American Journal of Analytical Chemistry, 2013, 4, 715-724
Published Online December 2013 (
Open Access AJAC
New Stability Indicating Method for Quantification of
Impurities in Amlodipine and Benazepril Capsules by
Validated HPLC
Rama Joga Venkata Eranki1,2*, Gopichand Inti1, Venkatasubramanian Jayaraman1,
Raghuram Pannala3, Sudhakar Rao Vidiyala1, Jadi Sree Ramulu2
1Quality & Analytical Development Department, InvaGen Pharmaceuticals, Inc., Hauppauge, USA
2Department of Chemistry, Sri Krishnadevaraya University, Anantapur, India
3Quality Assurance Department, ScieGen Pharmaceuticals, Inc., Hauppauge, USA
Email: *
Received October 21, 2013; revised November 25, 2013; accepted December 1, 2013
Copyright © 2013 Rama Joga Venkata Eranki et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
A stability indicating LC method was developed for the simultaneous determination of Amlodipine and Benazepril
capsules in pharmaceutical dosage form. Efficient chromatographic separation was achieved on Symmetry C18 station-
ary phase with simple combination of amobile phase containing 750 mL of DI Water, 250 mL of Acetonitrile and 2 mL
of Octylamine into suitable container with adjusted pH to 2.50 ± 0.05 with the aid of Ortho phosphoric acid delivered in
an isocratic mode and quantification was carried out using UV detection at 240 nm at a flow rate of 1.0 mL·min1 with
an injection volume of 20 μl and ambient column temperature. This method is capable to detect both the drug compo-
nents of Amlodipine and Benazepril in presence of their degradation products (Amlodipine Imp-A and Benazepril Im-
purity-C) with a detection level of 0.05%. Amlodipine/Benazepril in their combination drug product were exposed to
thermal, photolytic, hydrolytic and oxidative stress conditions, and the samples were analysed. Peak homogeneity data
of Amlodipine and Benazeprilis were obtained using PDA detector, in the stressed sample chromatograms, demonstrat-
ing the specificity. The method shows excellent linearity over a range of 0.05% - 2.0% for Amlodipine, Amlodipine
Impurity-A and 0.05% - 5.0% for Benazepril and Benazepril Impurity-C. The correlation coefficient for Amlodipine
and Benazepril is 1. The relative standard deviation was always less than 2%. The proposed method was found to be
suitable and accurate for quantitative determination and the stability study of Amlodipine and Benazepril in pharmaceu-
tical preparations. The developed HPLC method was validated with respect to linearity & range, accuracy, precision
and robustness.
Keywords: Column Liquid Chromatography; Method Validation; Stability Indicating Study Amlodipine and
1. Introduction
The amlodipine besylate component of amlodipine/bena-
zepril capsules is chemically described as 3-ethyl-5-me-
thyl (±)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,
4-dihydro-6-methyl-3,5-pyridinedicarboxylate, monoben-
zenesulphonate [1]. Amlodipine is a dihydropyridine cal-
cium antagonist (calcium ion antagonist or slow-channel
blocker) that inhibits the movement of calcium ions into
vascular smooth muscle cells and cardiac muscle cells.
Amlodipinebesylate is a white to pale yellow crystalline
powder, slightly soluble in water and sparingly soluble in
Amlodipine besylate has an empirical formula of
C20H25ClN2O5·C6H6O3S, and its molecular weight is
567.1, its structural formula is shown in Scheme 1.
Benazepril, the active metabolite of benazepril, is a no
sulfhydryl angiotensin-converting enzyme (ACE) inhibi-
tor. Benazepril is converted to benazeprilat by hepatic
cleavage of the ester group.
Benazepril hydrochloride is a white to off-white crys-
*Corresponding author.
Scheme 1. Amlodipine besy late c he mical str ucture.
talline powder, soluble (>100 mg/mL) in water, in etha-
nol, and in methanol. Benazepril hydrochloride’s chemi-
cal name is 3-[[1-(ethoxycarbonyl)-3-phenyl-(1S)-propyl]
1-acetic acid monohydrochloride
Benazepril Hydrochloride has an empirical formula of
C24H28N2O5·HCl, and its molecular weight is 460.96 and
its structural formula is shown in Scheme 2.
Amlodipine/Benazepril Capsules Brand name is called
as Lotrel [2]. Lotrel is a combination drug, containing
benazepril (Lotensin) and amlodipine (Norvasc) and is
used for treating high blood pressure. Benazepril is an
angiotensin converting enzyme (ACE) inhibitor. ACE is
an enzyme in the body that is important for the formation
of angiotensin II. Angiotensin II causes constriction of
the muscles surrounding arteries in the body, thereby nar-
rowing the arteries and elevating blood pressure. ACE
inhibitors such as benazepril lower blood pressure by
inhibiting the formation of angiotensin II, thus relaxing
the muscles and dilating the arteries. Relaxing the arter-
ies not only lowers blood pressure, but also improves the
pumping efficiency of a failing heart and improves the
pumping of blood by the heart in patients with heart fail-
ure. Amlodipine belongs to a class of medications called
calcium channel blockers. These medications block the
transport of calcium into the smooth muscle cells lining
the coronary arteries and other arteries of the body. Since
calcium is important in muscle contraction, blocking cal-
cium transport relaxes artery muscles and dilates coro-
nary arteries and other arteries of the body. Relaxing
muscles of arteries lowers blood pressure. The FDA ap-
proved Lotrel in March 1995.
Amlodipine is official in USP [3] and Benazepril is
also official in USP [4] and their combination drug prod-
uct also official in USP pharmacopoeia.
Lotrel is available in a variety of dosage combinations
that can be adjusted to best fit your needs. The Dosage
combinations are given as amlodipine besylate equiva-
lent to 2.5 mg, 5 mg or 10 mg of amlodipine, with 10 mg,
20 mg or 40 mg of benazepril hydrochloride providing
for the following available combinations: 2.5/10 mg,
5/10 mg, 5/20 mg, 5/40 mg, 10/20 mg and 10/40 mg.
Scheme 2. Benazepril hydroc hlor ide chemical structure.
Even though the products have been captured in USP,
an in-house method has been developed and validated as
per ICH guideline and monitoring of these impurities
with good separation of peaks and quantification of im-
purities in Amlodipine/Benazepril capsules as shown in
Schemes 3 and 4.
Stability testing forms an important part of the process
of drug product testing to provide evidence on how qual-
ity of a drug substance or drug product varies with time
under the influence of a variety of environmental factors
such as temperature, humidity, light and enables recom-
mendation of storage conditions, retest periods and shelf
life to be established. The two main aspects of drug
products that play an important role in shelf life deter-
minations are assay of active drug and degradants gener-
ated during the stability study. Stability-indicating meth-
ods have been reported for assays of various drugs in
drug products containing only one active drug substance.
Only few stability-indicating methods are reported for
the impurity assay of combination drug products con-
taining two or more active drug substances. The objec-
tive of this work was to develop an analytical LC proce-
dure, which would serve as stability-indicating impurity
assay method for combination drug products of Am-
lodipine and Benazepril.
The literature survey reveals that several methods were
reported for the individual estimation of Amlodipine and
Benazepril. Various methods using HPLC [5-7], RP-
HPLC [8,9], HPTLC [10,11], LC-MS [12], LCMS/MS [13]
and simultaneous UV spectrophotometric methods [14,15]
are reported for the estimation of Amlodipine alone or Be-
nazepril alone or in combination with other agents [16].
In USP, the reported analytical procedures describe a me-
thod for simultaneous determination of Amlodipine and Be-
nazepril in combined pharmaceutical dosage form in the
presence of their degradants with 110 minutes as runtime.
If the reported individual methods are applied for the
analysis of the capsules containing Amlodipine and Be-
nazepril, it would require UPLC to have shorter runtime
and it’s not possible for all to afford the same and the
method would not be rapid, less expensive, or economi-
cal, whereas the simultaneous determination of the in-
gredients of the Capsules would be rapid, stability in-
dicative and also economical and can be afforded by all.
In the present study, attempts were made to develop a
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R. J. V. ERANKI ET AL. 717
Scheme 3. Amlodipine impurity -A chem ic al str uc t ur e .
Scheme 4. Benazepril impurity-C chemical structure.
rapid, economical, precise and accurate method for the
simultaneous estimation of the ingredients of this com-
bination in the presence of their degradants.
2. Experimental
2.1. Chemicals & Reagents
Samples of amlodipine Impurity-A and the Benazepril
impurity-C were synthesized and characterized at Hetero
Drugs and Aarti Industries Limited, India. HPLC grade
acetonitrile & Methanol was procured from Honeywell:
Burdick & Jackson, Muskegon, MI 49442 and analytical
grade Octylamine was procured from Sigma Aldrich Com-
pany, 3050 spruce Street, St. Louis, MO-63103, High
purity water was generated in-house from Siemens water
purification system.
The product Amlodipine Tablets in Benazepril Cap-
sules consists of dibasic calcium phosphate, microcrys-
talline cellulose, crospovidone colloidal silicon dioxide,
magnesium stearate lactose monohydrate, pregelatinized
starch and hydrogenated vegetable oil as excipients. The
combination drug product samples were provided by
InvaGen Pharmaceuticals, Inc. for this work.
2.2. Chromatographic Conditions
The chromatographic system used was Shimadzu LC
2010 HPLC system comprised of degasser, quaternary
pump, auto injector, column compartment, UV detector
and the system was controlled through EZ chrome soft-
ware. Symmetry C18, (4.6 × 150 mm), 5 micron or equiva-
lent (Advance Chromatography, USA), maintained at
25˚C using a column oven, eluted with mobile phase at
the flow rate of 1.0 mL·min1 with Isocratic program.
Mobile Phase: Transferred 700 mL of DI Water, 200
mL of Acetonitrile and 100 mL of Methanol into suitable
container, Added 2.0 mL of Octylamine and mixed well.
Adjusted the pH to 2.50 ± 0.05 using Ortho Phosphoric
acid. Filtered through 0.45 µm nylon membrane filter
and degassed.
Measurements were made with injection volume 20 µl
and ultraviolet (UV) detection at 240 nm. For standard
and sample solution were prepared using the diluent
nothing but Mobile phase.
For analysis of forced degradation samples, the photo-
diode array detector (Model No. 2998) and Empower
Software was used in scan mode with a scan range of 200
- 400 nm. The peak homogeneity was expressed in terms
of peak purity and was obtained directly from the spec-
tral analysis report using the above-mentioned software.
2.3. Preparation of Solutions
Standard Stock Solutions:
Standard solutions were prepared by dissolving the
drugs in the diluent and diluting them to the desired con-
34.7 mg AmlodipineStandard (99.8%) was accurately
weighed, transferred into a 50 mL volumetric flask, and
dissolved with diluent.
100.0 mg Benazepril standard (99.7%) was accurately
weighed, transferred into a 100 mL volumetric flask, and
dissolved with diluent.
Low Level Standard Preparation:
Transferred 1.0 mL of each above solution in 200 mL
flask and diluted with diluent. The concentration of Low
levelstandard Preparation contains 0.0003472 mg/mL of
Amlodipine Besylate and 0.00050 mg/mL of Benazepril.
Detectability Level Standard Preparation:
Transferred 5.0 mL of the above Low level standard
solution in 50mL flask and diluted with diluent. The
concentration of Detectability level standard Preparation
contains 0.000347 mg/mL of Amlodipine Besylate and
0.00050 mg/mL of Benazepril.
Preparation of Sample:
Weighed 20 capsules and recorded the weight. Emp-
tied the capsules and cleaned the capsule shell with cot-
ton swabs. Weighed the empty capsules and recorded the
weight. Determined the average fill weight by subtract-
ing empty capsule weight from filled capsule weight ob-
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A quantity of powder equivalent to 50.0 mg of Am-
lodipine was transferred into a 100 mL volumetric flask.
To this flask, 75 mL of diluent were added, and the solu-
tion was sonicated for about 15 minutes with intermittent
shaking and with mechanical shaking for about 15 min-
utes. The solution was cooled to ambient temperature.
Then the volume was made up with diluent and centri-
fuged for about 15 minutes. Then the solution was used
for injection.
2.4. Optimization of the Chromatographic
To develop the stability-indicating method different sta-
tionary phases like C18, Discovery HS C18, different
mobile phases containing buffers like tri ethylamine in
water, Tetra butyl ammonium hydrogen sulfate with dif-
ferent pH (3 - 5) and organic modifier (acetonitrile) were
Our objective of the chromatographic method devel-
opment was to achieve a peak tailing factor <2, Signal to
Noise Ratio should be above 10, Theoretical plates should
be above 1000 for Amlodipine and Benazepril and %
RSD for 5 consecutive injection should be less than
NMT 10.0% and very good separation between am-
lodipine Impurity-A, Benazepril Impurity-C along with
drug peak amlodipine and benazepril. As this method is
used for quantifying impurities in drug product only
degradation products are monitored. This method is ca-
pable of separating other process related impurities also
but validation was done for only degradation products at
this time.
The chromatographic separation was achieved using a
Symmetry C18, (4.6 × 150 mm), 5 micron or equivalent.
Changing the composition of mobile phase optimized the
chromatographic method. Segregation of both peaks
(Amlodipine and Benazepril) was observed on any C18 or
CN column but it was difficult to separate both drug de-
gradants on these columns (amlodipine Impurity-A and
Benazepril Impurity-C). The Symmetry C18 column showed
better performance as compared to other columns.
3. Analytical Method Validation
The developed chromatographic method was validated
for selectivity, linearity, range, precision, accuracy, sen-
sitivity, robustness and system suitability.
3.1. Selectivity/Specificity
Selectivity of the developed method was assessed by
performing forced degradation studies [9-13]. According
to ICH [14] stress testing of the drug substance can help
the intrinsic stability of the molecule and validate the
stability indicating power of the analytical procedure
used. Photo stability testing should be an integral part of
stress testing. The standard conditions for photo stability
testing are described in ICH Q1B [11]. The specificity of
the developed LC method for Amlodipine and Benazepril
was determined in the presence of its related compounds
Amlodipine impurity-A, Benazepril impurity-C.
The stress conditions employed on Amlodipine and
Benazepril capsules as degradation study includes acid
hydrolysis (5 mL of Conc. HCl), base hydrolysis (2 mL
of 2N NaOH), oxidation (2 mL of 3% H2O2), photolysis
(ICH Q1B) and thermal (80˚C) study.
As such sample provided the Total Impurities level as
below reporting limit (BRL) which includes Amlodipine
Impurity-A and Benazepril Impurity-C as BRL whereas
in acid degration the sample subjected to 5 mL of Conc.
HCl for 2 hours and the Total degraded impurities were
found 14.85%.
In Base degradation the sample subjected to 2 mL of
2N NaOH for 2 hours which produces the total impurities
of 0.29% and the samples under Oxidation with 2 mL of
3% H2O2 provides the Total impurities result as BRL.
Under UV treatment of Sample on 3 days provides the
Total impurities as 0.05% and in Thermal condition at
80˚C the total impurities were found 0.10%.
For all the above, degradation studies results were pre-
sented in Tables 1(a) and (b) and related chromatograms
were represented in Figures 1 through 6.
Tables 1(a) and (b) result of analysis of forced degra-
dation study samples using the proposed method, indi-
cating percentage of degradation and peak purity of Am-
lodipine and Benazepril capsules. Figure 7 refers to Re-
lated compound and Assay chromatogram of the combi-
nation drug product. Figures 8 and 9 refer to peak purity
plot of Amlodipine and Benazepril respectively.
3.2. Results and Discussion
From the development studies, it was determined that
using mobile phase as 750 mL of DI Water, 250 mL of
Acetonitrile and 2 mL of Octylamine into a suitable con-
tainer adjusted the pH of the solution to 2.50 ± 0.05 with
the aid of Ortho phosphoric acid with Isocratic flow rate
of 1.0 mL/min and ambient temperature. The analytes of
this combination had adequate retentions, peak shape,
less tailing, more resolution between drug and it’s de-
gradantsand the chromatographic analysis time was about
60 minutes. In optimized conditions Amlodipine, Bena-
zepril and their degradants were well separated. Typical
retention times of Amlodipine and Benazepril were about
12.58 and 7.52 min and for amlodipine Impurity-A,
Benazepril Impurity-C and benzene sulphonic acid were
about 5.14 min, 3.57 min and 6.25 min respectively.
The retention time asper USP monograph method was
about 110 minutes but as per in-house validated method
the retention time was 60 minutes which is reduced to
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Table 1. (a) Summary on forced degradation results-percentage of degradation; (b) Summary on forced degradation re-
sults-peak purity.
Stress condition Time Amlodipine Imp-A
Benazepril Imp-C
Maximum individual
unknown impurity (%) Total impurities (%)
As such sample 0 hour BRL BRL BRL BRL
Conc. HCl (5 mL) 2 hours 7.87 3.19 3.37 14.85
2N NaOH (2 mL) 2 hours BRL 0.29 BRL 0.29
3% H2O2(2 mL) 2 hours BRL BRL BRL BRL
UV light 3 days BRL BRL 0.05 0.05
Thermal condition at 80˚C 7 days BRL BRL 0.05 BRL 0.10
ND: not detected; BRL: below reporting level (BRL = 0.05%), Imp: Impurity
Stress condition TimePeak Peak
time (min)Purity
threshold Match angle Match
Amlodipine 203802 11.66 0.707 1.499 0.498 2.145
Conc. HCl (5 mL) 2 hours Benazepril 448906 7.62 0.125 0.440 0.124 1.226
Amlodipine 232198 11.56 0.413 0.683 0.605 1.846
2N NaOH (2 mL) 2 hours Benazepril 464553 7.64 0.090 0.272 0.128 1.157
Amlodipine 232336 11.51 1.013 1.863 1.136 2.664
3% H2O2 (2 mL) 2 hours Benazepril 466682 7.65 0.178 0.514 0.158 1.234
Amlodipine 231330 11.49 0.415 0.680 0.685 1.851
UV light 3 days Benazepril 467746 7.66 0.090 0.277 0.117 1.158
Amlodipine 233186 11.48 0.706 1.138 0.772 1.968
Thermal condition at 80˚C 7 days Benazepril 467911 7.66 0.133 0.387 0.126 1.186
Figure 1. Chromatogram of 10/40 mg sample on as such.
fifty percent of total run time and at the same time the
separations were achieved to greater extent and the meth-
ods proved to be stability indicating. No EP methods are
available on this combination. Instead of regular HPLC if
we use UFLC the retention time can be reduced to an-
other 20 minutes with the same suitability parameters.
Development on UFLC is not an option in the present
study keeping in view this application for cost effective
generic products to help the people with low cost medi-
Figure 2. Chromatogram of 10/40 mg sample on acid degradation.
Figure 3. Chromatogram of 10/40 mg sample on Base degradation.
Figure 4. Chromatogram of 10/40 mg sample on oxidation degradation.
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R. J. V. ERANKI ET AL. 721
Figure 5. Chromatogram of 10/40 mg sample on UV treatment.
Figure 6. Chromatogram of 10/40 mg sample on Thermal treatment.
During the initial forced degradation experiments, it
was observed that acid hydrolysis was a fast reaction for
Amlodipine and Benazepril capsules and almost complete
degradation occurred when 5 mL of Conc. HCl solution
was used.Both drugs showed extensive degradation in
acidic condition and indicating homogeneous peaks and
thus establishing the specificity of the Impurity assay
3.3. Calibration and Linearity
Calibration curve obtained by the least square regression
analysis between average peak area and concentration
showed linear relationship with a regression coefficient
of 0.999 over the calibration ranges tested.
The results of linearity and range obtained for the two
potential impurities were tabulated. Linear calibration plot
for this chromatographic method was obtained over the
calibration ranges tested, i.e. 0.05% to 1.0% for Am-
lodipine impurity-A and 0.05% to 5.0% for Benazepril
impurity-C. The correlation coefficient obtained was greater
than 0.999 for the two impurities and the major com-
pounds Amlodipine and Benazepril (Figures 10 and 11).
The method exhibited good linearity with correlation
coefficient values greater than 0.999.
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Figure 7. Related substances and Assay chromatogram of amlodipine & benazepril 10/40 mg capsules.
Figure 8. Purity plot of amlodipine from amlodipine & benazepril 10/40 mg capsules.
Figure 9. Purity plot of benazepril from amlodipine & benazepril 10/40 mg capsules.
3.4. Precision (Repeatability)
The precision of the method was studied by determining
the concentrations of each0.13 and 0.16. The results of
the precision study indicate that the method is reliable
(RSD% < 10), in Tables 2(a) and (b).
Table 2(a): % RSD of six (6) replicate injections of
each impurity should be less than 10.0, theoretical plates
should be NLT 1000 and tailing factor should be NMT
2.0 for system precision.
Table 2(b): % RSD of six (6) sample preparations for
each impurity should be less than 10.0 for method preci-
3.5. Accuracy (Recovery Test)
The percentage recovery was established for all the ana-
lytes throughout the range concentration as explained
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R. J. V. ERANKI ET AL. 723
Slope (m) = 31102.932 ,Intercept (c) = 883.342, Correlation Co-efficient (r)
= 0.9999869.
Figure 10. Linearity for amlodipine impurity-A.
Slope (m) = 29317.229, Intercept = 3378.680, Correlation Co-efficient (r) =
Figure 11. Linearity for benazepril impurity-C.
Table 2. (a) System precision; (b) Method precision 10/40
Amlodipine Benazepril
% RSD 0.13 0.16
Theoretical plates (N) 6881.82 6142.15
Tailing factor (T) 1.074 1.094
Amlodipine Benazepril
impurity-A % RSD 0.0 Benazepril
impurity-C % RSD 0.0
under linearity studies and obtained results are tabulated
in Table 3.
3.6. Robustness
Robustness study was conducted by making small but
Table 3. Recovery studies results for amlodipine Imp-A and
benazepril Imp-C.
R % Spiking
level Average peak area % RecoveryMean
recovery %
Amlodipine Imp-A
Benazepril Imp-C
deliberate changes in the optimized method parameters.
To determine robustness of the method, experimental
conditions were purposely altered and chromatographic
resolution between Amlodipine and Benazepril were evalu-
The flow rate of the mobile phase was 1.0 mL·min1.
To study the effect of flow rate on the retention time of
Amlodipine and Benazepril it was changed to 0.9 ml/min
and 1.1 ml/min.
The effects of pH variation were studied at pH 2.40
and pH 2.60 instead of pH 2.50, while other mobile
phase components were held constant. At all conditions
the relative retention time found for Amlodipine Impu-
rity-A and Benazepril Impurity-C were found 0.47 and
0.42 respectively. See Table 4.
3.7. Determination of Limit of Quantification
Prepared Amlodipine and Benazepril LOQ solution as
per the method containing the concentration of about
0.331 µg/mL of Amlodipine and 0.5000 µg/mL of
Benazepril. Made five (5) replicate injections and re-
corded % RSD. Calculated S/N ratio of 0.05% to estab-
lish LOQ. See Table 5.
4. Conclusions
The Isocratic RP-LC method developed the analysis of
binary mixtures of Amlodipine and Benazeprilin. Their
pharmaceutical preparation is precise, accurate and with
a reasonable run time. This method is capable to detect
both the drug components of Amlodipine and Benazepril
in presence of their degradation products (Amlodipine
Imp-A and Benazepril Impurity-C) with a detection level
of 0.05%.
The method was fully validated showing satisfactory
data for all the method validation parameters tested. The
developed method is stability-indicating, separates de-
gradants and can be conveniently used by the quality
control department to determine impurity assay of phar-
maceutical preparations and also for stability sample
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Table 4. Summary on robustness paramete r study.
Parameters Amlodipine impurity-A Benazepril impurity-C
Flow rate of 1.0 mL/minute 0.46 0.43
Variation in flow rate of 0.9 mL/minute 0.47 0.42
Variation in flow rate of 1.1 mL/minute 0.47 0.42
Variation in pH of mobile phase—2.40 0.47 0.43
Variation in pH of mobile phase—2.60 0.47 0.42
Table 5. S/N ratio of amlodipine, benazepril and composite impurities at LOQ (0.05%) level.
Component name Concentration µg/mL % RSD Signal to noise ratio (S/N)
Amlodipine impurity-A 0.3310 0.27 78.915
Amlodipine 0.3469 0.21 188.860
Benazepril impurity-C 0.5010 0.13 197.081
Benazepril 0.5000 1.42 100.801
5. Acknowledgements
The authors wish to thank the management of InvaGen
Pharmaceuticals, Inc. for supporting this work. Coopera-
tion from colleagues of Quality and Analytical Research
and Development departments of InvaGen Pharmaceuti-
cals, Inc. and Sciegen Pharmaceuticals, Inc. is appreciated.
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