT in the ranges given in
RESULTS AND DISCUSSION
table 1. Regression analysis of the Beer’s law data using
the method of least squares was made to evaluate the
The proposed spectrophotometric methods are
slope (b), intercept (a) and correlation coefficient (r) for
indirect and are based on the determination of the residual
each system and the values are presented in table 1.
Table 1: Analytical and regression parameters of spectrophotometric methods. *Y = a+bX, where Y is the absorbance and X concentration in µg mL-1. Sa=Standard deviation of intercept, Sb=Standard deviation of slope. Table 2: Evaluation of accuracy and precision. RE: Relative error; RSD: Relative standard deviation * Mean value of seven determinations.
The optical characteristics such as Beer’s law limits
each day for five days with all solutions being prepared
and Sandell sensitivity values for both methods are given
afresh each day. The day-to-day relative standard
in table 1. The limits of detection (LOD) and quantitation
deviation values were less than 3.0 % and represent the
(LOQ) calculated according to ICH guidelines  are
best appraisal of repeatability of the proposed methods.
also presented in table 1 and reveal the very high
In order to check the validity of the proposed methods,
PNT was determined in some commercial tablets. Table 3
To evaluate the accuracy and intra-day precision of
gives the results of the determination from which it is
the methods, pure drug solution at three different
clear that there is close agreement between the results
concentration levels was analysed, each determination
obtained by the proposed methods and the label claim.
being repeated seven times. The relative error (%) and
The results were also compared statistically by a
relative standard deviation (%) were = 2 and indicate
Student’s t- test for accuracy and variance ratio F- test for
high accuracy and precision of the methods (table 2). For
precision with those of the literature method  at 95 %
a better picture of reproducibility on a day-to-day basis, a
confidence level. The calculated t- and F-values (table 3)
series of experiments were performed in which standard
did not exceed the tabulated values (t=2.77, F=6.39) and
drug solution at three different levels was determined
indicate that there was no significant difference between
Spectrophotometric Determination of … Table 3: Results of determination of PNT in tablets and statistical comparison with the reference method. *Mean value of five determinations #Marketed by: a. Alkem Ltd.; b. Cipla Ltd.; c. Aristo Ltd. Tabulated t-value at 95 % confidence level is 2.77 Tabulated F-value at 95 % confidence level is 6.39. Table 4: Results of recovery experiments by standard addition method. *Mean value of three determinations.
the proposed methods and the literature method in respect
Two useful micro methods for the determination of
The accuracy and validity of the proposed methods
PNT have been developed and validated. The methods
were further ascertained by performing recovery studies.
are simple and rapid taking not more than 20 min for
Pre-analysed tablet powder was spiked with pure PNT at
analysis. Besides, they are more sensitive than the
three different levels and the total was found by the
reported visible spectrophotometric methods [9,10] and
proposed methods. Each determination was repeated
also the chemometric methods reported by Wahbiet al.
three times. The recovery of the pure drug added was
. Whereas the already reported methods  have a
quantitative and revealed that co-formulated substances
working range of 0.5 -3.5 µg mL-1 with detection limit of
such as talc, starch, gelatin, gum acacia, calcium carbonate,
0.035 µg mL-1, the present methods (method A) has a
calcium gluconate, calcium dihydrogen orthophosphate,
detection limit of 0.02 µg mL-1 and has a wide linear
sodium alginate and magnesium stearate did not interfere
dynamic range of 0.1-2.0 µg mL-1. Precision wise the
in the determination. However, ascorbic acid was found
present methods (RSD, 0.54 -1.63 %) is comparable to
to interfere strongly in the assay. Drugs such as
that of the reported methods  (RSD, 0.5 %). The
omeprazole and lansoprazole were also found to interfere.
proposed methods rely on the use of simple and cheap
The results of recovery study are compiled in table 4.
chemicals and techniques but provide a sensitivity
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