Accepted Manuscript

Simultaneous Determination of metformin, captopril, lisinopril, and enalapril:

its application to Pharmacokinetics
Farhan Ahmed Siddiqui, Nawab Sher, Nighat Shafi, Saima Sher Bahadur
PII:
DOI:
Reference:

S1878-5352(13)00405-X
http://dx.doi.org/10.1016/j.arabjc.2013.11.035
ARABJC 1185

To appear in:

Arabian Journal of Chemistry

Received Date:
Accepted Date:

10 October 2012
18 November 2013

Please cite this article as: F.A. Siddiqui, N. Sher, N. Shafi, S.S. Bahadur, Simultaneous Determination of metformin,
captopril, lisinopril, and enalapril: its application to Pharmacokinetics, Arabian Journal of Chemistry (2013), doi:
http://dx.doi.org/10.1016/j.arabjc.2013.11.035

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Simultaneous Determination of metformin, captopril, lisinopril, and enalapril: its application

to Pharmacokinetics

Farhan Ahmed Siddiqui1*, Nawab Sher2, Nighat Shafi1, Saima Sher Bahadur2
1

Faculty of Pharmacy, Federal Urdu University Arts, Science and Technology, Karachi 75300
1

Department of Chemistry, University of Karachi, Karachi-75270, Pakistan.

*

(Email: farhanchemist@gmail.com
(0092-332 2524224)

Abstract

This study illustrates development and validation of a simple high performance liquid
chromatographic method for the simultaneous determination of metformin hydrochloride and
angiotensin-converting enzyme inhibitors (captopril, lisinopril, and enalapril) in bulk dosage form
and application in pharmacokinetics studies. The separation was achieved on a Purospher Star
RP-18 endcapped (250 mm x 4.6 mm id) column as stationary phase using methanol-water 50:50
(v/v) as mobile phase, adjusted to pH 3.1 with phosphoric acid. Effluent was monitored at a flow
rate of 1 mLmin-1 at room temperature (25oC), detector was set at 218 nm. The method was
validated according to ICH guidelines. The linearity was studied over the concentration range of
1010000 ngmL-1 for metformin and 3010000 ngmL-1 for captopril, lisinopril, and enalapril,
demonstrating good linearity with minimum r = 0.9964, respectively. The developed method was
successfully applied to pharmacokinetics studies of metformin, lisinopril, captopril and enalapril.

Keywords: Metformin, Captopril, Enalapril, Lisinopril, ACE inhibitors and RP-HPLC,

Pharmacokinetics

1.

Introduction

Sufferings of people from diabetes mellitus and hypertension is most common now adays and
these diseases has been the leading cause of morbidity. The generally it has been noted that patient
suffering from diabetes also suffers from sister disease hypertension, and the two coexists in
association. Angiotensin-converting enzyme are broadly used in order to treat hypertension and
heart failure. The inhibitors of the angiotensin-converting enzyme may be recommended along or
in combination in combination with other drugs [1]. Despite of the fact that ACEI (angiotensinconverting enzyme inhibitors) (Figure 1) possess contraindications, they are considered first-line
therapy owing to the overwhelming benefits it brings in to diabetes patients [2, 3].
Metformin(N,N-dimethylimidodicarbonimidic diamide) (Figure 1), has been the crown antidiabetics drug included in the World Health Organization Model List of Essential Medicines in the
year 2008. It can be rightly said that treatment of type 2 diabetes is not complete without the
administration of Metformin and it is the drug of choice particularly for obese patients. Metformin
helps in the reduction of LDL cholesterol and triglyceride and hence may aid weight loss [4].
Captopril chemically is (2S)-1-[(2S)-2-methyl-3-sulfanylpropanoyl] pyrrolidine-2-carboxylic acid
is a extensively prescribed antihypertensive drug and was the first inhibitor of angiotensin
converting enzyme. Enalapril, an ACE inhibitor used in the treatment of hypertension and some
types of chronic heart failure is chemically (2S)-1-[(2S)-2-{[(2S)-1-ethoxy-1-oxo-4-phenylbutan2-l]amino}propanoyl]pyrrolidine-2-carboxylic acid. The uniqueness of enalapril is that it was the
first member of its group (ACE inhibitors) that has two attached carboxylic acid groups. Another
orally active drug Lisinopril (N2-[(1S)-1-carboxy-3-phenylpropyl]-L-lysyl-L-proline) belonging to
ACE inhibitors is effective in lowering blood pressure, renovascular hypertension and congestive
heart failure. It is a lysine analog of enalaprilate [5].
A comprehensive literature survey done in this regard revealed few analytical reports for the
analysis of these drugs using HPLC alone or in combination [6-30]. None of these methods can be
used for the simultaneous estimation of these co-prescribed and co administered drugs. especially
at the therapeutic monitoring level. There was a need to develop a simultaneous LC method for
above mentioned. The focus of the present work described herein was to develop selective and
sensitive HPLC methods for the determination of these drugs in pharmaceutical formulation and in
human serum, using most common UV detection and having proven abilities for pharmacokinetics
studies. Robustness studies were completed to ensure continuous performance of the methods in
diverse analytical environments.

2.

Experimental

2.1.

Apparatus and Materials

Metformin hydrochloride, captopril, lisinopril and enalapril reference standards were benevolently
supplied by Sonaphy Aventis Limited, Efroze Chemical industries (Pvt.) Ltd., Atco Laboratories
(Pvt.) Ltd. and Nabi Qasim Industries (Pvt.) Ltd. Respectively. All the tablets were purchased from
local market. Analytical grade phosphoric acid (85% pure), and HPLC grade methanol were
purchased from Merck Marker Ltd. Water was distilled twice and was deionized by (Stedec CSW300). Ultrasonic bath (Elmasoni E 60 H), Jenway 3240 pH meter , stability Chamber (BINDER
KBF 720 (E5.2) ), Oven (Memmert D-91126) Schwabach FRG Germany and Sartorious TE2145
analytical balance was used in this work. Drug-free human plasma was obtained from the National
Institute of Cardiovascular Disease, Karachi, Pakistan (NICVD). Caffeine was purchased from
Merck.
2.2.

Chromatographic condition

The liquid chromatographic system of Shimadzu Corporation Tokyo Japan consisting of Pump
model (LC-10 AT VP), Detector model (SPD-20AV), Rheodyne manual injector fitted with a 20
L loop, was used. Chromatographic separation was obtained using Purospher Star RP-18 end
capped (25 cm x 4.6 mm id) analytical reversed phase column. Class GC-10 software was used for
data demand.
The mobile phase consisted of methanol-water 50:50 (v/v), pH was adjusted to 3.1 with
phosphoric acid (85%) and the diluent was methanol- water 60:40 (v/v). Before delivering into the
system, mobile phase was filtered through 0.45 m filter and degassed using an ultrasonic bath. The
analysis was carried out under isocratic conditions using a flow rate of 1.0 mLmin-1 at room
temperature. Chromatograms that were recorded at 218 nm (isobestic point) were considered
reasonable. This permitted the detection of all drugs with ample sensitivity.

3. Analytical Procedures

3.2.

Standard preparations

Stock solution of metformin 100 gmL1 was prepared by dissolving 10 mg of active drug in 100
mL flask and 250 gmL1 of captopril, enalapril and lisinopril were prepared by dissolving 25 mg
in 100 mL flask separately. Sequential dilution of these solutions were done to get a working

solution with a concentration range 1010000 ngmL-1 for Metformin and 30- 10000 ngmL-1 for
captopril, enalapril and lisinopril. Diluents were used to prepare these working solutions and were
scanned to get required data for the calibration curve.
3.3.

Assay Procedure for Pharmaceutical Formulations

10 tablets of each drug were ground in a mortar and pestle, and required amount were carefully
shifted to a 100 mL volumetric flask. The powder was dissolved in little diluent and was
mechanically shaken for 5 minutes, then the volume was adjusted to 100 mL using the same
diluent. Sequential dilutions were made to prepare solutions of different concentrations in the
working range and internal standard Caffeine was added. Filtration of the samples was done using
a 0.45-m membrane filter. Linear regression equations were used to calculate the amount of
Metformin HCl and ACE inhibitors per tablet.
3.4.

Preparation of Drug serum sample

As the plasma samples were stored in the freezer at -80oC therefore they were allowed to thaw at
room temperature before further processing. The plasma samples were centrifuged at 3000 rpm for
10 min. An aliquot of 1.0 mL was pipetted into a 10 mL polypropylene test tube and was added 2.0
mL of acetonitrile. The mixture was vortex mixed for a short while. It was then left to stand for 5
min at room temperature. The mixture was again centrifuged at 3000 rpm for 5 min. The
supernatant obtained was filtered through a 0.45

m filter. The worked out serum was then

mixed in ratio of 1:1 with drug solutions and was injected into the HPLC system.
3.5.

In-vitro Serum drug analysis

The availability of metformin, Lisinopril, enalapril and captopril from pooled human serum was
determined by the described chromatographic conditions. Blood samples of healthy volunteers
were collected. Volunteers (age ranging from 22-28 years) were non-smokers and were not taking
any other medicines. Pre written consent was obtained from all subjects. Multiple blood samples
(10 mL) were collected in evacuated glass tubes through an indwelling cannula placed in the
forearm veins. The blood was then slightly shaken and centrifuged at 10,000 rpm for 10 minutes
and the plasma separated [31]. The obtained plasma was processed as described above and stored
at -20 0C pending analysis.
3.6.

In-vivo Serum drug analysis

Blood samples were collected from subjects administered with drugs, in tubes containing heparin
before (0.5 h) and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.5,
12, 24, 30, 35, 40, 45 and 50 hrs intervals of administration of drug. Blood samples were
centrifuged at 10,000 rpm for 10 min and plasma was separated, stored at 80 C until use.
3.7. Preparation of Internal standard
10 mg of caffeine was transfer to 100 ml volumetric flask and some diluent was added to dissolve
and then diluted up to the mark, further dilution were made.

4.

Result and Discussion

4.1.

Method Optimization

The objective of this work was to develop an Rp-HPLC method for separation of these four drugs
in simultaneous isocratic condition. Various hurdles were systematically countered to get to
optimum conditions.
Mobile phase being the polar part of reverse phase HPLC system always has a profound effect on
the separation on drug molecules which are mostly polar in nature. So the selection of mobile
phase is cortically considered by chromatographers. For this purpose different ratios of methanol,
acetonitrile and water were use to inspect the separation between metformin and ACEI (captopril,
enalapril and lisinopril). We achieved the best separation in the mobile phase composition
methanol and water 50:50 (v/v) with pH maintained at 3.1 (1). It was observed that detector
responded well at wavelength 218.0 nm, which was found to be the most probable isobestic point
for all studied drug when scanned in various solvent including the diluent, thus it was selected as
optimum wavelength. The peak parameters such as height, asymmetry and tailing etc were
considered while maintaining flow rate, baseline drift etc. this analytical procedure does not
involve the use of internal standard as there was no extraction in simultaneous determination of
Metformin and ACE inhibitors in human serum and in pharmaceutical dosage forms. Individual
drug solutions (100 gmL1) were injected into the sample loop 20L, elution pattern and
resolution parameters were studied as a function of pH and water and organic modifiers ratios.

4.2.

Method validation

4.2.1. Specificity and Robustness

This LC method for the simultaneous determination of Metformin and captopril, lisinopril and
enalapril in human plasma and dosage forms is the most simplest and precise among other reported
methods. The proposed system is reasonably robust. Other ODS (octa deca silane) columns and
chromatographic systems were tested and it was found that there was a minimal effect on the
resolution of analytes. The best ruggedness and reproducibility was demonstrated by Purospher
Star RP-18 endcapped (25 cm 4.6 mm id) column, thus it is recommended for this assay. A
typical reference chromatogram, Metformin and ACEIs (lisinopril, captopril, enalapril of pure
standard mixture using the described condition is shown in Figure 2. Typical chromatograms of
pharmaceutical pharmaulation are shown in figure 3, humane serum in figure 4 and blank human
serum in figure 5.
4.2.2. Linearity and Range
For studying linearity standard solutions were prepared by serialy diluting stock solution at five
concentration level for standard refrence and at seven concentratin level in humane serum in the
range of 1010000 and 30-10000 ngmL-1 for Metformin and captopril, lisinopril, and enalapril
respectively which determined the linearity of the method (n=3). Plotting the mean peak areas of
Metformin and captopril, lisinopril, and enalapril as a function of drug concentration and gave
away the least-square regression calibration curves. The linearity and regression calibration curves
equation is represented in table 1 and 2 respectively.
4.2.3. Accuracy and precision
The accuracy of an analytical method is defined as the similarity of the results obtained by the
analytical method to the true value and precision is the degree of that similarity [32-33].
Repeatability of the method was performed as inter-day and intra-day accuracy and precision in
pharmaceutical dosage forms by injecting six replicates of three concentrations (80, 100 and
120%) into the system. Coefficient of variance (CV) and percentage recovery of each drug were
also evaluated as shown in table 3.
4.2.4

Recovery studies

Recovery studies were performed to check the interference of formulation additives. This was
done by adding a known amount of pure drug to already analyzed samples of commercial dosage
forms. For percent analytical recovery values following equation was used
% Recovery = [(Cv Cu)/Ca] 100

Where Cv was the total drug concentration measured after standard addition, Cu, drug
concentration in the formulation and Ca, drug concentration added to formulation. A separate
analysis for all the drugs was performed to test the developed methods precision and accuracy. All
the results were found ranging 98.2 to 101.6 %. Results prove good recovery of new developed
method.

4.2.5. Limit of Detection (LOD) & Limit of Quantitation (LOQ)

The criterion for the calculation of limits of detection (LOD) and quantitation (LOQ) was the use
of empirical formula i.e, 3.3(/s) and 10(/s) respectively, where is the standard deviation of the
peak area (for six replicates) for the sample and s is the slope obtained from calibration curve
equation [34-35]. Using the parameters mentioned above, LOD and LOQ were estimated to be
3.26, 1.14, 1.69 and 0.98 ngmL-1 while LOQ were 9.26, 6.17, 8.22 and 2.22 ngmL-1 for lisinopril,
enalapril, captopril and metformin respectively.
The uniformity of the system operation throughout the analysis was developed by initially
equilibrating the column with mobile phase prior to injection of the sample into the
Chromatographic system. Theoretical plates, tailing factor, resolution and, repeatability were
checked before start analytical work every day. All the factors were found satisfactory and were as
per ICH [36] guidelines.
4.3

Applicability

4.3.1

Application to human serum (in vitro study)

The applicability of the method in human serum was checked by spiking serum sample with
metformin and captopril, lisinopril, and enalapril simultaneously. The validated method was
effectively applied to an in-vitro study in human plasma samples for reference and test
formulations of metformin (500 mg), captopril 25mg, lisinopril 20mg, and enalapril 10 mg tablet
formulation. In order to evaluate the applicability of the proposed method in the perspective of
serum, serum samples were spiked with metformin, captopril, lisinopril, and enalapril at different
concentration levels and assayed in triplicate. The prepared concentrations were analogous to the
intermediate concentration level of the calibration points. The sample was prepared by adding 4
mL of the drug solution (containing Metformin (10 g mL-1) and captopril, lisinopril, and
enalapril (100 g mL-1) to 4 mL of drug-free human serum. This spiked serum was made up to 10
mL with diluent and injected in to the chromatograph. Further dilutions were made when and

where was require. No interference was observed in the analysis as shown in figure 4 and 5. The
obtained recoveries and %RSD are illustrated in table 2. Statistical analysis showed that no
considerable differences exist among the mean recoveries of all the drugs in serum samples.

4.3.2 Pharmacokinetic application ( in-vivo study)

The method was applied to a pharmacokinetics study of metformin, captopril, lisinopril, and
enalapril in 24 healthy Pakistani male volunteers with a mean age of (22.51.22 years and a mean
body mass index (BMI) of 21.22.12, after oral administration of a metformin (500 mg), captopril
25mg, lisinopril 20 mg, and enalapril 10 mg. Before the study the volunteers fasted overnight and
for 4 h after the dosing. Venous blood samples (8 ml) were collected 0.5 h before dosing and at
00.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.5, 12, 24, 30, 35, 40,
45 and 50 hrs interval following administration. The blood samples were treated as mentioned in
experimental section. The pharmacokinetic parameters for each volunteer were evaluated from the
plasma concentrationtime profile. Pre written consent was taken from all volunteers and all were
informed of the aim of the study and Ethics committee approved the study protocol. The study was
conducted strictly in accordance with guidelines laid down by International Conference on
Harmonization and USFDA [37]. Health check-up for all subjects was done by general physical
examination. All subjects were negative for HIV, HBSAg and HCV tests. They were orally
administered a single dose of test and reference drug with 240 ml of water. Drinking water was not
allowed and supine position was restricted 2 h post dose. Standardized meals were provided as per
requirement. The guidelines laid down by International Conference on Harmonization and USFDA
[37] were strictly followed in this study.

The peak plasma level of captopril was obtained at almost 0.5 hrs after the administration was in
good agreement with the values reported by Jankowski et al [38]. The Plasma levels of Lisinopril
obtained from healthy volunteers after the administration of a single dose was in good agreement
with the value reported by Feng et al [39]. Plasma concentration time profile of enalapril was in
good agreement with the one reported by Foda et. al [40]. Plasma level of metformin obtained
from healtjy volunteers after the administration of dose 500 mg was in good agreement with the
value reported by Hiren et. al [41-42]. The mean plasma concentration-time profile of four drugs in
volunteers is represented in Figure 6-9.

4.3.

Stability Studies

The stability studies were performed and six lots of commercially available drugs were obtained.
Accelerated and long term ambient condition studies were carried outs as described by ICH [36]. It
was found that the commercially available lots are stable up to six month. Two sets of studies were
done one on accelerated conditions and the other one at long term ambient conditions as described
by ICH [36]. Both the studies showed that all the drugs are stable in mentioned conditions. %
recoveries of all the drugs obtained were almost 100 %, table 4.
5.

Conclusion

The proposed HPLC method described in this paper provides a simple, universal, convenient and
reproducible approach for the simultaneous identification and quantification of metformin,
captopril, lisinopril and enalapril in biological fluids with good separation and resolution. In
addition, this method has the ability to be applied to pharmacokinetics studies in human volunteers
and Analytical results are accurate and precise with good recovery. This assay method was also
successfully applied in raw materials and pharmaceutical formulations. Linearity, accuracy,
precision, limit of detection and quantification, specificity were established. Hence this method is
beneficial for quality control laboratories as well as bio-analytical laboratories. This method can
not only be used to study dissolution and pharmacokinetics but also therapeutic monitoring of the
mentioned drugs can be studied in diabetes and hypertension patient having simultaneous drugs
prescription.

References
1.

Cocolas GH, Delgado JN, Remers WA (1998) Textbook of organic medicinal and
pharmaceutical chemistry, 10th edn. Lippincott- Raven, Philadelphia, pp 603607

2.

Vaughan, D.E., J.L. Rouleau, P.M. Ridker, J.M.O. Arnold, F.J. Menapace, and M.A.
Pfeffer, Effects of ramipril on plasma fibrinolytic balance in patients with acute anterior
myocardial infarction. Circulation, 1997. 96(2): p. 442-447.

3.

Di Pasquale, P., L. Valdes, V. Albano, V. Bucca, S. Scalzo, D. Pieri, G. Maringhini, and S.

Paterna, Early captopril treatment reduces plasma endothelin concentrations in the acute

and subacute phases of myocardial infarction: a pilot study. Journal of cardiovascular

pharmacology, 1997. 29(2): p. 202.

4.

Medicines, E., WHO Model List of Essential Medicines. WHO Medicines web: http://www.
who. int/medicines/publications/EML, 2007. 15.

5.

Lancaster, S.G. and P.A. Todd, Lisinopril. A preliminary review of its pharmacodynamic
and pharmacokinetic properties, and therapeutic use in hypertension and congestive heart
failure. Drugs, 1988. 35(6): p. 646.

6.

European Pharmacopoeia, in Council of Europe. 2001: Strasbourg, France.

7.

Amini, M., A. Zarghi, and H. Vatanpour, Sensitive high-performance liquid

chromatographic method for determination of captopril in plasma. Pharmaceutica Acta
Helvetiae, 1999. 73(6): p. 303-306.

8.

Bahmaei, M., A. Khosravi, C. Zamiri, A. Massoumi, and M. Mahmoudian, Determination

of captopril in human serum by high performance liquid chromatography using solid-phase
extraction. Journal of Pharmaceutical and Biomedical Analysis, 1997. 15(8): p. 1181-1186.

9.

Hillaert, S. and W. Van den Bossche, Determination of captopril and its degradation
products by capillary electrophoresis. Journal of Pharmaceutical and Biomedical Analysis,
1999. 21(1): p. 65-73.

10.

Huang, T., Z. He, B. Yang, L. Shao, X. Zheng, and G. Duan, Simultaneous determination
of captopril and hydrochlorothiazide in human plasma by reverse-phase HPLC from linear
gradient elution. Journal of Pharmaceutical and Biomedical Analysis, 2006. 41(2): p. 644648.

11.

Shimada, K., M. Tanaka, T. Nambara, Y. Imai, K. Abe, and K. Yoshinaga, Determination

of captopril in human blood by high-performance liquid chromatography with
electrochemical detection. Journal of chromatography, 1982. 227(2): p. 445.

12.

Tajerzadeh, H. and M. Hamidi, A simple HPLC method for quantitation of enalaprilat.

Journal of Pharmaceutical and Biomedical Analysis, 2001. 24(4): p. 675-680.

13.

Anzenbacherov, E., P. Anzenbacher, K. Macek, and J. Kvetina, Determination of enzyme

(angiotensin convertase) inhibitors based on enzymatic reaction followed by HPLC.
Journal of Pharmaceutical and Biomedical Analysis, 2001. 24(5-6): p. 1151-1156.

14.

United States Pharmacopoeia Convention, in The United States Pharmacopoeia, . 2006:

Rockville.

15.

Al-Momani, I.F., Determination of Hydrochlorothiazide and Enalapril Maleate in Tablet

Formulations by Reversed-Phase HPLC. TURKISH JOURNAL OF CHEMISTRY, 2001.
25(1): p. 49-54.

16.

Din, E., . stndag, A. zdemir, and D. Baleanu, A new application of chemometric

techniques to HPLC data for the simultaneous analysis of a two-component mixture.
Journal of liquid chromatography & related technologies, 2005. 28(14): p. 2179-2194.

17.

Wong, Y. and B.G. Charles, Determination of the angiotensin-converting enzyme inhibitor

lisinopril in urine using solid-phase extraction and reversed-phase high-performance
liquid chromatography. Journal of Chromatography B: Biomedical Sciences and
Applications, 1995. 673(2): p. 306-310.

18.

Sagirli, O. and L. Ersoy, An HPLC method for the determination of lisinopril in human
plasma and urine with fluorescence detection. Journal of Chromatography B, 2004. 809(1):
p. 159-165.

19

Rastkari N, Khoobi M, Shafiee A, Khoshayand MR, Ahmadkhaniha R. Development and

validation of a simple and sensitive HPLC-UV method for the determination of captopril in
human plasma using a new derivatizing reagent 2-naphthyl propiolate. J Chromatogr B
Analyt

Technol

Biomed

Life

Sci.

2013

Aug

1;932:144-51.

doi:

10.1016/j.jchromb.2013.06.019. Epub 2013 Jun 19.

20.

Sun Y, Zhang Z, Zhang X. Determination of captopril by high-performance liquid

chromatography with direct electrogenerated chemiluminescence. Spectrochim Acta A Mol
Biomol Spectrosc. 2013 Mar 15;105:171-5. doi: 10.1016/j.saa.2012.11.109. Epub 2012
Dec 20.

21.

uczak A, Zakrzewski R, Dobrogowski M. RP-HPLC-UV method coupled with postcolumn iodine-azide reaction for determination of free captopril in urine samples.
Bioanalysis. 2012 Jun;4(12):1481-9. doi: 10.4155/bio.12.113.

22.

Ramusovic S, Thielking G, Ler S. Determination of enalapril and enalaprilat in small

human serum quantities for pediatric trials by HPLC-tandem mass spectrometry. Biomed
Chromatogr. 2012 Jun;26(6):697-702. doi: 10.1002/bmc.1716. Epub 2011 Sep 23.

23.

Qin F, Wang D, Yang S, Jing L, Xiong Z, Li F. Quantitative determination of lisinopril in

human plasma by high performance liquid chromatography-tandem mass spectrometry and
its application in a pharmacokinetic study. Biomed Chromatogr. 2012 Jun;26(6):691-6. doi:
10.1002/bmc.1715. Epub 2011 Sep 19.

24.

Lima DM, Mundim IM, Jardim PC, Jardim TS, Diniz DG, Lima EM. A high performance
liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using solid phase

extraction for the simultaneous determination of plasma concentrations of enalapril and

enalaprilate in hypertensive patients treated with different pharmaceutical formulations.
Ther Drug Monit. 2009 Dec;31(6):710-6. doi: 10.1097/FTD.0b013e3181b8f1b6.
25.

Qin F, Wang D, Yang S, Jing L, Xiong Z, Li F. Quantitative determination of lisinopril in

human plasma by high performance liquid chromatography-tandem mass spectrometry and
its application in a pharmacokinetic study. Biomed Chromatogr. 2012 Jun;26(6):691-6. doi:
10.1002/bmc.1715. Epub 2011 Sep 19.

26.

Sun C, Zhu P, Hu N, Wang D, Pan Y. Differentiation of lisinopril and its RSS diastereomer
by

liquid

chromatography

combined

with

collision-induced

dissociation

mass

spectrometry. J Mass Spectrom. 2010 Jan;45(1):89-96. doi: 10.1002/jms.1694.

27.

Zhu PX, Wang DH, Sun CR, Shen ZQ. Characterization of impurities in the bulk drug
lisinopril by liquid chromatography/ion trap spectrometry. J Zhejiang Univ Sci B. 2008
May;9(5):385-90. doi: 10.1631/jzus.B0820031.

28.

Rezk MR, Riad SM, Mahmoud GY, Abdel Aleem AA. Simultaneous determination of
sitagliptin and metformin in their pharmaceutical formulation. J AOAC Int. 2013 MarApr;96(2):301-6.

29.

Li N, Deng Y, Qin F, Yu J, Li F. Simultaneous quantification of metformin and glipizide in

human plasma by high-performance liquid chromatography-tandem mass spectrometry and
its application to a pharmacokinetic study. Biomed Chromatogr. 2013 Feb;27(2):191-6.
doi: 10.1002/bmc.2768. Epub 2012 Jul 5.

30.

Sultana N, Arayne MS, Shafi N, Siddiqui FA, Hussain A. Development and validation of
new assay method for the simultaneous analysis of diltiazem, metformin, pioglitazone and
rosiglitazone by RP-HPLC and its applications in pharmaceuticals and human serum. J
Chromatogr Sci. 2011 Nov-Dec;49(10):774-9.

31.

FARHAN AHMED SIDDIQUI, M. SAEED ARAYNE, NAJMA SULTANA, FAIZA

QURESHI. Development and Validation of Stability-Indicating HPLC Method for the

Simultaneous

Determination

of

Paracetamol,

Tizanidine,

and

Diclofenac

in

Pharmaceuticals and Human Serum, JOURNAL OF AOAC INTERNATIONAL VOL. 94,

NO. 1, 2011, 150-58

32

Guideline, I. Validation of analytical procedures: Text and Methodology Q2 (R1). 2005.

33

Chow, S.C. and J. Shao, Statistics in drug research: methodologies and recent
developments. 2002: CRC Press.

34

Green, J.M., A practical guide to analytical method validation. Anal. Chem, 1996. 68: p.
305A-309A.

35

Guideline, I. Validation of analytical procedures: Methodology. 1996.

36.

ICH (2003) Stability Testing of New Drug Substances and Products, International
Conference on Harmonization, Geneva, Switzerland

37.

Guidance for Industry, Bioanalytical Method Validation, US Department of Health and

Human Services Food and Drug Administration,Center for Drug Evaluation and Research
(CDER), 2001.

38.

A. Jankowski, A. Skorek, K. Krzysko, P.K. Zarzycki, R.J. Ochocka and H. Lamparczyk,

Captopril: determination in blood and pharmacokinetics after single oral dose. Journal of
Pharmaceutical & Biomedical Analysis, Vol. 13, No. 4/5, pp. 655-660, 1995.

39.

Feng Qin, Dan Wang, Shuyan Yang, Lijuan Jing, Zhili Xiong and Famei Li, Quantitative
determination of lisinopril in human plasma by high performance liquid chromatography
tandem mass spectrometry and its application in a pharmacokinetic study. Biomed.
Chromatogr.2012;26: 691696]

40.

NH Foda, O Naeem, A A ELbary and G A ELbary. Simultaneous HPLC determination of

Enalapril and Hydrochlorothiazide in Human Plasma and its pharmacokinetic application.
J. Pharm. Sci. & Res. Vol.2 (11), 2010,786-794]

41.

Hiren N. Mistri, Arvind G. Jangid, Pranav S. Shrivastav, Liquid chromatography tandem

mass spectrometry method for simultaneous determination of antidiabetic drugs metformin
and glyburide in human plasma. Journal of Pharmaceutical and Biomedical Analysis 45
(2007) 97106.]

42.

Farhan Ahmed Siddiqui, Nawab Sher, Arif Zubair, Hina Shamshad, Nighat Shafi and Agha
Zeeshan Mirza. Analysis of metformin, glimepiride and pioglitazone in human serum and
its application to pharmacokinetics. Available online. DOI: 10.1039/c3ay40884a.

Table 1 Statistical regression analysis of proposed method

Lisinopril

Enalapri

Captopril

Metformin

30-10000

30-10000

30-10000

10-10000

ng/ml

ng/ml

ng/ml

ng/ml

Slope

37.99

2.8715

29.22

69.22

Intercept

199.66

165.59

102.37

111.97

LOD

3.26

1.14

1.69

0.98

LOQ

9.26

6.17

8.22

2.22

0.9964

0.997

0.9974

0.9986

Linearity

Table 2

Linearity accuracy and precision of proposed method

Reference sample
Injected

In Serum

Recovered %Recovery %RSD Recovered %Recovery %RSD

Conc.

Conc.

ngmL-1

ngmL-1

n=6

Conc.

n=6

ngmL-1

Lisinopril
30

29.66

98.86667

1.45

29.3

97.66667

2.24

100

99.41

99.41

1.17

98.33

98.33

1.69

500

501.22

100.244

1.34

492.5

98.5

1.77

1000

998.7

99.87

1.29

988.66

98.866

1.05

2000

2012.6

100.63

1.77

1978.5

98.925

1.35

5000

5044.9

100.898

1.43

4926.5

98.53

0.94

10000

1012.4

10.124

1.69

9876.5

98.765

1.06

Enalapril

2.09

30

30.11

100.3667

1.98

29.04

96.8

2.34

100

98.67

98.67

2.26

99.33

99.33

1.66

500

500.87

100.174

1.44

502.2

100.44

1.79

1000

989.4

98.94

1.69

993.4

99.34

1.24

2000

2014.5

100.725

0.88

1976.4

98.82

1.56

5000

5012.8

100.256

1.64

5012.8

100.256

1.38

10000

9982.8

99.828

1.35

1027.6

10.276

1.97

30

29.14

97.13333

2.09

29.14

97.13333

1.87

100

98.37

98.37

1.87

101.22

101.22

1.23

500

497.2

99.44

1.3

492.5

98.5

1.38

1000

988.6

98.86

1.46

976.5

97.65

1.45

2000

1994.4

99.72

1.29

1941.5

97.075

1.22

5000

4967.5

99.35

1.33

5032.5

100.65

1.72

Captopril

10000

9876.5

98.765

0.77

1056.8

10.568

1.69

10

9.82

98.2

2.29

9.76

97.6

2.27

50

49.37

98.74

2.61

48.69

97.38

2.33

500

496.7

99.34

1.74

488.6

97.72

1.25

1000

992.64

99.264

1.22

1055.2

105.52

1.61

2000

1987.5

99.375

0.88

1974.2

98.71

1.11

5000

5004.5

100.09

1.09

5012.8

100.256

1.38

10000

10121.5

101.215

1.22

10021.9

100.219

1.43

Metformin

Table 3

Recovery studies of proposed method

Concentrations
(%)

CV (n=6)

Recovery

Day 1

Day 2

Day 3

(%)

80

0.64

1.24

1.04

99.66

100

1.22

1.44

0.97

100.25

120

0.94

0.39

1.22

98.79

80

0.87

1.09

1.39

99.47

100

0.96

0.38

1.04

101.25

120

29

0.88

0.77

100.39

80

0.66

0.79

0.79

100.76

100

1.34

0.52

0.82

99.87

120

0.47

0.96

1.06

100.81

80

0.99

0.63

0.49

99.38

100

1.23

0.57

1.22

101.22

120

0.84

1.06

1.34

100.38

Metformin

Captopril

Enalapril

Lisinopril

Table 4

Month

Stability Studies using proposed method

Lisinopril

Enalapri

Captopril
Metformin

%Recovery RSD% %Recovery RSD% %Recovery RSD% %Recovery RSD%

40 0C/75% R.H
1st

99.98

0.66

100.94

0.39

99.96

1.02

101.29

0.49

2nd

99.46

0.94

100.67

0.89

99.39

0.86

100.98

1.34

3rd

99.06

0.88

100.22

1.02

99.09

0.71

100.76

1.06

6th

98.25

0.97

99.97

1.22

98.76

0.93

100.33

0.92

30 0C/65% R.H
1st

100.56

0.49

100.39

0.91

99.41

0.69

99.69

0.99

2nd

100.29

0.59

100.29

0.98

99.22

0.82

99.32

1.28

3rd

100.06

0.38

100.06

0.52

99.12

0.86

99.14

0.58

6th

99.89

0.92

99.76

0.64

98.85

0.84

98.69

1.08

CH3
H
N

NH2

H3C
NH

NH

Metformin
COOH

O
HS

N
H

CH 3

Captopril

H 3C

HOOC
CH 3
N

N
H
O

Enalapril

Lisinopril
Fig. 1 Chemical Structure of All Drugs

Fig. 2 Typical chromatogram of Metformin1, Internal Standard2 (Caffeine), lisinopril3,

Enalapril4 and captopril5 in reference standard

Fig.3 Typical chromatogram of Metformin1, Internal Standard2 (Caffeine), lisinopril3,

Enalapril4 and captopril5 in Pharmaceutical Formulation

Fig. 4 Typical chromatogram of Metformin1, Internal Standard2 (Caffeine), lisinopril3,

Enalapril4 and captopril5 in Human serum

Fig. 5 Typical chromatogram of Blank Human serum

Fig. 6 Mean plasma concentrationtime profile after oral administration of a 20 mg Lisinopril

Fig. 7 Mean plasma concentrationtime profile after oral administration of a 10 mg Enalapril

Fig. 8 Mean plasma concentrationtime profile after oral administration of a 25 mg Captopril

1000
900
800
700
600
500

Series1

400
300
200
100
0
0

10

20

30

40

Fig. 9 Mean plasma concentrationtime profile after oral administration of a 500 mg Metformin

Simultaneous Determination of metformin, captopril, lisinopril, and enalapril: its application

to Pharmacokinetics

Farhan Ahmed Siddiqui1*, Nawab Sher2, Nighat Shafi1, Saima Sher Bahadur2
1

Faculty of Pharmacy, Federal Urdu University Arts, Science and Technology, Karachi 75300
1

Department of Chemistry, University of Karachi, Karachi-75270, Pakistan.

*

(Email: farhanchemist@gmail.com
(0092-332 2524224)