FARMACIA, 2008, Vol.LVI, 4
363
UV-VIS SPECTROPHOTOMETRIC ASSAY OF
METOPROLOL.
NOTE 2. METHOD VALIDATION
MAGDALENA BĂDULESCU1*, DAN BĂLĂLĂU2,
IOANA CACOVEAN2, MIHAELA ILIE2, DANIELA LUIZA BACONI2
National Medicines Agency, 48 Aviator Sănătescu Str., Bucharest
”Carol Davila” University of Medicine and Pharmacy, Faculty of
Pharmacy, Toxicology Department, 6 Traian Vuia Str., Bucharest, Romania
*
corresponding author: magdalena.badulescu@anm.ro
1
2
Abstract
Metoprolol is a widely used beta-blocker mostly administrated in cardiovascular
diseases. Due to the highly probable overdosage, a rapid and easy to perform method for
the quantitative determination of metoprolol tartrate in biologic matrices would be useful in
its therapeutic monitoring as well as in intoxication cases.
The paper presents two validated methods for the UV-VIS quantitative assay of
metoprolol tartrate in tablets: the UV direct method and the oxidative method with
potassium permanganate. The parameters targeted for the validation were: specificity,
range, detection and quantification limits, accuracy, and precision.
Rezumat
Metoprololul este un beta-blocant cu utilizare frecventă, administrat cu precădere
în maladii cardiovasculare. Deoarece există posibilitatea supradozării, este necesară găsirea
unor metode rapide de determinare cantitativă a tartratului de metoprolol din matrice
biologică, atât pentru monitorizare terapeutică, cât şi pentru dozare în situaţia suspiciunii de
intoxicaţie.
Prezenta lucrare are ca scop validarea unor metode spectrofotometrice UV-VIS
de determinare a tartratului de metoprolol din comprimate: metoda UV directă şi metoda
oxidativă cu permanganat de potasiu. Parametrii avuţi în vedere la validare au fost:
specificitatea, domeniul de liniaritate, limitele de detecţie şi cuantificare, acurateţea şi
precizia.



metoprolol quantitative assay
UV-VIS absorption
validation
INTRODUCTION
Metoprolol tartrate is a selective beta-adrenergic antagonist, which
is used in the treatment of cardiovascular disorders such as hypertension,
angor pectoris, cardiac arrhythmias, congestive heart failure and myocardial
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infarction [1,2,3]. The drug is quite sensitive, even a small dose of the drug
giving a sufficient blockade of the beta-adrenergic receptors. Thus the
quantitative determination of the drug is important and a simple method
such as the UV-VIS absorption for the assay would be of a great interest
[4,5,6].
The paper presents the validated UV-VIS methods for the
metoprolol tartrate assay in tablets [6]. The validation was performed in
respect of the ICH Q2R1 regulations [7] for analytical procedures. The
following parameters were taken into account: specificity, linearity, limits of
detection and quantification, accuracy, precision.
MATERIALS AND METHODS
Reagents:
- metoprolol tartrate USP 30 (Sigma)
- metoprolol tablets Betaloc® ZOK (AstraZeneca)
- sodium hydroxyde (Merck),
- potassium permanganate (Chimopar).
Device
UV-Vis Cary 100 Bio spectrophotometer (Varian Inc.)
Procedure
a. General procedure
0.1 mg/mL metoprolol tartrate stock solution is prepared in double
distilled water to be further dilluted for the standard solutions.
Five tablets are analytically weighted (0.1 mg precision), triturated
and analytically transferred with 50 mL of double distilled water to a 100
mL Erlenmeyer flask. The content of the flask is filtered through analytical
filter paper in a 100 mL volumetric flask, the filter paper is washed with
another 40 mL of double distilled water, than the volumetric flask is
completed to the mark to give the stock sample solution. The working
solution is obtained by 1:100 dilution with double distilled water.
b. Direct UV method
First, a background correction of the spectrophotometer is
performed by using double distilled water. Then the spectra of a series of
working standards towards double distilled water, at least five
concentrations in the range of 0.5 – 10 μg/mL, in triplicate, is plotted, as
well as the spectra of the sample solution, by using the following
parameters: spectral range: 190 – 400 nm, spectral bandwidth 0.2 nm,
scanning speed 600 nm/min.
The absorbance of each solution at 222 nm [6] is measured.
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c. Oxidative method
First, a background correction of the spectrophotometer is
performed by using double distilled water.
Stock solutions of 0.6 M sodium hydroxyde and 0.015M potassium
permanganate are prepared ex temporae in double distilled water.
In 10 mL volumetric flasks aliquots of 0.2 to 1 mL of stock
standard solution and of 0.1 mL from the sample working solution are
added. Similary, the same aliquots of distilled water are added to 10 mL
volumetric flasks. To each flask 2 mL of NaOH stock solution and 2 mL of
0.015 M KMnO4 solution are added, then the flasks are completed to the
mark with double distilled water. As previous experiments showed that the
reaction is completed after 15 minutes, each standard and sample solution is
let to stand still for 15 min, than the spectrum of each sample and the
corresponding blank solution are plotted in the range 500 – 650 nm, spectral
bandwidth 0.2 nm, scanning speed 100 nm/min.
The absorbance at 610 nm is measured and the difference between
the solutions containing metoprolol tartrate and the corresponding blank
solution is computed to be further used.
Data analysis
For both methods the calibration curve absorbance vs.
concentration is plotted, and the concentration of the sample is computed by
interpolation on the corresponding calibration curve. The amount of
metoprolol tartrate is computed, taking into consideration the original
dillution (1:10000).
For the calculation of the detection (DL) and quantification (QL)
limits the following equations were used [7]:
DL 
QL 
3.3   A
B
10   A
B
(1)
(2)
where σA is the standard deviation of the intercept, and B – the slope of the
calibration line.
The recovery coefficient was computed following the equation:
R (%)=100 x (amount found)/(amount known)
(3)
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RESULTS AND DISCUSSION
1. Direct UV method
Specificity
In order to demonstrate the specificity of the method, UV spectra of
a standard solution and of the sample were recorded. As can be seen in
figure 1, no spectral interferences could be noticed from the excipients for
the 222 nm band of metoprolol tartrate.
Figure 1
Spectra of a 6 μg/mL metoprolol tartrate and a sample solution
Linearity, DL and QL
The linearity of the method was studied in the range of 1-30 μg/mL
using the 222 nm absorption band. The results are presented in Table I.
Table I
Linearity, limits of detection and quantification for the direct UV method
Parameter
Values
Linearity range
1-30 μg/mL
Intercept
0.02604±0.00291
Slope
0.03029±0.0005
Correlation coefficient (R)
0.99873
Probability (p)
<0.0001
Detection limit (DL)
0.317 μg/mL
Quantification limit (QL)
0.961 μg/mL
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Accuracy
The accuracy was studied using known amounts of metoprolol
tartrate in the range of 1 -20 μg/mL, via the recovery coefficient, which has
to be in the range of 99-101%. The obtained results are presented in Table
II, showing that method is accurate.
Concentration of
metoprolol tartrate
taken into analysis
(µg/mL)
2.00
6.00
10.00
14.00
20.00
Table II
Accuracy of the UV direct method
Concentration of the recovered
Recovery
metoprolol tartrate
coefficient
(µg/mL)
(%)
2.00
6.06
10.8
13.96
19.88
100.0
101.0
100.8
99.7
99.4
Precision
We investigated the precision of the method through
its
repetability and reproducibility. We repeated 5 times the assay of
metoprolol tartrate in the same day, by the same analyst, and 5 times the
assay of the content of metoprolol tartrate in the tablet by five different
analysts in different days, then we computed the relative standard deviation
(RSD). The acceptance criteria demands that RSD should be less than 2% .
The results are summarised in Table III, and show that the method is
precise.
Declared
amount
(mg/tablet)
50
Mean
SD
RSD (%)
Repetability
Amount found
Amount
(mg/tablet)
found (%)
49.2
49.7
50.6
50.2
50.2
50.5
50.067
0.5279
1.05
98.4
99.4
101.2
100.4
100.4
101
100.13
1.0558
1.05
Table III
Precision of the UV method
Reproducibility
Amount
Amount
found
found (%)
(mg/tablet)
51.0
102
50.3
100.6
49.0
98
49.7
99.4
50.0
100
50.2
100.4
50.033
100.067
0.6653
1.3307
1.33
1.33
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2. Oxidative method
Specificity
In order to prove the specificity of the method we registered spectra
of a sample, a 4 μg/mL standard and a blank solution (Figure 2) prepared as
mentioned above.
Figure 2
VIS absorption spectra of metoprolol tartrate – KMnO4 in alkaline conditions
As it can be seen in figure 2, in the presence of a sample or a
standard of metoprolol tartrate, the band at 610 nm is better differentiated
that in case of the blank sample, suggesting that in the redox reaction only
the active substance (metoprolol tartrate) is involved, not the excipients
present in the pharmaceutical formulation.
Linearity, DL and QL
The linearity of the method was studied in the range of 4-10
μg/mL. The results are presented in Table IV.
Table IV
Linearity, limits of detection and quantification for the oxidative method
Parameter
Values
Linearity range
4 – 8 μg/mL
Intercept
-0.102±0.0021
Slope
0.041±0.00036
Correlation coefficient (R)
0.99989
Probability (p)
<0.0001
Detection limit (DL)
0.566 μg/mL
Quantification limit (QL)
1.714 μg/mL
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Accuracy
The accuracy was studied using known amounts of metoprolol
tartrate in the range of 4 -8 μg/mL, via the recovery coefficient, which has to
be in the range of 99-101%. The obtained results are presented in Table V,
showing that the method is accurate.
Concentration of
metoprolol tartrate
taken into analysis
(µg/mL)
4.00
6.00
8.00
Table V
Accuracy of the oxidative method
Concentration of the recovered
Recovery
metoprolol tartrate
coefficient
(µg/mL)
(%)
4.03
5.99
7.96
100.7
99.8
99.5
Precision
We investigated the precision of the method through its repetability
and reproducibility. We repeated 5 times the assay of metoprolol tartrate in
the same day, by the same analyst, and 5 times the assay of the content of
metoprolol tartrate in the tablet by five different analysts in different days,
then we computed the relative standard deviation (RSD). The acceptance
criteria demands that RSD should be less than 2% . The results are
summarised in Table VI, and show that the method is precise.
Declared
amount
(mg/tablet)
50
Mean
SD
RSD (%)
Repetability
Amount found
Amount
(mg/tablet)
found
(%)
50.31
100.6
50.24
100.5
50.62
101.2
49.98
100.0
49.96
99.9
50.222
100.44
50.222
100.44
0.27096
0.52249
0.54
0.52
Table VI
Precision of the UV method
Reproducibility
Amount found
Amount
(mg/tablet)
found
(%)
51.20
102.4
50.71
101.4
49.86
99.7
50.06
100.1
51.08
102.1
50.582
101.18
50.582
101.18
0.6000
1.0759
1.19
1.06
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CONCLUSIONS
The validation of two methods for the quantitative assay of
metoprolol tartrate in tablets are presented: a direct UV absorption method
at 222 nm and an indirect method, using the redox reaction with potassium
permanganate.
The methods have been validated according to ICH Q2(R1)
recommendations, for selectivity, range, detection and quantification limits,
accuracy and precision.
While the direct method can be useful in the determination of
metoprolol in biological matrices, the oxidative method, due to the fact that
the measured physical parameter is the absorption of the manganate ion,
cannot be applied as mentioned for the metoprolol assay in biological
matrices.
REFERENCES
1. *** Martindale: The Complete Drug Reference. Metoprolol
Monograph – The Pharmaceutical Press 2007
2. *** AHFS Drug Information. Metoprolol Tartrate, Metoprolol
Succinate Monographs – American Society for Health-System
Pharmacists, 2007
3. Brunton LL, Lazo JS, Parker KI (eds.) – Goodman and Gilman’s
The Pharmacological Basis of Therapeutics, eleventh edition,
McGraw Hill, 2005
4. *** Clarke’s Analysis of Drugs and Poisons – Pharmaceutical Press,
2007
5. Rahman N., Rahman H., Aami S.N.H. – Validated Kinetic
Spectrophotometric Method for the Determination of Metoprolol
Tartrate in Pharmaceutical Formulations – Chem. Pharm. Bull.,
2005, 53(8): 942-948
6. Cacovean Ioana, Ilie Mihaela, Bădulescu Magdalena, Bălălău Dan,
Gubandru Miriana – UV-VIS Spectrophotometric Assay of
Metoprolol. Note 1. Comparison Between the Direct and the
Oxydative Methods – Farmacia, 2007, LV (5): 540-544
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ICH Q2 (R1) – International Conference on Harmonisation, Geneva,
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