Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2015 – vol. 119, no.

PHARMACY ORIGINAL PAPERS

A NEW SPECTROPHOTOMETRIC METHOD FOR THE ASSAY

OF LISINOPRIL

Mădălina Vieriu, Gladiola Țântaru*, M. Apostu, Alina Diana Panainte, Nela Bibire
University of Medicine and Pharmacy “Grigore T. Popa” – Iași
Faculty of Pharmacy,
Department of Analytical Chemistry
* Corresponding author. E-mail: gtantaru2@yahoo.com

A NEW SPECTROPHOTOMETRIC METHOD FOR THE ASSAY OF LISINOPRIL (Ab-

stract) Aim: A new spectrophotometric method for the assay of lisinopril using 2,4 -
dinitrophenol as reagent is described. Material and methods: The method involved the ad-
dition of 2,4-dinitrophenol to lisinopril in methanol followed by absorbance measurement at
400 nm. Experimental conditions that provide wide linear range, maximum sensitivity, sele c-
tivity, accuracy and precisions were optimized. Results and discussions: Beer’s law was
obeyed in the concentration range 2.0-14.0 μg/mL. Linear regression equation of calibration
graph was A = 0.005 + 0.045 · concentration, with a regression coefficient ( r) of 0.9995 (n =
8). The limits of detection (LOD) and quantification (LOQ) calculated according to the ICH
guidelines were 0.42 and 1.42 μg/mL, respectively. Accuracy and precision of the assays
were determined by computing the intra-day and inter-day variations at three different lis-
inopril concentrations; the intra-day and inter-day RSD was < 1.43% and accuracy was bet-
ter than 1.72 %. Conclusions: The proposed method is simple, easy to perform, sensitive,
linear, precise, accurate and robust. Keywords: LISINOPRIL, ASSAY, SPECTROPHO-
TOMETRY, 2,4-DINITROPHENOL.

Lisinopril (fig. 1) is chemically known
as N2-[(1S)-1-carboxy-3-phenylpropyl]-L-
lysyl-L-proline. It is a drug of the angioten-
sin converting enzyme inhibitor class that
is primarily used in the treatment of hyper-
tension, congestive heart failure, heart
attacks and also in preventing renal and
retinal complications of diabetes. Histori-
cally, lisinopril was the third angiotensin
converting enzyme inhibitor, after captopril
and enalapril, and it was introduced into
therapy in the early 1990s. Lisinopril has a
number of properties that distinguish it
from other angiotensin converting enzyme
inhibitors: it is hydrophilic, has a long half-
life and high tissue penetration and it is not
metabolized by the liver enzymes (1).
This paper presents a UV-Vis spectro-
photometric method for the assay of lis-
inopril using 2,4-dinitrophenol. The devel-
oped method was validated using the fol-
lowing criteria: linearity, detection and
quantification limits, precision, accuracy,
and robustness (2-5).
MATERIAL AND METHODS
The reagents used in the development of
the method were: lisinopril - 100.03% ref-
erence substance (Lupin, India), 2,4-
dinitrophenol (Fluka) and methanol

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 Mădălina Vieriu et al.
(Merck).
The following solutions were used:
- 1 mg/mL lisinopril stock solution: 100
mg lisinopril were dissolved in methanol in
a 100 mL volumetric flask;
- lisinopril working solutions with con-
centrations between 2 and 100 μg/mL lis-
inopril were obtained by diluting the stock
solution with methanol;
- 0.5% solution of 2,4-dinitrophenol:
500 mg 2,4-dinitrophenol were dissolved in
methanol in a 100 mL volumetric flask.
H tion prepared in similar conditions.
N
H RESULTS AND DISCUSSIONS
O
H From the analysis of absorption spectra
N (fig. 2), we observed a maximum absorb-
O
O O
N H
H O
Fig. 1. The chemical structure of lisinopril
A Hewlett Packard 8453 UV-Vis Spec-
trophotometer, a Kern 770 analytical bal-
ance and an Ultrasonic 08849-02 sonicator
were used.
Principle of the method: lisinopril forms
with 2,4-dinitrophenol in methanol a yel-
low compound which can be spectropho-
tometrically measured at 400 nm.
In order to establish the optimum detec-
tion wavelength 2 mL of the 50 μg/mL
lisinopril working solution were mixed
with 1 mL 0.5% 2,4-dinitrophenol solution
and 2 mL methanol. The UV-Vis absorp-
tion spectrum was recorded with 1 cm cell,
1200
after 15 minutes.
In order to establish the optimum con-
centration for the reagent solution, five
solutions of 2,4-dinitrophenol (0.1%,
0.25%, 0.5%, 0.75% and 1%) were used,
while the parameters of the method were
unchanged.
Procedure: 1 mL of lisinopril working
solutions with concentrations in the range
10-70 µg/mL were mixed with 1 mL 0.5%
2,4-dinitrophenol solution and 3 mL meth-
anol. The absorbance was measured after
15 minutes at 400 nm versus a blank solu-
ance for the reaction product at 400 nm.
That wavelength was used for all measure-
ments.
The best concentration of 2,4-
dinitrophenol reagent solution was estab-
lished to be 0.5%, because when using that
particular solution the maximum absorb-
ance measured at 400 nm had the greatest
value (tab. I). After studying the stability of
the samples it was established that the
chemical reaction between lisinopril and
2,4-dinitrophenol was final 15 minutes
after the reagent was added (tab. II). Also,
it was proved that the absorbance remained
almost the same for at least another 20
minutes, time sufficient enough for the
analysis to be performed.
Linearity was studied in the 0.4 - 20
μg/mL concentration range (fig. 3). The
obtained data were statistically evaluated
(tab. III) and the calibration curve was
obtained (fig. 4).
According to the experimental data, the
developed method was linear in 2 – 14
μg/mL concentration range. When this
 New spectrophotometric method for the assay of lisinopril

concentration range was compared with od had the following advantage: it did not
that of other published methods we found require the use of rare or complex reagents.
that it was very similar, but this new meth- It is simple and easy to perform (6-10).

0.6

0.5

0.4
Absorbance (AU)

0.3

0.2

0.1

0
400 500 600 700 800 900 Wavelength (nm)

Fig. 2. The spectrum of the reaction product for 20 μg/mL lisinopril

TABLE I
Study of reagent concentration
2,4- Lisinopril
dinitrophenol 2.0 μg/mL Lisinopril
14.0 μg/mL
% Absorbance (400 nm)
0.10 0.01222 0.35587
0.25 0.04415 0.56442
0.50 0.09014 0.64712
0.75 0.08708 0.62522
1.00 0.08294 0.60233

TABLE II
Stability study
Lisinopril
Time
2.0 μg/mL 14.0 μg/mL
(minutes)
Absorbance (400 nm)
5 0.08051 0.62298
10 0.09118 0.63274
15 0.09098 0.64022
20 0.09054 0.64152
25 0.08941 0.64191
30 0.08947 0.63145
35 0.08832 0.60203
40 0.08702 0.59157

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 Mădălina Vieriu et al.

Fig. 3. Study of method linearity

TABLE III
Linearity study of the method
Lisinopril Absorbance (400 nm)
(μg/mL) Ist Series IInd Series IIIrd Series IVth Series Average
0.4 0.01452 0.02440 0.00945 0.01147 0.01496
1.0 0.02245 0.01942 0.03144 0.01478 0.02202
2.0 0.08954 0.09314 0.09545 0.09291 0.09276
4.0 0.19056 0.20411 0.19412 0.18457 0.19334
6.0 0.28252 0.28147 0.27455 0.28990 0.28211
8.0 0.37046 0.37147 0.38412 0.37512 0.37529
10.0 0.46574 0.45892 0.47014 0.46025 0.46376
12.0 0.56559 0.57421 0.56142 0.56842 0.56741
14.0 0.63934 0.63412 0.64721 0.64455 0.64131
16.0 0.65435 0.64125 0.66120 0.64125 0.64951
18.0 0.64560 0.65744 0.64855 0.65470 0.65157
20.0 0.63547 0.65122 0.64177 0.67444 0.65073
Correlation and regression coefficients r = 0.9995, r 2 = 0.9991; Standard error = 0.0065; Intercept = 0.005;
Slope = 0.046

Fig. 4. Calibration curve

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 New spectrophotometric method for the assay of lisinopril

The calibration curve equation was es-
tablished:
- Absorbance = 0.046 x Concentration –
0.005.
- Detection and quantification limits
were calculated using the following formu-
las (2, 3):
- LD=3 x Standard error / Slope = 0.42
μg/mL
- LQ=10 x Standard error / Slope = 1.41
μg/mL.
Precision: three solutions of 6, 8 and 10
μg/mL lisinopril were used. Three assays
were performed for each concentration.
Two sets of assays were performed in dif-
ferent days in order to evaluate the inter-
mediary precision.
The sample concentrations were calcu-
lated using the calibration curve equation
(tab. VI). We observed that the relative
standard deviation for each data set and for
both data sets together was less than 2% (at
most RSD = 1.43%) (4), proving that the
proposed method was precise.
In order to establish the accuracy of the
method, lisinopril solutions of 6, 8 and 10
μg/mL were analyzed. For each concentra-
tion, three determinations were performed
(5).
The concentration of the samples was
calculated from the experimental values of
absorbance, using the regression curve equa-
tion (tab. IV). For the studied concentration
range recovery was 100.59%, the mean was
between 98.51% - 102.39%, and the relative
standard deviation was lower than 2% (RSD
= 1.42 %). Those values proved that the
proposed method was accurate.
The evaluation of robustness was per-
formed for system suitability to ensure the
validity of analytical procedure. That was
done by varying the instrument, analyst,
and time of study. The analysis was per-
formed also on a 1700 Shimadzu UV-
Visible spectrophotometer. Interday and
intraday analyses were performed by vari-
ous analysts. Reproducibility of the results
confirmed the robustness of the method.

TABLE IV
Study of the precision and the accuracy of the method
Precision Intermediate precision Accuracy
Concentration Real Real
(μg/mL) Recovery
concentration RSD % concentration RSD % Absorbance
(%)
(μg/mL) (μg/mL)
5.9650 5.965 0.28411 100.86
6 5.8428 1.768 6.0606 1.708 0.27455 97.40
6.0524 5.8571 0.28954 102.83
8.0089 7.9223 0.36841 98.56
8 7.9073 1.640 8.1393 1.639 0.37455 100.23
7.9506 8.1622 0.37276 99.74
10.0963 10.0815 0.47018 100.98
10 10.1894 1.440 9.9141 1.647 0.47855 102.80
9.9034 10.2463 0.46625 100.12
Mean 100.39

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 Mădălina Vieriu et al.

CONCLUSIONS limit was 0.42 μg/mL, the quantification

A UV-VIS spectrophotometric method
was developed for the assay of lisinopril
using 2,4-dinitrophenol. The reaction product
showed a maximum absorbance at 400 nm.
The analytical method was validated by
establishing the linearity domain in the
range of 2.0 – 14 μg/mL, with a correlation
coefficient of r = 0.9995, the detection
limit was 1.41 μg/mL lower than the lowest
concentration from the linearity domain,
method precision RSD% = 1.61% and the
accuracy evaluated through recovery was R
= 100.39%.
In conclusion, the proposed method is
simple, easy to perform, sensitive, linear,
precise, accurate and robust.

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