Determination
of Atenolol in Tablet Formulation by Analytical Spectrophotometry
Saad Antakli, Leon Nejem, Moustafa Alabo Joumaa
Department of Chemistry, Faculty of Science, University
of Aleppo, Syria.
*Corresponding
Author E-mail: antakli@scs-net.org
ABSTRACT:
Simple and rapid spectrophotometric method
has been developed for the determination of Atenolol in bulk and tables formulation.
The method is based on the formation of yellow ion-pair complex between Atenolol
and Bromocresol green in 1,2-dichloroethane medium. The maximum absorption of
the complex was found to be 414nm. Different parameters affecting the reaction were
optimized such as: effect of solvents, time,
reagent concentration, correlation ratio, etc. The formed complex
was quantified spectrophotometrically at maximum absorption. Linearity range was
2.66 - 26.63µg/mL. Regression analysis showed a good correlation coefficient R2
= 0.9999. The limit of detection (LOD) and limit of quantification (LOQ) were
to be 0.22µg/mL and 0.66 µg/mL. The average percent recovery was found to be (97.23 – 101.53)% for Atenolol. The method was successfully
applied for the determination of Atenolol in pharmaceutical tablets formulation
in six Syrian pharmaceutical trademarks: (Tenormin MPI 100, Tenormin MPI 50, Hypoten
UNIPHARMA 100, Hypoten UNIPHARMA 50, Normoten BARAKAT 100 and Normoten BARAKAT 50). The proposed method is simple, direct, sensitive and
do not require any extraction process. Thus, this method could be readily applicable
for the quality control and routine analysis.
KEYWORDS: Atenolol, Bromocresol
green, Spectrophotometric method.
INTRODUCTION:
Atenolol (ATN), chemically known as
4-(2-hydroxy-3-[(1-methylethyl) amino] propoxy) benzeneacetamide, is a
β1-selective (cardio selective) adrenoreceptor antagonist drug used for
antiangina treatment to relieve symptoms, improve tolerance, and as an antiarrhythmic
to help regulate heartbeat and infections. It is also used in management of
alcohol withdrawal, in anxiety states, migraine prophylaxis, hyperthyroidism,
and tremors1.
Several methods have been
reported in the literature for the analysis of Atenolol such as Thin-layer
chromatography (TLC)2, Liquid chromatography (LC)3-4, High
performance liquid chromatography (HPLC)5-6-7, Capillary
electrophoresis method8-9, Voltammetric method10-11-12, Spectrophotometric
method13-14-15, Gas
Chromatography (GC-MS) method16.
MATERIALS AND CHEMICAL REAGENTS:
Apparatus:
All spectral measurements were carried out using a T80+,
UV/Vis spectrophotometer PG instrument Ltd (UK), connected
to computer, quartz cells 1cm. Ultrasonic bath Daihan (China), and stirrer Velp Scientifica (Europe).
Chemical reagents:
1,2-dichloroethane from Surechem (England) was used to prepare the solutions. Methanol from Lobal Chemie (India) was used to dissolve
the tables. Atenolol, purity
100.42 % was obtained from India. Bromocresol
Green from Merck (Germany).
Standard preparation:
Atenolol stock solution:
Stock solution 10-3 M of Atenolol (MW = 266.341 g/mol) was prepared by
dissolving 13.317mg of Atenolol (raw
material) equivalent to 13.317mg (by taken the purity in consideration) in volumetric
flask 50 mL, diluted to volume by 1,2-dichloroethane to give concentration equivalent to 266.341𝜇g/mL. The
working standard solutions of Atenolol were prepared by appropriate dilutions among (100 - 1000)𝜇L of 266.341 𝜇g/mL in volumetric flask 10mL and added (100 - 1000)𝜇L of Bromocresol green 5×10-3M then completed
to volume with 1,2-dichloroethane to give concentrations between (2.66–26.63)𝜇g/mL of Atenolol.
Reagent stock solution:
Bromocresol green 5×10-3M was prepared by dissolving 174.5mg of pure Bromocresol green (MW = 698.04 g/mol) in volumetric flask 50mL and completing to volume with 1,2-dichloroethane.
Calibration Curve:
To construct the calibration curve, five standard
solutions between (2.66–26.63)𝜇g/mL of Atenolol
were prepared and the absorbance
was measured of each solution five times.
Sample
preparation:
Six Syrian products
were studied:
Ten tablets or Tenormin
MPI 50, Hypoten UNIPHARMA 50 or Normoten BARAKAT 50 products were finely
powdered and an accurate weight equivalent to 50mg Atenolol was taken
to volumetric
flask
10ml, completed to volume with Methanol and stirred, then 0.5mL of
the solution was taken to volumetric flask 10mL and diluted to volume
with 1,2-dichloroethane. 0.5mL of the last solution was taken to volumetric
flask 10mL and added 0.5mL of Bromocresol green 5×10-3M, then diluted to volume
with 1,2-dichloroethane, to obtain theoretically 12.5 𝜇g/mL of Atenolol.
Ten tablets Tenormin
MPI 100, Hypoten UNIPHARMA 100, Normoten BARAKAT 100 products finely powdered
and an accurate weight equivalent to 100mg Atenolol was
weighed accurately, in volumetric flask 25 ml, completed
to volume with Methanol and stirred, then 0.6mL of the solution was
taken to volumetric flask 10mL and diluted to volume with 1,2-dichloroethane.
0.5mL
of the last solution was taken to volumetric flask 10mL and added 0.5mL of
Bromocresol green 5×10-3M, then diluted to volume
with 1,2-dichloroethane, to obtain theoretically 12.00 𝜇g/mL 𝜇g/mL of Atenolol.
Stability of stock solution:
Time effect on standard stock solution of Atenolol in 1,2-dichloroethane was studied in three different concentrations 1.5×10-4M, 3×10-4M and 4.5×10-4M, at 278nm. We did not notice any significant changes during the absorption measurement within 12 weeks.
RESULTS
AND DISCUSSION:
The result complex solution was scanned in the range of wavelengths 350-550nm against a blank of BCG prepared in 1,2-dichloroethane. Maximum wavelength was at 414nm. We studied all the parameters of the colored result solutions to obtain the optimal conditions. Atenolol forms with Bromocresol green at 25±5ºC yellow ion-pair complex and Stability of the complex was 12 weeks.
Effect of reagent concentration:
To study the effect of reagent
concentration on the colored complex solution, we made a series of 10 mL of separated
volumetric flasks, by adding 0.4 mL of Atenolol 4×10-5M
equivalent to 40µM and
added between (0.01 - 1mL) of (BCG) 5 × 10-3M, equivalent
to (5 - 500µM), and completed
to 10mL by 1,2-dichloroethane. The absorbance at 414nm
for every added (BCG) reagent was measured against the blank of 1,2-dichloroethane. It was found that the completed colored
complex formation was 0.4mL of (BCG) solution as it is shown in Fig. 1. The
best concentration addition of (BCG) was 200µM , five times bigger than Atenolol concentration.
Fig. 1: Effect of reagent concentration.
Correlation ratios by molecular ratio:
We have prepared a series of complex solutions ATN-BCG in the medium of the 1,2-dichloroethane. The concentration of the reagent changes within the ratio (1 ×10-5 – 15×10-5) M while the concentration of Atenolol was constant in each solution and equal to 5×10-5 M. We measured the absorbance values of these solutions at the wavelength of the maximum absorbance 414 nm according to the used reagent percentage (using 1,2-dichloroethane as a blank). The absorption changes of the molecular ratio of the reagent to the Atenolol permitted us to determine correlation ratio, deducing from the curve A = f([BCG]/[ATN]) shown in Fig. 2 where the correlation ratio is (1:1).
Correlation ratios by continuous variation:
We have prepared a series of complex solutions ATN-BCG in the medium of the 1,2-dichloroethane. The concentration of the reagent and the concentration of Atenolol changes in solutions between (0.2×10-4–0.8 ×10-4M) where the sum of both concentrations remains constant and equal to 1×10-4 M. We plotted the spectral curves of each solution at the wavelength 414nm and plotted the absorption changes of the solutions of the formed complex in terms of molecular fraction of Atenolol. We obtained the curve A = f( [BCG] /{[BCG] + [ATN]}) shown in fig. 3, where the correlation ratio is (1:1).
Fig. 2: Correlation molecular ratios (1:1).
Fig. 3: Correlation ratio by continuous variation (1:1).
Calculation of formation constant for the studied complex:
The conditional stability constants (𝐾𝑓) of the ion-pair complexes were calculated from molecular ratio and the continuous variation curves. Data using the
following equation17-18-19:
Where 𝐴 and 𝐴𝑚 are the observed maximum absorbance and the absorbance value when all
the Atenolol standard material present is associated, respectively. 𝐶𝑀 is the mole concentration of Atenolol
standard sodium material at the maximum absorbance and 𝑛 is the stoichiometry with which dye ion
associates with Atenolol sodium standard material. The log 𝐾𝑓 value for ATN-BCG ion-pair associates at
correlation ratio (1:1) by molecular ratio was 8.195, and
by continuous variation was 8.198.
Range and linearity of Atenolol:
We studied the linearity of Atenolol concentrations
at the optimal conditions where we made a series of 10 mL of separated
volumetric flasks, each one contains a volume of BCG 5 × 10-3 M
equals to five times of Atenolol concentration, and variable concentrations
of Atenolol stock solution 1 × 10-3 M, and
completed to 10 mL with 1,2-dichloroethane, finally we measured the absorbance at 414
nm for each concentration against the blank of BCG in 1,2-dichloroethane. Fig. 4 presents the ATN-BCG complex spectra. The
range of linearity was obeyed to Beer’s law in concentration (2.66 – 26.63) μg/mL
and the linearity curve is presented in Fig. 5.
|
|
|
|
Fig. 4: spectra of (ANT-BCG):
C1: 2.66 𝜇g/mL, C2: 5.32 𝜇g/mL,
C3: 7.99 𝜇g/mL, C4: 10.65 𝜇g/mL,
C5: 13.32 𝜇g/mL, C6: 15.98 𝜇g/mL,
C7: 18.64 𝜇g/mL, C8: 21.30 𝜇g/mL,
C9: 23.98 𝜇g/mL, C10: 26.63 𝜇g/mL.
|
Fig. 5: Calibration curve for (ANT-BCG):
C1: 2.66 𝜇g/mL, C2: 5.32 𝜇g/mL,
C3: 7.99 𝜇g/mL, C4: 10.65 𝜇g/mL,
C5: 13.32 𝜇g/mL, C6: 15.98 𝜇g/mL,
C7: 18.64 𝜇g/mL, C8: 21.30 𝜇g/mL,
C9: 23.98 𝜇g/mL, C10: 26.63 𝜇g/mL.
n = 5 for each
concentration.
|
Accuracy and precision:
To determine the precision and accuracy of
the proposed method, five replicate determinations were carried out on three
different concentrations of standards (ANT). The accuracy and precision
results are presented in table 1.
Table 1: Precision and accuracy for
determination of Atenolol.
|
Pharmaceutically samples
|
Theoretical concentration (μg/mL)
|
 observed concentration (μg/mL)
|
SD
µg/mL
|
Precision
(RSD %)
|
Accuracy (%)
|
|
Atenolol
|
7.99
|
7.92
|
0.06
|
0.76
|
99.12
|
|
13.32
|
13.19
|
0.22
|
1.67
|
99.02
|
|
18.64
|
18.62
|
0.06
|
0.32
|
99.89
|
: mean of five replicated
determinations, Accuracy (%) = (observed concentration/theoretical
concentration) ˟ 100,
Precision (RSD %) = (standard deviation/mean
concentration) ˟100.
Analytical parameters:
Sensitivity of the proposed method for Atenolol was determined by calculating Sandell’s sensitivity
(SS), it was to be SS = 0.0298 µg/cm2.
The mean molar absorptivity ε was found equal to 17843
L/mol.cm.
Limit of detection (LOD) and limit of quantification
(LOQ) were calculated from the standard deviation of the absorbance
measurements obtained from Beer’s law and are presented in table 2.
|
|
;
|
|
Where SD, is the standard deviation of y intercepts of
regression lines and m is the slope of the calibration curve. The limit of
detection (LOD) and limit of quantification (LOQ) were to be 0.22 and 0.66 𝜇g/mL respectively.
Table 2: Analytical parameters of Atenolol determination.
|
Parameter
|
Practical
conditions
|
|
λ max
(nm)
|
414
|
|
Beer’s law limits
(μg/mL)
|
2.66 – 26.63
|
|
Stability of the complex
|
three months
|
|
Stability of stock standard solution
|
three months
|
|
Temperature
|
25
± 5 oC
|
|
Solvent
|
1,2-dichloroethane
|
|
CBCG:CANT
, M
|
≥ 5
|
|
Molar
absorptivity, ε (L/mol.cm)
|
17843
|
|
Regression
equation
|
Y = 0.0661 X + 0.0146
|
|
Slope (b)
|
0.0661
|
|
Intercept (C)
|
0.0146
|
|
R2
|
0.9999
|
|
LOD (µg/mL)
|
0.22
|
|
LOQ (µg/mL)
|
0.66
|
|
Sandell’s
sensitivity SS (µg/cm2)
|
0.0298
|
RECOVERY:
The recovery was studied by three addition
standards for every product. Table 3 presents the recoveries results for the six
products.
Table 3: Recoveries for Tenormin MPI 100, Tenormin MPI 50, Hypoten UNIPHARMA
100, Hypoten UNIPHARMA 50, Normoten BARAKAT 100 and Normoten
BARAKAT 50 Syrian products.
|
Recovery
Average
%
|
RSD%
|
SD
µg/mL
|
Recovery
%
|
Total
Found  µg/mL
|
Added
µg/mL
|
Sample
µg/mL
|
Pharmaceutical
dosage
|
Products
|
|
101.22
|
1.66
|
1.68
|
100.94
|
19.39
|
8.52
|
10.79
|
Atenolol
100 mg/tab.
|
Tenormin
MPI 100
|
|
0.96
|
0.97
|
100.75
|
21.52
|
10.65
|
|
1.06
|
1.08
|
101.96
|
23.82
|
12.78
|
|
101.42
|
2.53
|
2.66
|
105.16
|
19.73
|
8.52
|
10.77
|
Atenolol
50 mg/tab.
|
Tenormin
MPI 50
|
|
2.43
|
2.41
|
99.25
|
21.34
|
10.65
|
|
1.72
|
1.72
|
99.84
|
23.53
|
12.78
|
|
99.65
|
1.66
|
1.68
|
100.94
|
19.39
|
8.52
|
10.79
|
Atenolol
100 mg/tab.
|
Hypoten
UNIPHARMA
100
|
|
1.62
|
1.62
|
100.19
|
21.46
|
10.65
|
|
1.00
|
0.98
|
97.81
|
23.29
|
12.78
|
|
101.53
|
4.13
|
4.22
|
102.11
|
19.34
|
8.52
|
10.64
|
Atenolol
50
mg/tab.
|
Hypoten
UNIPHARMA
50
|
|
3.22
|
3.26
|
101.22
|
21.42
|
10.65
|
|
0.81
|
0.82
|
101.25
|
23.58
|
12.78
|
|
97.23
|
3.84
|
3.79
|
98.59
|
19.76
|
8.52
|
11.36
|
Atenolol
100
mg/tab.
|
Normoten
BARAKAT
100
|
|
1.88
|
1.83
|
97.09
|
21.70
|
10.65
|
|
3.85
|
3.70
|
96.00
|
23.63
|
12.78
|
|
98.09
|
4.62
|
4.47
|
96.83
|
19.17
|
8.52
|
10.92
|
Atenolol
50
mg/tab.
|
Normoten
BARAKAT
50
|
|
2.69
|
2.63
|
97.84
|
21.34
|
10.65
|
|
0.54
|
0.54
|
99.61
|
23.65
|
12.78
|
Mean for five replicated determinations were performed
and calculated the mean.
Application: Estimation of Atenolol in Syrian tablets products
The developed method was applied for quantitative
determination and identification of Atenolol in
Syrian pharmaceutical products. The samples were prepared as described in the
section of samples preparation and analyzed. Quantitative analysis was done by
using calibration curve. The obtained results are summarized in tables 4, for the
six products.
In general, the concentrations of the detected Atenolol
compounds in the two products were within in the allowed limits under USP
legislation) the tablets must contain not less than
90.00 % and not more than 110.00 % of labeled amount(. So the obtained results are conformed to
USP legislation20.
The relative standard deviations RSD % (n = 5) of the quantitative results were in the range of 0.62 -
3.72 %. Tables 4 present the determination results of Atenolol in six Syrian
Pharmaceutical products (Tenormin
MPI 100, Tenormin MPI 50, Hypoten Unipharma 100, Hypoten Unipharma 50, Normoten Barakat 100, Normoten Barakat 100), for five different batches for each one.
Table 7: Results of Atenolol in Tenormin
MPI 100, Tenormin MPI 50, Hypoten UNIPHARMA 100, Hypoten
UNIPHARMA 50, Normoten BARAKAT 100 and Normoten
BARAKAT 50 Syrian products
tablets.
|
Product
|
Tenormin MPI
100
|
|
1
|
2
|
3
|
4
|
5
|
|
Concentration  mg/ tab
|
100.68
|
102.07
|
99.34
|
101.33
|
100.85
|
|
Range mg/tab.
|
99.34 – 102.07
|
|
SD mg/ tab
|
1.50
|
1.41
|
1.28
|
1.94
|
1.31
|
|
RSD %
|
1.49
|
1.38
|
1.29
|
1.91
|
1.30
|
|
Range RSD %
|
1.29 – 1.91
|
|
Per %
|
100.68
|
102.07
|
99.34
|
101.33
|
100.85
|
|
Range Per %
|
99.34 – 102.07
|
|
Product
|
Hypoten
UNIPHARMA 100
|
|
Concentration
mg/ tab
|
100.83
|
101.12
|
100.70
|
101.09
|
99.47
|
|
Range mg/tab.
|
99.47 – 101.12
|
|
SD mg/ tab
|
0.63
|
1.20
|
1.42
|
2.15
|
1.25
|
|
RSD %
|
0.62
|
1.19
|
1.41
|
2.13
|
1.26
|
|
Range RSD %
|
0.62 – 2.13
|
|
Per %
|
100.83
|
101.12
|
100.70
|
101.09
|
99.47
|
|
Range Per %
|
99.47 – 101.12
|
|
Product
|
Normoten
BARAKAT 100
|
|
Concentration mg/ tab
|
98.24
|
102.22
|
101.12
|
101.52
|
102.42
|
|
Range mg/tab.
|
98.24 – 102.42
|
|
SD mg/ tab
|
2.72
|
3.80
|
2.01
|
3.26
|
1.65
|
|
RSD %
|
2.77
|
3.72
|
1.99
|
3.21
|
1.61
|
|
Range RSD %
|
1.61 – 3.72
|
|
Per %
|
98.24
|
102.22
|
101.12
|
101.52
|
102.42
|
|
Range Per %
|
98.24 – 102.42
|
|
Product
|
Tenormin MPI
50
|
|
Concentration mg/ tab
|
50.03
|
50.19
|
49.49
|
50.84
|
49.71
|
|
Range mg/tab.
|
49.49 – 50.84
|
|
SD mg/ tab
|
0.79
|
1.09
|
0.69
|
1.10
|
0.81
|
|
RSD %
|
158
|
2.17
|
1.39
|
2.16
|
1.63
|
|
Range RSD %
|
1. 39 – 2.17
|
|
Per %
|
100.06
|
100.38
|
98.98
|
101.68
|
99.42
|
|
Range Per %
|
98.98 – 101.68
|
|
Product
|
Hypoten UNIPHARMA
50
|
|
Concentration mg/ tab
|
50.74
|
50.31
|
51.85
|
52.17
|
49.44
|
|
Range mg/tab.
|
49.44– 52.17
|
|
SD mg/ tab
|
0.44
|
0.53
|
0.36
|
0.45
|
0.37
|
|
RSD %
|
0.87
|
1.05
|
0.69
|
0.86
|
0.75
|
|
Range RSD %
|
0.69 – 1.05
|
|
Per %
|
101.48
|
100.62
|
103.70
|
104.34
|
98.88
|
|
Range Per %
|
98.88 – 104.34
|
|
Product
|
Normoten
BARAKAT 100
|
|
Concentration
mg/ tab
|
50.46
|
50.57
|
49.66
|
50.16
|
51.49
|
|
Range mg/tab.
|
49.66 – 51.49
|
|
SD mg/ tab
|
0.44
|
0.45
|
0.78
|
0.38
|
1.51
|
|
RSD %
|
0.87
|
0.89
|
1.57
|
0.76
|
2.93
|
|
Range RSD %
|
0.76 – 2.93
|
|
Per %
|
100.92
|
101.14
|
99.32
|
100.32
|
102.98
|
|
Range Per %
|
99.32 – 102.98
|
Mean for five replicates.
CONCLUSION:
We developed a new method which is suitable for the
identification and quantification of Atenolol in Syrian tablets formulation. A
good percentage of recovery shows that the method can be successfully used in
routine analyses. The proposed method is simple, sensitive, rapid, specific, a
little cost and could be applied for quality control of Atenolol. The levels of Atenolol
compounds were within the permissible limits set by the USB legislation20.
ACKNOWLEDGEMENT:
The Ministry of High Education in Syria financially
and technically supported this work through department of Chemistry, Faculty of
Science, University of Aleppo, Syria.
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