2.1 introduction: letairis is the brand name fo that is selective for the
TRANSCRIPT
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2.1 Introduction:
Letairis is the brand name for Ambrisentan, an endothelin receptor antagoni
that is selective for the endothelin type
Ambrisentan (ABT) is (+)
diphenylpropanoic acid. It has a molecular formula of C
weight of 378.42. It contains a single chiral center determined to be the (S) configuration
and has the following structural formula.
Fig
Ambrisentan is a white to off
pKa of 4.0. Ambrisentan is practically insoluble in water and in aqueous solutions at low
pH. Solubility increases in aqueous solutions at higher pH. In the solid state Ambrisentan
is very stable, is not hygroscopic, and is not light sensitive. Letairis
and 10 mg film-coated tablets for once
following inactive ingredients: croscarmellose sodium, lactose monohydrate, magnesium
stearate and microcrystalline cellulose. The tablets are f
containing FD&C red #40 aluminum
alcohol, talc, and titanium dioxide. Each square, pale pink Letairis tablet contains 5 mg of
Letairis is the brand name for Ambrisentan, an endothelin receptor antagoni
that is selective for the endothelin type-A (ETA) receptor. The chemical name of
is (+)-(2S)-2-[(4,6-dimethylpyrimidin-2-yl)oxy]
diphenylpropanoic acid. It has a molecular formula of C22H22N2O4
.42. It contains a single chiral center determined to be the (S) configuration
and has the following structural formula.
Fig. 2.1: Chemical structure of Ambrisentan.
Ambrisentan is a white to off-white, crystalline solid. It is a carboxylic acid with
pKa of 4.0. Ambrisentan is practically insoluble in water and in aqueous solutions at low
pH. Solubility increases in aqueous solutions at higher pH. In the solid state Ambrisentan
is very stable, is not hygroscopic, and is not light sensitive. Letairis is available as 5 mg
coated tablets for once-daily oral administration. The tablets include the
following inactive ingredients: croscarmellose sodium, lactose monohydrate, magnesium
stearate and microcrystalline cellulose. The tablets are film-coated with a coating material
ontaining FD&C red #40 aluminum lake, lecithin, polyethylene glycol, polyvinyl
alcohol, talc, and titanium dioxide. Each square, pale pink Letairis tablet contains 5 mg of
Chapter-2
50
Letairis is the brand name for Ambrisentan, an endothelin receptor antagonist
A (ETA) receptor. The chemical name of
yl)oxy]-3-methoxy-3,3-
and a molecular
.42. It contains a single chiral center determined to be the (S) configuration
white, crystalline solid. It is a carboxylic acid with a
pKa of 4.0. Ambrisentan is practically insoluble in water and in aqueous solutions at low
pH. Solubility increases in aqueous solutions at higher pH. In the solid state Ambrisentan
is available as 5 mg
daily oral administration. The tablets include the
following inactive ingredients: croscarmellose sodium, lactose monohydrate, magnesium
coated with a coating material
lake, lecithin, polyethylene glycol, polyvinyl
alcohol, talc, and titanium dioxide. Each square, pale pink Letairis tablet contains 5 mg of
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Ambrisentan. Each oval, deep pink Letairis tablet contains 10 mg of Ambrisentan.
Letairis tablets are unscored.
Initiate treatment at 5 mg once daily with or without food, and consider increasing
the dose to 10 mg once daily if 5 mg is tolerated. Tablets may be administered with or
without food. Tablets should not be split, crushed, or chewed. Doses higher than 10 mg
once daily have not been studied in patients with pulmonary arterial hypertension (PAH).
Liver function tests should be measured prior to initiation and during treatment with
Letairis. Letairis is not recommended in patients with moderate or severe hepatic
impairment. There is no information on the use of Letairis in patients with mild hepatic
impairment; however, exposure to Ambrisentan may be increased in these patients. There
is no experience with over dosage of Letairis. The highest single dose of Letairis
administered to healthy volunteers was 100 mg and the highest daily dose administered to
patients with PAH was 10 mg once daily. In healthy volunteers, single doses of 50 mg
and 100 mg (5 to 10 times the maximum recommended dose) were associated with
headache, flushing, dizziness, nausea, and nasal congestion. Massive over dosage could
potentially result in hypotension that may require intervention [1-6].
The literature survey reveals that, some of the HPLC methods were reported for
the estimation of related substances, assay, enantiomeric purity and one bio analytical
HPLC method available, for the estimation of Ambrisentan in rat plasma by solid phase
extraction technique and characterization of degradation products of Ambrisentan by LC-
MS. Narayana et al. reported a validated specific stability indicating RP-HPLC assay
method for Ambrisentan and its related substances, in this HPLC method the authors
targeted totally four known impurities, these are impurity-1 (Sulphonyl pyrimidine
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impurity), impurity-2 (Hydroxy acid impurity), impurity-3 (Benzophenone impurity) and
impurity-4 (Vinyloxy impurity). But they did not targeted Pyrimidine ester and Hydroxy
ester impurities, but our research study was targeting total six impurities, which includes
above four impurities and Pyrimidine ester, Hydroxy ester impurities [7]. Nanjappan
Satheeshkumar et al. reported stability-indicating RP-HPLC method for Ambrisentan: an
endothelin receptor antagonist, this is a stability indicating assay method for the
determination of Ambrisentan in active pharmaceutical ingredient and no known
impurities have been reported [8]. Jayvadan K. Patel et al. reported stability-indicating
RP-HPLC method for the determination of Ambrisentan and Tadalafil in pharmaceutical
dosage form; this method is useful for the quantitative determination of Ambrisentan and
Tadalafil in drug product [9]. Nageswara Rao Ramisetti et al. reported LC-MS/MS
characterization of forced degradation products of Ambrisentan [10] and some of the
HPLC methods available for the assay and determination of related substances in drug
substance and drug product [11-17]. Michal Douša et al. reported rapid determination of
Ambrisentan enantiomers by enantioselective liquid chromatography using cellulose-
based chiral stationary phase in reverse phase mode. Ambrisentan is S-isomer, hence to
estimate the R-isomer impurity this method is useful [18]. Rucha Desai et al. reported bio
analytical method development and validation for estimation of Ambrisentan in rat
plasma by solid phase extraction technique: application to pharmacokinetic study [19].
No HPLC methods were reported in major pharmacopeia like USP, EP, JP and
BP. Therefore, it is felt to develop stability indicating HPLC method for determination of
six related substances and for quantitative estimation of Ambrisentan in bulk drugs.
Hence, an attempt has been made to develop an accurate, specific and reproducible
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method for the determination of Ambrisentan and six impurities in bulk drug samples and
along with method validation performed as per ICH guidelines [20-26].
2.2 Experimental:
2.2.1 Chemicals, reagents and samples:
Samples of active pharmaceutical ingredient standard and related impurities were
obtained from MSN laboratories private limited, R&D center (Hyderabad, India).
Acetonitrile (HPLC grade), potassium dihydrogen orthophosphate (KH2PO4; AR grade),
sodium hydroxide (NaOH; AR grade), hydrochloric acid (HCl; AR grade) and hydrogen
peroxide (30% w/v) (H2O2; LR grade) were purchased from Merck. Orthophosphric acid
(85%) (OPA) and formic acid (HPLC grade) were purchased from Rankem. High-purity
Milli-Q water was prepared by using a Milli-Q plus water purification system (Millipore;
Milford, MA).
2.2.2 Instrumentation:
Agilent 1200 series LC system equipped with quaternary pump (G1311A),
vacuum degasser (G1322A), column compartment (G1316A), auto sampler with
temperature control module (G1329A) and diode array detector (DAD) (G1315D) was
used for method development attempts (Agilent Technologies; Waldbronn, Germany).
Data was collected and processed by using Ez chrom Elite (3.3.2 SP2) software. Forced
degradation studies and method validation were performed on Waters e2695 separation
module LC system with having 2998 photodiode array detector (PDA) (Milford; MA,
USA). Data were collected, processed using Empower 2 software. Photo stability studies
were performed in a photo stability chamber (Atlas Suntest CPS+). Thermal stability
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studies were performed in a dry hot air oven (Cintex precision hot air oven; Mumbai,
India).
2.2.3 Chromatographic conditions:
The chromatographic separation was optimized using Waters symmetry C18
column with the dimension of 250 x 4.6 mm and 5 µm as particle size. A gradient elution
was involved with the 2.72 g potassium dihydrogen orthophosphate in 1000 mL of Milli-
Q water (100% v/v) and adjusted its pH to 3.0 with diluted orthophosphoric acid as a
mobile phase A and acetonitrile: water in the ratio of 90:10 v/v as mobile phase B. The
HPLC gradient program was set as: time/% mobile phase B: 0/55, 10/55, 25/65, 33/65,
46/75, 52/75, 53/55 and 60/55. The flow rate of the mobile phase and the column
temperature was set as 1.0 mL/min and 25°C respectively. The detection wave length was
optimized at 210 nm. The column loading was finalized as 8 µg of Ambrisentan in 10 µL
injection volume. A mixture of mobile phase A and acetonitrile in the same ratio was used
as diluent.
2.2.4 Preparation of standard solutions:
A stock solution of Ambrisentan (800 µg/mL) was prepared by dissolving an
appropriate amount of drug substance in diluent. A mixed stock solution (100 µg/mL) of
the impurities (six impurities) was also prepared in diluent. All working solutions were
prepared from this stock solution for determination of related substances.
2.2.5 Preparation of system suitability solution:
A mixture of Ambrisentan (800 µg/mL) and all six impurities (each 1.2 µg/mL)
was prepared by dissolving an appropriate amount in diluent.
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2.2.6 Preparation of sample solution:
800 µg/mL of Ambrisentan was prepared by dissolving an appropriate amount of
drug substance in diluent.
Structure and impurity name
Sulfonyl pyrimidine impurity
Hydroxy acid impurity
Hydroxy ester impurity
Benzophenone impurity
Pyrimidine ester impurity Vinyloxy impurity
Fig. 2.2: Chemical structures of impurities.
2.2.7 Generation of stress samples:
One lot of Ambrisentan drug substance was selected for stress testing. Different
types of stress conditions (i.e., acid, base, oxidative, water, heat and light) were used
N
N S
O
O O
OH
O
HO
O
OCH3
O
HO
O
O
OCH3
O
O
N N
N
N
O
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based on guidance available from ICH Stability Guideline (Q1AR2). The details of stress
conditions performed are as follows:
a) Acid degradation:
160 mg of Ambrisentan drug substance was transferred into a 100 mL volumetric
flask, to it added 50 mL of diluent to dissolve and then made up to the mark with of 3N
HCl and mixed well.
b) Base degradation:
160 mg of Ambrisentan drug substance was transferred into a 100 mL volumetric
flask, to it added 50 mL of diluent to dissolve and then made up to the mark with 5N
NaOH and mixed well.
c) Oxidation degradation:
160 mg of Ambrisentan drug substance was transferred into a 100 mL volumetric
flask, to it added 50 mL of diluent to dissolve and then made up to the mark with 10 %
H2O2 and mixed well.
d) Water degradation:
160 mg of Ambrisentan drug substance was transferred into a 100 mL volumetric
flask, to it added 50 mL of acetonitrile to dissolve and then added 50 mL of water and
mixed well.
e) Photolytic degradation:
Susceptibility of the drug substance to light was studied. 100 mg of Ambrisentan
substance for photo stability testing were placed in a photo stability chamber and exposed
to a white florescent lamp with an overall illumination of 1.2 million LUX hours (lxh)
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and near UV radiation with an overall illumination of 200 watt-hour per square meter
(Wh/m2). Following removal from the photo stability chamber, the sample was prepared
for analysis, as previously described.
f) Thermal degradation:
100 mg of Ambrisentan drug substance was transferred into a petri dish and
placed in a hot air oven at 60°C for 10 days. After 10 days, the petri dish was removed
and placed on the bench top to attain laboratory temperature. The sample was prepared
for analysis, as previously described.
g) Humidity degradation:
100 mg of Ambrisentan drug substance was transferred into a petri dish and
placed in a humidity chamber at 75% RH for 10 days. After 10 days, the petri dish was
removed and the sample was prepared for analysis, as previously described.
h) Sunlight degradation:
100 mg of Ambrisentan drug substance was transferred into a petri dish and
placed under sunlight for 50 h. After 50 h, the sample was prepared for analysis, as
previously described.
2.3 Method development and optimization of chromatographic conditions:
Forced degradation studies were carried out to develop a stability indicating
HPLC method for the quantitative determination and evaluation of purity in Ambrisentan
drug substance. Stressed samples obtained during forced degradation studies including
the samples of impurities six were used in the HPLC method development.
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2.3.1 Selection of wavelength:
A mixture of Ambrisentan (800 µg/mL) and all six impurities (each 1.2 µg/mL)
was prepared by dissolving an appropriate amount in diluent and scanned in HPLC PDA
detector, all the impurities and Ambrisentan were having UV maxima at around 210 nm
[Fig. 2.3]. Hence detection at 210 nm was selected for method development purpose.
2.3.2 Method development approach for the selection of suitable column and mobile
phase:
The HPLC method development carried out in this study aimed to develop a
suitable chromatographic system, which is capable of eluting and resolving Ambrisentan
from its process related impurities and degradation products that comply with the general
requirements for system suitability with good baseline. Following method development
conditions are selected based on physical and chemical properties (pKa value, solubility,
etc.) of Ambrisentan. Initial attempts for the method development were made in Inertsil
ODS-4V (250 × 4.6 mm i.d., particle size 5 µm) with mobile phase A as 0.01M of
potassium dihydrogen orthophosphate, at pH 3.0 adjusted with dilute orthophosphoric
acid and mobile phase B as acetonitrile: water in the ratio of 9:1 v/v. The gradient as
(time (min)/%(solution B)): 0/55, 10/55, 25/65, 35/65 45/90, 48/90, 49/55, 55/55 at flow
rate 1.0 mL/min. Column temperature 25ºC, injection volume 10 µL, sample
concentration 1 mg/mL, sample solvent selected as acetonitrile: buffer in the ratio of
50:50 v/v. With these conditions the separation of the impurities is good, but the baseline
was observed to be very poor at 210 nm in the gradient elution. After that tried with
different gradient conditions and finally good base line was observed at selected
programme with Intertsil ODS-4 (250 × 4.6 mm, 5 µm) column, but during different
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Sulfonyl pyrimidine impurity
Hydroxy ester impurity
Pyrimidine ester impurity
Fig. 2.3: Typical UV spectrums of
UV spectra and impurity name
yrimidine impurity
Hydroxy acid impurity
Hydroxy ester impurity
Benzophenone impurity
Pyrimidine ester impurity Vinyloxy impurity
Ambrisentan
2.3: Typical UV spectrums of Ambrisentan and its impurities.
Chapter-2
59
Hydroxy acid impurity
Benzophenone impurity
Vinyloxy impurity
and its impurities.
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batches analysis study it was observed that one of the unknown impurity merges with
Pyrimidine ester impurity. To separate the unknown impurity and Pyrimidine ester
impurity different stationary phases, various buffers, different pH and different
selectivities were used. Finally more than two resolution observed between unknown
impurity and Pyrimidine ester impurity with Symmetry C18 column with the dimension
of 250 x 4.6 mm and 5 µm as particle size. And also good peak shape with less peak
width and the resolution of all the related impurities were satisfactorily. The column and
acetonitrile were played a key role in the retention times and resolution between
impurities. The selected stationary phase is very stable at selected acidic pH 3.0. In the
optimized conditions it was observed that the Ambrisentan, Sulphonyl pyrimidine
impurity, Hydroxy acid impurity, Hydroxy ester impurity, Benzophenone impurity,
Pyrimidine ester impurity and Vinyloxy impurity were well separated with a resolution of
greater than 2.
a) Typical chromatographic pattern in Inertsil ODS-4, 250 x 4.6 mm, 5 µm.
Minutes
0 5 10 15 20 25 30 35
mA
U
0
10
20
mA
U
0
10
20
3.9
40
S
ulf
onyl P
yri
mid
ine
imp
9.1
60
H
ydro
xy a
cid im
p
16.8
13
A
mbri
senta
n
18.6
73
H
ydro
xy e
ster
im
p
22.0
27
B
enzo
phen
one
imp
31.3
53
P
yri
mid
ine
este
r im
p
31.4
67
U
nknow
n im
pDAD: Signal A, 210 nm/Bw:4 nmSST Solutiion(from degradation sample)
Retention TimeName
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b) Typical chromatographic pattern in Symmetry C18, 250 x 4.6 mm, 5 µm.
c) Typical chromatogram of system suitability solution.
Fig. 2.4: Typical chromatograms of column selection.
Minutes
0 10 20 30 40 50 60
mA
U
0
10
20
30
mA
U
0
10
20
30
3.0
33
Su
lfon
yl
pyri
mid
ine
imp
7.3
20
Hy
dro
xy a
cid
im
p
13.5
27
A
mb
rise
nta
n1
5.7
07
H
ydro
xy
est
er i
mp
18.1
00
B
enzo
ph
enon
e im
p
30.8
53
P
yri
mid
ine
este
r im
p3
2.0
20
Un
kno
wn
im
p
44.0
13
V
inylo
xy
im
pu
rity
DAD: Signal A, 210 nm/Bw:4 nmSST Solution
Retention TimeName
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2.4 Degradation behavior:
The LC studies on Ambrisentan under different stress conditions suggested the
following degradation behavior.
a) Degradation in acidic conditions:
Ambrisentan drug substance when exposed to 0.5
degradation was observed. Then increased the concentration to 3
degradation of about 7.5% was observed. [Fig. 2.5
Fig 2.5: Typical HPLC chromatogram of acid hydrolysis.
Fig. 2.5: Typical HPLC chromatogram of acid hydrolysis.
Fig. 2.6: Peak purity plot of acid hydrolysis.
2.4 Degradation behavior:
The LC studies on Ambrisentan under different stress conditions suggested the
degradation behavior.
cidic conditions:
Ambrisentan drug substance when exposed to 0.5 N HCl at
degradation was observed. Then increased the concentration to 3 N HCl at
degradation of about 7.5% was observed. [Fig. 2.5-2.6]
Typical HPLC chromatogram of acid hydrolysis.
2.5: Typical HPLC chromatogram of acid hydrolysis.
2.6: Peak purity plot of acid hydrolysis.
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62
The LC studies on Ambrisentan under different stress conditions suggested the
N HCl at 25°C, a slight
N HCl at 25°C, a
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b) Degradation in basic conditions:
Ambrisentan drug substance when exposed to 0.5 N NaOH at
degradation was observed. Then increased the concentration to 5
degradation of about 6.74% was observed. [Fig. 2.7
Fig. 2.7: Typical HPLC chromatogram of base hydrolysis.
Fig. 2.8: Peak purity plot of base hydrolysis.
asic conditions:
Ambrisentan drug substance when exposed to 0.5 N NaOH at
degradation was observed. Then increased the concentration to 5 N NaOH at
degradation of about 6.74% was observed. [Fig. 2.7-2.8]
2.7: Typical HPLC chromatogram of base hydrolysis.
2.8: Peak purity plot of base hydrolysis.
Chapter-2
63
Ambrisentan drug substance when exposed to 0.5 N NaOH at 25°C, no
N NaOH at 25°C, a
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c) Degradation in oxidation conditions:
Ambrisentan drug substance when exposed to 3.0% H
was observed. Then increased the concentration to 10.0% H
was observed.
d) Degradation in water hydrolysis:
No major degradation was observed at
temperature to 60°C and refluxed for 24
[Fig. 2.9-2.10]
Fig. 2.9: Typical HPLC chromatogram of water hydrolysis.
Fig. 2.10: Peak purity plot of water hydrolysis.
in oxidation conditions:
Ambrisentan drug substance when exposed to 3.0% H2O2 at 25°C
was observed. Then increased the concentration to 10.0% H2O2at 25°C
Degradation in water hydrolysis:
No major degradation was observed at 25°C after 24 h. Then increased the
temperature to 60°C and refluxed for 24 h. A degradation of about 9.11% was observed.
2.9: Typical HPLC chromatogram of water hydrolysis.
2.10: Peak purity plot of water hydrolysis.
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64
25°C, no degradation
25°C, no degradation
h. Then increased the
h. A degradation of about 9.11% was observed.
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e) Thermal degradation:
Ambrisentan was stable to thermal condition. When the drug substance was
exposed to 60°C for 10 days, no degradation was observed.
f) At 75% relative humidity degradation:
Ambrisentan was stable to 75% relative humidity condition. When the drug
substance was exposed to 75% relative humidity for 10 days, no degradation was
observed.
g) Under sunlight degradation:
Ambrisentan was stable to sunlight. When the drug substance was exposed to 50
h, no degradation was observed.
h) Photolytic degradation:
Ambrisentan drug substance was stable to effect of photolysis. When the drug
substance was directly exposed to light for an overall illumination of 1.2 lxh and an
integrated near to UV light energy of 200 Wh/m2 (in photo stability chamber), no
degradation of the drug was observed.
Peak purity test performed for the Ambrisentan peak using PDA detector and data
confirmed the purity of peak for all the stressed samples. Assay of all stressed samples
were calculated using qualified standard of Ambrisentan.
2.5 Mass balance:
The mass balance was calculated from the individual RS and assay
chromatograms of stressed samples (%assay + % deg + % imp). The mass balance of
each stressed sample was more than 99%. This clearly confirmed that the developed
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HPLC method was specific for Ambrisentan in presence of its impurities and degradation
products. [Table 2.1]
Table 2.1: Mass balance and forced degradation study results.
Stress conditions Degradation Assay Mass
balance
Purity
1 angle
Purity 1
threshold
Purity
flag
Normal 0.18% 99.74% NA 0.120 0.306 No
Acid hydrolysis (After 6 h) 7.50% 92.3% 99.8% 0.120 0.311 No
Base hydrolysis (After 26 h) 6.74% 93.41% 100.15% 0.143 0.342 No
Oxidation (After 48 h) 0.72% 99.34% 100.06% 0.121 0.316 No
Water hydrolysis (After 12 h) 9.11% 91.56% 100.67% 0.135 0.319 No
Photo
degradation
UV direct 0.22% 99.55% 99.77% 0.089 0.289 No
UV indirect 0.20% 99.60% 99.8% 0.118 0.310 No
Lux direct 0.19% 99.60% 99.79% 0.099 0.292 No
Lux indirect 0.18% 99.63% 99.8% 0.116 0.304 No
Thermal at 60oC (10 days) 0.21% 99.65% 99.86% 0.116 0.310 No
At 75% relative humidity (10
days) 0.22% 99.55%
99.77% 0.098 0.339 No
Under sunlight (50 h) 0.38% 99.45% 99.83% 0.108 0.303 No
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2.6 Analytical method validation- results and discussion:
The method that was developed and optimized in HPLC was considered for
method validation. The analytical method validation was carried out in accordance with
ICH guidelines.
2.6.1 System suitability test:
System suitability testing is an integral part of chromatographic method. The tests
are based to ensure that the equipment, analytical operations, electronics and samples to
be analysed make an integral system and it can be calculated as such.
The Ambrisentan was spiked with 0.15% impurity blend with respect to the test
concentration of Ambrisentan and injected for three times into HPLC system. Resolution
between Ambrisentan and Hydroxy acid, tailing factor, theoretical plates for Ambrisentan
was calculated. Good resolution was obtained between all impurities and Ambrisentan.
System suitability results were tabulated. [Table: 2.2]
Table 2.2: Results of system suitability.
Name Retention
time
Tailing Resolution Plate
count
Purity
1 angle
Purity 1
threshold
Sulphonyl
pyrimidine imp.
3.083 1.17 NA 10669 0.304 0.586
Hydroxy acid
imp.
6.937 1.45 19.34 10856 0.549 0.773
Ambrisentan 12.603 1.11 17.18 17789 3.024 3.159
Hydroxy ester
imp.
14.667 1.04 5.46 25541 0.598 0.834
Benzophenone
imp
16.793 1.04 5.77 34858 0.803 0.821
Pyrimidine ester
imp.
28.716 1.02 28.38 60446 0.704 0.911
Vinyloxy imp. 43.258 1.00 26.87 85112 0.473 0.632
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2.6.2 Limit of quantification (LOQ) and Limit of detection (LOD):
LOQ and LOD established for all impurities based on the impurities dilution method.
Methodology for establishment of LOQ and LOD:
Limits of detection (LOD) and quantification (LOQ) represent the concentration of
the analyte that would yield a signal-to-noise ratio of 3 for LOD and 10 for LOQ
respectively. LOD and LOQ were determined by measuring the magnitude of the
analytical back ground by injecting blank samples (mobile phase) and calculating the
signal-to-noise ratio for each compound by injecting a series of solutions until the S/N
ratio 3 for LOD and 10 for LOQ were obtained. The results have indicated good linearity.
Different dilutions of Ambrisentan and its impurities were injected to establish the limit of
detection and limit of quantification respectively. The results were recorded in Table 2.3.
Table 2.3: LOD and LOQ values of the impurities and Ambrisentan.
2.6.3 Precision at limit of quantification level:
The precision at LOQ level of the method was also ensured by injecting six
individual preparations of impurities at their quantification level with respect to the
S.No Name of the substance
S/N
ratio for
LOD
S/N
ratio for
LOQ
% level of
imp w.r.t. to
sample conc.
(LOD)
% level of
imp w.r.t. to
sample conc.
(LOQ)
1 Sulfonyl pyrimidine imp. 2.373 9.658 0.0022 0.0088
2 Hydroxy acid imp. 2.849 9.992 0.0035 0.0088
3 Hydroxy ester imp. 2.490 9.994 0.0028 0.0110
4 Benzophenone imp. 2.189 10.148 0.0017 0.0073
5 Pyrimidine ester imp. 2.377 10.332 0.0033 0.0132
6 Vinyloxy imp. 2.163 10.136 0.0039 0.0131
7 Ambrisentan 2.372 9.612 0.0024 0.0096
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Ambrisentan concentration. Upon repetitive injections at quantification limit, the peak
properties like retention time, area were not influenced. Results have shown negligible
variation in measured responses which revealed that the method was repeatable at LOQ
level with RSD below 8.8 %. [Table 2.4]
Table 2.4: Precision results for area of all impurities at LOQ level.
2.6.4 Accuracy at limit of quantification level:
Standard addition and recovery experiments were performed to evaluate accuracy
of the developed method for the quantification of all impurities in Ambrisentan sample at
LOQ level. The recovery study for impurities was carried out in triplicate at LOQ level of
Name of the
injection
Area of
Sulfonyl
pyrimi-
dine imp.
Area of
Hydroxy
acid imp.
Area of
Hydroxy
ester
imp.
Area of
Benzophenone
imp.
Area of
Pyrimidine
ester imp.
Area of
Vinyloxy
imp.
Area of
Ambrise
-ntan
LOQ
pre-1 976 2065 3358 3660 4493 5209 3243
LOQ
pre-2 1049 1923 3135 2907 4106 5265 3069
LOQ
pre-3 978 1967 3035 3103 3800 5352 2759
LOQ
pre-4 1054 1716 3581 3191 4291 5523 3167
LOQ
pre-5 978 1991 3031 3142 3840 5297 2651
LOQ
pre-6 1049 1917 2990 2907 4080 4748 3073
Avg.
area 1014 1930 3188 3152 4102 5232 2994
STDEV 40 118 234 276 264 260 235
%RSD 3.97 6.10 7.33 8.77 6.44 4.98 7.86
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the Ambrisentan target concentration (800 µg/mL). The percentage recovery of impurities
was calculated. [Table 2.5]
Table 2.5: Recovery at LOQ level for impurities.
S.No. Impurity name % of recovery
1 Sulfonyl pyrimidine imp. 100.7
2 Hydroxy acid imp. 101.0
3 Hydroxy ester imp. 98.4
4 Benzophenone imp. 95.0
5 Pyrimidine ester imp. 98.7
6 Vinyloxy imp. 95.4
2.6.5 Precision:
The precision of analytical method convey the closeness of agreement (degree of
scatter) between the series of measurements acquired from multiple sampling of the same
homogeneous sample under the prescribed conditions. Precision may be measured at
three levels: repeatability, intermediate precision and reproducibility. It is normally
expressed as RSD%.
a) Repeatability is the results of a method operated over a short interval of time under
the same conditions.
b) Intermediate precision is the end result from within-laboratories variations due to
random events that include different days, different analysts, different equipment,
etc.
c) Reproducibility is determined by testing the homogeneous samples in different
laboratories. It is a measure of precision between laboratories. The precision of
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related substances method was evaluated by injecting six individual preparations of
Ambrisentan (800 µg/mL) spiked with 0.15% of impurities with respect to
Ambrisentan analyte concentration. The %RSD for content of all impurities for six
consecutive determinations was below 7.8. [Table 2.7]
Table 2.6: Impurity content.
The method precision of assay study was calculated initially by performing system
precision, then by carrying out six independent assays of Ambrisentan test sample against
qualified reference standard. Results showed insignificant variation in measured response
which demonstrated that the method was repeatable with RSD’s below 0.03% [Table
2.8]. Intermediate precision for assay method was performed by carrying out six
independent assays of Ambrisentan test sample against qualified reference standard and
calculated RSD of six consecutive assays. Related substances method was performed by
injecting six individual preparations of Ambrisentan (0.8 mg/mL) and 0.15% of
impurities with respect to Ambrisentan analyte concentration over different days,
different instruments and with different analysts.
Prep.
no.
% of
Sulfonyl
pyrimidi
ne imp.
% of
Hydroxy
acid
imp.
% of
Hydroxy
ester imp.
% of
Benzophe-
none imp.
% of
Pyrimi-
dine ester
imp.
% of
Viny-
loxy
imp.
% of
S.M.U
mp.
% of
T. imp’s.
1 0.0 0.08 0.0 0.02 0.03 0.01 0.01 0.17
2 0.0 0.08 0.0 0.02 0.02 0.01 0.01 0.15
3 0.0 0.08 0.0 0.02 0.03 0.01 0.01 0.19
4 0.0 0.09 0.0 0.02 0.03 0.01 0.01 0.20
5 0.0 0.08 0.0 0.00 0.03 0.01 0.02 0.20
Avg. 0.0 0.08 0.0 0.02 0.03 0.01 0.01 0.18
Limit
in % 0.15 0.15 0.15 0.15 0.15 0.15 0.10 1.0
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Table 2.7: Area results for sample precision + 100% level impurities spiking.
Name of the
solution
Area of
Sulfonyl
pyrimidine
imp.
Area of
Hydroxy
acid
imp.
Area of
Hydroxy
ester
imp.
Area of
Benzophenone
imp.
Area of
Pyrimidine
ester imp.
Area of
Vinyloxy
imp.
Sample +
100% imp’s-1 19650 67131 47871 67958 66091 62716
Sample+
100% imp’s-2 20126 68023 48459 68233 56850 65550
Sample+
100% imp’s-3 19242 70256 46340 77231 54825 65720
Sample+
100% imp’s-4 19387 67669 47532 77089 55794 62519
Sample+
100% imp’s-5 19356 70057 47344 77853 54321 62756
Sample+
100% imp’s-6 19254 70957 47266 76994 55070 63649
Avg. area 19503 69016 47469 74226 57159 63818
STDEV 339.09 1596.40 703.87 4759.15 4463.30 1461.21
% RSD 1.74 2.31 1.48 6.41 7.81 2.29
Table 2.8: Precision results of the assay method.
Assay no % assay
Set-I assay 99.66
Set-II assay 99.64
Set-III assay 99.71
Set-IV assay 99.65
Set-V assay 99.65
Set-VI assay 99.61
Average 99.65
STDEV 0.03266
% RSD 0.03
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Table 2.9: Results of intermediate precision study.
Parameter Variation %RSD
assay
(n=6)
%RSD for
related
substances
Resolution
b/w ABT
and Ester
imp
ABT
theoretical
plates
ABT
tailing
factor
Different
system
(a) Waters
alliance
(b) Agilent
1200 series
VWD
0.03%
0.15%
<7.81
< 5.12
5.46
5.56
25541
32301
1.11
1.08
Different
column
(1) LC09054
(2) LC10004
0.03%
0.15%
<7.81
< 5.12
5.46
5.56
25541
32301
1.11
1.08
Different
analyst
Analyst-1
Analyst-2
0.03%
0.15%
<7.81
< 5.12
5.46
5.56
25541
32301
1.11
1.08
2.6.6 Linearity:
Linearity of the related substances method
The linearity of an analytical method is the ability to attain test results which are
directly proportional to the concentration of analyte within the given range. Detector
response linearity experiments were carried out by preparing the Ambrisentan sample
solution containing impurities covering the range from LOQ–150% (LOQ, 0.0375, 0.075,
0.1125, 0.15, 0.1875 and 0.225) with respect to specification limit (0.15%), was assessed
by injecting seven separately prepared solutions of the normal test sample concentration
(800 µg/mL). The correlation coefficients, slopes and Y-intercepts of the calibration
curve were determined The calibration curve was drawn by plotting average area of the
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impurities on the Y-axis and concentration on the X-axis which has shown linear
relationship with a correlation coefficient greater than 0.998 for all impurities.
Table 2.9: Linearity of Sulfonyl pyrimidine impurity.
Fig. 2.11: Linearity graph for Sulfonyl pyrimidine impurity.
Sulphonyl pyrimidine impurity
0
5000
10000
15000
20000
25000
30000
5.9 25 50 75 100 125 150
Concentration
Are
a
Area
Sulfonyl pyrimidine impurity
Concentration in % Average area
5.9 1066
25 5648
50 8457
75 14370
100 17611
125 22460
150 26578
Slope 175
Y-intercept 491
Correlation coefficient 0.9979
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Table 2.10: Linearity of Hydroxy acid impurity.
Fig. 2.12: Linearity graph for Hydroxy acid impurity.
Hydroxy acid impurity
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
5.8 25 50 75 100 125 150
Concentration
Are
a
Area
Hydroxy acid impurity
Concentration in % Average area
5.8 23219
25 31661
50 42672
75 54710
100 64373
125 75105
150 85387
Slope 432
Y-intercept 21104
Correlation coefficient 0.9997
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Table 2.11: Linearity of Hydroxy ester impurity.
Fig. 2.13: Linearity graph for Hydroxy ester impurity.
Hydroxy ester impurity Linearity
0
10000
20000
30000
40000
50000
60000
70000
7.3 25 50 75 100 125 150
Concentration
Are
a
Area
Hydroxy ester impurity
Concentration in % Average area
7.3 3253
25 11496
50 20672
75 32480
100 42475
125 53537
150 63730
Slope 424
Y-intercept 305
Correlation coefficient 0.9998
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Table 2.12: Linearity of Benzophenone impurity.
Fig. 2.14: Linearity graph for Benzophenone impurity.
Benzophenone impurity
0
20000
40000
60000
80000
100000
120000
4.8 25 50 75 100 125 150
Concentration
Are
a
Area
Benzophenone impurity
Concentration in % Average area
4.8 9901
25 23728
50 34838
75 51375
100 66790
125 80507
150 96275
Slope 590
Y-intercept 7240
Correlation coefficient 0.9994
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Table 2.13: Linearity of Pyrimidine ester impurity.
Fig. 2.15: Linearity graph for Pyrimidine ester impurity.
Pyrimidine ester impurity
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
8.8 25 50 75 100 125 150
Concentration
Are
a
Area
Pyrimidine ester impurity
Concentration in % Average area
8.8 12568
25 21766
50 32238
75 45415
100 55151
125 67578
150 78307
Slope 462
Y-intercept 9469
Correlation coefficient 0.9994
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Table 2.14: Linearity of Vinyloxy impurity.
Fig. 2.16: Linearity graph for Vinyloxy impurity.
Linearity of the assay method
The linearity of the assay method was ascertained by injecting test sample at level
of 50%, 75%, 100%, 125% and 150% of Ambrisentan assay concentration (i.e.50
µg/mL). Each solution was injected in triplicate into LC system, and at each
Vinyloxy impurity
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
8.7 25 50 75 100 125 150
Concentration
Are
a
Area
Vinyloxy impurity
Concentration in % Average area
8.7 5181
25 16835
50 29276
75 42752
100 56734
125 65477
150 78148
Slope 509
Y-intercept 3269
Correlation coefficient 0.9976
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80
concentration level the average area was calculated. A calibration curve attained by least
square regression analysis between average peak areas and concentration showed, linear
relationship with a correlation coefficient of greater than 0.999.
Table 2.15: Linearity results of Ambrisentan in the assay method.
Fig. 2.17: Linearity graph of Ambrisentan in for assay method.
AMBRISENTAN
1087407
1580968
2146041
2717645
3272380
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
50 75 100 125 150
Concentrsation in %
Are
a
Concentration in % Average area
50 1087428
75 1580968
100 2146041
125 2717645
150 3272380
Slope 22026.48
Y-intercept -41761.33
Correlation coefficient 0.99967
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2.6.7 Accuracy:
The accuracy of an analytical method is measure of the closeness of test results obtained
to the true value.
Accuracy of the related substances method
The accuracy of the related substances method calculated at 50%, 75%, 100%,
125% and 150% to the impurities specification limit (0.15%). Recovery experiments
were performed at 50%, 75%, 100%, 125% and 150% levels. The test solution prepared
in triplicate (n=3) with impurities at the level of 0.075%, 0.1125%, 0.15%, 0.1875% and
0.225% (w.r.t 800 µg/mL test concentration) and each solution was injected thrice (n=3)
into LC system. The mean % recovery of impurities was determined in the spiked test
solution by using the area of impurities in the standard solutions at 0.15% level with
respect to Ambrisentan analyte concentration.
Table 2.16: Recovery results.
Conc. in %
% of rec
Sulfonyl
pyrimidine
imp.
% of rec
Hydroxy
acid imp.
% of rec
Hydroxy
ester imp.
% of rec
Benzophenone
imp.
% of rec
Pyrimidine
ester imp.
% of rec
Vinyloxy
imp.
50 96.2 103.3 98.0 103.3 101.6 105.5
75 109 107.0 102.6 106.6 105.5 104.8
100 100.2 103.2 100.6 106.4 100.1 105.4
125 102.2 103.0 101.5 103.9 101.5 97.7
150 100.8 102.1 100.7 104.6 99.9 97.6
Avg. %
recovery 101.7 103.7 100.7 105.0 101.7 102.2
The related substances method has been found consistent and with high recoveries
at all the five concentration levels i.e 50%, 75%, 100%, 125% and 150%, which convey
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the absolute recovery ranging from 100.7% to 105.0%. The Ambrisentan recovery study
specified that the related substances by LC method were appropriate for
determination/quantification of impurities of Ambrisentan drug substance.
Accuracy of the assay method
Accuracy of the assay was performed by injecting three preparations of test
sample at the level of 50%, 75%, 100%, 125% and 150% of analyte (Ambrisentan test
concentration) i.e 50 µg/mL. The study was performed in triplicate (n=3), the solution
was injected into HPLC system and the mean peak area of Ambrisentan analyte peak was
calculated for assay determination. Assay (%w/w) of test solution was calculated against
three injections (n=3) of qualified Ambrisentan reference standard.
Table 2.17: Recovery of the assay method for drug substance.
Fig. 2.18: Linearity plot for % recovery.
Ambrisentan
49.86
74.54
99.62
124.69
149.51
0
50
100
150
200
50 75 100 125 150
Concentration
Avera
ge R
ecovery
Concentration in % Average % recovery
50 49.86
75 74.54
100 99.62
125 124.69
150 149.51
Slope 1.00
Y-intercept -0.14
Correlation coefficient 1.00000
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2.6.8 Establishment of response factor and relative retention time:
Response factor
The response (e.g. peak area) of drug substance or related substances per unit weight.
RF= peak area / concentration (mg/mL)
Relative response factor (RRF):
RRF=RF impurity / RF API (or) RRF=slope impurity / slope API.
Relative retention time
The use of the relative retention time (RRT) reduces the effects of some of the variables
that can affect the retention time. RRT is an expression of impurity retention time,
relative to the standard’s retention time.
RRT = Impurity RT / Sample RT
Response factors and relative retention times were calculated by injecting three different
concentrations (0.10%, 0.15% and 0.20% w.r.t to Ambrisentan test concentration (800
µg/mL)) of six impurities and Ambrisentan.
Table 2.18: Average RRF, RF&RRT on the basis of three different concentrations.
S.No. Name of impurity RRF RF RRT
1.0 Sulfonyl pyrimidine impurity 0.39 2.56 0.26
2.0 Hydroxy acid impurity 0.87 1.15 0.55
3.0 Hydroxy ester impurity 0.89 1.12 1.20
4.0 Benzophenone impurity 1.21 0.83 1.37
5.0 Pyrimidine ester impurity 0.96 1.04 2.35
6.0 Vinyloxy impurity 1.09 0.92 3.55
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2.6.9 Solution stability:
The solution stability of Ambrisentan in diluent in the assay method was
performed by leaving the test solutions of sample in tightly capped volumetric flasks on a
laboratory work table at room temperature for 48 h. The sample solution was assayed for
every twelve hours interval up to the study time and freshly prepared reference standard
was used each time to estimate the assay of sample. The % RSD of assay of Ambrisentan
during solution stability experiments were less than 0.2. The solution stability of
Ambrisentan in diluent in the related substances method was performed by leaving the
spiked test solutions of sample in tightly capped volumetric flasks on a laboratory work
table with room temperature for 48 h (two days). No considerable change was observed
in the impurity content when compared to the initial values during solution stability
experiments up to study period. Hence, Ambrisentan sample solution is stable for a
minimum of 48 h with the same diluent.
Table 2.19: Summary of RS content obtained at different intervals in solution
stability.
Duration
in hours
% of
Sulfonyl
pyrimidi-
ne imp.
% of
Hydroxy
acid
imp.
% of
Hydroxy
ester
imp.
% of
Benzophe-
none imp.
% of
Pyrimidine
ester imp.
% of
Vinyloxy
imp.
% of
S.M.U.
imp.
% of
T. imp’s.
0 0.00 0.08 0.00 0.02 0.02 0.01 0.01 0.16
12 0.00 0.08 0.00 0.02 002 0.02 0.01 0.16
24 0.00 0.08 0.00 0.02 0.02 0.03 0.01 0.17
36 0.00 0.08 0.00 0.02 0.03 0.05 0.01 0.17
48 0.00 0.08 0.00 0.02 0.03 0.07 0.01 0.16
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Table 2.20: Summary of assay content obtained at different intervals in solution
stability.
Duration in hours Assay %
0 99.74
8 99.32
16 99.36
24 99.78
32 99.72
40 99.73
48 99.71
Average 99.62
STDEV 0.19
% RSD 0.20
2.6.10 Mobile phase stability:
The mobile phase stability of Ambrisentan in diluent in the assay method was
carried out by fresh test solutions of sample and mobile phase was kept constant up to 48
h. The fresh same Ambrisentan sample solutions were assayed for every twelve hours
interval up to the study period, each time freshly prepared reference standard was used to
estimate the assay of sample. The %RSD of assay of Ambrisentan during mobile phase
stability experiments were less than 0.13. The mobile phase stability of Ambrisentan in
diluent in the related substances method was carried out by fresh spiked test solution
leaving the mobile phase at the room temperature for two days and impurities checked for
every twelve hours interval up to the study period. No major change was observed in the
impurity content during mobile phase stability study experiments. Hence Ambrisentan
mobile phase solution is stable for at least 48 h in the above stated analytical method
developed.
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Table 2.21: Summary of RS content obtained at different intervals in mobile phase
stability.
Duration
in hours
% of
Sulfonyl
pyrimidine
imp.
% of
Hydroxy
acid
imp.
% of
Hydroxy
ester
imp.
% of
Benzoph-
enone
imp.
% of
Pyrimidine
ester
imp.
% of
Vinyloxy
imp.
% of
S.M.U.
imp.
% of
T. imp’s.
0 0.00 0.08 0.00 0.02 0.02 0.01 0.01 0.16
12 0.00 0.08 0.00 0.02 0.02 0.00 0.01 0.15
24 0.00 0.08 0.00 0.02 0.02 0.01 0.01 0.18
36 0.00 0.07 0.00 0.02 0.03 0.00 0.01 0.16
48 0.00 0.07 0.00 0.02 0.02 0.02 0.01 0.19
Table 2.22: Summary of assay content obtained at different intervals in mobile
phase stability.
Duration in hours Assay %
0 99.74
8 99.59
16 99.38
24 99.49
32 99.58
40 99.72
48 99.55
Average 99.58
STDEV 0.13
% RSD 0.13
2.6.11 Robustness:
To determine the robustness of developed method experimental conditions were
intentionally altered and the resolution between critical pairs, tailing factor and
theoretical plates were evaluated in each deliberately altered chromatographic condition.
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Table 2.23: System suitability- Robustness.
Parameter Conditions
Resolution
between ABT
and Hydroxy
acid imp
Tailing
factor of
ABT
Theoretical
plates of ABT
Temperature
20°C 6.35 1.15 16234
25°C 6.28 1.06 15698
30°C 6.10 1.04 15454
Flow
0.9 mL/min 6.41 1.21 16141
1.0 mL/min 6.28 1.06 15698
1.1 mL/min 6.18 1.09 15567
pH
2.8 5.92 1.24 14826
3.0 6.28 1.06 15698
3.2 6.35 1.08 15897
Organic
composition
95% 6.45 1.28 16548
100% 6.28 1.06 15698
105% 6.16 1.07 16298
2.7 Summary and conclusion:
The gradient RP-HPLC method developed for quantitative estimation of
Ambrisentan and other impurities and degradation products is accurate, precise, linear,
robust and specific. Acceptable results were obtained from validation of the method. This
method revealed an excellent performance in terms of sensitivity and resolution. The
method is proven as stability-indicating and can be used for routine analysis of
production samples and to ensure the stability of samples of Ambrisentan in drug
substances.
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Table 2.24: Summary of the validation results.
Parameter Related substances results
ABT Sulphonyl
pyrimidine
imp.
Hydroxy
acid
imp.
Hydroxy
ester
imp.
Benzoph
-enone
imp.
Pyrimi-
dine
imp.
Vinyloxy
imp.
Precision
(%RSD) NA 1.74 2.31 1.48 4.8 3.9 2.29
Intermediate
Precision
(%RSD)
NA 1.63 0.81 1.98 1.64 1.91 2.18
LOD (%) 0.0024 0.0022 0.0035 0.0028 0.0017 0.0033 0.0039
LOQ (%) 0.0096 0.0088 0.0088 0.0110 0.0073 0.0132 0.0131
Linearity (R2
value) 0.9979 0.9997 0.9998 0.9994 0.9994 0.9997 0.9976
Accuracy (%) NA 101.7 103.7 100.7 105.0 101.7 102.2
Robustness Rs>2.0 Rs >2.0 Rs >2.0 Rs >2.0 Rs >2.0 Rs >2.0 Rs >2.0
Solution
stability
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Mobile phase
stability
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
Stable up
to 48 h
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