Simultaneous UV Spectrophotometric
Methods for Estimation of Carvedilol and
Hydrochlorothiazide in Bulk and Tablet Dosage Form
Audumbar Mali1*,
Vikas Kekan2, Rohan
Dongare2, Sachin Gholve2, Ritesh Bathe1
1Department of Pharmaceutics, Sahyadri
College of Pharmacy, Methwade, Sangola-413307, Solapur, Maharashtra, India
2Department of Quality Assurance, Channabasweshwar
Pharmacy College, Latur.
Dist. Latur-413512
Maharashtra, India
*Corresponding
Author E-mail: maliaudu442@gmail.com
ABSTRACT:
Two
simple, precise, economical, fast and reliable two UV methods have been
developed for the simultaneous estimation of Carvediloland
Hydrochlorothiazide in bulk and pharmaceutical dosage form. Method A is
Absorbance maxima method, which is based on measurement of absorption at
maximum wavelength of 290 nm and 271 nm for Carvedilol
and Hydrochlorothiazide respectively. Method B is area under curve (AUC), in
the wavelength range of 260-308 nm for Carvedilol and
2246-292 nm for Hydrochlorothiazide. Linearity for detector response was
observed in the concentration range of 5-25μg/ml for Carvedilol
and 5-25 μg/ml for Hydrochlorothiazide. The
accuracy of the methods was assessed by recovery studies and was found to be
100.13% and 102.28% for Carvedilol and 99.04% and
99.89% Hydrochlorothiazide by using method A and B respectively. The developed
method was validated with respect to linearity, accuracy (recovery), precision
and specificity. The results were validated statistically as per ICH Q2
R1guideline and were found to be satisfactory. The proposed methods were
successfully applied for the determination of for Carvedilol
and Hydrochlorothiazide in commercial pharmaceutical dosage form.
KEY WORDS: Carvedilol, Hydrochlorothiazide, Simultaneous estimation,
Accuracy, Absorbance maxima method, Area under curve.
INTRODUCTION:
Carvedilol is a combined alpha- and nonselective beta blocker. Carvedilol chemically, 2-Propanol, 1-(9H-carbazol-
4-yloxy)-3-[[2-(2- methoxy phenoxy)
ethyl amino]-, (±)-; (±)-1-(Carbazol-4-yloxy)-3-[[2-(o-methoxy
phenoxy) ethyl] amino]-2-propanol. It is a non-selective beta blocker indicated in the
treatment of mild to moderate congestive heart failure (CHF). It blocks beta-1
and beta-2 adrenergic receptors as well as the alpha-1 adrenergic receptors. Carvedilol
is official drug in British Pharmacopoeia.
It has been prescribed as an antihypertensive agent and an angina
agent. It is first beta blocker labeled in United States especially for the
treatment of heart failure of ischemic or cardiomyopathic
origin with significant antioxidant activity. Relative to other beta blocker, Carvedilol (CAR) has minimal inverse agonist indicating a
reduced negative chronotropic and inotropic
effect, which decreases its potential to worsen symptoms of heart failure. At
high dosage, it exerts Calcium channel blocking activity. The benefits of using
CAR in patient with CHF in both single center and multicenter trial have been
reported in the literature. It prevents vitamin E, glutathione and SH protein
depletion induced by oxidation stress, the main defense
mechanism against tissue injury caused by free radical. [1-4]
Fig. 1: chemical structure of Carvedilol
Hydrochlorothiazide (HTZ)
chemically6-chloro-3, 4-dihydro-2, 4-1, 2, 4-benzothiadiazine-7- sulphonamide
1, 1-dioxide (Fig. 1b) is a widely used thiazide
diuretic [1-3]. [5-8]
Fig. 2: chemical structure of
Hydrochlorothiazide
A survey of pertinent literature revealed that in estimation of
individual [9] as well as
combination of Carvedilol and Hydrochlorothiazide.
Simultaneous determinations of Carvedilol and Hydrochlorothiazide dosage form were
also reported like HPLC [10, 11],
RP-HPLC [12-15], HPTLC [16, 17] and UV-Spectroscopy [18, 19]. Therefore an attempt was made
to develop a new rapid and sensitive UV Spectrophotometric method and to
validate as per ICH-guidelines. A comprehensive literature research reveals the
lack of a Spectrophotometric analytical method for simultaneous estimation of Carvedilol and Hydrochlorothiazide in
pharmaceutical formulations. A successful attempt was made to develop accurate,
precise and simple method of analysis for estimation of both the drugs in
combined dosage form.
MATERIALS AND METHODS:
Apparatus
and instrumentation:-
A Shimadzu 1800
UV/VIS double beam spectrophotometer with 1cm matched quartz cells was used for
all spectral measurements. Single Pan
Electronic balance (Contech, CA 223, India) was used
for weighing purpose. Sonication of the solutions was carried out using an
Ultrasonic Cleaning Bath (Spectra lab UCB 40, India).Calibrated volumetric
glassware (Borosil) was used for the validation
study.
Materials:
Reference
standard of Carvedilol and Hydrochlorothiazide API was supplied as gift sample by Lupin Laboratory Park Aurangabad, Maharashtra, India. The
commercial formulation Co-Dilatrol® as
purchased from the local market Solapur, Maharashtra,
India.
Method
development:
Preparation of standard stock solution: -
Stock solution was prepared by diluting
10 mg of each drug in sufficient quantity of methanol in separate volumetric
flask and volume was made up to 100 ml to get the concentrations of
100μg/ml for each drug. Dilutions from stock solution were prepared in the
range of 5-25 μg/ml for Carvedilol and 5-25 μg/ml
for Hydrochlorothiazide. Methanol was used as a blank solution.
Method A: Absorption Maxima Method:
For the selection of analytical wavelength, standard solution of Carvedilol and Hydrochlorothiazidewere
scanned in the spectrum mode from 400 nm to 200 nm separately. From the spectra
of drug λmax of Carvedilol 290 nm [Fig.3], and λmax of Hydrochlorothiazide, 271 nm [Fig.4], were selected for the analysis.
Aliquots of standard stock solution were made and calibration curve was
plotted. [20]
Fig. 3: It shows λmax of
Carvedilol
Fig. 4: It shows λmax of
Hydrochlorothiazide
Simultaneous estimation of Carvedilol and
Hydrochlorothiazide:
The wavelength maxima of Carvedilol and Hydrochlorothiazidewere determined and found to be 290 nm (λ1) and
271 nm (λ2) respectively where there was no interference among the drugs.
The overlain spectrum is shown in Fig.5.
Fig. 5 Isobestic point of Carvedilol and Hydrochlorothiazide
Method B: Area under Curve Method:
From the spectra of drug obtained after scanning of standard solution
of Carvedilol and Hydrochlorothiazideseparately,
area under the curve in the range of 260-308 nm and 246-292 nm was selected for
the analysis. The calibration curve was prepared in the concentration range of
5-25 μg/ml for Carvedilol and 5-25 μg/ml for Hydrochlorothiazide at their respective AUC range. Both drugs followed
the Beer-Lambert’s law in the above mentioned concentration range. The
calibration curves were plotted as absorbance against concentration of Carvedilol and Hydrochlorothiazide. The coefficient of correlation (r), slope and intercept
values of this method are given in Table 2.
Area calculation: (α+β)
=
Where, α is area of portion bounded
by curve data and a straight line connecting the start and end point, β is the
area of portion
bounded by a
straight line connecting
the start and
end point on curve data and horizontal axis λ1 and λ2 are wavelength range start and end point of
curve region. [21, 22, 23]
Application of the proposed methods
for the determination of Carvedilol and Hydrochlorothiazide in tablet dosage form:
For the estimation of drugs in the tablet formulation, 20 tablets were
weighed and weight equivalent to 25mg of Carvedilol and 12.5mg of Hydrochlorothiazide was transferred
to 100 ml volumetric flask and ultra sonicated for 20
minutes and volume was made up to the mark with methanol. The solution was then
filtered through a Whatmann filter paper (No.42). The
filtrate was appropriately diluted further.
In Method-A, the concentration of Carvedilol and Hydrochlorothiazide
was determined by measuring the
absorbance of the sample at 290nm and 271 nm respectively in zero order
spectrum modes. By using the calibration curve, the concentration of the sample
solution was determined.
Fig.6: It shows AUC of Carvedilol
Fig.7: It shows AUC of Hydrochlorothiazide
Table 1: Table shows Results of
Analysis of Tablet Formulation
|
Method
|
Drug
|
Label
Claim (mg)
|
Sample Solution
Concentration (µg/ml)
|
Amount
found (%)*±
|
%
Recovery
|
%RSD
|
|
A
|
Carvedilol
|
25
mg
|
20
|
98.12±1.87
|
100.13
|
0.8731
|
|
B
|
Carvedilol
|
25
mg
|
20
|
100.56
±0.84
|
102.28
|
|
A
|
Hydrochlorothiazide
|
12.5
mg
|
20
|
102.04±1.51
|
99.04
|
0.8624
|
|
B
|
Hydrochlorothiazide
|
12.5
mg
|
20
|
100.76±1.22
|
99.89
|
*n=3, % RSD = %
Relative Standard Deviation.
In Method-B, the concentration of Carvedilol and Hydrochlorothiazide was determined by measuring area under curve in the
range of 217-247 nm and 213-239nm. By using the calibration curve, the
concentration of the sample solution was determined.
Validation of the Developed Methods: [24-27]
The methods were validated with respect to accuracy, linearity,
precision and selectivity.
Fig.8:
Calibration curve for Carvedilol at 290 nm
Accuracy:
Accuracy of an analysis was determined by systemic error involved.
Accuracy may often be expressed as% Recovery by the assay of known, added
amount of analyte. It is measure of the exactness of
the analytical method. Recovery studies carried out for
both the methods by spiking standard drug in the powdered formulations80%,
100%, 120% amount of each dosage content as per ICH guidelines.
Linearity:
The linearity of measurement was evaluated by analyzing different
concentration of the standard solution of Carvedilol and Hydrochlorothiazide. Result should be expressed in terms of correlation
co-efficient.
Fig.9: Calibration curve for Hydrochlorothiazide at 271 nm
Precision:
The reproducibility of the proposed method was determined by performing
tablet assay at different time intervals (morning, afternoon and evening) on
same day (Intra-day assay precision) and on three different days (Inter-day
precision). Result of intra-day and inter-day precision is expressed in % RSD.
Sensitivity:
The limit of detection (LOD) and limit of quantitation
(LOQ) were calculated by using the equations LOD = 3xσ/ S and LOQ =
10xσ/S, where σ is the standard deviation of intercept, S is the
slope. The LOD and LOQ were found to be 0.5482 μg/ml
and 1.6458μg/ml respectively of Carvedilol and
0.5749 μg/ml and 1.7285
μg/ml of Hydrochlorothiazide.
Table 2: Optical Characteristics and Precision
|
Sr. No.
|
Parameter
|
Carvedilol
|
Hydrochlorothiazide
|
|
1
|
λ range
|
200-400 nm
|
200-400nm
|
|
2
|
Regression Equation (y=mx+c)
|
Y=0.0239x+0.0076
|
Y=0.0231x+0.0135
|
|
3
|
Measured wavelength
|
290 nm
|
271 nm
|
|
4
|
Linearity range
|
5-25µg/ml
|
5-25µg/ml
|
|
5
|
Slope
|
0.0239
|
0.0231
|
|
6
|
Intercept
|
0.0076
|
0.0135
|
|
7
|
Correlation coefficient (R2)
|
0.9994
|
0.9982
|
|
8
|
Limit of Detection (LOD) µg/ml
|
0.5482
|
0.5749
|
|
9
|
Limit of Quantitation
(LOQ)µg/ml
|
1.6458
|
1.7285
|
Table 3: Results of drug content and
analytical recovery of Carvedilol and Hydrochlorothiazide
|
Excess drug added to the analyte (%)
|
Drug
|
% Recovery
|
% RSD
|
|
Method A
|
Method B
|
Method A
|
Method B
|
|
80
|
Carvedilol
|
101.27
|
98.17
|
0.5214
|
0.5217
|
|
100
|
99.56
|
100.28
|
0.4563
|
0.8389
|
|
120
|
102.13
|
100.74
|
0.6950
|
0.6694
|
|
|
|
80
|
Hydrochlorothiazide
|
99.11
|
102.18
|
0.8547
|
0.6854
|
|
100
|
100.25
|
100.28
|
0.7421
|
0.8749
|
|
120
|
99.33
|
98.59
|
0.5842
|
0.7481
|
Table
4: Results of Intra-day and Inter-day Precision
|
Method
|
Drug
|
Intra-day Precision
|
Inter-day Precision
|
|
SD
|
%RSD
|
SD
|
%RSD
|
|
A
|
Carvedilol
|
0.9842
|
0.6412
|
0.7548
|
0.4368
|
|
B
|
0.8438
|
0.5769
|
0.7123
|
0.3402
|
|
|
|
A
|
Hydrochlorothiazide
|
0.9650
|
0.6741
|
0.4217
|
0.2148
|
|
B
|
0.7423
|
0.7759
|
0.3361
|
0.3587
|
RESULTS AND DISCUSSION:-
The methods discussed in the present work provide a convenient and
accurate way for analysis of Carvedilol and
Hydrochlorothiazidein its bulk and pharmaceutical dosage form. Absorbance maxima of Carvedilolat290nm and Hydrochlorothiazideat 271nm were
selected for the analysis. Linearity for detector response was observed in the
concentration range of 5-25 μg/ml for Carvedilol and 5-25 μg/ml
for Hydrochlorothiazide. Percent amount found for Carvedilol and
Hydrochlorothiazide in tablet analysis was found in the range of 98.12%, 100.56 % and
102.04 %, 100.76 %respectively [Table 1]. Standard deviation and coefficient of
variance for three determinations of tablet formulation was found to be less
than ± 2.0 indicating the precision of the methods. Accuracy of proposed
methods was ascertained by recovery studies and the results are expressed as
%recovery. % recovery for Carvedilol and Hydrochlorothiazidewas found in the range of 101.27% and 99.11% values of
standard deviation and coefficient of variation was satisfactorily low
indicating the accuracy of all the methods.% RSD for Intraday assay precision
for Carvedilol was found to be 0.6412 and 0.5769 for
Method A and B, and for Hydrochlorothiazide, 0.6741 and 0.7759 for Method A and B. Interday assay
precision for Carvedilol was found to be 0.4368 and 0.3402 for
Method A and B and for Hydrochlorothiazide 0.2145 and 0.3587 for Method A and B. The LOD and LOQ
were found to be 0.5482 μg/ml
and 1.6458 μg/ml respectively of Carvedilol and 0.5749 μg/ml
and 1.7285 μg/ml of Hydrochlorothiazide. Based on the results obtained, it is found that the
proposed methods are accurate, precise, reproducible and economical and can be
employed for routine quality control of Carvedilol and Hydrochlorothiazidein bulk drug and its pharmaceutical dosage form.
CONCLUSION:
UV spectrophotometric methods for Carvedilol and Hydrochlorothiazidewere developed separately in bulk and tablet dosage form
by, Absorbance maxima method and Area under curve method. Further, UV
Spectrophotometric methods for the simultaneous estimation of Carvedilol
and Hydrochlorothiazide were in bulk and combined dosage form. The methods were validated as
per ICH guidelines. The standard deviation and % RSD calculated for these
methods are <2, indicating high degree of precision of the methods. The
results of the recovery studies showed the high degree of accuracy of these
methods. In conclusion, the developed methods are accurate, precise and
selective and can be employed successfully for the estimation of Carvedilol
and Hydrochlorothiazidein bulk and pharmaceutical dosage form.
ACKNOWLEDGEMENT:
The authors are highly thankful to the Sahyadri
College of Pharmacy, Methwade, Sangola,
Solapur, Maharashtra, India
for proving all the facilities to carry out the research work successfully.
REFERENCES:
1.
Mali Audumbar, Nagargoje Rajendra, Hake Gorakhnath, Tamboli Ashpak. Zero Order and Area under Curve Spectrophotometric
Methods for Determination of Carvedilol in
Pharmaceutical Formulation. Inventi Rapid: Pharm
Analysis and Quality Assurance. 2015; 2: 1-5.
2.
Tarek S. Belal, Rasha A. Shaalan, Fawzy A. E Yazbi and Sohila M. Elonsy. Application of a new simple spectrophotometric
method for determination of the binary mixtures of hydrochlorothiazide with
either carvedilol or losartan
potassium in tablets dosage forms. Scholars Research Library Der Pharma Chemica.
2014; 6(4):120-129.
3.
Audumbar Digambar Mali, Tukaram Kedar. Determination of Carvedilol and
Hydrochlorothiazide by Third Order Derivative Spectrophotometric Method in Pharmaceutical
Preparations. International Journal of
Analytical, Pharmaceutical and Biomedical Sciences. 2015; 4(7): 18-27.
4.
Mali Audumbar and Gavade Shivaji. Estimation of Carvedilol
in bulk and formulation by second order derivative area under curve UV
spectrophotometric methods. International Journal of Pharmaceutical and Interdisciplinary Sciences.
2015; 1(2): 1-13.
5.
Chaitali Thube, Jyoti Dhagude and P.Y. Pawar. Simultaneous
estimation and validation of losartan potassium and
hydrochlorothiazide in bulk and tablet dosage form by using different
spectrophotometric method. Scholars Research Library Der
Pharma Chemica. 2014; 6(2):
24-30.
6.
Audumbar Digambar Mali. Simultaneous
Determination Of Carvedilol And Hydrochlorothiazide
In Pharmaceutical Dosage Form By First Order Derivative UV Spectrophotometry. International Journal of Pharmacy and Pharmaceutical
Sciences.2015;
7(9): 371-374.
7.
K. Ilango, Shiji Kumar.P.S. Simultaneous
Determination of Olmesartan Medoxomil
and Hydrochlorothiazide by Area under Curve and Dual Wavelength
Spectrophotometric Methods. Journal of Pharmaceutical Sciences and Research.
2012; 4(10): 1946 – 1949.
8.
A.T. Hemke, M.V. Bhure, K.S. Chouhan, K.R. Gupta
and S.G. Wadodkar. UV Spectrophotometric Determination of Hydrochlorothiazide and Olmesartan Medoxomil in
Pharmaceutical Formulation. E-Journal of Chemistry. 2010; 7(4): 1156-1161.
9.
Rajan V. Rele and Prathamesh P. Tiwatane. UV spectrophotometric estimation of first order
derivative and area under curve methods in bulk and pharmaceutical dosage form.
Pelagia Research Library Der
Pharmacia Sinica. 2014; 5(6): 29-35.
10. Kuldeep Singh, Anirbandeep
Bose, Gurubasavaraja Swamy
PM and Divakar Goli.Method
Development and Validation of Simultaneous Analysis of Olmesartan
Medoxomil and Hydrochlorothiazide by UV and HPLC,
Their Cross Validation. World Journal of Pharmacy and Pharmaceutical Sciences.
2015; 4(7): 905-917.
11. Jain R and Jain C I.
Simultaneous quantification of Captopril and Hydrochlorothiazide using HPLC.
Indian Drugs. 1991; 28(18): 380-382.
12. Kareem M. Younes. Spectrophotometric and
Chromatographic Methods for the Simultaneous Determination of Carvedilol and Hydrochlorthiazide
in Their Pharmaceutical Formulation. International Journal of Pharmacy and
Pharmaceutical Sciences. 2014; 6(4): 362-368.
13. Basaveswara Rao
M.V, Nagendrakumar A. V. D, Yedukondalu
M, Raman B.V. New Validated RP–HPLC Method for the Estimation of Carvedilol in Pharmaceutical Formulation. International
Journal of Pharmacy and Pharmaceutical Sciences.2012; 4(2): 253-358.
14. Patel LJ, Suhagia BN, Shah PB, Shah RR. RP-HPLC and HPTLC methods for
the estimation of Carvedilol in bulk drug and
pharmaceutical formulations. Ind J pharma sci. 2006; 68(6): 790-793.
15. Chouhan KS, Bhure MV, Hemke AT, Gupta KR, Wadodkar SG. Development of RP-HPLC method for estimation
of Hydrochlorothiazide and Olmesartan Medoxomil in Pharmaceutical Formulation. Res. J Pharma. Biol. Chem. Sci. 2010; 1:78 -84.
16. Damle MC, Topagi KS, Bothara KG.
Development and validation of a stability-indicating HPTLC method for analysis
of Nebivolol hydrochloride and Hydrochlorothiazide in
the bulk material and in pharmaceutical dosage forms. Acta
Chromatographica. 2010; 22:433-443.
17. S. T. Kumbhar, G. K. Chougule, V. S. Tegeli, G. B. Gajeli, Y. S. Thorat, U. S. Shivsharan. A
Validated HPTLC Method for Simultaneous Quantification of Nebivolol
and Hydrochlorothiazide in Bulk and Tablet Formulation. International Journal
of Pharmaceutical Sciences and Drug Research. 2011; 3(1): 62-66.
18. Shah K, Desai TR, Behera A, Pal RS, Girhepunje KM.
Development of spectrophotometric methods for estimation of Nebivolol
hydrochloride and Hydrochlorothiazide simultaneously, in bulk and tablet dosage
form. Res. J Pharma. Biol. Chem. Sci.2010; 1:451-457.
19. Veerasekaran V, Katakdhond SJ, Kadam SS, Rao JR. Simultaneous spectrophotometric estimation of
Hydrochlorothiazide and Metoprolol tartrate from combined dosage form. Ind. Drugs. 2001;
38:187-189.
20. Mali Audumbar,
Jadhav Santosh, Tamboli
Ashpak, Bathe Ritesh, Patil Manoj. Development and Validation of
UV-Spectrophotometric Method for the Estimation of Quinapril
Hydrochloride in Bulk and In Its Formulation. World Journal of Pharmaceutical Research. 2015; 4(1): 1839-1846.
21. Mali Audumbar
Digambar, Hake Gorakhnath, Patil Manojkumar, Bathe Ritesh, Tamboli Ashpak. Zero Order and Area under Curve
Spectrophotometric Methods for Determination of Ampicillin
Trihydrate in Pharmaceutical Formulation.GCC Journal of Science and
Technology. 2015; 1(1): 6-12.
22. Mali Audumbar
Digambar, Hake Gorakhnath, Bathe Ritesh. Zero
Order and Area under Curve Spectrophotometric Methods for Determination of
Aspirin in Pharmaceutical Formulation. Indian E-Journal of
Pharmaceutical Sciences. 2015; 1(1): 40-44.
23.
Audumbar Digambar Mali. Estimation of Domperidone in Bulk and Formulation by First Order
Derivative Area under Curve UV-Spectrophotometry Methods. International Journal
of Pharmacy and Technology. 2015; 7(1): 8040-8048.
24.
Audumbar Digambar Mali. Estimation of Ranitidine in Bulk and
Formulation by Second Order Derivative Area under Curve UV-Spectrophotometric
Methods. International Journal of
Analytical, Pharmaceutical and Biomedical Sciences. 2015; 4(6): 7-16.
25.
Audumbar Digambar Mali. Zero,
First, Second Order Derivative and Area under Curve Spectrophotometric Methods
for Determination of Cefixime Trihydrate
in Pharmaceutical Formulation. International
Journal of Pharmacy and Pharmaceutical Sciences. 2015; 7(6): 321-325.
26.
Audumbar Digambar Mali, Ritesh
Bathe, Manojkumar Patil and
Ashpak Tamboli. Zero order and area under
curve spectrophotometric methods for determination of Levocetirizine
in pharmaceutical formulation. International
Journal of Advances in Scientific Research. 2015; 1(06): 270-275.
27.
International Conference on Harmonization (ICH) of
Technical Requirements for the registration of Pharmaceuticals for Human use,
Validation of Analytical Procedures Methodology. ICH-Q2 (R1), Geneva.1996, 1-8.
Asian J. Pharm. Tech. 2016; Vol. 6: Issue 1, Jan. - Mar., Pg 15-20