Two validated stability-indicating chromatographic methods for the separation of two anti-hypertensive combinations in the presence of their degradation products or impurities

Article abstract of DOI:10.1007/s11696-019-00792-z

Two RP-HPLC methods were developed, optimized, and validated for the determination of two different anti-hypertensive combinations in the presence of their degradation products or impurities and in their pharmaceutical formulations. The first mixture is Ramipril (RAM) in combination with Amlodipine besylate (AML) [mixture I], while the second one is a combination of Ramipril (RAM), Atorvastatin (ATV), and Aspirin (ASP) [mixture II].The proposed combinations were successfully separated on X-bridge C₁₈column (250 × 4.6?mm i.d, 5?μm p.s.), using a mobile phase of 0.05?M phosphate buffer-acetonitrile-THF (60:40:0.1% by volume) pH 2.5 and an isocratic mobile phase formed of acetonitrile-0.05?M phosphate buffer-THF (60:40:0.1% by volume) pH 2.5 for mixture (I) and (II) at a flow rate of 1?mL/min and 1.2?mL/min, respectively. The compromising components of the mixtures were detected at 218?nm. For the best separation of the mentioned components different parameters were examined and optimized. The two suggested methods were validated in compliance with the ICH guidelines and were successfully applied for the quantification of the cited components in presence of their obtained degradation products as well as in their commercial pharmaceutical formulations. For both methods the obtained results were statistically analyzed and compared to those of the official and reported methods; using Student’s t test and F test showing no significant difference with high accuracy and good precision.

Full text of DOI:10.1007/s11696-019-00792-z

Chemical Papers
https://doi.org/10.1007/s11696-019-00792-z
ORIGINAL PAPER
Two validated stability‑indicating chromatographic methods
for the separation of two anti‑hypertensive combinations
in the presence of their degradation products or impurities
Noha Samy Mostafa1 · Ghada AbdElHamid1 · Hala Elsayed Zaazaa1 · Sawsan Mohamed Amer1
Received: 17 December 2018 / Accepted: 19 April 2019
© Institute of Chemistry, Slovak Academy of Sciences 2019
Abstract
Two RP-HPLC methods were developed, optimized, and validated for the determination of two diferent anti-hypertensive
combinations in the presence of their degradation products or impurities and in their pharmaceutical formulations. The
frst mixture is Ramipril (RAM) in combination with Amlodipine besylate (AML) [mixture I], while the second one is a
combination of Ramipril (RAM), Atorvastatin (ATV), and Aspirin (ASP) [mixture II].The proposed combinations were
successfully separated on X-bridge C18column (250×4.6 mm i.d, 5 µm p.s.), using a mobile phase of 0.05 M phosphate
bufer-acetonitrile-THF (60:40:0.1% by volume) pH 2.5 and an isocratic mobile phase formed of acetonitrile-0.05 M phos-
phate bufer-THF (60:40:0.1% by volume) pH 2.5 for mixture (I) and (II) at a fow rate of 1 mL/min and 1.2 mL/min, respec-
tively. The compromising components of the mixtures were detected at 218 nm. For the best separation of the mentioned
components diferent parameters were examined and optimized. The two suggested methods were validated in compliance
with the ICH guidelines and were successfully applied for the quantifcation of the cited components in presence of their
obtained degradation products as well as in their commercial pharmaceutical formulations. For both methods the obtained
results were statistically analyzed and compared to those of the ofcial and reported methods; using Student’s t test and F
test showing no signifcant diference with high accuracy and good precision.
Keywords Anti-hypertensive combination · Stability-indicating · Liquid chromatography
Introduction
Fig. 1b. It is a dihydropyridine calcium channel blocker
which is used for the management of hypertension and
angina (Mofat et al. 2011; Brunton et al. 2006).
Ramipril (RAM) [2S,3aS,6aS)-1-[(2S)-2-[[(1S)-1-
(ethoxycarbonyl)-3-phenylpropyl]amino]-1-oxopropyl]
octahydrocyclopenta[b]pyrrole-2-carboxylic acid)], Fig. 1a,
is an angiotensin-converting enzyme (ACE) inhibitor used
in the management of mild to severe hypertension(Mofat
et al. 2011). It is a prodrug which after absorption undergoes
rapid metabolic ester hydrolysis to the active diacidic form
ramiprilat (Evoy 2009).
Atorvastatin (ATV) [R-(R*,R*)]-2-(4-fuorophenyl)-β,δ-
dihydroxy-5-(1–methylethyl)-3-phenyl-4-[(phenylamino)-
carbonyl]-1H-pyrrole-1-heptanoic acid, Fig. 1c, is an HMG-
CoA reductase inhibitor used to reduce LDL-cholesterol and
triglycerides (Mofat et al. 2011) (Brunton et al. 2006).
Aspirin (ASP) is 2-acetyloxybenzoic acid, Fig. 1d, which
is an acetyl derivative of salicylic acid useful in the relief of
headache and muscle and joint aches. Aspirin is also efec-
tive in reducing fever and swelling (Mofat et al. 2011). It
is often used as an analgesic, antipyretic, anti-infammatory
and antiplatelet agent (The Merk index).
Amlodipine (AML) is chemically known as [3-O-
ethyl-5-O-methyl-2-(2-aminoethoxymethyl)-4-(2-chloro-
phenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate],
Cardace am 5® launched by Sanof is a combination of
two medicines Ramipril (RAM) and Amlodipine (AML)
which lowers blood pressure efectively. It acts by relaxing
blood vessels so that blood fows more smoothly and the
heart can pump blood more efciently.
* Noha Samy Mostafa
noha.ayesh@pharma.cu.edu.eg
1
Analytical Chemistry Department, Faculty of Pharmacy,
Cairo University, Kasr Elaini St, P.O. box 11562, Cairo,
Egypt
Vol.:(0123456789)
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Chemical Papers
Fig. 1 Structural formula of, a
RAM, b AML, c ATV and d
ASP
Ramipril (RAM), Atorvastatin (ATV), and Aspirin (ASP)
were co formulated together in Ramitorva® manufactured
by Zydus cardiva. It can be used for the treatment of high
cholesterol, high blood pressure, prevention of heart attack,
and risk of strokes.
however, the possible stated degradation products were nei-
ther isolated nor characterized (Shetty et al. 2011).
This manuscript aims to establish and validate a stability-
indicating HPLC method for the simultaneous determination
of RAM either in combination with AML or ATV and ASP
in the presence of their hydrolytic degradation products or
impurities in their bulk powder and in their pharmaceutical
formulations.
A literature survey revealed that RAM, AML, ATV, and
ASP are all ofcial drugs in the British Pharmacopoeia
(British Pharmacopoeia, Medicines and Healthcare Prod-
ucts Regulatory Agency (MHRA), London 2013). Up to our
recent knowledge diferent methods have been applied for
simultaneous estimation of the binary mixture of RAM and
AML (Mixture I) in their bulk powder and pharmaceutical
formulation; namely UV spectroscopic method (Patil et al.
2009), thin-layer chromatographic separation (Gupta et al.
2007), high-performance liquid chromatography (Babu et al.
2011; Kumar et al. 2012; Maste et al. 2011; Patel and Patel
2014; Rajput et al. 2012; Ramadevi et al. 2013) and one in
the presence of stated impurities which are not the main
degradation products (Dai et al. 2013). Also, for the ternary
mixture of RAM, ATV, and ASP (Mixture II), diferent tech-
niques have been applied for their simultaneous determi-
nation, including chemometrics-assisted UV-spectroscopic
techniques (Sankar et al. 2011a, b) and HPLC (Panchal et al.
2009; Sharma et al. 2012; Patole et al. 2010) in their bulk
powder and pharmaceutical formulated capsules. Moreover,
they were detected by LC/MS in plasma (Gajula et al. 2012),
another stability-indicating UPLC method was reported for
a quaternary mixture of RAM, ATV, ASP, and Metoprolol;
The stability-indicating behavior of the proposed drugs
was studied by subjecting them to diferent stress condi-
tions. The stressed samples were analyzed using the pro-
posed (RP)-HPLC methods, which were able to separate
the drugs from the compounds produced during the forced
degradation studies.
Experimental
Instruments
The liquid chromatography system consisted of an isocratic
pump, Model G1310A (Agilent 1100 series liquid chroma-
tography, Germany), an ultraviolet (UV) variable wave-
length detector (Model G1314A, Agilent 1100 Series, Ger-
many), a Rheodyne injector, Mode 7725 I (Sigma-Aldrich,
Taufkirchen bei Mu¨nchen, Germany) equipped with a
20 mL injector loop (Agilent Technologies, Germany).
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The stationary phase was X-bridge RP-C18 column
(250×4.6 mm i.d, 5 µm p.s.).
Degraded samples
TLC plates 20×20 cm (Sigma-Aldrich, Germany) coated
with a 0.2-mm silica gel 60 F254 layer. The sample was
applied to the plates using micro-droppers and a UV lamp
at 254 nm was used for visualization of the spots, for deg-
radation tracing.
RAM was exposed to acidic and alkaline stress conditions.
It showed complete degradation upon refuxing with 1 N
NaOH and 2 N HCl for 5 h producing two degradation prod-
ucts namely ramiprilat, the active form, and Ramipril DKP
(Aschar et al. 2015).
Jenway 3505 pH meter (Staffordshine, UK), for pH
adjustment.
For AML, upon alkaline hydrolysis with 1 N NaOH for
1 h it gives out AML deg, the same degradation product
is formed after 2 h of exposure to 2 N HCl. All the hydro-
lyzed solutions were cooled and neutralized to pH 7.0 and
the solutions were further tested for complete degradation
by TLC using 33% ethyl acetate–methanol–toluene—33%
ammonia solution (6.5:2:1:0.5 by volume) as a developing
system (Zaazaa et al. 2012).
Sonix TVss-series ultrasonicator (USA).
UPLC MS/MS “Waters” 3100 “USA”, the sample
injected directly on TQ Detector (Acquity ultra performance
LC) coupled with Software Mass lynx V4 (programmed on
+ve mode).
Materials and reagents
ATV showed a complete degradation using 6 N HCl for
3 h and resulting into two degradation products ATV deg1
(aniline) and ATV deg2 (Darwish et al. 2016; Hassan et al.
2016).
Pure standard
Standard RAM, AML, ATV, and ASP, were supplied by
Rameda Pharmaceutical Company (Cairo, Egypt), their
purities were assessed according to the ofcial methods
(British Pharmacopoeia, Medicines and Healthcare Prod-
ucts Regulatory Agency (MHRA), London 2013) and found
to be (99.85% 0.71), (100.17% 0.99), (99.88% 0.97),
and (99.68% 0.45); respectively. Salicylic acid as aspirin’s
main impurity was purchased from Sigma Aldrich, where its
certifed purity is (99.95%).
ATV-degraded solution was then cooled and neutralized
to pH 7.0 with NaOH and the solutions were tested for com-
plete degradation by TLC using toluene–methanol (70:30,
V/V) as a developing system for ATV (Hassan et al. 2016).
All the degradation product solutions were extracted with
10 mL methanol three times to ensure complete extraction
and purifcation, and then the solutions were evaporated.
Standard solutions
Phamaceutical formulations
Stock standard solutions of Ramipril and Amlodipine
(0.1 mg/mL), and their degradation products derived from
the complete degradation of RAM and AML (1 mg/mL),
were prepared in methanol then further diluted using the
mobile phase of 0.05 M phosphate bufer–acetonitrile–tet-
rahydrofuran (60: 40: 0.1% by volume) and adjusted to a pH
of 2.5 0.2 with 85% phosphoric acid for mixture [I].
Meanwhile for mixture [II]; Ramipril, Atorvastain, and
Aspirin (0.1 mg/mL) and stock standard solutions of the
degradation products derived from the complete degradation
of standard solutions of both RAM and ATV (1 mg/mL) and
salicylic acid—Aspirin main impurity—were all prepared in
methanol then further dilutions were made up using mobile
phase acetonitrile-0.05 M phosphate bufer solution—tet-
rahydrofuran (60: 40: 0.1% by volume) and adjusted to a pH
of 2.5 0.2 with 85% phosphoric acid.
– Cardace am 5® 5/5 mg tablets of RAM and AML were
manufactured by Sanof, India.
– Ramitorva® capsules of 5/10/75 mg RAM, ATV and
ASP, respectively, were manufactured by Zydus Cardiva,
India.
Both formulations were purchased from the Indian
market.
Chemicals and reagents
All chemicals used throughout this work were of high ana-
lytical grade, and the solvents were of HPLC grade. These
included concentrated hydrochloric acid, 33% ammonia
solution (0.88 gm/mL), toluene, ethyl acetate, sodium
hydroxide, methanol and chloroform (El-Nasr Pharmaceu-
tical Chemicals, Egypt), tetrahydrofuran (THF), acetonitrile
(Sigma-Aldrich, Germany), O-phosphoric acid (85%) (BDH,
Poole, England), de-ionized water, and bi-distilled from an
Aquatron Automatic Water Still A4000 (Bibby Sterillin Staf-
fordshire, UK).
All stock standard solutions were freshly prepared and
stored in the refrigerator to be used for up to 3 weeks.
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mixture was sonicated for 30 min. Volumes were completed
with the same solvent and then fltered. Suitable dilution
was achieved using the appropriate mobile phase to obtain
concentrations of (5 µg/mL) of each of RAM and AML for
analysis of mixture [I].
Procedures
Chromatographic conditions
HPLC was conducted on an X-bridge RP-C18 column (250
× 4.6 mm i.d, 5 µm p.s). The mobile phase for mixture [I]
consisted of 0.05 M phosphate bufer–acetonitril–tetrahy-
drofuran (60:40:0.1% by volume) and acetonitrile-0.05 M
phosphate bufer solution—tetrahydrofuran (60:40:0.1% by
volume) for mixture [II] where the fnal pH of both mobile
phases was adjusted to pH 2.5 0.2 using O-phosphoric
acid, fltered through a 0.45 mm Millipore membrane flter
(Billerica, MA) and were degassed for 30 min in an ultra-
sonic bath prior to usage. The system was operated at ambi-
ent temperature with a fow rate of 1 mL/min and 1.2 mL/
min for mixture [I] and [II], respectively, and UV detection
at 218 nm. Seeking good equilibrium, the analysis was usu-
ally performed after passing 50–60 mL of the mobile phase,
for conditioning and pre-washing of the stationary phase.
The contents of 5 capsules of Ramtrov® claimed to con-
tain (5/10/75 mg of RAM, ATV, and ASP, respectively)
was quantitatively transferred into 100 mL volumetric fask,
50 mL acetonitrile was added, the mixture was sonicated for
30 min. Volume was completed and then fltered. Suitable
dilution was achieved using the appropriate mobile phase to
obtain concentrations of (5, 10, and 7.5 µg/mL) for RAM,
ATV, and Aspirin, respectively, for mixture [II]. Then the
previously detailed procedures were followed.
Results and discussion
Ramipril was combined in two diferent formulations, the
frst combination is with Amlodipine, to manage severe
hypertensive cases, and the second one is with Atorvastatin
and Aspirin that plays a great role in hypertensive cases
with the risk of hyperlipidemia and strokes. It gives a rise to
more compliance routes of treatment that the patient instead
of taking two or three dosage forms, he takes all in one.
From here arises the importance of our work, which gives
rise to two simple, rapid, precise and time-saving stability-
indicating HPLC methods, that could be applied in everyday
quality control assay of the above-mentioned combinations,
moreover, it can check for any extent of degradation in their
pharmaceutical formulations.
Linearity
For (Mixture I), standard stock solutions (0.1 mg/mL) of
RAM and AML were further diluted with the specifc mobile
phase to obtain dilutions of RAM and AML in the ranges of
(5–50 µg/mL). Triplicates of 20 mL volumes were injected
and chromatographed for each solution. The relative peak
area ratios, using 30 µg/mL of RAM and AML as a divisor
for RAM and AML, respectively, were calculated and plot-
ted against the corresponding concentrations to obtain the
calibration graph for each component.
Stability-indicating methods are an integral part of the
process of drug product quality control. Thus in the present
work a stability study was carried out with complete sepa-
ration and elucidation of the major degradation products of
two commonly used anti-hypertensive combinations as rec-
ommended by the ICH guidelines (ICH, Q1A (R2) 2003)
and USP (2007).
For (Mixture II), standard stock solutions (0.1 mg/mL) of
RAM, ATV, and ASP were further diluted with the specifed
mobile phase to obtain dilutions of RAM in the range of
(5–50 µg/mL), while both ATV and ASP working solutions
were in the range of (2–16 µg/mL). A volume of 20 mL of
each solution was injected in triplicate and chromatographed
under the previously mentioned conditions. Using 30 µg/mL
of RAM and 10 µg/mL of either ATV or ASP as a divisor,
relative peak areas were calculated and plotted against the
corresponding concentrations to obtain the calibration graph
for each. And the regression equations were computed for
each component in both mixtures.
In this study Ramipril was subjected to alkaline, acidic,
and oxidative hydrolysis using 1 N NaOH, 2 N HCl, and 30%
H2O2, respectively, under refux for 5 h. All of them resulted
in the formation of two degradation products namely ram-
iprilate (RAM deg1) which was found to be RAM active
metabolite, and ramipril DKP (RAM deg2) (Aschar et al.
2015).
Both degradates are stated in the British Pharmaco-
poeia as main impurities; impurity E and impurity D for
ramiprilate and RAM DKP, respectively (British Pharma-
copoeia, Medicines and Healthcare Products Regulatory
Agency (MHRA), London 2013). Although it is one of the
most widely used ACE inhibitors, the drug has considerable
degree of instability, and the degradation is shown by two
major pathways: hydrolysis, forming ramipril-diacid then
Assay of pharmaceutical formulations
Ten tablets of Cardac am 5® claimed to contain 5 mg of both
RAM and AML were weighed, fnely powdered and thor-
oughly mixed. An accurately weighted portion of the powder
equivalent to the weight of one tablet was transferred into
100 mL volumetric fask, 50 mL acetonitrile was added, the
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cyclization by inter-nucleophilic attack leading to the forma-
tion of diketopiperazine (DKP) (Elshanawane et al. 2008; De
Diego et al. 2010).
2011), it is stated in the British pharmacopeia as one of
Aspirin main impurities.
Two simple, accurate, and precise RP-HPLC methods
were investigated and validated for quantitative analysis of
RAM in combination either with AML or with ATV and
ASP. The procedures were carried out with a view to develop
stability-indicating methods in a reasonable time and with
high resolution for the proposed components. Mixture [I] is
supposed to contain fve components; namely RAM, rami-
prilate, ramipril DKP, AML, and AML deg1. While Mixture
[II] is supposed to contain eight components; namely RAM,
ramiprilate, ramipril DKP, ATV, ATV deg2, aniline, ASP
and its main impurity salicylic acid.
But oxidative degradation resulted only in partial hydrol-
ysis which was confrmed by the appearance of intact RAM
peak under the mentioned HPLC condition. The elucida-
tion of Ramipril degradation products was confrmed by LC/
MS programmed on +ve mode, showing the disappearance
of Ramipril peak at 417.41 m/z and the appearance of the
two main degradation products with peak at 389.28 m/z and
399.60 m/z, Fig. 2, for ramiprilate and ramipril DKP, respec-
tively. The suggested pathway of degradation is summarized
in Fig. 3a.
Amlodipine was subjected to alkaline hydrolysis by 1 N
NaOH for 1 h under refux and acid hydrolysis using 2 N
HCl for 2 h where the complete degradation was confrmed
by TLC using ethyl acetate–methanol–toluene–ammo-
nia solution, 33% (6.5:2:1:0.5 by volume) as the develop-
ing solvent, where a spot other than the intact AML was
observed, which ensures the formation of the same degra-
dation product AML deg. under both hydrolytic conditions.
The suggested degradation pathway is presented in Fig. 3b,
which indicates hydrolysis of the ester linkages and release
of the free alcohols, mass was preformed and compared to
the reported one (Zaazaa et al. 2012; Darwish et al. 2016;
A.Hassan et al. 2016).
Parameters affecting the efficiency of the chromato-
graphic separation were tested and optimized in a trial to
obtain the best separation for the cited components of both
mixtures. The pH of the mobile phase was found to have a
great infuence on the separation of the components; mainly
Ramipril, where pH 2.5 was found to be optimum. Many
trials for obtaining the optimum ratio for acetonitrile and
phosphate bufer to obtain the best resolution for all mixture
components and a reasonable run time were performed. For
Mixture [I] the best resolution was achieved upon using a
mobile phase consisting of 0.05 M phosphate bufer–ace-
tonitrile–tetrahydrofuran (60:40:0.1% by volume) with pH
2.5 on X-bridge column (250 × 4.6 mm i.d, 5 µm p.s.) under
fow rate of 1 mL/min. Scanning was tried at 230, 225, and
218, the scanning at 218 nm which is RAM λmax has showed
reasonable sensitivity for the active components. Under
these chromatographic conditions, RAM, AML, ramiprilate,
ramipril DKP and AML deg. were eluted at 6.23, 7.65, 3.90,
4.85, and 3.40 min 0.2, respectively, as shown in Fig. 4.
For Mixture [II] the best resolution was achieved when
using a mobile phase consisting of acetonitrile–0.05 M
phosphate bufer–tetrahydrofuran (60:40:0.1, by volume)
adjusted to pH 2.5, using X-bridge column (250 × 4.6 mm
i.d, 5 µm p.s.) under fow rate of 1.2 mL/min at 218 nm.
Under the stated chromatographic conditions; peaks for
RAM, ATV, ASP, ramiprilate, ramipril DKP, aniline, ATV
deg. 2, and salicylic acid appeared at 2.9, 6.29, 3.41, 4.84,
5.28, 2.43, 3.89, and 4.10 min+0.2, respectively, as shown
in Fig. 5.
But upon oxidation of AML using 30% H2O2 for 5 h,
and its application on TLC using the previously mentioned
system, no degradation was characterized.
For ATV degradation was carried out under refux with
6 N NaOH, 6 N HCl for 3 h and oxidative hydrolysis with
30% H2O2 for 5 h. Only 6 N HCl showed the complete deg-
radation of ATV which was resulted by the cleavage of the
amide bond-producing aniline (ATV deg1) and (ATV deg2)
with free carboxylic acid group as shown in Fig. 3c, where
the degradation process was monitored by spotting on TLC
plates using toluene: methanol (7.0: 3.0 v/v) as develop-
ing solvent, and it was found that ATV degrades completely
after 3 h. The solid ATV deg2 was extracted from NaCl, after
neutralization and evaporation of the solution, by methanol,
while ANL was collected with the evaporated solution and
extracted after heating at 100 °C to expel the water, confr-
mation by mass was found identical to the published litera-
ture (Darwish et al. 2016). ASP is an ester moiety, which
is very susceptible to hydrolysis under diferent hydrolytic
conditions. ASP is known to undergo decomposition by
hydrolysis into salicylic acid, when exposed to moisture. It
is reported that the decomposition reaction is promoted at
high temperature, in alkaline solutions (Zoglio et al. 1986).
Salicylic acid is the precursor and Aspirin main degrada-
tion product, it has keratolytic action and bactericidal efect
which leads to its usage in topical preparations (Mofat et al.
To validate the performed chromatographic methods,
overall system suitability tests (Snyder et al. 2011) were
conducted to evaluate the performance of the applied meth-
ods; Table 1. ICH guidelines for method validation [ICH,
Q1A (R2) 2003] were accomplished for validation of the
suggested methods.
Under the previously described experimental conditions,
linear relationships were obtained by plotting the drug con-
centrations versus the relative peak areas for each drug. The
corresponding concentration ranges, calibration equations,
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Fig. 2 Mass spectra of, a RAM and b ramiprilate and ramipril DKP
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Fig. 3 Suggested degradation pathways for a RAM, b AML and c ATV
Fig. 4 HPLC chromatogram showing the separation of RAM (6.23 min), AML (7.65 min), RAM deg1 (3.90 min), RAM deg2 (4.85 min), and
AML deg at (3.40 min), using phosphate bufer (pH 2.5, 0.05 M)-acetonitril-THF (60: 40: 0.1% by volume) as mobile phase
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Fig. 5 HPLC chromatogram showing the separation of RAM
(2.90 min), ATV (6.30 min), ASP (3.41 min), RAM deg1 (4.83 min),
RAM deg2 (5.28 min), ATV deg1 (ANL) at (2.43 min), ATV deg2
(3.89), and salicylic acid at (4.10 min) using acetonitril-phosphate
bufer (pH 2.5, 0.05 M)-THF (60: 40: 0.1% by volume) as mobile
phase
limit of detection (LOD) and limit of quantifcation (LOQ)
and other statistical parameters are listed in Table 2.
Achievement of method specifcity was taken with analy-
sis of diferent laboratory prepared mixtures of RAM and
AML, RAM, ATV, and ASP for Mixture I and II, respec-
tively, spiked with diferent levels of degradation products,
ranging from 10–70% degradation Table 2.
am 5® tablets 5/5, also Ramipril, Atorvastatin and Aspirin in
Ramitorva® capsules using the previously mentioned condi-
tions stated for each mixture, Table 5.
The results obtained for the analysis of RAM, AML,
ATV, and ASP in their pure form by the proposed HPLC
methods were statistically compared to those obtained by
applying the ofcial methods [British Pharmacopoeia, Medi-
cines and Healthcare Products Regulatory Agency (MHRA),
London 2013]. The calculated t and F values were less than
the tabulated ones, which revealed that there is no signifcant
diference between the two methods with respect to accuracy
and precision, Table 6.
The accuracy of the investigated methods was validated
by analyzing pure samples of RAM and AML for mixture
[I], and RAM, ATV, and ASP for mixture [II], while preci-
sion was evaluated by calculating intra-day and inter-day
precision. Good results are shown in Table 2.
To determine the robustness of the developed HPLC
methods, experimental conditions were subjected to minor
changes in fow rate, pH value and acetonitrile composition
and the tailing factor, capacity factor and resolution between
the studied drugs were recorded for mixture I and II, respec-
tively (Tables 3, 4).
To investigate the accuracy of the pharmaceutical for-
mulation analysis, the results obtained were compared sta-
tistically to those from reported methods (Patel and Patel
2014; Sharma et al. 2012) for Mixtures [I] and [II]; respec-
tively, using Student’s t test and the variance ratio F-test.
The results showed no signifcant diferences between the
results obtained from these methods and the published ones
Table 7.
The two proposed HPLC methods were successfully
applied to determine Ramipril and Amlodipine in Cardac
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Table 1 Statistical analysis of parameters required for system suitability testing of hplc method for Mixture I and II
Parameters
Mixture I
RAM
Mixture II
RAM
Reference values
AML
ASP
ATV
tR (relative retention
time)
N (column efciency)
6.23 0.03
5013.91
7.65 0.02
2.9 0.03
2592
3.4 0.03
6.3 0.04
> 1
6907.2
3317.76
6881.3
N>2000
Increases with
efciency
the efciency of
the separation
1–10 acceptable
> 1
K’ (capacity factor)
α (separation factor)
3.24
1.29
4.20
1.00
1.34
1.34
2.49
3.34
RAM
RAM
AML
AML RAM RAM
ASP SA
ATV ATV deg1
deg2
deg deg1
1.41
3.08
0.00217
2.33
0.00579
1.31
0.00452
2.01
0.00218
HETP (height equivalent 0.00299
to theoretical plates)
The smaller
the value, the
higher the col-
umn efciency
T (tailing factor)
1.06
1.00
0.88
2.48
0.94
1.06
T<2
T=1 for sym-
metric peak
Rs (experimental resolu- 4.11
5.24
Rs>2
tion)
RAM
RAM
deg2
AML
10.46
AML RAM RAM
ASP
3.33
SA ATV ATV deg1
deg
deg1
11.36
9.92
11.26
Table 2 Validation parameters
for the simultaneous
Parameters
Mixture (I)
RAM
Mixture (II)
determination of the sited
components in mixture I and II
by the proposed HPLC methods
AML
RAM
ATV
ASP
Specifcity (recovery SD, %)a 100.45 1.12 100.09 0.73 99.68 0.87 100.29 1.20 99.72 1.14
Accuracy (recovery, %)b
Precision, RSD (%)
Repeatabilityc
100.56
101.33
100.22
100.35
99.98
0.714
0.759
0.54
0.542
0.580
0.69
0.687
0.868
0.43
0.435
0.924
0.3
0.215
0.439
0.6
Intermediate precisiond
LOD (μg/mL)e
LOQ (μg/mL)e
1.91
2.05
1.84
1.56
1.41
Regression line
Slope
Intercept
Correlation coefcient (R)
Linearity range (μg/mL)
0.0286
0.0328
0.9999
5–50
0.0085
0.0334
0.999
5–50
0.0337
0.0321
0.999
5–50
0.0043
0.0995
0.999
2–16
0.0485
0.0935
0.999
2–16
aMean SD (n=3) of laboratory prepared mixtures of each mixture spiked with diferent levels of degra-
dation
products, ranging from 10 to 70% degradation
bMean (n=4) of three concentrations (15, 25, 35, and 45 μg/mL) for RAM and AML (3. 5, 9, and 15) for
ATV and ASP
cIntraday (n=3), three concentrations (10, 30, and 50 μg/mL) for RAM and AML, (4, 8, and 16 μg/mL) for
ATV and ASP repeated three times within the day
dInterday (n=3), three concentrations (10, 30, and 50 μg/mL) for RAM and AML, (4, 8, and 16 μg/mL) for
ATV and ASP repeated three times in three days
eLOD and LOQ were calculated using the following equations: LOD=3.3 σ/S and LOQ=10 σ/S
1 3

Chemical Papers
Table 3 Robustness of the
proposed HPLC methods for
mixture I
Drug
RAM
Robustness parameter
Flow rate
Ta
K’a
Rsb
% Assayc
1.0+0.1 mL/min
1.0 − 0.1 mL/min
2.5+0.1 units
2.5 − 0.1 units
40+2%
1.05
1.07
1.09
1.08
1.09
1.07
0.90
1.10
1.01
1.0
3.01
3.03
3.13
3.15
2.84
3.09
4.18
4.22
4.21
4.22
4.23
4.22
–
–
–
–
–
–
4.12
4.13
4.11
4.12
4.14
4.13
100.59
99.92
99.45
100.71
99.19
100.83
100.24
99.41
99.09
99.73
100.33
100.92
pH values
Acetonitrile composition
Flow rate
40 − 2%
AML
1.0+0.1 mL/min
1.0 − 0.1 mL/min
2.5+0.1 units
2.5 − 0.1 units
40+2%
pH values
Methanol composition
1.03
1.05
40 − 2%
Table 4 Robustness of the
proposed HPLC methods for
mixture II
Drug
RAM
Robustness parameter
Flow rate
Ta
K’a
Rsb
% Assayc
1.2+0.1 mL/min
1.2 – 0.1 mL/min
2.5+0.1 units
2.5 – 0.1 units
60+2%
0.87
0.89
0.88
0.87
0.89
0.87
0.92
0.96
0.95
0.93
0.94
0.93
1.08
1.04
1.09
1.05
1.10
1.05
1.01
1.02
1.02
1.01
1.05
1.03
1.32
1.31
1.33
1.31
1.35
1.34
3.32
3.35
3.35
3.36
3.33
3.35
–
–
–
–
–
–
2.49
2.47
2.42
2.43
2.45
2.43
5.26
5.22
5.23
5.25
5.21
5.24
99.38
100.12
100.54
99.61
99.79
99.65
100.32
100.21
99.42
100.71
99.32
99.73
99.51
pH values
Acetonitrile composition
Flow rate
60 – 2%
ASP
ATV
1.2+0.1 mL/min
1.2 – 0.1 mL/min
2.5+0.1 units
2.5 – 0.1 units
60+2%
pH values
Acetonitrile composition
Flow rate
60 – 2%
1.2+0.1 mL/min
1.2 – 0.1 mL/min
2.5+0.1 units
2.5 – 0.1 units
60+2%
100.72
99.49
100.15
100.31
99.82
pH values
Acetonitrile composition
60 – 2%
*Robustness (n=3), three concentrations (10, 30, and 50 μg/mL) for RAM and AML, (4, 8, and 16 μg/mL)
for ATV and ASP
aTailing factor and capacity factor determined for individual peak
bResolution factor determined between each drug peak and the previous one
c% assay was calculated from the regression equation
Table 5 Quantitative determination of RAM and AML in Cardac® tablets, and RAM, ATV, and ASP in Ramitorv®capsules by the proposed
HPLC methods
Pharmaceutical formula-
tion
RAM
AML
Claimed taken (µg/mL) Found% SD*
Claimed taken (µg/mL)
Found % SD*
99.64 0.78
Cardac® tablets
5
99.46 0.99
5
Ramitorv®capsules
RAM
ATV
ASP
Claimed taken (µg/mL) Found % SD* Claimed
taken (µg/
mL)
10
Found % SD* Claimed taken (µg/mL)
Found
%
SD*
5
99.62 0.78
100.69 0.81
7.5
100.48 0.95
*Average of fve experiments
1 3

Chemical Papers
Table 6 Statistical comparison of the results obtained by the proposed HPLC methods and the ofcial methods for the analysis of the proposed
components in their pure forms
Parameters
Proposed HPLC method
Mixture I
Ofcial method**
Mixture II
RAM
RAM
AML
ATV
ASP
RAM
AML
100.17 99.88
0.99 0.97
ATV
ASP
Mean
SD
Variance
n
100.01
1.12
1.2544
99.89
1.35
1.8225
6
99.75
0.80
0.6400
6
100.42
0.58
0.3364
100.10
0.75
0.5625
99.85
0.71
0.5041
5
99.68
0.45
0.9801 0.9409 0.2025
6
8
8
5
5
5
Student’s t test 0.063 (2.262)* 0.041 (2.262)* 0.055 (2.262)*
0.344 (2.201)* 0.211 (2.201)*
F value
2.488 (5.192)* 1.860 (5.192)* 1.2700 (5.192)* 2.800 (4.120)* 2.777 (4.120)*
RAM, potentiometric titration using 0.1 N NaOH
AML, HPLC method using C18 column, methanol: ammonium acetate (70:30 v/v) as a mobile phase, fow rate 1.5 mL/min, and UV detection at
237 nm
ATV, HPLC method using C18 column, acetonitrile: ammonium acetate: THF (67:21:12 by volume) as a mobile phase, fow rate 1.5 mL/min,
and UV detection at 244 nm
ASP, by acid–base titration using 0.5 M HCl for the back titration of the excess unreacted 0.5 M NaOH, using 0.2 ml phenolphthalein as an indi-
cator
*These values represent the corresponding tabulated values of t and F at P=0.05
**The British pharmacopoeial
Table 7 Statistical analysis of the proposed HPLC methods and the reported methods for determination of the proposed components in their
pharmaceutical formulations
Parameter
HPLC method
RAM
Reported method**a
RAM
AML
AML
Cardac® Tablets
Mean %
SD
99.46
0.99
99.64
0.78
101.09
1.06
100.82
1.10
Variance
n
0.9801
5
0.6084
5
1.1236
5
1.2100
5
Student’s t test (2.306)*
F value (6.388)*
0.801
1.146
HPLC method
0.721
1.988
Reported method**b
RAM
ATV
ASP
RAM
ATV
ASP
Ramitorv® Capsules
Mean %
SD
Variance
99.62
0.78
0.6084
5
100.69
0.81
0.6561
5
100.48
0.95
0.9025
5
98.94
0.93
0.8649
5
100.63
0.86
0.7396
5
101.04
1.12
1.2544
5
n
Student’s t test (2.306)*
F value (6.388)*
0.421
1.422
0.036
1.127
0.289
1.389
*These values represent the corresponding tabulated values of t and F at P=0.05
**a HPLC method The column used is Hypersil BDS C8 with mobile phase consisting of phosphate bufer (pH 2.5):acetonitrile (70:30).
(150×4.6 mm, 5 μm) with fow rate of 1.2 mL/min with UV detection at 210 nm
**b HPLC method was performed on C18 column the mobile phase is mixture of (A) acetonitrile methanol (65:35) and (B) phosphate bufer (pH
3.0)in the ratio of (60:40 v/v) at a fow rate of 1.5 ml min−1, UV detection was performed at 230 nm
1 3

Chemical Papers
ICH, Q1A (R2) (2003) Stability testing of new drug substances and
products. International Conference on Harmonization, IFPMA,
Geneva, Switzerland
Conclusion
The proposed chromatographic methods were satisfyingly
used for the analysis of the two anti-hypertensive formula-
tions under study, especially in quality control laboratories.
The method furnished benefcial linearity and being trusted
to selective determination of the studied drugs in presence
of their degradation products resulted from diferent stress
conditions.
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Maste MM, Kalekar MC, Kadian N, Bhat AR (2011) Development and
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Mofat AC, Osselton MD, Widdop B (2011) Clarke’s analysis of drugs
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Conflict of interest All the authors working in this manuscript have no
confict of interest.
Panchal HJ, Suhagia BN, Patel NJ, Rathod IS, Patel BH (2009) Simul-
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