New Stability Indicating Method for Quantification of Impurities in Amlodipine and Benazepril Capsules by Validated HPLC

doi:10.4236/ajac.2013.412086

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American Journal of Analytical Chemistry, 2013, 4, 715-724

Published Online December 2013 (http://www.scirp.org/journal/ajac)

http://dx.doi.org/10.4236/ajac.2013.412086

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: *[email protected]

Received October 21, 2013; revised November 25, 2013; accepted December 1, 2013

tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly

cited.

ABSTRACT

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·min−1 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

Benazepril

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

ethanol.

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.

R. J. V. ERANKI ET AL.

716

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]

amino]-2,3,4,5tetrahydro-2-oxo-1H-1-(3S)-benzazepine-

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·min−1 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-

centration.

Amlodipine:

34.7 mg AmlodipineStandard (99.8%) was accurately

weighed, transferred into a 50 mL volumetric flask, and

dissolved with diluent.

Benazepril:

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-

tained.

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R. J. V. ERANKI ET AL.

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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

Conditions

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

used.

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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R. J. V. ERANKI ET AL.

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Table 1. (a) Summary on forced degradation results-percentage of degradation; (b) Summary on forced degradation re-

sults-peak purity.

(a)

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

(b)

Stress condition TimePeak Peak

area

Retention

time (min)Purity

angle

Purity

threshold Match angle Match

threshold

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-

cines.

R. J. V. ERANKI ET AL.

720

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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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

method.

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-

sion.

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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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) =

0.99999.

Figure 11. Linearity for benazepril impurity-C.

Table 2. (a) System precision; (b) Method precision 10/40

mg.

(a)

Amlodipine Benazepril

% RSD 0.13 0.16

Theoretical plates (N) 6881.82 6142.15

Tailing factor (T) 1.074 1.094

(b)

Amlodipine Benazepril

Amlodipine

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

0.4300

4.3000

17.2000

0.05

0.5

2.0

18,083

157,352

578,724

121.3

105.6

97.1

108.0

Benazepril Imp-C

1.0020

16.475

164.75

0.05

0.5

1.0

14,099

585,209

5,344,103

97.4

101.0

92.3

96.9

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-

ated.

The flow rate of the mobile phase was 1.0 mL·min−1.

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

analysis.

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R. J. V. ERANKI ET AL.

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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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