chapter-4 118 4.1 introduction: edarbi (azilsartan medoxomil), a

Chapter-4

118

4.1 Introduction:

Edarbi (Azilsartan medoxomil), a prodrug, is hydrolyzed to Azilsartan in the

gastrointestinal tract during absorption. Azilsartan is a selective AT1 subtype angiotensin

II receptor antagonist. The substance used in the drug product formulation is the

potassium salt of Azilsartan medoxomil, also known by the US accepted name of

Azilsartan kamedoxomil (AZL) and is chemically described as (5-Methyl-2-oxo-1,3-

dioxol-4-yl)methyl2-ethoxy-1-{[2'-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl)biphenyl-

4yl]methyl}-1H-benzimidazole-7-carboxylatemono-potassium salt. Its empirical formula

is C30H23KN4O8 and its structural formula is:

Fig. 4.1: Chemical structure of Azilsartan kamedoxomil.

AZL is a white to nearly white powder with a molecular weight of 606.62. It is

practically insoluble in water and freely soluble in methanol. Edarbi is available for oral

use as tablets. The tablets have a characteristic odour. Each Edarbi tablet contains 42.68

or 85.36 mg of AZL, which is equivalent to containing 40 mg or 80 mg respectively, of

Azilsartan medoxomil and the following inactive ingredients: mannitol, fumaric acid,

sodium hydroxide, hydroxypropyl cellulose, croscarmellose sodium, micro crystalline

cellulose and magnesium stearate. Azilsartan medoxomil is hydrolyzed to Azilsartan, the

Chapter-4

119

active metabolite, in the gastrointestinal tract during absorption. Azilsartan medoxomil is

not detected in plasma after oral administration. Dose proportionality in exposure was

established for Azilsartan in the Azilsartan medoxomil dose range of 20 mg to 320 mg

after single or multiple dosing. The estimated absolute bioavailability of Azilsartan

following administration of Azilsartan medoxomil is approximately 60%. After oral

administration of Azilsartan medoxomil, peak plasma concentrations (Cmax) of

Azilsartan are reached within 1.5 to 3 hours. Food does not affect the bioavailability of

Azilsartan. limited data are available related to over dosage in humans. During controlled

clinical trials in healthy subjects, once daily doses up to 320 mg of Edarbi were

administered for 7 days and were well tolerated. In the event of an overdose, supportive

therapy should be instituted as dictated by the patient's clinical status. Azilsartan is not

dialyzable. The recommended dose in adults is 80 mg taken orally once daily. Consider a

starting dose of 40 mg for patients who are treated with high doses of diuretics. If blood

pressure is not controlled with Edarbi alone, additional blood pressure reduction can be

achieved by taking Edarbi with other antihypertensive agents. Edarbi may be taken with

or without food [1].

In order to have cost-effective process AZL was synthesized from two different

routes. Hence, the present work was taken up to have a suitable HPLC method that can

quantify the process related and degradation impurities from two different route of

synthesis [2-3]. An extensive literature survey reveals that, few HPLC methods have

been reported, for the quantification of AZL in pharmaceutical dosage forms. One HPLC

method available for the quantification of AZL in human plasma using solid phase

extraction procedure by RP-HPLC method. Some stability-indicating RP-LC methods

Chapter-4

120

were reported for determination of AZL and Chlorthalidone (CLT) in pharmaceutical

dosage forms. UV spectrophotometric and HPTLC methods were also reported for

determination of AZL in bulk and pharmaceutical dosage forms.

Walid et al. reported, stability-indicating RP-LC method for determination of

AZL and CLT in pharmaceutical dosage forms [4]. This is an isocratic elution method,

with a flow rate of 0.8 mL/min at ambient condition. Mobile phase containing a mixture

of methanol and dipotassium hydrogen phosphate (pH: 8±0.1, 0.05M) (40:60, v/v) was

used. Eclipse XDB C18 (150 x 4.6 mm, 5 µm) column was used. Run time was about 10

min. CLT and AZL was separated well with the resolution 3.88 and tailing factors were

1.22 and 1.19 respectively. This method is suitable for estimation of CLT and AZL in

pharmaceutical dosage forms (Estimation of assay). But, this method is not suitable for

estimation of related compounds in AZL drug substance. Naazneen et al. and some other

researchers reported stability-indicating RP-HPLC methods for the simultaneous

estimation of AZL and CLT in drug substance and drug product [5-9]. These methods

also not suitable for the estimation of related compounds in AZL drug substances.

Srinivasan et al. and some other researchers reported, stability indicating RP-HPLC

method for determination of AZL in bulk and its dosage forms [10-12]. Walid M. Ebeid

et al. and some other researchers reported UV spectrophotometric methods for

determination of AZL in bulk and pharmaceutical dosage forms [13-15]. Raja Gorla et al.

reported a simple and sensitive stability-indicating HPTLC assay method for the

determination of AZL [16]. Paras et al. reported RP-HPLC method for AZL

quantification in human plasma by solid phase extraction procedure [17]. AZL has not

been listed in major pharmacopeia like United States Pharmacopeia (USP), European

Chapter-4

121

Pharmacopeia (EP), Japanese Pharmacopeia (JP) and British Pharmacopeia (BP). It is,

therefore, felt necessary to develop a new stability indicating method, suitable for two

different synthetic routes with short runtime. Hence, an attempt has been made to

develop an accurate, specific and reproducible method for the determination of eight

impurities in AZL drug substance. This method was successfully validated according to

the ICH guidelines (validation of analytical procedures: test and methodology Q2) [18-

24]. To the best of our knowledge, the present study is the first reported stability-

indicating HPLC method for the quantitative estimation of related substances and

degradation products in AZL drug substance.

4.2 Experimental:

4.2.1 Materials and reagents:

Samples of active pharmaceutical ingredient standard and eight related impurities

were obtained from MSN laboratories private limited, R&D centre (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. A anhydrous 1-

octanesulphonicacid sodium salt, orthophosphric acid (85%) (OPA) and formic acid

(HPLC grade) were obtained from Rankem. High-purity Milli-Q-water was prepared by

using a Milli-Q Plus water purification system (Millipore; Milford, MA).

4.2.2 Instrumentation:

Agilent 1200 series LC system equipped with quaternary pump (G1311A),

vacuum degasser (G1322A), column compartment (G1316A), auto sampler with

Chapter-4

122

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

studies were performed in a dry hot air oven (Cintex precision hot air oven; Mumbai,

India). The LC-MS analysis was performed by using an Agilent 1200 series liquid

chromatography coupled with Agilent 6150 single quadrupole mass spectrometer

consisting of a binary pump (G4220A) with a degasser, auto sampler (G4226A), column

compartment (G1316C), diode array detector (DAD) (G4212A) and mass detector

(G2710BA) (Agilent Technologies; Waldbronn, Germany). Data was collected and

processed by using chemstation software.

4.2.3 Chromatographic conditions:

A YMC Pack Pro C18 column (150 x 4.6 mm, S-3 µm, 12 nm) (YMC karasuma-

Gojo Bldg, kyoto, Japan) was used as stationary phase. Mobile phase containing two

components. Mobile phase A contains 2.72 g of potassium dihydrogen orthophosphate

and 4 g of 1-octane sulphonic acid sodium salt anhydrous dissolved in 1000 mL of Mill-

Q water, the pH adjusted to 2.5 with diluted orthophosphoric acid (OPA and Milli-Q-

water in the ratio of 1:1) and the mobile phase B contains a mixture of water and

acetonitrile in the ratio 10: 90 v/v in gradient mode. The flow rate of mobile phase was

1.0 mL/min. The HPLC gradient program (Time (min) (T) /% mobile phase B (%B)) was

Chapter-4

123

set as 0.01/50, 3/50, 15/70, 17/90, 21/90, 21.5/50 and 25/50. The column and auto

sampler temperatures were maintained at 25 and 5ºC respectively. The detection was

measured at wavelength 220 nm. The injection volume was 5.0 µL. Acetonitrile is used

as diluent.

4.2.4 LC-MS conditions:

LC–MS system was used for the identification of known and unknown

compounds formed during forced degradation studies. Xterra RP 18 column (250 x 4.6

mm, 5 µm) (Waters, Ireland) was used as stationary phase. Mobile phase A contains

0.1% Formic acid in Milli-Q water and mobile phase B contains mixture of water and

acetonitrile in the ratio 10:90 v/v. The gradient program (T/%B) was set as 0.01/50, 3/50,

15/70, 17/90, 21/90, 21.1/50 and 25/50. Acetonitrile was used as diluent. The flow rate

was 1.0 mL/min. The analysis was performed on both atmospheric pressure chemical

ionization (APCI) and electrospray ionization (ESI) positive and negative modes.

Capillary voltage was 4000V, nebulizer gas pressure was 40 psi, drying gas flow was 10

L/min, drying gas temperature was 300°C, vaporizer temperature was 200°C, fragmentor

was 120 V and gain EMV was 1.0.

4.2.5 Solution preparations:

Preparation of standard solutions

A stock solution of AZL (500 µg/mL) was prepared by dissolving an appropriate

amount of drug substance in acetonitrile. A mixed stock solution (100 µg/mL) of the

impurities (eight impurities) was also prepared in acetonitrile. All working solutions were

Chapter-4

124

prepared from this stock solution for determination of related substances.

Preparation of system suitability solution

A mixture of AZL (500 µg/mL) and eight impurities (each 0.75 µg/mL) were

prepared by dissolving an appropriate amount in acetonitrile.

Preparation of sample solution

500 µg/mL of AZL was prepared by dissolving an appropriate amount of drug

substance in acetonitrile.

4.2.6 Generation of stress samples:

One lot of AZL drug substance was selected for stress testing. Different types of

stress conditions (i.e., acid, base, oxidative, water, heat and light) were used based on

guidance available from ICH Stability Guideline (Q1AR2). The details of stress

conditions performed are as follows:

a) Acid, base and oxidation degradation:

50 mg of AZL drug substance was transferred into a 100 mL volumetric flask, to

it added 50 mL of acetonitrile to dissolve and then made up to the mark with 0.2N HCl

or 0.02N NaOH or 2% H2O2 and mixed well. The flask was placed at 25 °C and sample

solution was collected and injected after 16 h for acid, 10 min for base and 1 h for

oxidation.

b) Water degradation:

50 mg of AZL drug substance was transferred into a 100 mL volumetric flask, to

it added 50 mL of acetonitrile to dissolve and then made up to the mark with water and

mixed well. The flask was placed at 60°C in a water bath for 1 h. After 1 h, the flask was

Chapter-4

125

removed and placed on the bench top to attain laboratory temperature and sample

solution was collected and injected after 1 h.

Fig. 4.2: Formation of Azilsartan kamedoxomil from two different routes of

synthesis and degradation path way of acid and base degradations.

Chapter-4

126

Azilsartan kamedoxomil

Chemical name: (5-Methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-ethoxy-1- {[2'-(5-oxo-4,5-

dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl]methyl}1H-benzimidazole-7-carboxylate

monopotassium salt.

Impurity-1 (Hydroxy acid impurity)

Chemical name: 2-Ethoxy-1-((2’-(N’-(hydroxyamino) iminomethyl) biphenyl- 4-yl)

methyl)-1H-benzimidazole-7-carboxylic acid.

Impurity-2 (Amidoximedioxolene ester impurity)

Chemical name: (Z)-(5-methyl-2-oxo-1, 3-dioxol-4-yl) methyl 2-ethoxy-1- ((2’-(N’-

Hydroxycarbamimidoyl) biphenyl-4-yl) methyl)-1H-benzo[d]imidazole-7-carboxylate.

Chapter-4

127

Impurity-3 (Acid impurity)

Chemical name: 2-Ethoxy-1-[[(2’-2,5-dihydro-5-oxo-1,2,4-oxadiazol-3-yl) biphenyl-4-

yl] methyl] benzimidazole-7-carboxylic acid.

Impurity-4 (Desethoxy impurity)

Chemical name: (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl 2-hydroxy-1-((2'-(5-oxo-4,5-

dihydro-1,2,4-oxadiazol-3-yl)biphenyl-4-yl)methyl)- 1H-benzo [d] imidazole-7-

carboxylate.

Impurity-5 (Amide impurity)

Chemical name: (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl1-((2'- carbamoylbiphenyl-4-

yl) methyl)-2-ethoxy-1H- benzo[d]imidazole-7-carboxylate.

Chapter-4

128

Impurity-6 (Imidazole carbonyl dioxolene ester impurity)

Chemical name: (Z)-(5-methyl-2-oxo-1,3-dioxol-4-yl) methyl 2-ethoxy-1-((2'-(N'-

hydroxy-N-(1H-imidazole-1-carbonyl) carbamimidoyl) biphenyl-4-yl)methyl)-1H-benzo

[d] imidazole-7-carboxylate.

Impurity-7 (Ester impurity)

Chemical name: Methyl 2-ethoxy-1-((2’-(5-oxo-4, 5-dihydro-1,2,4-oxadiazol-3-yl)

biphenyl-4-yl) methyl)-1H-benzo[d]imidazole-7-carboxylate.

Impurity-8 (Dimer impurity)

Chemical name: (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl2-ethoxy-1-((2’-(4-((5-methyl-

2-oxo-1,3-dioxol-4-yl) methyl)-5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl)biphenyl-4-

yl)methyl)-1H benzo [d]imidazole-7-carboxylate.

Fig. 4.3: Chemical structures and chemical names of Azilsartan kamedoxomil and

eight related impurities.

Chapter-4

129

c) Photolytic degradation:

Susceptibility of the drug substance to light was studied. 100 mg of AZL drug

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)

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.

d) Thermal degradation:

100 mg of AZL 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.

e) Humidity degradation

100 mg of AZL drug substance was transferred into a petri dish and placed in a

humidity chamber at 75% RH for 5 days. After 5 days, the petri dish was removed and

the sample was prepared for analysis, as previously described.

f) Sunlight degradation:

100 mg of AZL drug substance was transferred into a petri dish and placed under

sunlight for 30 h. After 30 h, the sample was prepared for analysis, as previously

described.

Chapter-4

130

4.3 Method development and optimization:

A literature survey reveals that no stability indicating related substances by HPLC

method available for AZL drug substance. Hence, it is necessary to develop a rapid,

accurate and validated method of related substances and degradation compounds of AZL

drug substance. From the literature it was found that the pKa of the molecule is 6.1. By

considering the molecule pKa value, initial method development attempts were

performed by using 0.01M dipotassium hydrogen phosphate (pH adjusted to 6.5 with

OPA) as mobile phase A and acetonitrile: water in the ratio of 90:10 v/v as mobile phase

B. The blend containing 500 µg/mL of AZL and 5 µg/mL of each individual impurity

(Eight) was prepared in the acetonitrile. AZL spiked solutions were subjected to

separation by reverse-phase LC on a Waters Symmetry C18 (150 x 4.6 mm, 3.5 µm)

column. Flow rate was set to 1.0 mL/min. The HPLC gradient program (T/%B) was set

as 0.01/40, 20/80, 25/80, 25.5/40 and 30/40. Column temperature was maintained at

25°C. By applying above conditions impurity-2 was not retained well. So efforts were

made to get good retention time for impurity-2, 0.1% OPA in Milli-Q water was used as

mobile phase A, and kept the remaining chromatographic conditions as above mentioned.

With this buffer impurity-2 was well retained, but in these conditions the impurity-3 and

impurity-4 were not separated. To separate these impurities column was changed from

Waters Symmetry C18 (150 x 4.6 mm, 3.5 µm) to YMC Pack pro C18 (150 × 4.6 mm, 3

µm). In this column the impurity-3 and impurity-4 were separated well, but the impurity-

4 was very closely eluted with impurity-5 and all remaining impurity peaks also closely

eluted with each other. Hence, to change the chromatographic pattern, ion pair reagent

was introduced into the mobile phase, i.e, 0.1% OPA and 4 g of 1-octane sulphonic acid

Chapter-4

131

sodium salt (anhydrous) in 1000 mL of Milli-Q water. It was used as mobile phase A,

with this conditions all peaks were well separated with more than resolution of 2.5 and

met other system suitability parameters. But, impurity-2 peak shape was not good. In

order to obtain good peak shape for impurity-2, 2.72 g of potassium dihydrogen

orthophosphate and 4.0 g 1-Octane sulphonic acid sodium salt anhydrous dissolved in

1000 mL of mill-Q water and adjusted pH to 2.5 with dilute OPA was used as mobile

phase A. With this buffer, eight impurities and AZL were well resolved with more than

3.0 resolution and good peak shapes obtained for all eight impurities and AZL. The

tailing factor for AZL and its eight impurities found less than 1.5.

Fig. 4.4: AZL spiked with its eight impurities.

4.4 Degradation behavior:

The LC studies on AZL under different stress conditions suggested the

following degradation behavior.

Minutes

0 2 4 6 8 10 12 14 16 18 20 22 24

mA

U

0

25

50

75

100

mA

U

0

25

50

75

100

2.5

2

Impuri

ty-1

4.7

3

Impuri

ty-2

5.6

7 Im

puri

ty-3

6.3

6 Im

puri

ty-4

8.1

4

Impuri

ty-5

9.3

8

Impuri

ty-6

10.7

7

Impu

rity

-7

11.8

0 A

zils

arta

n k

amed

oxo

mil

15.9

3 Im

pu

rity

-8

VWD: Signal A, 220 nm

Retention TimeName

Chapter-4

132

a) Degradation in acidic conditions:

AZL drug substance when exposed to 0.5N HCl at 25ºC, highly degradation was

observed. When the concentration was decreased to 0.1N HCl at 25ºC, degradation of

about 9.38% was observed, majorly impurity-4 was formed. [Fig. 4.5-4.6]

Fig. 4.5: Typical HPLC chromatogram of acid hydrolysis.

Fig. 4.6: Typical LC-MS spectra of acid hydrolysis.

Chapter-4

133

b) Degradation in basic conditions:

AZL drug substance when exposed to 0.5 N NaOH at 25ºC, high degradation was

observed. Then a decrease in the concentration to 0.01N NaOH at 25ºC, degradation of

about 75.94% was observed, majorly impurity-3 formed. [Fig. 4.7-4.8]

Fig. 4.7: Typical HPLC chromatogram of base hydrolysis.

Fig. 4.8: Typical LC-MS spectra of base hydrolysis.

Chapter-4

134

c) Degradation in oxidation conditions:

AZL drug substance when exposed to 3.0% H2O2 at 25ºC, high degradation was

observed. Then decrease in the concentration to 1.0% H2O2 at 25ºC, degradation was

observed, majorly impurity-3 formed. [Fig. 4.9-4.10]

Fig. 4.9: Typical HPLC chromatogram of peroxide.

Fig. 4.10: Typical LC-MS spectra of peroxide degradation.

d) Degradation in water hydrolysis:

Degradation was observed during water hydrolysis at 60ºC, after 1

of about 6.09 % was observed, majorly impurity

Fig. 4.11: Typical HPLC chromatogram of water hydrolysis.

Fig. 4.12: Typical LC

e) Thermal degradation:

Slight degradation was observed at 60ºC, after 10 days. A degradation of about 0.58

% was observed.

Degradation in water hydrolysis:

Degradation was observed during water hydrolysis at 60ºC, after 1

about 6.09 % was observed, majorly impurity-3 formed. [Fig. 4.11- 4.12]

4.11: Typical HPLC chromatogram of water hydrolysis.

4.12: Typical LC-MS spectra of water hydrolysis.

Thermal degradation:


Chapter-4

135

h. A degradation

4.12]


Chapter-4

136

f) At 75% relative humidity degradation:

Degradation was observed at 75% relative humidity degradation, after 5 days. A

degradation of about 10.22% was observed.

g) Under sunlight degradation:

Degradation was observed under sunlight, after 30 h. A degradation of about 5.91%

was observed.

h) Photolytic degradation:

AZL drug substance was slightly degraded to effect of photolysis, when the drug

substance was directly and indirectly exposed to light for an overall illumination of 1.2

million LUX hours (lxh) and near to UV light energy of 200 Wh/m2 (in photo stability

chamber).

Slight degradation was observed when the drug was subjected to photo

degradation [UV direct for 200 (Wh/m2) and LUX direct for 1.2 million (lxh)].

Degradation was observed when the drug was subjected to 75% relative humidity (5

days), sunlight (30 h) and water (at 60°C for 1 h) and significant degradation was

observed with acid (0.1 N HCl at 25°C for 16 h), base (0.01 N NaOH at 25°C for 10 min)

and peroxide (1% H2O2 at 25°C for 1 h). Acid degradation leads to the formation of

impurity-4. Base, peroxide, water and 75% relative humidity degradations lead to the

formation of impurity-3. AZL was well resolved from all its related substances and

degradants, proving the stability-indicating power of the method.

Identification of major degradation compounds by LC-MS

An LC–MS study was carried to determine the m/z value of the major degradation

product formed under acid, base, peroxide and water degradation conditions. The ESI

Chapter-4

137

mass spectrum of the acid degradation major peak showed [M+H] + at m/z 541.2, which

corresponds to impurity-4. The APCI mass spectrum of base, peroxide and water

degradation studies showed major peaks [M-H] - at m/z 455.3, 455.2 and 455.3,

respectively, which corresponding to impurity-3.

4.5 Mass balance:

Table 4.1: Mass balance and forced degradation study results.

Stress conditions Total

degra-

dation

Assay Mass

balance

Normal 0.26% 99.86% NA

Acid hydrolysis (0.1N HCl at

25ºC after 16 h) 9.38%

90.05% 99.43%

Base hydrolysis

(0.01N NaOH at 25ºC after 10

min)

75.94%

Sample is highly sensitive to

base. Hence, assay test not

performed.

NA

Oxidation

(1% H2O2 at 25ºC after 1 h) 16.23%

Sample is highly sensitive to

peroxide. Hence, assay test

not performed.

NA

Water hydrolysis

(at 60°C after 1 h) 6.09%

92.72% 98.81%

Photo

degra-

dation

UV direct (200 Watt

hours/square meter) 1.42%

97.88% 99.3%

UV indirect (200 Watt

hours/square meter) 0.46%

99.35% 99.81%

Lux direct (1.2 million

LUX hours) 1.01%

98.70% 99.71%

Lux indirect (1.2

million LUX hours) 0.47%

99.40% 99.87%

Thermal at 60oC (10 days) 0.58% 99.31% 99.89%

At 75% relative humidity (5

days) 10.22%

89.19% 99.41%

Under sunlight (30 h) 5.91% 92.87% 98.78%

Chapter-4

138

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 98%. This clearly confirmed that the developed

HPLC method was specific for AZL in presence of its impurities and degradation

products.

4.6 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.

4.6.1 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 Azilsartan kamedoxamil was spiked with 0.15% impurity blend with respect

to the concentration of Azilsartan kamedoxamil and injected for three times into HPLC

system. Resolution between Azilsartan kamedoxamil and ester impurity, tailing factor

and theoretical plates for Azilsartan kamedoxamil was calculated. Good resolution was

obtained between Azilsartan kamedoxamil and ester impurity. System suitability results

were tabulated. [Table: 4.2]

Chapter-4

139

Table 4.2: Results of system suitability

Name of the compound Resolution between Ester

impurity and AZL (Rs)

Theoretical

plates (N)

Tailing

factor (T)

Azilsartan kamedoxamil - 59163 0.983

Ester impurity 4.93 - -

4.6.2 Limit of quantification (LOQ) and limit of detection (LOD):

LOQ and LOD established for all impurities based on the impurities signal to

noise ratio 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 Azilsartan kamedoxamil and its impurities were injected to establish

the limit of detection and limit of quantification respectively. The results are recorded in

Table 4.3.

Chapter-4

140

Table 4.3: LOQ and LOD values of the impurities

S.No Name of the

substance

S/N ratio

for LOD

S/N ratio

for LOQ

% level of

impurity w.r.t. to

sample conc.

(LOD)

% level of

impurity w.r.t. to

sample conc.

(LOQ)

1 Imp-1 2.200 10.000 0.00420 0.0126

2 Imp-2 2.870 10.120 0.00280 0.0112

3 Imp-3 2.450 9.680 0.00210 0.0084

4 Imp-4

2.680 9.580 0.00360 0.0152

5 Imp-5

2.790 9.980 0.00250 0.0100

6 Imp-6

2.760 9.540 0.00415 0.0134

7 Imp-7

2.170 10.290 0.00225 0.0090

8 Imp-8 2.350 10.040 0.00225 0.0109

9 AZL 2.310 10.310 0.00265 0.0074

4.6.3 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 AZL

concentration. Upon repetitive injections at quantification limit, the peak properties like

retention time and 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 4.05%.

Chapter-4

141

Table 4.4: Precision results for area of all impurities at LOQ level.

Name of the

injection

Imp-1

area

Imp-2

area

Imp-3

area

Imp-4

area

Imp-5

area

Imp-6

area

Imp-7

area

Imp-8

area

AZL

LOQ prep-1 18809 25229 18041 19905 18858 18895 18884 20564 23403

LOQ prep-2 18326 24566 18507 18181 19515 18833 18545 19487 23871

LOQ prep-3 18973 24730 18313 18148 18913 18412 18548 19071 25483

LOQ prep-4 18711 25471 19164 18505 18905 18244 18767 20201 24622

LOQ prep-5 19282 25474 18916 18684 19042 19950 18526 20822 25888

LOQ prep-6 18433 25422 18807 19010 18724 18676 18015 19715 25543

% RSD 1.87 1.60 2.23 3.50 1.45 3.19 1.61 3.35 4.05

4.6.4 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 AZL sample at LOQ level.

The recovery study for impurities was carried out in triplicate at LOQ level of the target

AZL concentration (500 µg/mL). The percentage recovery of impurities was calculated.

Table 4.5: Recovery at LOQ level for impurities.

S.No. Impurity name % of recovery

1 Imp-1 99.7

2 Imp-2 101.4

3 Imp-3 102.4

4 Imp-4 99.8

5 Imp-5 96.4

6 Imp-6 101.3

7 Imp-7 103.3

8 Imp-8 103.6

Chapter-4

142

4.6.5 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%.

Repeatability is the results of a method operated over a short interval of time under the

same conditions.

Intermediate precision is the end result from within-laboratories variations due to

random events that include different days, different analysts, different equipment, etc.

Reproducibility is determined by testing the homogeneous samples in different

laboratories. It is a measure of precision between laboratories.

The precision of related substances method was evaluated by injecting eight

individual preparations of AZL (0.5 mg/mL) spiked with 0.15% of impurities with

respect to AZL analyte concentration. The % RSD for content of all impurities for eight

consecutive determinations was below 1.77. [Table 4.6]

Table 4.6: Precision results of the RS method.

Name of the

injection Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

Sample + imp’s-1 15456 14988 86581 16335 21279 10846 27753 16243

Sample + imp’s-2 15752 14840 86419 16112 21277 10356 27819 16230

Sample + imp’s-3 15613 14929 85531 16273 21371 10509 28022 16514

Sample + imp’s-4 15358 14694 85437 15952 20944 10565 27694 16332

Sample + imp’s-5 15506 14616 86080 16593 21079 10461 27933 16228

Sample + imp’s-6 15693 15075 87211 16488 21428 10767 28233 16680

Average area 15563 14857 86210 16292 21230 10584 27909 16371

STDEV 149.56 175.96 672.13 236.32 183.46 187.21 198.49 186.64

% RSD 0.96 1.18 0.78 1.45 0.86 1.77 0.71 1.14

Chapter-4

143

The method precision of assay study was calculated initially by performing

system precision, then by carrying out six independent assays of AZL test sample against

qualified reference standard. Results showed insignificant variation in measured response

which demonstrated that the method was repeatable with RSDs below 0.02%. [Table 4.7]

Table 4.7: Precision results of the assay method.

Assay no. % assay

Set-I Assay 99.75

Set-II Assay 99.74

Set-III Assay 99.77

Set-IV Assay 99.80

Set-V Assay 99.78

Set-VI Assay 99.76

Average 99.77

STDEV 0.02

%RSD 0.02

Intermediate precision for assay method was performed by carrying out six

independent assays of AZL test sample against qualified reference standard and

calculated %RSD of six consecutive assays. Related substances method was performed

by injecting six individual preparations of AZL (0.5 mg/mL) and 0.15% of impurities

with respect to AZL analyte concentration over different days, different instruments and

with different analysts. [Table 4.8]

Chapter-4

144

Table 4.8: Results of intermediate precision study.

Parameter Variation %RSD

assay

(n=6)

%RSD for

related

substances

Resolution

between

Ester and

AZL (Rs)

AZL

theoretical

plates

AZL

tailing

factor

Different

systems

(a) Shimadzu

LC-2010CHT

(b) Agilent

1200 series

VWD

0.02%

0.05%

<1.77

< 1.26

4.93

5.32

59163

58485

0.983

1.08

Different

column

1) ARLC14012

2) ARLC14030

0.02%

0.05%

< 1.77

< 1.26

4.93

5.32

59163

58485

0.983

1.08

Different

analyst

Analyst-1

Analyst-2

0.02%

0.05%

< 1.77

< 1.26

4.93

5.32

59163

58485

0.983

1.08

4.6.6 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 AZL

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%) and assessed by injecting eight separately prepared solutions of the normal test

sample concentration (500 µg/mL). The correlation coefficients, slopes and Y-intercepts

of the calibration curve were determined. [Table 4.9-4.16]

Chapter-4

145

The calibration curve was drawn by plotting average area of the impurities on the Y-

axis and concentration on the X-axis which has shown linear relationship with a

regression coefficient greater than 0.998 for all impurities.

Table 4.9: Linearity of imp-1 (Hydroxy acid impurity).

Fig. 4.13: Linearity graph for Hydroxy acid impurity.

18655

56489

114924

173201

229112

282241

342341

0

50000

100000

150000

200000

250000

300000

350000

400000

0 50 100 150 200

Aver

age

Are

a

Concentration in %

Hydroxy acid impurity

Hydroxy acid impurity

Concentration in % Average area

12.6 18655

25 56489

50 114924

75 173201

100 229112

125 282241

150 342341

Slope 2316

Y-intercept -3994

Correlation coefficient 0.99950

Chapter-4

146

Table 4.10: Linearity of imp-2 (Amidoxime dioxolene ester impurity).

Fig. 4.14: Linearity graph for Amidoxime dioxolene ester impurity.

25191

54647

110541

168301

222587

277160

331750

0

50000

100000

150000

200000

250000

300000

350000

0.00 50.00 100.00 150.00 200.00

Aver

age

Are

a

Concentration in %

Amidoxime dioxolene ester impurity

Amidoxime dioxolene ester impurity


11.20 25191

25 54647

50 110541

75 168301

100 222587

125 277160

150 331750

Slope 2216

Y-intercept 274


Chapter-4

147

Table 4.11: Linearity of imp-3 (Acid impurity).

Fig. 4.15: Linearity graph for Acid impurity.

259066

540762

834734

1121943

1426245

1729572

2058784

0

500000

1000000

1500000

2000000

2500000

0.00 50.00 100.00 150.00 200.00

Aver

age

Are

a

Concentration in %

Acid impurity

Acid impurity


1.68 259066

25 540762

50 834734

75 1121943

100 1426245

125 1729572

150 2058784

Slope 12042

Y-intercept 232690


Chapter-4

148

Table 4.12: Linearity of imp-4 (Desethoxy impurity).

Fig. 4.16: Linearity graph for Desethoxy ester impurity.

32519

63721

111138

160656

211130

261866

315508

0

50000

100000

150000

200000

250000

300000

350000

0.00 50.00 100.00 150.00 200.00

Aver

age

Are

a

Concentration in %

Desethoxy impurity

Desethoxy impurity


10.13 32519

25 63721

50 111138

75 160656

100 211130

125 261866

150 315508

Slope 2009

Y-intercept 11612


Chapter-4

149

Table 4.13: Linearity of imp-5 (Amide impurity).

Fig. 4.17: Linearity graph for Amide impurity.

30122

92966

174364

255501

336715

420808

505654

0

100000

200000

300000

400000

500000

600000

0.00 50.00 100.00 150.00 200.00

Aver

age

Are

a

Concentration in %

Amide impurity

Amide impurity


6.67 30122

25 92966

50 174364

75 255501

100 336715

125 420808

150 505654

Slope 3301

Y-intercept 8748


Chapter-4

150

Table 4.14: Linearity of imp-6 (Imidazole carbonyl dioxolene ester imurity).

Fig. 4.18: Linearity graph for Imidazole carbonyl dioxolene ester impurity.

18955

35241

68589

105341

141643

172757

215706

0

50000

100000

150000

200000

250000

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00

Aver

age

Are

a

Concentration in %

Imidazole carbonyl dioxolene ester impurity

Imidazole carbonyl dioxolene ester impurity


13.40 18955

25 35241

50 68589

75 105341

100 141643

125 172757

150 215706

Slope 1423

Y-intercept -1129


Chapter-4

151

Table 4.15: Linearity of imp-7 (Ester imurity).

Fig. 4.19: Linearity graph for Ester impurity.

29410

97519

195781

289437

383564

470690

576423

0

100000

200000

300000

400000

500000

600000

700000

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00

Aver

age

Are

a

Concentration in %

Ester impurity

Ester impurity


6.00 29410

25 97519

50 195781

75 289437

100 383564

125 470690

150 576423

Slope 3776

Y-intercept 5366


Chapter-4

152

Table 4.16: Linearity of imp-8 (Dimer imurity).

Fig. 4.20: Linearity graph for Dimer impurity.

20409

68349

136234

204273

273814

339258

412498

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 160.00

Aver

age

Are

a

Concentration in %

Dimer impurity

Dimer impurity


7.27 20409

25 68349

50 136234

75 204273

100 273814

125 339258

150 412498

Slope 2737

Y-intercept -248


Chapter-4

153

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 AZL assay concentration (i.e 200 µg/mL). Each

solution was injected in triplicate into LC system and at each concentration level, the

average area was calculated. A calibration curve attained by least square regression

analysis between average peak areas and concentration show [Fig.4.21], linear

relationship with a regression coefficient greater than 0.999.

Table 4.17: Linearity results of AZL in assay method.

Fig. 4.21: Linearity graph for AZL in assay method.

2477169

37628124729718

58991526993099

010000002000000300000040000005000000600000070000008000000

0 50 100 150 200

Av

era

ge

are

a's

Concentration

AZILSARTAN KAMEDOXOMIL

Conc. in % Average area

50 2477169

75 3762812

100 4729718

125 5899152

150 6993099

Slope 44673

Y-intercept 305110


Chapter-4

154

4.6.7 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 RS 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 500 µg/mL test concentration) and each solution was injected thrice

(n=3) into LC system.

The mean % recovery of impurities determined in the spiked test solution by

using the area of impurities in the standard solutions at 0.15% level with respect to AZL

analyte concentration [Table 4.18].

Table 4.18: Recovery at 50%, 75%, 100%, 125% and 150% level.

Conc. in % Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

50 % 101.4 101.0 101.8 99.3 100.4 97.4 102.2 100.2

75 % 101.9 102.5 100.7 99.9 100.3 99.7 102.5 100.1

100 % 101.1 101.6 101.6 100.7 100.3 100.5 102.8 100.7

125 % 99.6 101.2 102.0 101.3 101.0 98.1 101.4 99.8

150 % 100.7 101.0 103.8 102.6 101.6 102.1 103.9 101.1

Avg.

% recovery 100.9 101.5 102.0 100.8 100.7 99.6 102.6 100.4

Chapter-4

155

The related substance method have revealed consistent and high recoveries at all

the five concentration levels i.e, 50%, 75%, 100%, 125% and 150%, which convey the

absolute recovery ranging from 99.6% to 102.6%. The AZL recovery study specified that

the related substances by LC method were appropriate for determination/quantification of

impurities of AZL 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 (AZL test

concentration) i.e 200 µg/mL. The study was performed in triplicate (n=3), the solution

was injected into HPLC system and the mean peak area of AZL analyte peak was

calculated for assay determination. Assay (%w/w) of test solution was calculated against

three injections (n=3) of qualified AZL reference standard. [Table 4.19]

Table 4.19: Recovery of the assay method for drug substance.

Conc. in % % Average assay

50 49.82

75 74.76

100 99.65

125 124.60

150 149.37

Slope 0.9958

Y-intercept 0.0640


Chapter-4

156

Fig. 4.22: Linearity graph for average % assay versus concentration.

The method has shown consistent and high recoveries at all the three

concentration (50%, 75%, 100%, 125% and 150%) levels. The above accuracy/recovery

study indicated that the method was suitable for determination of AZL drug substances.

4.6.8 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 a impurities retention time,

relative to the standard’s retention time.

49.82

74.76

99.65

124.60

149.37

0.0020.0040.0060.0080.00

100.00120.00140.00160.00

0 50 100 150 200

Av

era

ge

% a

ssa

y

Concentration

AZILSARTAN KAMEDOXOMIL

Chapter-4

157

RRT = Impurity RT / Sample RT

Response factors and relative retention times were calculated by injecting four

different concentrations (0.10%, 0.20%, 0.30% and 0.50% w.r.t. to AZL test concentration

(500 µg/Ml)) of eight impurities and AZL.

Table 4.20: Average RRF, RF & RRT on the basis of four different concentrations.

S.No. Impurity RRF RF RRT

1 Imp-1 1.16 0.86 0.20

2 Imp-2 0.99 1.01 0.40

3 Imp-3 1.11 0.90 0.48

4 Imp-4 0.92 1.09 0.54

5 Imp-5 1.01 1.00 0.69

6 Imp-6 0.88 1.13 0.77

7 Imp-7 1.17 0.85 0.92

8 Imp-8 0.88 1.14 1.35

4.6.9 Solution stability:

The solution stability of AZL in 157imultan in the assay method was performed

by leaving the test solutions of sample in tightly capped volumetric flasks on a

laboratory work table with room temperature for about 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 AZL during solution stability experiments were less than 0.03.

The solution stability of AZL in 157imultan in the related substances method was

performed by leaving the sample solution and spiked test solutions of sample in tightly

capped volumetric flasks on a laboratory work table with room temperature for about

48 h (two days). The content of impurities is checked in sample solution and test spiking

solutions for every twelve hours interval up to the study period. Sample solution is stable

Chapter-4

158

up to 38 h at auto sampler temperature 5°C, after 38 h impurity-3 was increasing; no

other significant changes are observed during solution stability study. Hence AZL sample

and spiked sample solution was stable for a minimum of 38 h with the same 158imultan.

[Table 4.21- 4.25]

Table 4.21: Summary of RS content obtained at different intervals in solution

stability.

Duration in

hours

Resolution between

ester impurity and

AZL system

suitability solution

Theoretical plates

for AZL peak

from system


Tailing factor for AZL

peak from system


0 5.32 58485 1.08

49 5.28 58531 1.07

Table 4.22: % of impurities for sample solution stability from 0 to 49 hours.

Duration

in hours

Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 S.M.U.

imp

T.

imp’s

0 0.00 0.00 0.12 0.01 0.01 0.00 0.00 0.00 0.02 0.21

13 0.00 0.00 0.12 0.01 0.01 0.00 0.00 0.00 0.02 0.21

24 0.00 0.00 0.13 0.01 0.01 0.00 0.00 0.00 0.02 0.23

38 0.00 0.00 0.15 0.01 0.00 0.00 0.00 0.00 0.02 0.25

49 0.00 0.00 0.17 0.01 0.01 0.00 0.00 0.00 0.02 0.27

Chapter-4

159

Table 4.23: % of impurities for sample+100% all imp’s from 0 to 49 hours.

Duration

in hours Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

0 0.10 0.11 0.61 0.10 0.16 0.07 0.20 0.13

13 0.10 0.12 0.62 0.10 0.16 0.07 0.20 0.13

24 0.10 0.12 0.62 0.10 0.16 0.07 0.20 0.14

38 0.10 0.11 0.65 0.10 0.16 0.07 0.20 0.15

49 0.10 0.12 0.66 0.10 0.16 0.07 0.19 0.15

Table 4.24: Summary of assay content obtained at different intervals in solution

stability.

Duration

%RSD Theoretical plates Tailing factor

1st 2

nd First Last First Last

0 Hours 0.40 0.39 15382 15393 1.02 1.02

8 Hours 0.70 0.66 15533 15831 1.02 1.02

38 Hours 0.09 0.33 15362 15400 1.02 1.02

48 Hours 0.60 0.61 15178 15248 1.02 1.02

Chapter-4

160

Table 4.25: 0 to 48 hours sample solution stability assay results.

Duration in hours Assay %

0 99.71

8 99.77

38 99.72

48 99.73

Average 99.73

STDEV 0.03

% RSD 0.03

4.6.10 Mobile phase stability:

The mobile phase stability of AZL in 160imultan 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 AZL 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 AZL during mobile phase stability experiments

were less than 0.1.

The mobile phase stability of AZL in 160imultan 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 are 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 AZL mobile phase solution is stable for at least

48 h in the above stated analytical method developed. [Table 4.26- 4.27]

Chapter-4

161

Table 4.26: Summary of RS content obtained at different intervals in mobile phase

stability.

% of impurities for mobile phase stability from 0 to 49 hours.

Duration

in hours

Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 S.M.U.

imp

T. imp’s

0 0.00 0.00 0.12 0.01 0.01 0.00 0.00 0.00 0.02 0.21

13 0.00 0.00 0.11 0.00 0.01 0.00 0.00 0.00 0.02 0.19

24 0.00 0.00 0.10 0.01 0.01 0.00 0.01 0.00 0.02 0.20

38 0.00 0.00 0.10 0.01 0.01 0.00 0.00 0.00 0.02 0.19

49 0.00 0.00 0.10 0.01 0.01 0.00 0.00 0.00 0.02 0.18

Table 4.27: Summary of assay content obtained at different intervals in mobile

phase stability.

“0” to “48” hours mobile phase stability assay results.

Duration in hours % assay

0 99.71

38 99.70

48 99.79 Average 99.73

STDDEV 0.05 %RSD 0.05

4.6.11 Robustness:

To determine the robustness of developed method experimental conditions were

intentionally altered and the resolution between critical pairs, tailing factor and

Chapter-4

162

theoretical plates were evaluated in each deliberately altered chromatographic conditions.

[Table 4.28]

Table 4.28: System suitability-Robustness.

Parameters

Conditions

Resolution

between Ester

impurity and

AZL (Rs)

Tailing factor

of AZL

Theoretical

plates of AZL

Column

temperature

20ºC 5.08 0.990 63482

25ºC 4.93 0.983 59163

30ºC 4.79 0.977 58408

Different flow

0.9 Ml 4.79 0.983 66880

1.0 Ml 4.93 0.983 59163

1.1 Ml 5.064 0.986 55392

Ph

2.3 5.10 0.987 60748

2.5 4.93 0.983 59163

2.7 4.90 0.996 60443

Organic

composition

95% 5.06 0.976 62214

100% 4.93 0.983 59163

105% 4.93 0.988 59662

4.7 Summary and conclusion:

The quick gradient RP-HPLC method developed for quantitative estimation of

AZL 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 speed. 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 AZL in drug substances.

Chapter-4

163

Table 4.29: Summary of the validation results.

Test

parameter Related substances results

AZL Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

Precision

(%RSD) NA 0.96 1.18 0.78 1.45 0.86 1.77 0.71 1.14

Intermediate

precision

(%RSD)

NA 0.34 0.59 0.79 1.16 0.60 0.31 0.39 1.26

LOD (%) 0.003 0.004 0.003 0.002 0.004 0.003 0.004 0.002 0.002

LOQ (%) 0.007 0.013 0.011 0.008 0.015 0.010 0.013 0.009 0.011

Linearity

(R2 value)

NA 0.99950 0.99996 0.99983 0.99987 0.99997 0.99951 0.99984 0.99995

Accuracy

(%) NA 100.9 101.5 102.0 100.8 100.7 99.6 102.6 100.4

Robustness >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0 >2.0

Solution

stability

Stable

up to

36 h

Stable

up to 36

h

Stable

up to 36

h

Stable

up to

36h

Stable

up to 36

h

Stable up

to 36 h

Stable

up to 36

h

Stable

up to 36

h

Stable

up to 36

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

Stable

up to 48

h

Stable

up to 48

h

Chapter-4

164

References:

1 Takeda pharmaceuticals America, Inc. Deerfield, IL, http://www. Accessdata.

Fda.gov/ drugsatfda_docs/label/2011/200796s000lbl.pdf.

2 Rajan ST, Nagaraju C, Ramprasad AK, Rama Subba Reddy K; Process for

preparation of Azilsartan medoxomil and its salts; WO 2013186792 A3; (2014).

3 Rajan ST, Nagaraju C, Ramprasad AK, Rama Subba Reddy K; Process for the

preparation of (5-methyl-2-oxo-l,3-dioxoy-4-yl) methyl 2-ethoxy-l-f[20-(5-oxo-4,5-

dihydro-l,2,4-oxadiazol-3-yl)biphenyl-4- yl]164imult-1H-benzimidazole-7-

carboxyiate and its salts; WO 2013186792 A2; (2013).

4 Walid ME, Ehab FE, Asmaa AE, Ramzia IE, Gabor P; Stability-indicating RP-LC

method for determination of Azilsartan medoxomil and Chlorthalidone in

pharmaceutical dosage forms: application to degradation kinetics; Analytical and

Bioanalytical Chemistry; (2014), 406(26), 6701–6712.

5 Naazneen S, Sridevi A; Stability-indicating RP-HPLC method for the simultaneous

estimation of Azilsartan medoxomil and Chlorthalidone in solid dosage forms;

International Journal of Pharmacy and Pharmaceutical sciences; 2014, 6(6), 235-243.

6 Sravani P, Rubesh Kumar S, Duganath N, Devanna N; Method development and

validation for the simultaneous estimation of Azilsartan and Chlorthalidone by RP-

HPLC in pharmaceutical dosage Form; International Journal of Pharma Sciences;

2014, 4(5), 725-729.

7 MadhuBabu K, Bikshal Babu K; Reverse phase HPLC method development and

validation for the simultaneous estimation of Azilsartan medoxomil and

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