J Pharm Sci Bioscientific Res. 2015 5(5):487-493 ISSN NO.

2271-3681

Development and Validation of UV- Spectrophotometric Method for

Simultaneous Estimation of Amlodipine Besylate and Hydrochlorothiazide
in Combined Dosage Form Including Stability Study
Z. M. Sayyed*, S. A. Shinde, V. J. Chaware, B. P. Chaudhari, K. R. Biyani
Department of Quality Assurance, Anuradha College of Pharmacy, Chikhli. Dist. - Buldana, Pin – 443201, Maharashtra, India

ABSTRACT:

Two simple, rapid, precise and accurate spectrophotometric methods have been developed for determination of
Amlodipine Besylate (AMB) and Hydrochlorothiazide (HCTZ) by simultaneous equation method and stability study
method in combined tablet dosage form. The simultaneous equation method is based on measurement of absorbance
at 238 nm and 271 nm as two wavelengths selected for quantification of Amlodipine Besylate and
Hydrochlorothiazide. The method obeyed Beer’s law in the concentration range of 5-30 μg/ml for Amlodipine and 2.5-
15 μg/ml for Hydrochlorothiazide. Amlodipine and Hydrochlorothiazide (Marketed Formulation) were subjected to
stress degradation under different conditions recommended by ICH. The proposed methods were validated and can
be applied successfully for routine quality control analysis of AMB and HCTZ in bulk and pharmaceutical formulation.

KEYWORDS: Simultaneous Equation Method, λmax, Validation, Amlodipine Besylate, Hydrochlorothiazide, Stability
Study, Stress Degradation.

Article history: INTRODUCTION:

Received 23 Sept 2015
Revised 04 Oct 2015 Amlodipine Besylate, 3-Ethyl 5-methyl (4RS)-2-[(2-aminoethoxy) methyl]-4-
Accepted 06 Oct 2015 (2-chlorophenyl)-6-methyl-1, 4-dihydropyridine-3, 5-dicarboxylate
Available online 15 Oct 2015 [1,2]
benzenesulfonate, is a antihypertensive and antianginal drug. Amlodipine
Besylate belongs to a class of medications called calcium channel blockers
Citation:
Sayyed Z. M., Shinde S. A., Chaware V. J., (CCBs).[3]Amlodipine Besylate having molecular formula C20H25ClN2O5 and
[4,5]
Chaudhari B. P., Biyani K. R. Development molecular weight 408.879 g/mol. It is official in IP, BP, EP and USP (Fig. No.
and Validation of UV- Spectrophotometric 1).
Method for Simultaneous Estimation of
Amlodipine Besylate and
Hydrochlorothiazide, 6-chloro-3, 4-dihydro-2H-1, 2, 4-benzothiadiazine-7-
Hydrochlorothiazide In Combined Dosage
Form Including Stability Study. J Pharm Sci Sulphonamide 1, 1- dioxide, is a diuretic, which inhibits active chloride
Bioscientific Res. 2015 5(5):487-493 reabsorption at the early distal tubule via the Na-Cl co-transporter, resulting
[6,7]
in an increase in the excretion of sodium, chloride, and water. It has been
used in the treatment of several disorders including edema, hypertension,
*For Correspondence:
diabetes insipidus, and hypoparathyroidism.[8,9] Hydrochlorothiazide having
Mr. Zuber Maheboob Sayyed molecular formula C7H8ClN3O4S2 and molecular weight 297.739 g/mol. It is
Department of Quality Assurance, [10]
official in IP, BP and USP (Fig. No. 2).
Anuradha College of Pharmacy, Chikhli.
Dist.- Buldana, Pin – 443201, The literature review reveals that few analytical techniques like RP-HPLC,
Maharashtra (India). HPTLC, HPLC, Spectroscopic methods have been reported for the
Email: Patelsanket2312@gmail.com
determination of Amlodipine Besylate and its combination with other drugs
(www.jpsbr.org) [11-13]
in pharmaceutical dosage forms. Estimation of

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Hydrochlorothiazide in combination with other drugs by The aliquot portion of stock standard solutions of AMB
spectrophotometric methods, LC-MS, HPLC and stability and HCTZ were diluted appropriately with 0.1 N sodium
study has been reported Hydrochlorothiazide in hydroxide to obtain concentration 5 μg/ml and 12.5
combination with other drugs by spectrophotometric μg/ml respectively. The solutions were scanned in the
methods, LC-MS, HPLC and stability study has been range of 200-400 nm in 1 cm cell against blank. The λmax
[14,15]
reported. But there are no methods reported in any was determined on double beam UV – Visible
pharmacopoeia for simultaneous estimation of Spectrophotometer using 0.1 N sodium hydroxide as
Amlodipine Besylate and Hydrochlorothiazidein blank. The λmax was found to be 238 nm and 271 nm
combined dosage form including stability study using 0.1 respectively. The Overlain UV absorbance spectrum of
N NaOH as a solvent. AMB and HCTZ is shown in Fig.No. 3.

MATERIALS AND METHODS Preparation of Calibration Curve or study of Beer-

Lambert’s Law:
Instruments:
The aliquot portion of stock standard solutions of AMB
(a) Spectrophotometer -Double beam UV –Visible and HCTZ were diluted appropriately with 0.1 N sodium
spectrophotometer with 1 cm matched quartz cell Model hydroxide as solvent to get a series of concentration
UV-1601 Shimadzu. between 5- 30 μg/ml of AMB and 2.5 – 15 μg/ml of HCTZ.
(b) Hot Air Oven- Dolphin. The absorbance of each solution was measured at 238
(c) Electronic Balance-Shimadzu. nm and 271 nm in 1 cm cell against 0.1 N sodium
hydroxide as blank. The graphs plotted as concentration
Reagents and Chemicals: Vs absorbance at selected wavelengths for AMB and
HCTZ are shown in Fig. No. from 4 and 5.
Amlodipine Besylate and Hydrochlorothiazide reference
standard were kindly provided by Yarrow Chem. Products Determination of A (1%, 1 cm) of drugs at selected
Mumbai. 0.1 N Sodium Hydroxide of analytical grade was wavelengths:
used as a solvent throughout the analysis. All chemicals Aliquot portions of AMB from stock solution were
and reagents used were of analytical reagent grade. transferred to five 10 ml volumetric flasks; volume was
adjusted to mark to obtain the concentration of 5 μg/ml.
Marketed Preparation: Similarly, aliquot portions from HCTZ stock solution were
The brand name of marketed combined tablet transferred to 10 ml volumetric flasks; volume was
formulation is Amlong-H containing Amlodipine Besylate adjusted to mark to obtain concentration of 12.5 μg/ml.
5.0 mg and Hydrochlorothiazide 12.5 mg manufactured Absorbance of these solutions were recorded at two
by Micro Labs Ltd. South Sikkim. wavelengths 238 nm and 271 nm. A (1%, 1 cm) values of
drugs were calculated using following formula-
Preparation of standard stock solution:
A (1%, 1 cm) = Absorbance / Concentration (g / 100 ml)
(a) Amlodipine Besylate:- Standard stock solution of Results of A (1 %, 1 cm) of drugs are given in Table No. 1.
Amlodipine besylate was prepared by dissolving 10 mg
Cx= A2ay1 – A1ay2 / ax2ay1 – ax1ay2 ---------- (I)
Amlodipine besylate in 50 ml of 0.1N sodium hydroxide
and sonicated for 20 min and then diluted upto 100 ml to Cy= A1ax2 – A2ax1 / ax2ay1 – ax1ay2 ---------- (II)
produce a concentration of 100 μg/ml which was
Where,
standard stock solution.
A1 and A2 are the absorbance of the sample solution
(b) Hydrochlorothiazide: -Standard stock solution of measured at 238 nm and 271 nm respectively.
Hydrochlorothiazide was prepared by dissolving 10 mg of Cx and Cy are concentration of AMB and HCTZ
Hydrochlorothiazide in 100 ml of 0.1N sodium hydroxide respectively.
to produce a concentration of 100 μg/ml which was ax1 and ax2 are absorptivity of AMB at 238 nm and 271
standard stock solution. nm respectively.
ay1 and ay2 are absorptivity of HCTZ at 238 nm and 271
Determination of λ max of Amlodipine Besylate and nm respectively.
Hydrochlorothiazide:

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Simultaneous Estimation of drugs in standard C = Amount of pure drug added. Results are shown in
laboratory mixture: Table No. 4.

In order to study the practicability of proposed method

Validation of Proposed Method:
for simultaneous estimation of AMB and HCTZ in
marketed pharmaceutical formulations, the method was Validation of method was done as per ICH guidelines.
first tried for estimation of drugs in standard laboratory Method was validated for various parameters such as
mixture. Accurately weighed 10 mg AMB and 10 mg HCTZ accuracy, precision, linearity, repeatability and
were transferred to 100 ml volumetric flask individually ruggedness.
containing 40 ml 0.1 N sodium hydroxide, shake manually
for 10 minute and the volume was adjusted to the mark Accuracy: Accuracy of the proposed method was
with the same solvent. Appropriate aliquot portion of ascertained on the basis of recovery studies performed
these solutions were mixed to get the concentration 5 by the standard addition method. The results of recovery
μg/ml AMB and 12.5 μg/ml of HCTZ. Absorbance was studies are shown in Table No. 4.
measured at 238 nm and 271 nm against 0.1 N sodium
hydroxide as blank. Amount of each drug was estimated Precision: Precision was determined by intra-day and
using (I) and (II) equation and results are given in Table inter-day precision. Intra-day precision was determined
No. 2. by analyzing the 2.5, 5, 10 μg/ml of AMB and 10, 12.5, 15
μg/ml of HCTZ drug solution for three times in the same
Simultaneous Estimation of drugs in tablets: day. Inter-day precision was determined by analyzing the
Twenty tablets each containing 5 mg of AMB and 12.5 mg same concentration at three different days. The results
of HCTZ were weighed and average weight was are given in Table No.5.
calculated. The tablets were crushed to fine powder. The
powder equivalent to 5 mg of AMB and 12.5 mg of HCTZ Linearity: The study of linearity and range was performed
was transferred to 100 ml volumetric flask containing 70 as per ICH guidelines. AMB and HCTZ was found to be
ml of 0.1 N sodium hydroxide by intermittent shaking linear at a concentration range of 5-30 µg/ml and 2.5-15
2
followed by sonication for 10 min and then the volume µg/ml respectively with R = 0.99 at selected wavelength
was made upto 100 ml with 0.1 N sodium hydroxide. The for both the methods.
solution was diluted further with 0.1 N sodium hydroxide
to obtain 5 µg/ml of AMB and 12.5 µg/ml of HCTZ. The Repeatability: Repeatability was determined by analyzing
solution was filtered through a Whatman filter paper AMB (5 μg/ml) and HCTZ (12.5 μg/ml) of drug solutions
(No. 41). The absorbances were recorded. The for five times and results are given in Table No. 6.
concentrations of two drugs in sample were determined
using equation No. (I) and (II), results are given in Table Ruggedness: Analysis of aliquots from homogenous slot
No. 3. Brand Name: Amlong-H (Composition- Amlodipine by two analyst using same operational and environment
Besylate: 5.0 mg, Hydrochlorothiazide: 12.5 mg), Average conditions was performed for determination of
Weight: 250 mg. Ruggedness of proposed method. The results are given
in Table No. 7.
Recovery Study:
In recovery study to the preanalysed sample solutions (5 Method for Performing Stability Study:
μg/ml AMB and 12.5 μg/ml of HCTZ) a known amount of Stress or Forced Degradation Studies: The International
standard solutions of pure drugs (AMB and HCTZ) was Conference on Harmonization (ICH) guideline entitled
added at different level. The % recovery was calculated stability testing of new drug substances and products
by using formula, requires that stress testing be carried out to elucidate the
inherent stability characteristics of the active substance.
% Recovery = (A / B + C) X 100 The present work was performed on the stress
degradation studies of Amlodipine and
Where,
Hydrochlorothiazide (Tablet).
A = Total amount of drug estimated,
B = Amount of drug found on preanalysed basis and

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Preparation of stock solution (tablet): An accurately Record the absorbance of stressed sample then compare
weighed tablet powder equivalent to 5 mg of AMB and it with absorbance of unstressed sample to determine
12.5 mg of HCTZ was transferred to 100 ml volumetric the % degradation.
flask containing about 40 ml 0.1 N sodium hydroxide, ( ) ( )
sonicated for 10 min and volume was made upto the
mark with the same solvent and further diluted to obtain
5 μg/ml of AMB and 12.5 μg/ml of HCTZ and filtered Results of the Stability studies are given in Table No. 8.
through Whatman filter paper (No. 41).
RESULTS AND DISCUSSION
Stress degradation by hydrolysis under acidic condition: The wavelength of Amlodipine Besylate and
To 1 ml of stock solution of Amlodipine and Hydrochlorothiazide were found to be 238 nm and 271
Hydrochlorothiazide (tablet), 5 ml of 1N HCl was added in nm respectively in the solution of 0.1 N Sodium
10 ml of volumetric flask and kept for 3 hours. After 3 Hydroxide. In this method drugs obeyed Beer’s law in the
hours solution was diluted with 0.1 N sodium hydroxide concentration range of 5-30 µg/ml of AMB and 2.5-15
up to the mark and the solution was taken in cuvette and µg/ml of HCTZ. The results showed an excellent
analysed in UV spectrophotometer at 238 nm and 271 correlation between absorbance’s and concentration of
nm of AMB and HCTZ respectively. the drugs. Standard calibration curves for AMB and HCTZ
2
were linear with correlation coefficients R =0.99 at all the
Stress degradation by hydrolysis under basic condition: selected wavelengths. The results of analysis of the
To 1 ml of stock solution of Amlodipine and marketed formulation by Simultaneous Equation Method
Hydrochlorothiazide (tablet), 5 ml of 1 N NaOH was are shown in Table No.3. Method validations were done
added in 10 ml of volumetric flask and kept for 3 hours. as per ICH Validation parameters like Accuracy,
After 3 hours solution was diluted with 0.1 N sodium Precision,Linearity, Repeatability and Ruggedness. The
hydroxide up to the mark and the solution was taken in method showed accuracy in the range of 96-100 % .
cuvette and analysed in UV spectrophotometer at 238 Results of Validation parameters are shown in Table
nm and 271 nm of AMB and HCTZ respectively. No.from 4-7. The stress degradation studies showed that
Amlodipine Besylate and Hydrochlorothiazide (Marketed
Oxidative degradation:To 1 ml of stock solution of Formulation) i.e. tablet undergoes degradation in acidic
Amlodipine and Hydrochlorothiazide (tablet), 5 ml of 6% and oxidative conditions whereas it is relatively stable
Hydrogen Peroxide (H2O2) was added in 10 ml of when exposed to basic and dry heat conditions. Results
volumetric flask and kept for 3 hours. After 3 hours of stress degradation studies of AMB and HCTZ are
solution was diluted with 0.1 N sodium hydroxide up to shown in Table No.8.
the mark and the solution was taken in cuvette and
analysed in UV spectrophotometer at 238 nm and 271 CONCLUSION
nm of AMB and HCTZ respectively. A simple, accurate, precise and rapid UV-visible
spectrophotometric method was developed for
Dry heat induced degradation: Amlodipine and simultaneous estimation of AMB and HCTZ in combined
Hydrochlorothiazide (tablet powder) sample was taken in tablet dosage form. The method was validated for AMB
a Petri plate and exposed to a temperature of 50°C for 48 and HCTZ in pharmaceutical dosage form. The present
hours in an oven. After 48 hours, 10 mg of the sample study was concluded to understand the degradation
was diluted with 0.1 N NaOH in order to make the behavior of AMB and HCTZ under ICH recommended
volume upto 100 ml. From this solution, dilutions were stress conditions. This study is also an example of the
carried out and the solution was taken in cuvette and development of a stability indicating assay method for
analysed in UV spectrophotometer at 238 nm and 271 AMB and HCTZ where forced degradation was carried out
nm of AMB and HCTZ respectively. under all stress conditions. These drugs showed
degradation in all of the studied conditions. However the
Determination of % Degradation:- extent of degradation was different. The method
developed for quantitative determination of AMB and
HCTZ is rapid, precise, accurate and selective. The results

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of validation tests were found to be satisfactory and Dosage Form, Journal of Applied Pharmaceutical
therefore, these methods can be applied successfully for Sciences, 2011; 1(4)-46-49.
routine quality control analysis of AMB and HCTZ in bulk 9. Haque MA, Bakshi V and Srikanth G. Development
and pharmaceutical formulation. The developed method and Validation of Analytical Method for Simultaneous
can be conveniently used for the assay determination of Estimation of Quinapril and Hydrochlorothiazide by
AMB and HCTZ in bulk drugs and pharmaceutical dosage RP-HPLC, International Journal of Innovative
form. Pharmaceutical Science& Research, 2014; 2(11):2757-
64.
ACKNOWLEDGEMENT 10. Rathee P, Rathee S and Chaudhary H. Stability
Indicating UV-Methods for Simultaneous
The authors wish to thank Yarrow Chem. Products
Determination of Losartan and Hydrochlorothiazide in
Mumbaifor providing gift samples of Amlodipine Besylate
Pharmaceuticals, Eurasian Journal of Analytical
and Hydrochlorothiazide.
Chemistry, 2009; 4(1):98-09.
11. Kranthi M and Srinivas A. Analytical Method
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TABLES AND FIGURES

Table No. 1: Absorptivity values of AMB and HCTZ at 238 Table No. 5: Results of Precision Studies (Intra-day and
nm and 271 nm Inter-day)
Sr. Drugs Conc. Intra-day Inter-day
AMB AMB HCTZ HCTZ No. (µg/ml) amount amount
Sr. No. (238 nm) (271 (238 nm) (271 nm) found (n=5) found (n=5)
nm)
1 149 88.3 60 158 Mean % Mean %
2 149.1 87 63 157.3 RSD RSD
3 151 93.8 57.09 158 1 AMB 2.5 2.36 0.88 2.20 1.20
4 157.5 91 41.3 149.5
5 4.89 0.65 4.01 0.92
5 160.7 91.4 49.71 151
Mean 153.46 90.3 54.22 154.76
10 9.77 0.29 8.50 1.25
± SD 0.475 0.832 1.03 0.884
2 HCTZ 10 9.58 0.94 9.27 0.41

Table No. 2: Analysis of AMB and HCTZ in Standard 12.5 12.40 0.49 12.43 0.17
Laboratory Mixture
15 14.93 0.22 14.70 0.31
Sr. Drugs Conc. of Amount % % RSD
No. std found Amount (n=5)
(µg/ml) (µg/ml) found
1 AMB 5 4.905 98.1 0.68
Table No. 6: Results of Repeatability Studies
2 HCTZ 12.5 12.41 99.28 0.55 Sr. Drugs Conc. Mean % RSD
No. (µg/ml) conc. (n=5)
found
(µg/ml)
Table No. 3: Analysis of AMB and HCTZ in Tablet
1. AMB 5 4.921 0.39
Sr. Drugs Conc. of Mean % % RSD 2. HCTZ 12.5 12.41 0.47
No. tablet amount Amount (n=5)
(µg/ml) found found
(mg/tab) (mean)
1 AMB 5 4.88 97.6 0.72
Table No. 7: Results of Ruggedness studies

2 HCTZ 12.5 12.39 99.12 0.49

Sr. Amount found % RSD (n=3)
No. (%)
AMB HCTZ AMB HCTZ
Table No. 4: Results of Recovery Studies
Sr Pre- Pure Drug % % RSD 1 Analyst-I 99.11 99.92 0.87 0.75
. analyse drug recover Recover (n=5) 2 Analyst-II 98.13 97.3 0.95 1.16
N d added ed y
o. samples (µg/ml) (µg/ml)
(µg/ml)
A HC A HC A HC A HC A HC
M TZ M TZ M TZ M TZ M TZ
B B B B B
1 5 12 2. 10 2. 9.6 97 96. 0. 1.
.5 5 44 8 .6 8 76 11
2 5 12 5 12 4. 12. 98 98. 0. 0.
.5 .5 90 37 96 52 49
3 5 12 10 15 9. 14. 96 99. 1. 1.
.5 61 86 .1 06 09 38

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Table No. 8: Results of Stability Studies

Sr. Stress Condition Conc. taken Conc. found % % Assay of active
No. (µg/ml) (µg/ml) Degradation substance of drugs
AMB HCTZ AMB HCTZ AMB HCTZ AMB HCTZ
1 Acidic Degradation 5 12.5 4.49 11.29 10.11 9.66 89.89 90.34
(1 N HCl, 3 hours)
2 Basic Degradation 5 12.5 4.97 12.39 0.58 0.81 99.42 99.19
(1 N NaOH, 3 hours)
3 Oxidative Degradation 5 12.5 4.30 10.58 13.86 15.3 86.14 84.7
( 6% H2O2, 3 hours)
4 Dry Heat Induced 5 12.5 4.91 12.13 1.79 2.96 98.21 97.04
Degradation
(50°C, 48 hours)

Fig. No. 1: Chemical Structure of Amlodipine

Besylate

Fig. No. 4: Plot of Beer- Lambert’s study for

AMB at 238 nm and 271 nm

Fig. No. 2: Chemical Structure of

Hydrochlorothiazide

AMB
HCTZ

Fig. No. 5: Plot of Beer- Lambert’s study for

HCTZ at 238 nm and 271 nm

Fig. No. 3: Overlain spectra of AMB and HCTZ

(5: 12.5 µg/ml)

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