6. Brief Resume of Intended Work:

6.1: Need for the Study:

Lisinopril is a lysine analog of the nonsulphydryl angiotensin-

converting enzyme (ACE) inhibitor. It is used for the treatment of

hypertension, congestive heart failure and cataract11, 16.

Lisinopril is chemically 1-[6-amino-2- (1-carboxy-3-phenyl-propyl) amino-hexanoyl]

pyrrolidine- 2-carboxylic acid dehydrate which is official in B.P & U.S.P 14, 15. There are

various analytical methods such as potentiometric and HPLC methods are official.

Lisinopril can be determined by UV spectrophotometry2,5,7. spectrofluorometric3,

chromatography9, combined spectrophoto-fluorimetry 3, 4, 6 capillary

electrophoresis4 and radio immunoassay fluoroimmunoassay 1, methods are

available to estimate the lisinopril in single or multicomponents3, 4., in formulation or

determination of impurities in lisinopril1. There is need to develop rapid, economical,

accurate, precise, sensitive analytical method to determine the lisinopril.

This work is an effort to study fluorescent intensity of the lisinopril in

NaOH solution, and to develop new fluorimetric method to estimate the lisinopril in various

alkaline and acidic solutions and to study the proper modifications or coupling with

fluorophores on non-fluorescent lisinopril to get derivative which is fluorescent. The study

of effect of various alkalis and substituents on florescent intensity of lisinopril is essential

to develop a simple, accurate method of estimation. The developed method is validated as

per the ICH guidelines to assure Accuracy, Precision, Repeatability, Intermediate Precision,

Specificity, Linearity, and, Range.

6.2: Review of literature

Yuan AS, Gilbert JD1. Time-resolved fluoroimmunoassay for the determination of

lisinopril and enalapril. A solid-phase immunoassay with detection based on time-

resolved fluorescence (TR- FIA) has been developed for the determination of lisinopril

and enalaprilat in human serum. The immunogen was prepared by coupling lisinopril to

bovine serum albumin through a two-step reaction with difluorodmitrobenzene. An

antiserum specific to both lisinopril and enalaprilat was used. The assay is based on the

competitive immunoassay principle in which the drug competes with biotin-labeled

drug for a limited quantity of primary antibody bound via sheep anti-rabbit globulin to

the wells of microtitration strips. At the end of the first incubation, the unbound biotin-

labeled drug is washed away in the second step, europium-labeled streptavidin {specific

to biotin) reacts with the biotin ahead} bound to the solid-phase antibody. After a

washing step, the addition of tin enhancement solution dissociates the europium ions

from the labeled streptavidin into solution. The fluorescence from each sample is

inversely proportional to the concentration of the drug in the sample.

_Durisehvar Ozer, Hulya Senel2. Determination of lisinopril from pharmaceutical

preparations by derivative UV spectrophotometry. In this article the authors have

observed that the lisinopril derivatives show intensive spectrophotometric peaks in 1N

NaOH solution and the values obtained from second derivative spectrum which are in

linearity range of 30–2000 ppm between 220 and 340 nm at ∆λ=4.

Fawzy A, El-Yazbi, Heba H, Abdine, Rasha A, Shaalan3. Spectrophotometric and

spectrofluorometric methods for the assay of lisinopril in single and multicomponent

pharmaceutical dosage forms. Here the four coupling methods are used to estimate the

lisinopril and lisinopril in combination with hydrochlorothiazide. The first method is

based on the reaction of the drug with chloranil in aqueous solution of pH 9.5 which is

maintained by using Borax buffer pH 9.5 and Walpole acetate buffer pH 3.6 to give

yellow color measured at 346 nm. The second method is based upon the interaction of

lisinopril with dichlone resulting in the formation of an intense purple color measured at

580 nm. The third method depends on the reaction of the drug with acetyl acetone and

formaldehyde to form a colored condensation product measured at 356 nm and also has

a strong fluorescence at 475 nm (λ max 410 nm). This method is extended to determine

lisinopril in binary mixtures with hydrochlorothiazide. The last method depends on

measuring the first and second derivative spectra of lisinopril3.

Gotti R, Andrisano V, Cavrini V, Bertucci C, Furlanetto S.4 Analysis of ACE-inhibitors

by CE using alkylsulfonic additives. Capillary electrophoresis (CE) was applied to the

determination of ACE inhibitors in pharmaceuticals. Since a free solution CE system

failed to reach a complete separation of closely related compounds (lisinopril, ramipril,

benazepril, and quinapril), alkylsulfonic additives (sodium heptansulfonate and (+)-10-

camphorsulphonic acid) were added to the running buffer: improved separations were

obtained suggesting a favorable effect of ion-pairing interactions between analytes and

additives.

Alaa El-Gindy, Ahmed Ashour, Laila Abdel-Fattah, Marwan M. Shabana6.

Spectrophotometric, septrofluorimetric and LC determination of lisinopril. Three

methods are described for the determination of lisinopril in the pharmaceutical tablets.

The spectrophotometric method depends on the reaction of the lisinopril with sodium

hypochlorite and phenyl hydrazine to form a condensation product measured at 362 nm.

The spectrophotometric method was extended to develop a stability indicating method.

The spectrofluorimetric method depends on reaction of the lisinopril with o-

phthalaldehyde in the presence of 2-mercaptoethanol in borate buffer pH 9.5.

Paraskevas J, Atta Politou, M Kouppar7. Spectrophotometric determination of lisinopril

in tablets using 1-fluoro-2, 4-dinitrobenzene reagents. A spectrophotometric method for

the determination of lisinopril in single and multicomponent tablets also containing

hydrochlorothiazide based on the derivatization reaction with 1-fluoro-2, 4-

dinitrobenzene (FDNB, Sanger reagent) is described. Aqueous solutions of Lisinopril

(4.5–27.2×10−5 M) react with FDNB (in acetonitrile) at pH 8.2 (borate buffer) in dark

at 60°C for 45 min. After acidification with HCl to decolorize 2, 4–dinitrophenolate

(the alkaline hydrolysis product of FDNB), the Lisinopril-derivative is measured at

356.5 or 405.5 nm (only at 405.5 nm if hydrochlorothiazide is present). The calibration

curves are linear (r_0.996 at both wavelengths) with a between days precision of slopes

of 1.8 and 2.3% at 405.5 and 356.5 nm respectively.

Devi PA, Rao GPV Mallikarjuna, Prasad KMM Krishna, Sastry C SP8. Four simple

spectrophotometric determinations of lisinopril in pure state and in tablets. Four

spectrophotometric methods developed.

Ali A, El-Emama, Steen Honore Hansen, Mohamed A. Moustafa, Saadia M. El-Ashry ,

Dina T. El-Sherbiny9. Determination of lisinopril in dosage forms and spiked human

plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl)

followed by spectrophotometry or HPLC with fluorimetric detection. In this case

lisinopril present in dosage form as well as spiked human plasma has been determined

using solid phase extraction procedures which are based upon spectrophotometric and

reversed-phase HPLC with fluorimetric detection. The methods are based on the

derivatization of lisinopril with 7-chloro-4-nitrobenzo-2-oxa-1, 3 diazole (NBD-Cl) in

borate buffer of pH 9 to yield a yellow, fluorescent product. The spectrophotometric

method depends on measuring the formed yellow color at 470 nm after optimization of

the reaction conditions. The HPLC method is based on measurement of the derivatized

product using fluorescence 9.

Asad Raza, Tariq Mahmood, Ansaria, Atta-ur-Rehmanb10. Spectrophotometric

Determination of Lisinopril in Pure and Pharmaceutical Formulations. The method is

based on the reaction of ninhydrine with primary amine present in the lisinopril the

presence of DMF.

Langade DG, Rao G, Girme RC, Patki PS, Bulakh PM11. In vitro prevention by ACE

inhibitors of cataract induced by glucose. Lenses treated with lisinopril or enalapril in

concentrations of 1, 5, and 10 ng/ml showed higher protein (total and water soluble

proteins) content and prevented formation and progress of cataract by glucose, as

evidenced by biochemical parameters

D Ivanovic, M Medenica, B. Jancic, N Knezevic, Malenovic A, Milic J 12. Validation of

an analytical procedure for simultaneous determination of hydrochlorothiazide,

lisinopril and their impurities. Here evaluation of a chromatographic method for

simultaneous determination of hydrochlorothiazide, lisinopril, and their impurities.

Chlorothiazide and disulfonamide, as impurities in hydrochlorothiazide, and

diketopiperazine,

Vikas Shinde, Anurag Trivedi, PR Upadhayay, NL Gupta, DG Kanase,

Rajeev Chikate13. Identification of a new impurity in lisinopril. This is

about detection of new impurity in lisinopril. LC–UV scan of lisinopril

revealed the presence of an unknown impurity at a relative retention

time of 3.26 employing phosphate buffer-acetonitrile as binary

gradient system while LC–MS analysis with binary gradient system

comprising of a ammonia ammonium acetate buffer (pH 5.0) and

acetonitrile indicated it to be C31H41N3O7. The impurity isolated by

HPLC utilizing a linear gradient of water and acetonitrile. The

structural analysis of the isolated product by 1H, 13C NMR, mass

spectroscopy and FT IR revealed it to be a 4- phenyl butanoic acid

derivative of lisinopril13.

6.3 Main objective of the study :

The aim of the present work is to carry out the

Fluorimetric estimation of lisinopril by a simple coupling and report the effect

of alkaline conditions along with comparative study of existing methods of

fluorimetric estimation.

* validation of new method as per the ICH guidelines

7.

Materials & methods

7.1 Source of the data:

The preliminary data required for the experimental study was obtained from

1. Library of Govt. College of pharmacy, Bangalore.

2. JRD TATA Memorial Library IISc, Bangalore.

3. Journals and e-journals.

4. Library and Information Centre, Rajiv Gandhi University of Health Sciences,

Bangalore.

5. Internet Sources.

6. http://www.rguhs.ac.in/HelinetHome/other electronic media.

7.2 Method of collection of data:

Department of Pharmaceutical Chemistry laboratories, Govt. College of Pharmacy,

Bangalore.

7.3 Does the study require any investigations or interventions to be

Conducted on patients or other humans or animals? If so, please describe in brief.

-Not applicable-

7.4 Has ethical clearance been obtained from your institute in case 7.3?

- Not applicable-

8.

List of references:

1. Yuan AS, Gilbert JD. Time-resolved fluoroimmunoassay for the determination of

lisinopril and enalaprilat in human serum, J.of Ph.ceutical and Biomed.

Analysis.1996; 14:773 -81.

2. Durisehvar Ozer, Hulya Senel. Determination of lisinopril from pharmaceutical

preparations by derivative UV spectrophotometry. J, of Ph.ceutical and Biomed.

Analysis.1999; 21:691–95.

3. Fawzy A, El-Yazbi Heba H, Abdine, Rasha A, Shaalan. Spectrophotometric and

spectrofluorometric methods for the assay of lisinopril in single and multicomponent

pharmaceutical dosage forms. J,of Ph,ceutical and Biomed. Analysis.1999; 19:819–

27.

4. Gotti R, Andrisano V, Cavrini V, Bertucci C, Furlanetto S. Analysis of ACE-

inhibitors by CE using alkylsulfonic additives. Journal of Pharmaceutical and

Biomedical Analysis. 2000; 22:423–31.

5. Jain HK. Agrawal RK. Spectrophotometric method for simultaneous estimation of

amlodipine besylate and lisinopril in tablet. Indian Drugs. 2000; 37(4):196-99.

6. Alaa El-Gindy, Ahmed Ashour, Laila Abdel-Fattah, Marwan M, Shabana.

Spectrophotometric, septrofluorimetric and LC determination of lisinopril J. of

Ph.ceutical and Biomed. Analysis.2001; 25:913-22.

7. Paraskevas JG, Atta Politou, Koupparis M. Spectrophotometric determination of

lisinopril in tablets using 1-fluoro-2, 4-dinitrobenzene reagent J. of Ph.ceutical and

Biomed. Analysis. 2002; 29 ;( 5):865-72.

8. Devi PA, Mallikarjuna Rao GPV, Krishna Prasad KMM, Sastry C SP. Four simple

spectrophotometric determinations of lisinopril in pure state and in tablets. Indian

J,of Ph,ceutical sci 2003; 65;(3) :296-99.

9. Ali A, El-Emama, Steen Honore, Hansen Mohamed A, Moustafa, Saadia M, El-

Ashry Dina T, El-Sherbiny. Determination of lisinopril in dosage forms and spiked

human plasma through derivatization with 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole

(NBD-Cl) followed by spectrophotometry or HPLC with fluorimetric detection. J.of

Ph.ceutical and Biomed.Analysis. 2004; 34:35–44.

10. Asad Raza, Tariq Mahmood Ansaria, Atta-ur-Rehmanb. Spectrophotometric

Determination of Lisinopril in Pure and Pharmaceutical Formulations. J. of Chinese

Chem.Soci.2005; 52:1055-59.

11. Langade DG, Rao G, Girme RC, Patki PS, Bulakh PM.In vitro prevention by ACE

inhibitors of cataract induced by glucose.Indian J. Pharmacol.2006; 38; 2:107-10.

12. D Ivanovic, M Medenica, B. Jancic, N Knezevic, A Malenovic, J Milic.. Validation

of an analytical procedure for simultaneous determination of hydrochlorothiazide,

lisinopril, and their impurities. Acta Chromatographica, 2007; 18.

13. Vikas Shinde, Anurag Trivedi, Upadhayay PR, Gupta NL, Kanase DG, Rajeev

Chikate. Identification of a new impurity in lisinopril. J. of Ph.ceutical and Biomed.

Analysis. 2007; 43; (1): 381-86.

14. British Pharmacopoeia. 2005; 2: p.1199.

15. United States Pharmacopoiea-2000; USP-24, NF-19, p. 979.

16. Martindale, ‘THE COMPLETE DRUG REFERENCE’ 33: P. 921.1.