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J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 1
Research Article
Spectrofluorometric Determination of Lisinopril dihydrate and
Methyl- Dopa in Bulk and Pharmaceutical Formulation by Using
Dansyl Chloride
Sobhy M. El-Adl1, Mohamed El.Hossinny El-Sadek1, Nariman M. Saeed2*
1Department of Medicinal Chemistry, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt
2Pharmacist, Ministry of Health, Egypt
*Corresponding Author: Nariman M. Saeed, Pharmacist, Ministry of Health, Egypt, Tel: (20-2) 7951821; E-
mail: nariman_saeed@yahoo.com
Received: 17 January 2018; Accepted: 30 January 2018; Published: 01 February 2018
Abstract
A novel spectrofluorometric method has been developed for quantitative determination of Lisinopril dihydrate and
methyldopa either in pure form or in pharmaceutical preparation. The principle of the method based on the reaction
of each drug with dansyl chloride [5-(Dimethyl Amino) Naphthalene-1-Sulfonyl chloride] by the aid of sodium
hydroxide solution of pH 8.5 resulted in a product of a high fluorescent intensity which was then measured at 511
nm and 516 nm, for lisinopril and methyldopa respectively after excitation at 323 nm. Factors affecting reaction
development were studied and optimized. The proposed method was found to be sensitive, selective and
reproducible. Calibration curves were linear over the concentration ranges of 3–20 for lisinopril and 7–25 µg/mL for
methyldopa. Results obtained from tablets analysis were compared statistically with reported reference methods.
Keywords: Spectrofluorometric; Dansyl chloride; Lisinopril; Methyldopa
1. Introduction
Lisinopril dihydrate [LIS] (S)-1-[N2-(1-carboxy-3-phenylpropyl)-lysyl-proline dihydrate (Figure 1). It has a
molecular formula of C21H31N3O5 .2H2O and a molecular weight of 441.52 g/mol, official in BP and USP [1-2].
Lisinopril is non sulfhydryl long-acting angiotensin converting enzyme inhibitor used as antihypertensive agent,
prophylactic after myocardial infarction, it exerts a haemodynamic action and allows natriuresis, it was also found to
be useful in preventing diabetic retinopathy [3-4]. Several analytical methods, including spectrophotometric
methods [5-7], spectrofluorometric [8-10], chromatographic methods [11-14], polarographic methods [15] and
titrimetric method [16] have already been reported for its determination alone or in combination with other drugs.
Methyl-dopa [MD] L-3-(3, 4- Dihydroxyphenyl)-2-methylalanine sesquihydrate (Figure 1), it is a catechol
derivative with a molecular formula of C10H13NO4.1 ½ H2O and a molecular weight of 238.24 g/mol, official in USP
J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
2
[2]. “It is the drug of choice for hypertension in pregnancy”. It acts centrally on alpha2-adrenoreceptor leading to a
decrease in sympathetic tone causing blood pressure fall [3]. Several analytical methods, including
spectrophotometric methods [17-22], spectrofluorometric [23], chromatographic methods [24, 25], titrimetric
method [26] and Volta metric method [27] have been reported for its determination alone or in combination.
Figure 1: Chemical structure of [a-lisinopril], [b- methyldopa] and [c-dansyl-Cl].
2. Experimental
2.1. Apparatus
Fluorescence measurements were carried out on a FP-6300 spectro-fluorimeter (Jasco, Japan) equipped
with a 150 W xenon lamp and 1 cm quartz cells.
Consort P400
®
digital pH-meter for pH adjustment.
2.2. Materials and reagents
All solvents and reagents used throughout the work were of analytical grade and double distilled water was used.
Lisinopril pure authentic sample (99%, purity) was kindly supplied by Rameda, Egypt. Sinopril
®
tablets, labeled to
contain 10 mg of lisinopril, batch no. b23205 (GNP, Egypt). Methyl dopa pure authentic sample (99.6%, purity)
was kindly supplied by Eipico, Egypt. Aldomet
®
tablets, labeled to contain 250mg of Methyldopa batch no.
S420171 (KAHIRA, Egypt). Dansyl chloride (DNS-Cl) was purchased from Cornell Lab Company, solution of
DNS-Cl was freshly prepared at 3.0 mg/ml in acetone. (Stable for one week). Sodium hydroxide was purchased
from Research lab fine chemical industry, solution of NaOH was freshly prepared at 20 mg/ml in distilled water and
pH was adjusted by 1M of (34%) HCl to 8.5.
2.3. General procedures
2.3.1. Preparation of stock standard drug solution: A stock standard solution of lisinopril (1mg/ml) was prepared
by weighing accurately 0.1 gm of pure drug and dissolving in 100 ml distilled water. stock standard solution of
methyl dopa (1mg/ml) was prepared by weighing accurately 0.1 gm of pure drug and dissolving in 100 ml methanol.
2.3.2. Preparation of working standard solution and construction of calibration curve: Aliqout containing from
1 to 25 μg/mL of (LIS) and (MD) was prepared by transferring certain increasing volume from each drug to a series
of 10 ml volumetric flasks, followed by adding 0.3 ml of standard NaOH, mixed well, then 0.5 ml of standard
(DNS-Cl) solution was added, mixed and left for 30 minutes for [LIS] and 35 minutes for [MD] in dark at 35 °C.
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018.
J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
3
The flasks were then cooled and the volume was completed to10 ml with methanol. The fluorescence intensity of the
resulting solution was measured at 511 and 516 nm for (LIS) and (MD) respectively after excitation at 323 nm
against reagent blank that had been treated similarly. The fluorescence intensity was plotted versus the final drug
concentrations to get the calibration curve. The corresponding regression equation was computed.
2.4. Application on pharmaceutical preparations
Ten tablets of Sinopril
®
and five tablets of Aldomet
®
were crushed, powdered and the average weight of one tablet
was determined. Then specific weight from each powdered drug equivelant to 100 mg was dissolved in 100 ml
distilled water for sinopril® and methanol for aldomet® then filtered through whatman filter paper to give a final
stock concentration of 1mg/ml for each drug. Working solutions were prepared by serial dilution from stock
solution. The procedures were then completed as previously mentioned using standard addition technique.
3. Results and Discussion
3.1. Fluorescence spectra
Both (LIS) and (MD) possess amino groups which have the ability to react with dansyl chloride in alkaline medium
resulted in formation of a high fluorescent product, while the reagent blank exhibited a weak fluorescence intensity
at the selected excitation and emission wavelengths under the optimized experimental conditions. Scheme 1 and 2)
illustrates the suggested reaction path way for both drugs with dansyl chloride. Figure 2 and 3 explain the
Fluorescence spectra for both drugs.
Scheme 1: Suggested reaction pathway between Lisinopril and dansyl chloride.
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018.
J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
emm = 516 nm).
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 4
Scheme 2: Suggested reaction pathway between methyl dopa and dansyl chloride.
Figure 2:Excitation (A/black) and emission (B/blue) spectra of the LIS-DNS product (λ ex = 323 nm and λ
em = 511 nm).
Figure 3:Excitation (A/black) and emission (B/blue) spectra of the MD-DNS product (λ ex = 323 nm and λ
J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 5
3.2. Optimization of reaction conditions
Different experimental factors including pH, volume of buffer, amounts of reagent, temperature and reaction time
were studied and optimized by changing each factor individually keeping the others constant.
3.2.1. Effect of pH: The influence of pH on the fluorescence intensity of the reaction product was examined using
sodium hydroxide solution over the pH range from 6 to 11.0 since DNS-Cl reacts under alkaline conditions. The
maximum fluorescence intensity was obtained when the reaction was carried out with NaOH solution of pH 8.5.
Figure 4 illustrate the effect of pH.
Figure 4: Effect of the pH on the development of the reaction product of (LIS) and (MD) (10 µg/mL) with DNS-Cl.
3.2.2. Effect of sodium hydroxide volume: The influence of volume of NaOH on the fluorescence intensity of the
reaction product was examined using different volume over the range from 0.1 to 0.7 ml. Maximum fluorescence
intensity was obtained when the reaction was carried out using 0.3 ml NaOH solution of pH 8.5 as shown in Figure
5.
Figure 5: Effect of volume of NaOH solution on development of the reaction product of (LIS) and (MD) (10
µg/mL) with DNS-Cl.
0
50
100
150
200
250
0 0.2 0.4 0.6 0.8 1 1.2
intensity
volume of NaOH
LIS
MD
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 6
3.2.3. Effect of dansyl chloride volume: The influence of the volume of dansyl chloride solution was examined by
addition of different volumes of 3.0 mg/ml reagent in the range of 0.2 to 1 ml. A maximum fluorescence intensity
was obtained when 0.5 ml of dansyl chloride solution was utilized as depicted in Figure 6.
Figure 6: Effect of volume of DNS-Cl solution on development of the reaction product of (LIS) and (MD) (10
µg/mL).
3.2.4. Effect of time and temperature: In this study, the reaction between the two drugs and dansyl chloride was
performed using pH 8.5 at different temperatures (25°C, 35°C, 40°C, and 50°C) for various time intervals (10, 20,
30, 35 and 40 min). As it is seen in Figure 7 and 8, the reaction was found to be completed after 30 and 35 min for
(LIS) and (MD) respectively at 35°C.
Figure 7:Effect of time on the reaction completion of (LIS) and (MD) (10 μg/mL) with DNS-Cl.
0
50
100
150
200
250
0 0.2 0.4 0.6 0.8 11.2
intensity
volume of DNS‐Cl
LIS
MD
0
50
100
150
200
250
0 10 20 30 40 50
INTENSITY
TIME (MIN)
lis
MD
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 7
Figure 8:Effect of temperature on the reaction completion of (LIS) and (MD) (10 μg/mL) with DNS-Cl.
3.3. Stoichiometry of the reaction
The molar ratio of the reagent and the two drugs in the reaction was studied by using the continuous variation
method (Job ҆s method) [28]. The molar ratio was found to be 1:2 (drug: reagent) and 1:1 (drug: reagent) for
Lisinopril and methyl- dopa respectively as seen in Figure 9.
Figure 9: Continuous variation plots for the reaction between: (A) 2.26 × 10-4 M of DNS-Cl and 2.26 × 10-4 M of
Lisinopril and (B) 4.2 × 10-4 M of DNS-Cl and 4.2 × 10-4 M of Methyl-dopa.
3.4. Method validation
The validity of the proposed method was tested regarding linearity, range, limits of detection, limits of
quantification, accuracy, precision, robustness and specificity according to ICH recommendations [29].
3.4.1. Linearity: Under the optimum experimental conditions, standard calibration curves were constructed at five
concentration levels by plotting the values of florescence intensity versus the final concentrations (μg/ml) as shown
in Figure 10. The correlation coefficient was 0.9999 and 0.9997 for (LIS) and (MD) respectively, indicating good
0
50
100
150
200
250
0 10 20 30 40 50 60
INTENSITY
TEMPREATURE Cº
LIS
MD
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 8
linearity over the concentration range of 3.0 to 20.0 and 7.0 to 25.0 μg/ml for (LIS) and (MD) respectively. The
intercept, slope, Correlation coefficient for the calibration data are summarized in Table 1. Calibration graph is
described by the equation (Y= a+bX), (Where Y= florescence intensity, a= intercept, b= slope and X= concentration
in μg.ml
-1
).
Figure 10:Calibration curves for the reaction between (DNS-CL) and (LIS) (A) and (MD) (B) respectively.
Parameters Lisinopril Methyl dopa
Excitation wave length, nm 323 323
Emission wave length, nm 511 516
pH 8.5 8.5
Volume of NaoH (ml) 0.3 0.3
(DNS-Cl) volume (ml) 0.5 0.5
Time for derivatization reaction 30 35
Temperature (
o
C) 35 ± 2°C 35 ± 2°C
Solvent water Methanol
Beer’s law limits (µg.ml
-1
) 3-20 7-25
Regression equation* Slope (b) 1.0815 79.373
Intercept (a) 21.553 27.17
Correlation coefficient 0.9999 0.9997
Table 1: Statistical and analytical parameters of (LIS) and (MD) determination.
y=21.553x ‐1.0815
R² =0.9999
0
50
100
150
200
250
300
350
400
450
500
010 20 30
area under curve
A
concentration
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 9
3.4.2. Limits of detection and limits of quantification: The limit of detection (LOD) was calculated according to
the equation [LOD = 3.3 S /K]. The limit of quantification (LOQ) was calculated by the equation [LOQ = 10 S/K]
Where S is the standard deviation of the three replicate determination and K is the slope of calibration graph. The
results are summarized in Table 2.
Parameters
Lisinopril Methyl dopa
Conc.take
n Conc.found Recovery
% Conc.taken Conc.found Recovery
%
µg/ml µg/ml µg/ml µg/ml
32.959 98.64 7 6.8779 98.2559
55.03788 100.757 10 10.124 101.241
10 10.0506 100.506 15 15.0958 100.638
15 14.922 99.482 20 19.8757 99.3785
20 20.0288 100.144 25 25.0266 100.106
Mean* 99.9 99.92
N 5 5
SD 0.85 1.1575
RSD 0.85 1.1584
SE 0.427 0.5787
Variance 0.729 1.3399
LOD,µgml-1 0.264 0.2097
LOQ,µgml-1 0.88 0.699
Apparent
Molar
absorptivity** 9435577 4957090
L Mol-1 cm-1
*Mean of three different experiments; ** Calculated in the basis of molecular weight of the drug.
Table 2: Statistical data for the reaction of lisinopril and methyl dopa with dansyl chloride.
3.4.3. Accuracy and precision: Accuracy of the proposed methods was checked by performing recovery
experiments through standard addition technique. The results are shown in Table 3 indicates good accuracy. The
precision of the method was calculated in term of intermediate precision (intraday and inter-day). Three different
concentrations five times of lisinopril and methyl dopa were analyzed during the same day (intra-day precision) and
five consecutive days (inter-day precision). The standard analytical errors, relative standard deviations (RSD) and
recoveries obtained by the proposed method were found to be acceptable. The results are summarized in Table 4.
J Pharm Pharmacol Res 2018; 2 (1): 001-014 DOI: 10.26502/jppr.0005
Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 10
Sinopril ® tablets Aldomet ® tablet
Take from Sinopril
tablet (µg/ml)
Added from pure
drug (µg/ml)
Conc. found
(µg/ml)
Recovery* %
Taken from
aldomet tablet
(µg/ml)
Added from pure
drug (µg/ml)
Conc. found
(µg/ml)
Recovery* %
30 2.936 98 70 6.879 98.28
32 5.0237 100.474 73 10.1218 101.218
37 10.07 100.735 78 15.095 100.639
312 14.9 99.399 7 13 19.874 99.372
317 20.056 100.281 718 25.027 100.108
321 23.95 99.796 - - - -
Mean* -- - 99.76 - - - 99.923
N -- - 6 -- - 5
S.D. -- - 1.03595 - - - 1.14
R.S.D. -- - 1.0384 - - - 1.14
V -- - 1.07 - - - 1.3
S.E. -- - 0.5179 - - - 0.57
*Mean of three different experiments.
Table 3: Application of standard addition technique for the determination of Sinopril ® and Aldomet® tablets form
through reaction with DNS-Cl.
Item
Concentration
µg/ml
Intraday Inter-day
Mean ±SD RSD Mean ±SD RSD
Lisinopril
10 µg/ml 100.5 ± 0.15 0.15 100.46 ± 0.28 0.28
15 µg/ml 99.655 ± 0.244 0.245 99.2 ± 1.23 1.24
20 µg/ml 100.117 ± 0.1688 0.1686 100.108 ± 0.29 0.29
Methyldopa
10 µg/ml 101.156 ± 0.4 0.39 101.2 ± 0.63 0.62
15 µg/ml 100.465 ± 0.197 0.196 100.6 ± 0.208 0.206
20 µg/ml 99.19 ± 0.185 0.1867 99.349 ± 0.637 0.642
Table 4: Results of the intraday and inter-day precision for the determination of lisinopril and methyl dopa using
DNS-Cl.
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 11
3.4.5. Robustness: Robustness was achieved by making small incremental change in volume of sodium hydroxide
and volume of reagent. The effect of changes was studied on the percent recovery of drugs proved that all changes
had a negligible influence on the results as seen in Table 5.
Table 5: Results of the robustness for the determination of lisinopril and methyl dopa using DNS-Cl.
3.4.6. Analysis of pharmaceutical preparations: The proposed methods were applied to the analysis of the drug in
dosage forms and the results were statistically compared with reported reference methods by calculating Student’s t-
and F-values. The evaluated t-and F-values were less than the tabulated values at the 95% confidence level. Results
listed in Table 6 showing that there is no statistical significance difference between the proposed and reference
methods.
Statistics
Sinopril® tablet Aldomet ®tablet
Reported reference
method number [133]
Proposed
method
Reported reference
method number
[153]
Proposed
method
Mean recovery* ± SD 100.33 ± 0.7 99.76 ± 1.0359 100.1 ± 1.2 99.9 ± 1.14
N 6 6 5 5
Variance 0.49 1.07 1.44 1.3
t-test** -1.1 (2.23)a - 0.27(2.3)a
F-ratio** -2.18 (5.05)
b
- 1.1 (6.39)
b
*Average of three experiments; a and b are Theoretical Student t-values and F- ratio at p=0.05.
Table 6: Statistical data for the determination of pharmaceutical tablets Sinopril® and Aldomet® through the
proposed methods using DNS-Cl compared with the reference methods.
4. Conclusion
The proposed method was simple, fast, selective, sensitive, specific, reproducible and not very expensive. Validation
experiments proved that results were linear over the mentioned working range. No interference was observed from
the described drugs and their common excipients. Also there was no need for extraction procedure.
Robustness
% of recovery ± SD
Methyl dopa Lisinopril Item
99.9±1.65
99.9±0.814 NaOH+0.05ml
99.9± 1.15
99.99± 0.716 Na0H-0.05 ml
99.9±0.888
99.8±1.17 DNS-Cl+0.05ml
99.948± 0.7
99.88± 0.8 DNS-Cl-0.05ml
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Journal of Pharmacy and Pharmacology Research Vol. 2 No. 1 - Mar 2018. 12
5. Acknowledgment
Authors are grateful to all members of staff of Medicinal chemistry department (Zagazig University) also to all
members of staff of analytical chemistry department and laboratories staff (Tanta University)
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This article is an open access article distributed under the terms and conditions of the
Creative Commons Attribution (CC-BY) license 4.0
Citation: Sobhy M. El-Adl, Mohamed El.Hossinny El-Sadek, Nariman M.
Saeed. Spectrofluorometric Determination of Lisinopril dihydrate and Methyl- Dopa in
Bulk and Pharmaceutical Formulation by Using Dansyl Chloride. J Pharm Pharmacol Res 2
(2018): 001-014.