899

Priyanka Bendapudi. et al. / I nternational J ournal of Biological & Pharmaceutical Research. 2012; 3(7): 899-903.

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International Journal of Biological
&
Pharmaceutical Research
Journal homepage: www.ijbpr.com


DEVELOPMENT OF RP- HPLC METHOD FOR THE
SIMULTANEOUS ESTIMATION OF PROPRANOLOL
HYDROCHLORIDE AND HYDROCHLOROTHIAZIDE IN
COMBINED DOSAGE FORM

Priyanka Bendapudi
1*
, Venketeswara Rao. P
2
, Sudhakar babu A.M.S
3
, Pramod. N
2

2
Department of Pharmaceutical Chemistry,
3
Department of Pharmaceutics,
1
A.M.Reddy Memorial College of Pharmacy,
Narasaraopet, Guntur district, Andhra Pradesh, India.

ABSTRACT
A simple, precise, economic, accurate high Performance Liquid Chromatographic (HPLC) method was developed and
validated for the analysis of hydrochlorothiazide and Propranolol hydrochloride. Chromatographic separation achieved
isocratically on a C18 column [Use Inertsil C18, 250 mm x 4.6 mm,5m] utilizing a mobile phase of Methanol: Acetonitrile
:Water in the ratio of 50:30:20(v/v/v)] at a flow rate of 1.0 ml/min with UV detection at 232nm. The retention time of
hydrochlorothiazide and propranolol was 4.5, 9.02 respectively. The developed method was validated in terms of accuracy,
precision, linearity, limit of detection, limit of quantitation. This study aimed at developing and validating an HPLC method,
being simple, accurate and precise, and the proposed method can be used for the estimation of these drugs in dosage form
formulation INDERIDE (hydrochlorothiazide : propranolol hydrochloride=50:80ppm).

Key Words: Hydrochlorothiazide, Propranolol hydrochloride, RP-HPLC, Validation.

INTRODUCTION
Propranolol hydrochloride (Reiter MJ, 2004) is a
non-selective beta blocker mainly used in the treatment of
hypertension. Propranolol hydrochloride is used in the
treatment or prevention of many disorders including acute
myocardial infarction, arrhythmias, angina pectoris,
hypertension, hypertensive emergencies, hyperthyroidism,
migraine, pheochromocytoma, menopause, and anxiety.
Chemically propranolol hydrochloride is 1-naphthalen-1-
yloxy-3-(propan-2-ylamino) propan-2-ol hydrochloride
(Fig.1a).
Hydrochlorothiazide (Deppler HP, 1981), is a first
line diuretic drug of thiazide class that acts by inhibiting

Corresponding Author

Priyanka Bendapudi
Email: priyankabendapudi@gmail.com
the kidneys ability to retain water. This reduces the volume
of the blood decreasing blood return to the heart and thus
cardiac output and, by other mechanisms, is believed to
lower peripheral vascular resistance. Chemically
hydrochlorothiazide is 6-chloro-3,4-dihydro-2H-1,2,4-
benzothiadiazine-7-sulfonamide1,1-dioxide (Fig. 1b).
Propranolol and hydrochlorothiazide combination
is used to treat high blood pressure (hypertension).
Literature survey reveals a few spectrophotometric and
chromatographic methods for the estimation of both drugs
as a single component and in combination with other drugs
(Anna et al., 2001; Michael et al., 1987; Jain SK et al.,
2002; Silvana EV et al., 2006; Nikitha AS et al., 2012).
The objective of present work is to develop simple, rapid,
and precise RP-HPLC method for the estimation of
propranolol hydrochloride and Hydrochlorothiazide using
methanol : acetonitrile : water (50 : 30 : 20)v/v.
IJBPR
900
Priyanka Bendapudi. et al. / I nternational J ournal of Biological & Pharmaceutical Research. 2012; 3(7): 899-903.

MATERIALS AND METHODS
Materials
HPLC grade Water, Methanol, acetonitrile-
procured from Merck, India. Formulations of Propranolol
hydrochloride and Hydrochlorothiazide are purchased from
local market. Standard drugs of Propranolol hydrochloride
and Hydrochlorothiazide procured from Dr. Reddys
laboratories.

Equipment
The instrument used was Peak LC-7000 series
HPLC instrument. The instrument is equipped with a LC -
7000 pump and variable wavelength programmable UV
detector and a 20L Rheodyne Inject port. The other
equipment used is UV-2301 UV-Visible
spectrophotometer.Sonication was done using Loba
ultrasonic bath sonicator. Weighing was done on Denwar
weighing balance.

Chromatographic Conditions
Inertsil C18, 250 mm x 4.6 mm,5 ) was used for
separation. The mobile phase containing Methanol:
Acetonitrile: Water in the ratio of 50:30:20(v/v/v) was
delivered at a flow rate 1.0 ml/min and the elution was
monitored at 232 nm. Injection volume was 20l and the
analysis was performed at ambient temperature.

Standard stock solution
Stock solutions of propranolol hydrochloride and
Hydrochlorothiazide (1 mg/ml) were prepared separately
using mobile phase as solvent. From the standard stock
solutions, mixed standard solutions of different
concentrations ranging from 25 to 150 ppm of propranolol
hydrochloride and 40 to 240ppm of hydrochlorothiazide
were prepared by diluting with mobile phase.

Preparation of Sample solution
10 tablets were weighed and powdered. Weighed
accurately a quantity equivalent to 25mg of
hydrochlorothiazide and 40mg of propranolol
hydrochloride transferred into a 100ml volumetric flask
and diluted to the volume with diluent. Further diluted
2.5ml of the above solution to 50ml with diluent and mixed
to get 50:80ppm concentration of hydrochlorothiazide and
propranolol respectively. The above solution was filtered
through 0.45m nylon filter/0.45m PVDF membrane
filter.

Method validation
The method was validated as per ICH
guidelines14, 15 (ICH guidelines 1994 and 1996; USP
1995).

Accuracy: The accuracy of the method was determined by
recovery experiments. The recovery studies were carried
out three times and the percentage recovery were
calculated and presented in table no 1.

Precision: The precision of the method was demonstrated
by inter day and intraday variation studies. In the intraday
studies, six repeated injections of standard solutions were
made and the response factor of drug peaks and percentage
RSD were calculated. In the inter day variation studies, six
repeated injections of standard solutions were made for
three consecutive days and response factor of drug peaks
and percentage RSD were calculated.

Linearity: The linearity of the method was determined at
concentration levels ranging from 25 to 150ppm for
hydrochlorothiazide and 40 to 240ppm for propranolol
hydrochloride. The calibration curve was constructed by
plotting response factor against concentration of drugs.

LOD and LOQ: The Limit of Detection (LOD) and Limit
of Quantification (LOQ) of the developed method were
determined by injecting progressively low concentrations
of the standard solutions using the developed RP-HPLC
method. Determination of the signal-to-noise ratio was
performed by comparing measured signals from samples
with known low concentrations of analyte with those of
blank samples and establishing the minimum concentration
at which the analyte can be reliably detected.

Robustness: Robustness of the method was determined by
creating variations in flow rate, mobile phase composition
and changes in wavelength and determining percentage of
change in assay.

RESULTS
In order to achieve simultaneous elution of the
two components, initial trails were performed with the
objective to select adequate and optimum chromatographic
conditions. Parameters, such as ideal mobile phase and
their proportions, detection wavelength, optimum pH,
different columns and concentration of the standard
solutions were carefully studied. Several solvents were
tested by using different proportions. Methanol:
Acetonitrile: Water in the ratio of 50:30:20(v/v/v) was
selected as the optimum mobile phase and a flow rate of
1.0 mL/min. Under these conditions, the analyte peaks
were well resolved and were free from tailing. The tailing
factor was <2.0 for both the analytes. The retention times
of Hydrochlorothiazide and Propranolol hydrochloride
were found to be 4.5 min and 9.02 min, respectively.

Optimized Chromatographic conditions
Mobile phase: MeOH: Acetonitrile:Water(50:30:20)v/v
Diluent : Acetonitrile
Flow rate : 1.0ml/min
Column : Inertsil , C
18
column (250 x 4.6 mm, 5 )
Detector wave length : 232 nm
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Priyanka Bendapudi. et al. / I nternational J ournal of Biological & Pharmaceutical Research. 2012; 3(7): 899-903.

Column temperature : Ambient
Injection volume : 20 l
Accuracy: The recovery studies yielded the mean results
within 98 to 102 % of true concentration of each drug
indicating that the test method has an acceptable level of
accuracy.

Linearity: The calibration plot was constructed by plotting
response factor (RF) versus concentration (g/ml) of
hydrochlorothiazide and propranolol hydrochloride which
were found to be linear in the range of 25:40ppm to
150:240ppm for hydrochlorothiazide (r1=0.999 and
propranolol hydrochloride (r2=0.999) respectively.

LOD and LOQ: Limit of detection (LOD) values of
hydrochlorothiazide and propranolol hydrochloride were
experimentally verified to be 0.2ppm and 0.5ppm,
respectively. Limit of quantitation (LOQ) values of
hydrochlorothiazide and propranolol hydrochloride were
found to be 0.6ppm and 0.15ppm, respectively.
Robustness: % of change and relative standard deviation
was calculated and found to be less than 2%.

Assay
The proposed method was applied to the
simultaneous estimation of Hydrochlorothiazide and
Propranolol hydrochloride in tablets. The assay results
show that the proposed method was selective for the
simultaneous determination of Hydrochlorothiazide and
propranolol hydrochloride without interference from the
excipients used in the tablet dosage form. The values were
shown in Table 6.

Formulation: INDERIDE (HC 25 and PR 40mg)

Fig 1a. 1-naphthalen-1-yloxy-3-(propan-2-ylamino)
propan-2-ol hydrochloride

Fig 1b. 6-chloro-3,4-dihydro-2H-1,2,4-
benzothiadiazine-7-sulfonamide 1,1-dioxide

Fig 2a. Linearity Graph of hydrochlorothiazide

Fig 2b. Linearity Graph of Propranolol hydrochloride


Table 1. Recovery studies
Sample
Level %
Hydrochlorothiazide Propranolol
Conc., (ppm) Conc.,Obtained % recovery Conc., (ppm) Conc., Obtained % recovery
50% 75 74.42 99.22 120 118.68 98.9
75 73.67 98.22 120 118.65 98.87
75 75.34 100.45 120 119.43 99.52
100% 100 100.32 100.32 160 160.55 100.34
100 99.98 99.98 160 158.36 98.97
100 99.39 99.39 160 160.1 100.06
150% 125 125.52 100.41 200 201.8 100.9
125 125.91 100.72 200 197.69 98.84
125 123.21 98.56 200 201.34 100.67
Precision: Precision studies (intraday and interday) has shown the result within acceptance limit, % RSD below 2.0

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Priyanka Bendapudi. et al. / I nternational J ournal of Biological & Pharmaceutical Research. 2012; 3(7): 899-903.

Table 2. Intraday Precision
S.No Hydrochlorothiazide Concentration Area Propranolol Area
1


50ppm
66253


80ppm
108159
2 66274 108354
3 67635 109338
4 66530 109371
5 65736 108985
6 65708 105060
RSD 1.06 1.74

Table 3. Interday precision
S.No Hydrochlorothiazide Concentration Area Propranolol Area
1

50ppm
66639

80ppm
108087
2 66082 102472
3 66206 106464
4 65856 106234
5 68753 107350
6 68649 106600
RSD 1.96 1.02

Table 4. Linearity results
S.No Hydrochlorothiazide Concentration (ppm) Area Propranolol Concentration (ppm) Area
1 25 33912 40 62059
2 50 68453 80 101571
3 75 99600 120 151344
4 100 131328 160 204176
5 125 162685 200 251005
6 150 199106 240 304103
Result
C.C
Intercept
Slope
0.999
968.5
1311
C.C
Intercept
Slope
0.999
4236
1243

Table 5. Robustness
S.No Condition
Hydrochlorothiazide
(50ppm)
Area
% of
Change
Propranolol
(80ppm)
Area % of Change
1 Flow (0.9ml/min) 68690 100.34 0.34 100267 98.71 1.29
2 Flow (1.1ml/min) 67739 98.95 1.05 100065 98.51 1.49
3 MP (M.A.W 48:32:20) 68401 99.92 0.08 100834 99.27 0.73
4 MP (M.A.W 52:28:20) 67409 98.47 1.53 102575 100.98 0.98
5 WL 230nm 67763 98.99 1.01 101614 100.04 0.04
6 Wl 234nm 68612 100.23 0.23 100764 99.2 0.8

Table 6. Assay Results
S.No Drug Sample Area % of Assay
1 Hydrochlorothiazide (50ppm) 68229 99.67
2 Propranolol (80ppm) 100242 98.69

DISCCUSSION
The developed RP-HPLC method was accurate,
precise, reproducible and robust. The percentage recoveries
of Hydrochlorothiazide and Propranolol hydrochloride
were found to be in the range of 98.22-100.72% and 98.84-
100.67%, respectively. The results were shown in Table1
which indicates that the method is accurate. Precision
studies has shown the result within acceptance limit, %
RSD below 2.0, indicating reproducibility of the method
shown in tables 2 and 3.The developed method has been
found to be better, because of its wide range of
linearity(25-150ppm for propranolol,40-240ppm for
hydrochlorothiazide), robust under the influence of small
variations in flow rate, wavelength, mobile phase
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Priyanka Bendapudi. et al. / I nternational J ournal of Biological & Pharmaceutical Research. 2012; 3(7): 899-903.

composition as shown in table 5, use of a readily available
mobile phase, lack of extraction procedure and low
retention times. The assay results and low %RSD values
indicated that the developed method can be used for
routine analysis of Hydrochlorothiazide and Propranolol
hydrochloride in pharmaceutical dosage form. All these
factors make the proposed method suitable for the
quantification of Hydrochlorothiazide and Propranolol
hydrochloride in bulk drugs and in table dosage form. The
method can be successfully used for the routine analysis of
hydrochlorothiazide and propranolol hydrochloride in
pharmaceutical dosage forms without interference.

ACKNOWLEDGEMENT
The author are thankful to smt.Santhi Secretary of
A.M. Reddy Memorial College of Pharmacy,
Narasaraopeta, Andhra Pradesh through the principal for
providing necessary facilities to carry out research work. A
special thanks to Dr.P.Venkateswara rao for his valuable
guidance.

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