2.1 introduction: letairis is the brand name fo that is selective for the

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



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


stearate and microcrystalline cellulose. The tablets are film-coated with a coating material

ontaining FD&C red #40 aluminum lake, lecithin, polyethylene glycol, polyvinyl


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



is available as 5 mg

daily oral administration. The tablets include the


coated with a coating material

lake, lecithin, polyethylene glycol, polyvinyl


Chapter-2

51

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

Chapter-2

52

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

Chapter-2

53

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

Chapter-2

54

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.

Chapter-2

55

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

Chapter-2

56

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:


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:


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:


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)

Chapter-2

57

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:


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:


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.

Chapter-2

58

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



Pyrimidine ester impurity

Fig. 2.3: Typical UV spectrums of

UV spectra and impurity name

yrimidine impurity




Pyrimidine ester impurity Vinyloxy impurity

Ambrisentan

2.3: Typical UV spectrums of Ambrisentan and its impurities.

Chapter-2

59



Vinyloxy impurity

and its impurities.

Chapter-2

60

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

Chapter-2

61

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

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:


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.

Chapter-2

62


N HCl at 25°C, a slight

N HCl at 25°C, a

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

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.

Chapter-2

64

25°C, no degradation

25°C, no degradation

h. Then increased the

h. A degradation of about 9.11% was observed.

Chapter-2

65

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

Chapter-2

66

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

Chapter-2

67

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

Chapter-2

68

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

Chapter-2

69

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

Chapter-2

70

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

Chapter-2

71

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

Chapter-2

72

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

Chapter-2

73

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

Chapter-2

74

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


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

Chapter-2

75

Table 2.10: Linearity of Hydroxy acid impurity.

Fig. 2.12: Linearity graph for 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



5.8 23219

25 31661

50 42672

75 54710

100 64373

125 75105

150 85387

Slope 432

Y-intercept 21104


Chapter-2

76

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



7.3 3253

25 11496

50 20672

75 32480

100 42475

125 53537

150 63730

Slope 424

Y-intercept 305


Chapter-2

77

Table 2.12: Linearity of Benzophenone impurity.

Fig. 2.14: Linearity graph for Benzophenone impurity.


0

20000

40000

60000

80000

100000

120000

4.8 25 50 75 100 125 150

Concentration

Are

a

Area



4.8 9901

25 23728

50 34838

75 51375

100 66790

125 80507

150 96275

Slope 590

Y-intercept 7240


Chapter-2

78

Table 2.13: Linearity of Pyrimidine ester impurity.

Fig. 2.15: Linearity graph for 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



8.8 12568

25 21766

50 32238

75 45415

100 55151

125 67578

150 78307

Slope 462

Y-intercept 9469


Chapter-2

79

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


8.7 5181

25 16835

50 29276

75 42752

100 56734

125 65477

150 78148

Slope 509

Y-intercept 3269


Chapter-2

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


50 1087428

75 1580968

100 2146041

125 2717645

150 3272380

Slope 22026.48

Y-intercept -41761.33


Chapter-2

81

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

Chapter-2

82

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


Chapter-2

83

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

Chapter-2

84

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

Chapter-2

85

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.

Chapter-2

86

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.

Chapter-2

87

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.

Chapter-2

88

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

Chapter-2

89

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2.1 introduction: letairis is the brand name fo that is selective for the

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