E-ISSN: 2278-3229

IJGHC, June 2014August -2014; Vol.3, No.3, 931-938.

International Journal of Green and

Herbal Chemistry
An International Peer Review E-3 Journal of Sciences

Available online atwww.ijghc.com

Section A: Green Chemistry

Research Article

CODEN (USA): IJGHAY

Development and validation of analytical method for

estimation of Enalapril maleate in oral solution
Rachana DP, Jigisha Patel, Mayank Bapna*
*

Department of Quality assurance, Shivam Pharmaceutical Studies and Research Centre,

Valasan-Anand, Gujarat
Received: 18 May 2014; Revised: 03 June 2014; Accepted: 16 June 2014

Abstract: A simple, fast, accurate and precise reverse phase high performance liquid
chromatographic method has been developed for the estimation of Enalapril maleate
in oral solution containing Sodium benzoate as excipient. The chromatographic
separation was achieved on Zorbax Eclipse Plus C8 (250 x 4.6) mm; 5m column
with an isocratic mixture of phosphate buffer (2.1pH): acetonitrile (75:25) v/v. The
mobile phase was set at a flow rate of 1.3 ml/min with injection volume 20 l,
wavelength of detection 215 nm and column temperature 60 C. The retention times
for Enalapril maleate, and Sodium benzoate were found to be 6.8360.1 min, and
5.876 0.1 min, respectively. The linearity was obtained in the range of 40-400
g/mL and 80 -800 g/mL with correlation coefficient 0.998 and 0.999 for Enalapril
maleate and Sodium benzoate, respectively. The proposed methods was validated as
per ICH guidelines and successfully applied for the determination of investigated
drugs in oral solution.
Keywords: Enalapril maleate, sodium benzoate, RP-HPLC, forced degradation

INTRODUCTION
Enalapril maleate (EM)1: (S)-1[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-L-alanyl]-L-proline,(Z)-2butenedioate salt (1:1), is an antihypertensive (ACE Inhibitor) drug. Enalapril, after hydrolysis to
enalaprilat, inhibits angiotensin-converting enzyme (ACE) in human subjects and animals. ACE is a
peptidyl dipeptidase that catalyzes the conversion of angiotensin I to the vasoconstrictor substance,
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Bapna et al.

angiotensin II. Angiotensin II also stimulates aldosterone secretion by the adrenal cortex. The
beneficial effects of (EM) in hypertension and heart failure appear to result primarily from
suppression of the renin-angiotensin-aldosterone system. Inhibition of ACE results in decreased
plasma angiotensin II, which leads to decrease in vasopressor activity and taldosterone secretion.
Sodium benzoate (SB)2 : It is used as preservative and food additive. It is bacteriostatic and
fungistatic in acidic conditions. The mechanism starts with the absorption of benzoic acid into the
cell. If the intracellular pH falls to 5 or lower, the anaerobic fermentation of glucose through
phosphofructokinase decreases sharply which inhibits the growth and survival of microorganisms that
cause food spoilage.
Rationale
The drug EM is official in IP 2007 and BP 2009. Detailed literature survey for EM and SB revealed
that several methods have been reported for their estimation in combination with other drugs like,
difference spectrophotometric estimation of EM from tablet dosage form3, spectrophotometric
methods for the determination of EM in commercial dosage forms 4, determination
of saccharin, sodium benzoate, and caffeine in beverages byRP-HPLC5, RP-HPLC method for the
analysis of sodium benzoate and potassium sorbate in foods6, validation of stability indicating HPLC
method for the determination of EM in tablet formulations 7, determination and rotamer separation of
enalapril maleate by capillary electrophoresis8, EM and lercanidipine HCl in synthetic
mixture9,simultaneous estimation of losartan potassium, EM and hydrochlorothiazide in
pharmaceuctial formulations 10, simultaneous estimation of EM and amlodipine besylate in combined
dosage forms11, EM and hydrochlorothiazide in pharmaceutical dosage forms 12, simultaneous
estimation of EM and hydrochlorothiazide and paracetamol in pure and its pharmaceutical dosage
form 13 , simultaneous estimation of EM and ramipril in bulk and tablet dosage forms 14 , EM and
statin's in pharmaceutical formulations by RP-HPLC 15, high-performance liquid chromatographic
determination of EM and felodipine in pharmaceutical-dosage form16 , simultaneous determination of
metformin, captopril, lisinopril, and enalapril: its application to pharmacokinetics 17. No single
method has been reported, for estimation of EM and SB in combined liquid dosage form.
In the proposed method forced degradation of drug substances and drug product will also be carried
out under different stress conditions (thermal, photolytic, UV exposure, acidic and basic), and the
stressed samples will be analyzed by the developed and validated method.
MATERIALS AND METHOD
Materials: The formulation EM oral solution (label claim: Enalapril maleate 200 mg and Sodium
benzoate 400 mg), manufactured by Thames laboratory ltd. was procured from Analytical Research
laboratory, Ahmedabad. All the chemicals used were of analytical grade purchased from MERCK
Chem. Ltd., Mumbai. Potassium dihydrogen phosphate and orthophosphoric acid of AR grade were
obtained from S.D. Fine Chemicals Ltd., Mumbai, India.
Instruments: The following instruments with given specification were used for estimation of EM
from oral solution. HPLC (Shimadzu) LC2010CHT with SPDM20A diode detector is used for
estimation. LC solution software was applied for data collecting and processing. pH meter (ELICO),
digital balance (Sartorius-0.1 mg205 gm). FT-IR Spectrophotometer (Brukeroptics).
Method: The chromatographic separation was performed with isocratic elution on a ZORBAX
ECLIPSE PLUS C8 (250 x 4.6) mm; 5m column as a stationary phase with mobile phase -phosphate

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buffer (pH 2.1) : acetonitrile (75:25), pumped at a flow rate of 1.3 ml/min. The samples were
analysed by a PDA detector at 215 nm with the injection volume of 20L.
Preparation of standard solution for EM: EM, 200 mg, was accurately weighed and transferred to a
100 ml volumetric flask. Fifty ml of mobile phase was added, shaken well and then the volume was
made up to the mark with mobile phase to obtain a solution containing 2000 g/ml of EM. From these
solutions 20ml of EM taken in a 50ml volumetric flask and diluted up to 50ml with mobile phase to
obtain a solution containing 800 g/ml EM. Further, 2.5ml of the above solution was taken and
diluted up to 10 ml with mobile phase to give working standard solutions containing 200 g/ml EM.
Preparation of standard solution for SB: SB 400 mg was accurately weighed and transferred to a
100ml volumetric flask. Then 50 ml of mobile phase was added, shaken well and then the volume was
made up to the mark with mobile phase to obtain a solution containing 4000 g/ml of SB. From these
solutions 20 ml of SB was taken in a 50ml volumetric flask and diluted up to 50ml with mobile phase
to obtain a solution of 1600 g/ml of SB. Further, 2.5ml of the above solutions were taken and diluted
up to 10ml with mobile phase to give a working standard solutions containing 400 g/ml of SB.
Preparation of sample solution: Accurately measure 4 ml of sample solution and dilute up to 20 ml
with the diluent. According to label claim 4 ml of solution is containing 4 mg of EM and 8 mg of
sodium benzoate, therefore after dilution of 4 ml of solution in 20 ml of diluent a sample solution
containing 200g/ml and 400g/ml of EM and SB is obtained.
Method optimization: The chromatographic separation was achieved after various trials with
different ratios of solvents and the chromatogram obtained is shown in Figure 1.
Calibration curve: Calibration curves constructed were linear over the concentration range of 40400g/ml and 80-800g/ml. EM and SB, respectively. Calibration curves were prepared using ratio of
analyte peak area to internal standard peak versus concentration of analytes. The calibration curves
are shown in Figure 2 and 3.

Chromatogram obtained after optimized

conditions

Figure 1: Typical chromatogram of standard for EM and SB

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Linearity for Enalapril maleate

10000000

Area

8000000
6000000
y = 21722x + 101687
R = 0.9987

4000000
2000000
0
0

50

100

150

200

250

300

350

400

450

Concentration
Figure 2: Linearity for Enalapril maleate

Linearity for Sodium benzoate

25000000

Area

20000000
15000000
10000000

y = 24985x + 275466
R = 0.9999

5000000
0
0

100

200

300

400

500

600

700

800

900

Concentration

Figure 3: Linearity for Sodium benzoate

METHOD VALIDATION
The proposed method was validated in accordance with ICH guidelines in terms of accuracy,
precision, LOD, LOQ, linearity and percentage recovery.
Linearity: Linearity study was carried out for EM and SB for six different concentration levels.
Calibration curves constructed were linear over the concentration of 40-400g/ml and 80-800g/ml
for EM and SB, respectively. Evaluation of both the drugs was performed with UV detector at 215 nm
and peak area was recorded for all the peaks. The correlation coefficient for EM was found to be
0.998 and 0.999 for SB.
Accuracy: The accuracy of the method was assessed by recovery studies of EM and SB in combined
dosage form at three concentration levels. A fixed amount of pre-analyzed sample was taken and
standard drug was added at 50%, 100% and 200% levels. Each level was repeated for three times as
shown in Table-1. The percentage mean recoveries of EM and SB were 100.6% and 99.61% which
shows that there is no interference from excipients and the lower values of RSD of assay indicate the
method is accurate.
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Precision: The precision for the developed method was determined in terms of intraday and inter-day
precision. For intraday precision evaluation a standard solution of fixed concentration was injected at
various time intervals on a particular day. The %RSD for EM and SB was found to be 0.70% and
0.16%, respectively (limit %RSD < 2.0%). In addition, the inter-day precision was studied by
injecting the same concentration of standard solution on consecutive days and the %RSD for EM and
SB were found to be 1.013% and 0.28% respectively (limit %RSD < 2.0%). The results are shown in
Table 3.

Table-1: Accuracy studies for the proposed method

Spiked level

Amount added (g/ml)

Found (g/ml)

%Recovery

EM

SB

EM

SB

EM

SB

50%

100

200

00.4

200.9

100.4

100.5

100%

200

400

198.7

392.7

99.35

98.16

200%

400

800

399.3

800.8

99.83

100.1

Table 2: Inter-day and Intraday precision for the analysis of Enalapril maleate and Sodium benzoate

Sr. No.

Intraday precision (mau)*

Inter-day precision (mau)*

EM

SD

EM

SD

1.

4455438

9925364

4456856

1007983

2.

4397694

9926252

4456869

1007017

3.

4409840

9945868

4356458

1005671

Avg.

4420991

9932495

4423394

1006890

SD

30444.14

11590.15

57968.57

11547.86

%RSD

0.68

0.44

1.013

0.28

*milli absorbance unit

Limit of detection and limit of quantification: The LOD and LOQ for the developed
method were determined by injecting progressively low concentration of the standard
solutions using the developed HPLC method. The LOD for EM and SB was found to be
17.4754 g/ml and 52.9486 g/ml, respectively. The LOQ for EM and SB were found to be
11.20 g/ml and 33.94g/ml, respectively.
Assay: 10 l of each standard and sample solution were injected and from the peak area of EM and
SB amount of each drug in samples were computed. The results of the assay (Table-3) undertaken,
yielded 100.67% and 100.65% of label claim of EM and SB, respectively.
STABILITY INDICATING STUDY
Force degradation studies: Whole stability indicating RP-HPLC assay method for simultaneous
determination of SPIRO, MP and PP were done using above developed RP-HPLC method. In order to
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establish stability-indicating nature of the method, standards of drugs, drug product and diluent were
subjected to various stress conditions to conduct force degradation studies. Stress studies were carried
out under the conditions of acidic, basic, oxidative, thermal and UV exposure. Several trials with
different severity of each stressed condition were conducted, the results of which are shown in
Table-4.
Table-3: Assay results of proposed methods
Drug

Label claim(mg)

Amount found(mg)

%Assay

EM

200

201.34

100.67

SB

400

402.61

100.65

Table 4: Force degradation study of EM and SB

% Assay

% Degradation

Degradation Condition
EM

SB

EM

SB

0.1N HCl

94.3

95.99

5.7

4.01

0.1N NaOH

93.0

96.95

7.0

4.05

3% H2O2

82.7

91.17

17.3

8.83

Photo/UV

98.83

98.56

1.17

1.44

Thermal

98.80

98.60

1.2

1.4

RESULTS AND DISCUSSION

The HPLC procedure was optimized with a view to develop an accurate assay method for estimation
of EM and SB in oral solution. The samples were analyzed by a PDA detector at 215 nm with the
injection volume of 20L with. resultant peaks in good shape and resolution. The method was linear
in the range of 40-400 g/ml and 80-800 g/ml for both EM and SB, respectively.
The percentage mean recoveries for EM and SB were found to be 99.3% and 99.53% which shows
that there is no interference from excipients and the lower values of RSD of assay indicate the method
is accurate. The results are shown in Table-1. The %RSD for EM and SB for intraday precision study
were found to be 0.68% and 0.44%, respectively. Percentage RSD for EM and SB for inter-day
precision study were found to be 1.013% and 0.28%, respectively. The precision results are shown in
Table-2.
The retention time of EM and SB was found to be 6.77 min and 5.81 minutes, respectively with an
asymmetry factor of 1.27 for EM and 1.22 for SB, which indicates efficient performance of the
column. The LOD for EM and SB were found to be 17.4754 g/ml and 52.9486g/ml and LOQ for
EM and SB were found to be 11.20g/ml and 33.94g/ml, respectively, which indicates good
sensitivity of the proposed method. Assay studies of the proposed method indicate 100.67% and
100.65% recovery for EM and SB, respectively and the assay results were shown in Table-3. No
interfering peaks were found in the chromatogram of the formulation within the run time indicating
that excipients used in formulations did not interfere with the estimation of the drug by the proposed
HPLC method.
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CONCLUSION
The developed HPLC method is simple, specific, accurate and precise for the simultaneous estimation
of EM and SB in oral liquid solution. The developed method provides good resolution between EM
and SB. It was successfully validated in terms of linearity, accuracy, precision, LOD, LOQ and
recovery in accordance with ICH guidelines. Thus the described method is suitable for routine
analysis and quality control of pharmaceutical preparations containing these drugs in combinations.
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*Corresponding author: Mayank Bapna; Department of Quality assurance,

Shivam Pharmaceutical Studies and Research Centre, Valasan-Anand, Gujarat, India

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