Development of a New, Simple,
Sensitive and Cost-Effective Method for Estimation of Atenolol
in Formulation and Bulk
Mohamed Khaleel*,
Nirmal T Havannavar, Sukhen Som and Vaseeha Banu TS
Department of
Pharmaceutical Chemistry,
ABSTRACT
Atenolol is selective β1- adrenergic
receptor blocking agent with insignificant partial agonist activity and weak
membrane stabilizing properties. Atenolol is official
in Indian Pharmacopoeia (IP) and British Pharmacopoeia (BP) and the official
method for its assay is by non-aqueous titration. Literature survey revealed
non-aqueous titration used for the assay of pure drug and in formulations, High
Performance Liquid Chromatography (HPLC) and Gas Liquid Chromatography (GLC)
methods for the determination of this drug from serum & urine and
Colorimetric and Spectrophotometric methods to estimate this drug in its
formulations. But the titrimetric method suffers from
various drawbacks and is not satisfactory for pharmaceutical products. This
prompted us to develop a newer, simple and cost-effective method for estimation
of Atenolol in formulation and bulk. This method is
based upon the reaction of Atenolol with dinitrofluorobenzene in acetone in presence of borax and dioxane to develop a yellow colour which is then determined
spectrophotometrically at 389 nm (λ max of the complex formed). A series
of dilutions containing atenolol 2, 4, 6, 8, 10, 12,
14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 μg
/ml were prepared among which linearity showed at the range of 2-24 µg/ml.
Calibration plot was obtained by using above dilutions. By using the
calibration plot the amount of atenolol present in
tablet formulation and bulk was found out and the results were satisfactory and
encouraging.
KEYWORDS: Atenolol, Spectrophotometric determination, λ
max, calibration curve
INTRODUCTION
Atenolol is belonging to the class of aryloxyisopropanolamines and chemically it is
(RS)-4-(2-hydroxy-3-isopropylaminopropoxy)-phenylacetamide
with molecular formula C14H22N2O3
and half life of 6-9 hours. Due to its hydrophilic nature little atenolol penetrates the brain. It is a widely
used antihypertensive agent.
Various methods were
reported for the estimation of atenolol in bulk,
finished products and in biological fluids. Extensive literature survey
revealed that HPLC (1- 7) and GLC (8, 9) methods can be
used for the same. Besides some researchers also found out suitable fluorimetric (10) method to estimate atenolol in bulk and in formulations. Although non-aqueous
titration method is the official one for estimation of atenolol
according to IP and BP (end point determination potentiometrically),
it suffers from various disadvantages like the volume measurements are
incorrect due to the formation of meniscus, it requires large amount of sample
and also time consuming and the colour change at the end point can not be
detected exactly. Due to these reasons a need was realized to develop a newer,
simple and sensitive method for quantitative estimation of atenolol
and an attempt was made to do so for the drug in bulk and in formulation using
2, 4-dinitro-1-fluorobenzene (DNFB). The method is based on the reaction of atenolol at 75-80 0C with DNFB which gave a
light yellow colour stable for upto 2 hours.
MATERIALS AND METHOD:
Atenolol was procured as gift sample.
Before we found out the key reagent for colour
development, a number of other reagents were tried to develop newer methods for
the estimation. These include 6% Isoniazid & 30%
w/v potassium hydroxide, 0.1% w/v ninhydrin solution
in 0.05 N hydrochloric acid (HCl), 1% ferric chloride
in 0.05 N HCl, 3% w/v sodium nitrite in water and HCl, 1mg/ml cupric acetate in methanol, potassium ferricyanide 1% in distilled water and 0.4% of p-benzoquinone in ethanol. All the above reagents selected
were based upon the fact that they could react by virtue with the functional
groups present in the drug molecule i.e. an amide group, a secondary amino and
alcoholic group to give a suitable coloration. But unfortunately all these
efforts proved unsuccessful due to either one of the following reasons- no
colour difference between blank and sample or no difference in absorbance
between blank and sample or no development of colour at all in the reaction
subjected for.
Spectrophotometric Method:
DNFB reacts with compounds containing an easily
replaceable hydrogen atom like amines (11, 12, 13). The proposed
method is based on the reaction of DNFB with atenolol
to give yellow colour at 75-80 0C.
Determination of absorption
maxima:
0.5 ml and 1.0 ml of atenolol
(20 mg/ 100ml of distilled water) solution was pipetted
out in two different volumetric flasks. 1ml of DNFB reagent (prepared freshly
by mixing 1% v/v DNFB in acetone and 2.5% w/v borax solution in water in the
ratio of 1:9) was then added and the solutions were then heated for 30 minutes
at 75-80 0C. Cooled and then added 4 ml of HCl-dioxane
reagent (prepared by mixing 5% v/v HCl in 1, 4 dioxane) and the volume was made up with distilled water.
The resulting yellow colour (proposed reaction shown in scheme 1) was scanned
in the range of 300-500 nm against a reagent blank prepared in the similar way
omitting atenolol solution. The spectra obtained
shown in figure 1. From the spectra it is clear that the absorption maxima
(λ max) of the product obtained by interaction of atenolol
with DNFB is 389 nm.
FIGURE 1- Absorption maxima of
DNFB-atenolol complex
SCHEME- 1: Proposed reaction
between atenolol and DNFB
Confirmation of reaction
between DNFB and atenolol:
This was carried out to confirm the reaction between
DNFB and atenolol which gave a yellow colour and the
absorption maxima was only due to colour obtained but not due to the reagent
used. DNFB and atenolol were reacted to obtain yellow
colour in the similar method described earlier. A blank was prepared using 1ml
of 2.5% w/v of borax, 4ml of 5% v/v HCl-Dioxane. A
reagent blank was prepared in the similar method described as before. A spectra
was obtained for atenolol using DNFB against the
reagent blank (Fig 2, graph A). Subsequently another spectra was obtained using
reagent blank against the solvents (Fig 2, graph B). From the spectral analysis
it is clear that the absorption maxima at 389 nm are only for atenolol when DNFB used as the reagent.
FIGURE 2- Absorption maxima of
A.
DNFB-atenolol
versus blank
B.
DNFB versus solvents
Table 1- Validation parameters
for atenolol
|
Parameters |
Values (for bulk sample) |
Values (for tablet) |
|
Linearity range |
2-24 µg/ml |
2-24 µg/ml |
|
Precision (% RSD) |
0.342 |
0.427 |
|
Intraday (n = 3) |
0.284-1.012 |
0.247-0.985 |
|
Interday (n = 3) |
0.381-0.964 |
0.365-1.017 |
|
Accuracy (%) |
99.16-101.00 |
99.04-101.66 |
|
Specificity |
Specific |
Specific |
Parameters fixation:
Effect of DNFB reagent
concentration and other reagents:
A set of solutions each containing 10 µg/ml of atenolol and varying volumes of DNFB reagent (0.1%) ranging
from 0.1 ml to 1.5 ml in a total volume of 10 ml containing 4 ml of HCl-Dioxane reagent and water was prepared and absorbances were measured against the corresponding reagent
blank at 389 nm. The absorbances for solutions
containing 0.7 to 1.0 ml of the reagent were found to be the same. Hence 1.0 ml
of DNFB reagent (0.1%) was selected for further work. HCl-Dioxane
reagent was added to remove the excess of DNFB. It was found by experimentation
that 4 ml of 5% HCl-Dioxane reagent is necessary to
decolorize 1 ml of DNFB reagent.
Effect of solvent:
DNFB reagent was prepared in borax solution. Borax
solution renders the medium alkaline. This is necessary to neutralize the
hydrogen fluoride produced during the reaction. The other basic substances like
sodium bicarbonate, sodium hydroxide etc. was found to be not satisfactory.
Effect of time:
The absorbances of the
colored solution were measured at different intervals of heating after addition
of the reagent. The maximum absorbance was observed after 30 minutes of
heating.
The colour developed was found to be stable up to 2
hours. This was studied by measuring the absorbances
at regular interval.
Determination of concentration
range:
Aliquots containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6 and 1.7 ml of atenolol
solution (20 mg of pure atenolol in 100 ml of water)
were taken in 10 ml volumetric flasks. 1 ml DNFB reagent was added to each of
these flasks. The solutions were heated for 30 to 35 minutes at a temperature
75-80 0C. 4 ml of HCl-Dioxane reagent was
added to each and the volume was made upto 10 ml with
distilled water. Similarly reagent blank was also prepared without atenolol solution. The absorbance of the resultant yellow
solution was measured at 389 nm against the reagent blank. From the absorbance
noted it is clear that Beer’s Law is obeyed in the concentration range of 2-24
µg/ ml and a deviation is seen above it.
Preparation of calibration
curve:
Aliquots containing 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0, 1.1 and 1.2 ml of atenolol solution
(20 mg in 100 ml) were taken into a series of 10 ml volumetric flasks and
reagents added in a similar manner mentioned in determination of concentration
range and measured at 389 nm (Fig 3)
FIGURE 3- Calibration plot
Analysis of atenolol in formulation:
20 tablets (purchased from local market) each
containing 50 mg of atenolol were weighed and average
weight of each tablet was calculated. Then the tablets were ground to a fine
powder and a quantity of powder equivalent to 20 mg of atenolol
was weighed and transferred to a flask. About 50 -60 ml of distilled water was
added and shaken well, contents were then filtered into a 100 ml volumetric
flask and the residue was washed with two portions of 5 ml each of distilled
water and the volume finally made upto 100 ml. 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml of the above sample solution
were pipetted out in different 10 ml volumetric flask
and the analysis was carried out as described earlier and the results given in
table 2.
TABLE 2- Estimation of purity
of atenolol in formulation *
|
Sr. no |
Amount of pure drug calculated from label (µg/ml) |
Atenolol
found (µg/ml) |
% purity |
|
1 |
2 |
2.00 |
100.00 |
|
2 |
4 |
4.00 |
100.00 |
|
3 |
6 |
6.00 |
100.00 |
|
4 |
8 |
8.13 |
101.66 |
|
5 |
10 |
10.00 |
100.00 |
|
6 |
12 |
12.13 |
101.00 |
|
7 |
14 |
13.86 |
99.04 |
|
8 |
16 |
16.13 |
100.83 |
|
9 |
18 |
17.86 |
99.26 |
|
10 |
20 |
19.86 |
99.33 |
*
Average of 3 determinations
Analysis of pure atenolol bulk sample:
From a 200 µg/ml atenolol
solution. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 ml were
taken into a series of 10 ml volumetric flasks. 1 ml of DNFB reagent was added
to each flask and the colour was developed and procedure was followed as
mentioned earlier. The drug content in bulk sample and percentage purity was
calculated (Table 3).
Recovery experiments:
To study the accuracy, reproducibility and precision of
the above proposed method, recovery study was carried out by addition of
standard drug solution to preanalysed tablet sample
solution at different concentration taking into consideration percentage purity
of added bulk drug sample. The results of the recovery studies were found to be
satisfactory and shown in table 4.
\
TABLE 3- Estimation of purity
of atenolol in bulk*
|
Sr. no. |
Volume taken from 200 µg/ml atenolol
solution (ml) |
Amount taken of atenolol
(µg/ml) |
Amount found of atenolol
(µg/ml) |
% of atenolol |
|
1 |
0.1 |
2 |
2.00 |
100.00 |
|
2 |
0.2 |
4 |
4.00 |
100.00 |
|
3 |
0.3 |
6 |
6.00 |
100.00 |
|
4 |
0.4 |
8 |
8.05 |
100.62 |
|
5 |
0.5 |
10 |
10.1 |
101.00 |
|
6 |
0.6 |
12 |
12.0 |
100.00 |
|
7 |
0.7 |
14 |
14.13 |
100.95 |
|
8 |
0.8 |
16 |
15.86 |
99.16 |
|
9 |
0.9 |
18 |
18.00 |
100.00 |
|
10 |
1.0 |
20 |
20.13 |
100.66 |
|
11 |
1.1 |
22 |
21.86 |
99.39 |
|
12 |
1.2 |
24 |
24.13 |
100.55 |
* Average
of 3 determinations
TABLE 4- Results of recovery
studies *
|
Sr no |
Volume of 200 µg/ml of sample solution taken (ml) |
Volume of 200 µg/ml of standard
solution taken (ml) |
Calculated quantity of drug (µg) |
Estimated quantity of drug (µg) |
Percentage recovery |
|
1 |
0.1 |
0.2 |
6 |
5.9 |
98.33 |
|
2 |
0.2 |
0.2 |
8 |
8.0 |
100.00 |
|
3 |
0.3 |
0.2 |
10 |
10.0 |
100.00 |
|
4 |
0.4 |
0.2 |
12 |
12.1 |
100.08 |
|
5 |
0.5 |
0.2 |
14 |
14.2 |
101.43 |
* Average
of 3 determinations
Validation of method:
Validation of developed method was done according to
ICH Q2 (R1), 2005 guideline 14.
RESULTS AND DISCUSSION:
The proposed method for estimation of atenolol in bulk and tablet dosage form was found to be
simple, economical, accurate and sensitive.
Absorption maxima
The scanning of the atenolol-DNFB
complex at 300-500 nm revealed the λ max at 389 nm (Fig 1). At this
wavelength drug obeys Beer’s law over the concentration range of 2-24 µg/ml.
Above this concentration there is a deviation.
Calibration curve:
The data obtained for calibration curve by proposed
method indicate that response is linear over the concentration range of 2-24
µg/ml (Fig 3). Percentage RSD (Relative Standard Deviation) is less than 2% at
the wavelength selected.
Validation of method:
Precision:
The precision of the analytical method is determined by
assaying a sufficient number of aliquots of homogenous sample to be able to
calculate statistically valid estimate of % RSD. Repeatability of a standard
solution was carried out using six replicates of same solution. It showed RSD
of 0.342 and 0.427 for bulk and tablet dosage form respectively. This confirms
that method is precise as RSD is well below 2%. Intermediate precision of the
method was determined for same sample by 3 different analysts on different time
duration. Validation parameters for analysis of atenolol
are presented in table 1.
Accuracy:
Accuracy of the method was determined by spiking
working standard into tablet solution. Recovery study was carried out by
standard addition method. Percent recovered was calculated by comparing the
absorbance before and after the addition of working standard. Recovery of atenolol in the range of 98.33-101.43 % (Table 4) shows
method may be used for routine analysis of atenolol
in tablet dosage form. The % recovery indicates the accuracy of the developed
method.
Specificity:
Results of specificity studies show no interference of
excipients.
Analysis of marketed
formulation
The developed method was applied to the analysis of atenolol in tablet dosage form. Results given in table 2.
The content of atenolol in marketed tablet dosage
form was found to be in the range of 99.04-101.66% with RSD less than 2% which
indicates the suitability of the proposed method for routine analysis of atenolol in tablet dosage form.
CONCLUSION:
The proposed study describes new UV Spectrometric
method for the estimation of atenolol in bulk and
tablet dosage form. The method was validated and analysis proves that the
method is simple, sensitive, economical, accurate and precise. % of recovery
shows that the method is free from interference of the excipients used in the
formulation. As proposed previously the reaction of DNFB is taking place with
the free –NH2 group (of amide) of atenolol,
there is a very scope to develop any other reagent which can react with the
secondary alcoholic group present in the molecule to give a colour which can
then be determined spectrophotometrically. And as the method described is easy
to understand, accurate, sensitive, simple, reliable and less time consuming,
we hope that in near future it can be adopted successfully in the routine quantitative
estimation of atenolol in formulation or in the raw
material as bulk.
REFERENCES:
1. Modamio P, Lastra C F and Marino
E L. Error structure for the HPLC analysis for atenolol,
metoprolol and propranolol:
a useful weighting method in parameter estimation. J of Pharm Biomedical Analysis.
1998; 17, 3:
July, 507-513
2. Ravi S S, Nanjan M.
J, Vasudevan M, Shaat N,
Suresh B. Simultaneous estimation of atenolol
and amlodipine in formulations by reversed phase:
HPLC.
3. Morris R G.; Saccoia
N C.; Sallustio B C.; Zacest
R. Improved High-Performance Liquid Chromatography Assay for Atenolol in Plasma and Urine Using Fluorescence Detection.
Therapeutic Drug Monitoring, 1991; 13: 4
4. Leite, F S; Pereira V A, Omosako
C E, Carvalho C M J, Costa A, José O J. Cavani J S, Silvia R. A Micromethod
for the Quantification of Atenolol in Plasma Using
High-Performance Liquid Chromatography with Fluorescence Detection: Therapeutic
Drug Monitoring of Two Patients with Severe Coronary Insufficiency before
Cardiac Surgery. Therapeutic Drug Monitoring. 2006; 28: 2. 237-244
5. Gantala V
S, Ramanathan S.M, Mansor
N.K.N, Mvar A S, Simon L. C and Navaratnam
V. Development and validation of RP-HPLC-UV method for simultaneous
determination of buparvaquone, atenolol,
propranolol, quinidine and verapamil: A tool for the standardization of rat in situ
intestinal permeability studies. J of Pharm and Biomed Analysis.
2007; 43: 4.
1546-1551
6. Rontogianni
M; Markopoulou C; Koundourellis J. HPLC and Chemometrically‐Assisted Spectrophotometric
Estimation of Two Binary Mixtures for Combined Hypertension Therapy. J of Liquid
Chromatography & Related Technologies, 2006; 29; 18. 2701-2719
7. Prasad C V N, Parihar
C, Sunil K and Parimoo
P. Simultaneous determination of amiloride HCl,
hydrochlorothiazide and atenolol in combined
formulations by derivative spectroscopy. J of Pharm and Biomedical
Analysis. 1998; 17:4-5.
877-884
8. Peng J, Tu J, Xin J. Determination of Atenolol
in Human Plasma by Gas Chromatography. J of China Pharmaceutical University, 1995-06
9. Lho, D, Hong J
K, Paek H K, Lee J A, Park J. Determination of Phenolalkylamines,
Narcotic Analgesics, and Beta-Blockers by Gas Chromatography/Mass Spectrometry.
J of Analytical Toxicology,
1990; 14: 2. 77-83(7)
10. Morris R G. Improved Liquid Chromatographic
Fluorescence Method for Estimation of Plasma Sotalol
Concentrations. Therapeutic Drug Monitoring. 1989; 11: 1
11. Stanley H. P. organic chemistry.
12. Robert T M and Robert N B. organic
Chemistry. Pearson education,
13. Arun B and Bahl B S.
Advanced Organic Chemistry. S Chand and Company Ltd.
14. ICH-Q2 (R1) (2005), Guideline on Validation
of Analytical Procedure. Text and Methodology, International Conference on
Harmonization.
Received on 03.12.2009
Accepted on 26.01.2010
© A&V Publication all right reserved
Research Journal of Pharmaceutical Dosage
Forms and Technology. 2(1): Jan. –Feb. 2010, 72-76