13 chapter-iii fexofenadine odt method development and method...

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CHAPTER III

FEXOFENADINE ODT METHOD DEVELOPMENT AND

METHOD VALIDATION

This chapter describes about the fexofenadine formulation sample development and

validation of a stability indicating Revere phase-Ultra performace LC procedure fexofenadine

in pharmaceutical formulations. This chapter also describes about materials used and scope of

the present study. It describes the reagents, solvents, glass-ware, standards and test samples

(dosage forms) used in this present study, brief description of equipment and methods

employed in the present study.

3.1 REAGENTS AND CHEMICALS

Sodium hydroxide (NaOH):

Merck supplied AR grade Sodium hydroxide was utilized in this study.

Hydrogen Peroxide (H2O2):

Merck supplied Analytical grade (AR) Hydrogen peroxide (30%) was used for this

study.

Ortho-phosphoric acid (H3PO4):

J.T.Baker, Mumbai supplied analytical grade (AR) of ortho-phosphoric acid was used

for the analysis.

Hydrochloric acid (HCl):

Rankem supplied hydrochloric acid was used for the analysis.

Tri-ethyl amine:

J. T. Baker, Mumbai supplied analytical grade (AR) of Tri-ethyl amine was used for

the analysis.

3.2 SOLVENTS

Acetonitrile (CH3CN):

HPLC grade solvent was used for the analysis, manufactured by Rankem and Merck

India Ltd.

Methanol (CH3OH):

HPLC grade solvent was used for the analysis, manufactured by Merck India Ltd and

Rankem Ltd.

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3.3 GLASSWARE:

Brand new of Borosil and Glaxo of class-A, grade glassware like pipette, volumetric

flask, conical flask, glass bottle, test tubes, measuring cylinder and glass beaker were used for

analysis.

3.4 STANDARDS:

Working standards of Fexofenadine hydrochloride and its impurity was used for this

study.

3.5 TEST SAMPLE (DOSAGE FORMS)

Finished products: Fexofenadine orally disintegrating tablet

3.6 EQUIPMENTS AND INSTRUMENTS

Ultra performance liquid chromatography (UPLC):

To develop and validate the chromatographic method Acquity UPLCTM was used. The

LC system has, a auto sampler. The signals recorded by Empower2 software.

Liquid Chromatography-Mass Spectroscopy (LC-MS):

To determine the mass by charge ratio of potential degradant in specificity (forced

degradation) experiment the LC-MS/MS system was utilized. Agilent make LC system which

is connected to Biosystem 4000 Q trap used for study.

Nuclear magnetic resonance (NMR):

The proton and carbon Nuclear magnetic resonance (NMR) experiment were

performed for potential degradant in drug product. Deuterated methanol used as solvent.

Frequency for 1H and 13C was 500 megahertz and 125 megahertz correspondingly applied.

Bruker make INOVA was used for the spectrum.Tetramethylsilane is used as internal

standard which chemical shift considered as Zero ppm.

Analytical balance:

Mettler Toledo make AB265-5/fact model is used for this research work.

Centrifuge:

Remi limited make centrifuge instrument is used for samples preparation.

pH Meter:

Mettler-Toledo, Switzerland make pH meter seven easy model is used to measure pH

of the solution for this work.

Sonicator:

Partech scientific suppliers manufactured 25 Liter sonicator and Life care

manufactured 10 Liter was used for analysis.

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Degasser:

Borosil manufactured glass degasser apparatus is used and Millipore 0.45 micron

filter paper is used for degassing.

Milli-Q water purification system:

Milli pore manufactured water purification system which is having 0.22µm membrane

filter is used for analysis.

Water bath:

Water bath, make Cintex, which is manufacture in India, was used for hydrolysis

study.

Photo stability chamber:

Photo-stability experiments were performed in this equipment which is from Sanyo,

Leicestershire, UK.

3.7 SCOPE OF THE PRESENT STUDY:

Keeping in view of the importance of active ingredients and related substances

(process and degradant related impurities) present in finished product to be monitored the

scope of the present study is designed. The present research work focuses on the

identification of an oxidative degradant impurity and the method development and validation

of quantification methods for active ingredients and its impurities present in drug products by

RP-UPLC which are sophisticated analytical techniques. FEX is an antihistamine active

substance utilized in the curing of allergy indication and fever urticaria. Terfenadine is

another choice of fexofenadine which is similar therapeutic effect. Fexofenadine used as an

antagonist to the H1 receptor.

The author selected the following active pharmaceutical drug substances and its

impurity for the present study. The Literature study reveals that no proposed validated test

procedure as available for separation and quantification of the below stated active ingredients

& its impurities present in pharmaceutical dosage forms by the proposed techniques.

Table- 3.7.a: Active Pharmaceutical Ingredients & Its Impurity Structures

3.8 INTRODUCTION:

Fexofenadine, structure is given in

HO

Fig-3.8.a.It shows Structure and Chemical name of fexofenadine hydrochloride

In the literature survey there were few HPLC assay and dissolution methods have

been given. Besides the reported literature for N

method as per our present knowledge no literature specifies about a new potential degradant

of fexofenadine as N-Oxide impurity in fexofenadine dosage form. This degradation product

was formed in formal stability study and forc

elucidated by advanced spectroscopic techniques ESI

indicating chromatographic method is not reported for this impurity. The current research

study explains about isolation and

developmeint and validatiron of selective RP

degradants of fexofenadine and other relate

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structure is given in Fig. 3.8.a.,

O

OH

N

O

OH H Cl

3.8.a.It shows Structure and Chemical name of fexofenadine hydrochloride


Besides the reported literature for N-oxide impurity of fexofenadine and selective


Oxide impurity in fexofenadine dosage form. This degradation product

was formed in formal stability study and forced degradation study and its structure was

elucidated by advanced spectroscopic techniques ESI-MS/MS,1H and 13C NMR. The stability


study explains about isolation and characterization of this impurity. It is also describes

on of selective RP-LC test procedure for the determination of

degradants of fexofenadine and other relatead substaances (Imp.-A, Imp.-B and Imp.

3.8.a.It shows Structure and Chemical name of fexofenadine hydrochloride


f fexofenadine and selective


Oxide impurity in fexofenadine dosage form. This degradation product

ed degradation study and its structure was

C NMR. The stability


characterization of this impurity. It is also describes

LC test procedure for the determination of

B and Imp.-C). The

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optimized liquid chromatography procedure method validation was performed for specificity,

linearity, precision, recovery, LOD, LOQ and robustness. Force degradation study in

different stress conditions was performed on placebo and finished dosage forms to ensure the

selectivity of the developed test procedure. These parameters were studied in accordance with

ICH guidelines [17-18].

3.9 OPTIMIZED TEST PROCEDURE

Chromatographic Conditions:

Column: 100 x 2.1 millimeter ,Waters Aquity BEH C18 column, 1.7

micron

Mobile phase:

Solvent-A: pH 7.00±0.05 of water adjusted with OPA with 0.05%TEA

Solvent-B: water: Acetonitrile (10:90)

Gradient program: 0-10min, sol-B: 25-25; 10-15min, sol-B: 25-35; 15-33min, sol-

B: 35-60; 33-35min, sol-B: 60-80; 35-36min, sol-B: 80-25 and

36-40min, sol-B: 25-25.

Flow rate: 0.40 Milliliter per minute

Wavelength: 220nm

Injection volume: 1.5μL

Column Temperature: 30°C

Diluent:

Diluent was 50% Acetonitrile and 50% water mixture with 1 mL of OPA.

System suitability Solution:

Transferred an accurately weighed amount of 60 mg fexofenadine hydrochloride WS

to a 50 milliliter flask and added 35 milliliter of diluent. This sample is sonicated to dissolve

the material totally and then added 1.2 milliliter of impurity solution (0.1 milligram per

milliliter solution of impurity A, B and C) and added diluent to the mark.

Standard solution:

960 μg/mL standard stock solutions were prepared in diluent. About 96 mg of

fexofenadine hydrochloride WS was taken to 100 milliliter flask. It is dissolved in diluent

with vigorous shaking and sonication. Above 5.0mL solution was diluted 20 times in 100mL

volumetric flask with diluent. Further this diluted 20 times in 100mL volumetric flask with

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diluent to obtain 2.4 ppm concentration. Five replicate standard injections were made to

determine system suitability.

Test solution preparation:

Weighed separately not less than 20 tablets of fexofenadine ODT and triturated in a

mortar to get fine and homogeneous sample. Separately weighed the powdered sample of

fexofenadine hydrochloride equivalent to 120mg and transferred in to 100 milliliter flasks,

sonicated for 25 minutes with intermediate shivering and sample were diluted volumetric

flask till mark with diluent, centrifuged the sample solutions at 10000 RPM for 10minutes.

Procedure:

Separately injected equal 1.5μL volume of blank, system suitability solution, standard

preparation, test solution in UPLC to record the chromatograms and measured the areas of

peaks in the chromatograms and calculated the results using below formula.

System Suitability:

Fexofenadine peak tailing in standard solution was NMT two, the USP plate count

was not more than 5000 for fexofenadine peak forstd preparation, The Resolution between

Impurity B peak and fexofenadine from system suitability preparation was NLT 3.0 and the

ratio of fexofenadine peak areas from two standard injections was between 0.9 to 1.1.

Calculation:

Percentage (%) of known of impurity =

At*WS*5*5*100*P*Average weight of tablet *100 As*100*100*100*WT*100*L*RRF

Here, “As” is the peak response of fexofenadine in STD. “At” is the peak response of

known impurity in sample preparation.“WT” is fexofenadine content weight taken for sample

preparation in milligram. “WS” is the fexofenadine hydrochloride standard weight taken for

standard preparation in milligram. “P” is fexofenadine potency as fexofenadine

hydrochloride. “L” is the Label claim of fexofenadine hydrochloride in mg per tablet and

''RRF'' is relative response factor of each know impurity.

Percentage (%) of unknown of impurity =

Au * WS * 5 * 5 * 100 * P * Average weight of tablet * 100 As * 100 * 100 * 100 * WT * 100 * L

Where in “As” is the peak response of fexofenadine in STD preparation. “Au” is the

peak response of unknown impurity in sample preparation. “WT” = fexofenadine content

weight taken for sample preparation in milligram. “WS” is the fexofenadine hydrochloride

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standard weight taken for STD preparation in milligram. “P” is fexofenadine potency as

fexofenadine hydrochloride and “L” is the Label claim of fexofenadine hydrochloride in mg

per tablet.

3.10 METHOD DEVELOPMENT:

IR Spectra:

Fexofenadine samples were separately scanned in FT-IR range from 4000 to 450cm-1

and the individual IR Spectra’s are represented as Spectrum- 3.10.a

Spectrum- 3.10.a: FTIR spectrum of Fexofenadine

Selection of wavelength:

Fexofenadine hydrochloride and its impurities were scanned across the UV range

from 200-400nm using 2996 PDA Detector. All Fexofenadine hydrochloride and its

impurities have UV maximum absorbance. Spectrum represents the absorption spectra which

are presented in Figure 3.10.a.

Fig 3.10.a:

The above UV spectra indicate that, fexofenadine an

Absorbance at 220 nm. The results were found satisfactory and reproducible at this

wavelength.

Selection of UPLC Column:

Preliminary phase of trial for method development started by isocratic method and

100mm x 2.1 millimeter Acquity BEH C8 which has particle size of 1.7µm column but Imp

B peak was merged with fexofenadine peak and baseline disturbance found. To resolve the

imp-B peak using same column, mobile phase used as per pH 3.0 chlorate buffer as sol

acetonitrile as sol-B with gradient elution experiment was done. To achieve resolution

column stationary phase changed to C18 and Ethylene bridged hybrid (

0.21centimeter) 1.7 micron column. Finally separation was achieved in sol

0.05% TEA, 7.0 pH with OPA and sol

column was proved for Reproducibility, and Ruggedness.

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Fig 3.10.a: UV spectrum of Fexofenadine and its impurities

The above UV spectra indicate that, fexofenadine and its impurities have maximum



Acquity BEH C8 which has particle size of 1.7µm column but Imp


B peak using same column, mobile phase used as per pH 3.0 chlorate buffer as sol

B with gradient elution experiment was done. To achieve resolution

column stationary phase changed to C18 and Ethylene bridged hybrid (

meter) 1.7 micron column. Finally separation was achieved in sol

pH with OPA and sol-B consists of 10 % H2O and 90% ACN mixture. This

column was proved for Reproducibility, and Ruggedness.

UV spectrum of Fexofenadine and its impurities

d its impurities have maximum



Acquity BEH C8 which has particle size of 1.7µm column but Imp-


B peak using same column, mobile phase used as per pH 3.0 chlorate buffer as sol-A and

B with gradient elution experiment was done. To achieve resolution

column stationary phase changed to C18 and Ethylene bridged hybrid (10centimeter x

meter) 1.7 micron column. Finally separation was achieved in sol-A which has

B consists of 10 % H2O and 90% ACN mixture. This

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Selection of Diluent:

Selection of diluent for fexofenadine and its impurity was a major challenge in the

method development as it is unreservedly soluble in CH3CH2OH and CH3OH, insoluble in

hexane & in water its slightly soluble. All of molecules are soluble in Acetonitrile and

Methanol and solubility is very less in water. Initially water and acetonitrile mixture was used

for dilution but because of peak shape of impurity 0.1% orthophosphoric acid added in

diluent. Finally, Acetonitrile and water in the proportion of 1:1, added 1 mL of ortho

phosphoric acid was used as diluents.

Selection of Run Time and Flow Rate:

With this developed method the retention times for fexofenadine and its impurities

were found within 40 min where mobile phase FR is 0.4 milliliter per minute. Above 0.4

milliliter per minute flow rates leads to early retention and merging of impurities it causes

quantification problems for fexofenadine impurities.

Selection of Standard and Sample Solution Concentration:

The standard concentration was based on 0.2% concentration of test concentration.

The sample concentration selected as 1.2 mg/mL to get LOD and LOQ of impurity below

reporting threshold as per ICH guideline.

Selection of Other Chromatographic Parameters:

The test procedure development was initiated with the prime focus on the resolution

of the impurities from main peak. The aim also predefined that less chromatographic time

non-polar Imp-C should properly elute. The solution having 1.2mg/milliliter of fexofenadine,

2.4 microgram/milliliter of all impurities was as used for development. A gradient method

used with sol-A which has 50 Millimolar NaH2PO4 + 10 millimolar NaoCl pH three and

100% of acettonitrile used as Sol-B. UPLC column was used as Ethylene bridged hybrid C8,

100 millimeter x 0.21 centimeter 1.7 micron column particle size. The FR was 0.5 milliliter

per minute on UPLC equipped with PDA detector. Imp-B peak was merged with

fexofenadine peak and at RT of Imp-C baseline disturbance found. To achieve resolution

column stationary phase changed to C18 and Ethylene bridged hybrid (10 centimeter x 0.21

centrimeter) 1.7 micron column. Finally separation was achieved in sol-A which has 0.05%

TEA, 7.0 pH with OPA and sol-B consists of 10 % H2O and 90% ACN mixture. After

altering of gradient, column and MP, all peaks were well resolved, satisfactory results were

obtained also confirm the column lot to lot variation. Based on above study below mentioned

method conditions were further optimized.

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Ethylene bridged hybrid C-18 (10 centimeter x 0.21 centrimeter) 1.7 micron column

was as UPLC column. Sol-A which has 0.05% TEA, 7.0 pH with OPA and sol-B consists of

10 % H2O and 90% ACN mixture used as mobile phase. The FR was 0.4 milliliter per min

with a gradient program (Time/%B) was set 0/25, 10/25, 15/35, 33/60, 35/80, 36/25 and

40/25. The column temp was kept at 30 degree centigrade. The output signals were recorded

at 220 nanometer. The vol of inj. was 1.5µL.

3.11 IDENTIFICATION AND CHARACTERIZATION OF OXIDATIVE

IMPURITY:

Semi-Preparative LC Parameter:

Isolation and resolution of degradant was performed on Inertsil octa decyl sillane 3V

(1 centimeter × 25 centimeter, particle size 5 μm) LC column. MP solvent sol-A which has

10 Milli molar ammonium formate solution and sol-B consists of 10 % 10 Milli molar

ammonium formate and 90% ACN mixture. The FR was 4.0 milliliter per minute. The

gradient (Time/%B) programmed 0/20, 10/35, 25/50, 35/90, 40/100, 60/100, 65/20 and 75/20.

The detection wavelength was 220 nanometer. The column oven temp was set at 25°C.

Liquid Chromatography-Mass Spectrometry (LC-MS) Conditions:

To establish the mass by charge ratio of potential degradant in specificity (forced

degradation) experiment the LC-MS/MS system was utilized. Agilent make LC system which

is connected to Biosystem 4000 Q trap used for study. The test procedure was optimized on

Zorbax Stable bond Phenyl, 25centimeter X 0.46 centimeter, five micron column. MP was

having a gradient program. MP solvent sol-A which has 10 Milli molar ammonium formate

solution and Sol-B consists of 10 % 10 Milli molar ammonium formate and 90% ACN

mixture. Both the solvent mixture was degassed before use. The gradient elution program

was set as (Time (min)/% sol-B) 0/30, 7/40, 20/50, 25/90, 27/100, 50/100, 52/30 and 60/30.

The FR was kept as 1 milliliter per min. The sample was injected as 20 micro liter. The

column oven temp was set as 25 degree centigrade. +ve ESI mode was used for analysis.

NMR Conditions

NMR and HRMS Conditions:

The proton and carbon Nuclear magnetic resonance (NMR) experiment were

performed for potential degradant in drug product. Deuterated methanol used as solvent.

Frequency for 1H and 13C was 500 megahertz and 125 megahertz correspondingly applied.

Bruker make INOVA was used for the spectrum.Tetramethylsilane is used as internal

standard which chemical shift considered as Zero ppm.

Identification of N-Oxide Impurity

During the drug product stability evaluation and specificity study, especially in

peroxide degradation experiment 0.71 RRT impurity was found potential. In order to enhance

the impurity, fexofenadine sample oxidation study was performed at 80

eight hours. The impurity has been found to enhance up to 65

impurity was performed on semi

impurity was and evaporated by using rotavapour with 40 d

resultant aqueous layer was lyophilized. The purity of the lyophilized impurity was checked

in HPLC and found highly pure. Further it is utilized to identify the impurity by advanced

technique.

The positive electro-spray ioniza

mass to charge ratio 518 amu [M+H]

fexofenadine. The m/z result reveals the existence of extra ‘O’ oxygen in potential degradant.

Employ HRMS found C32H39NO

Fig. 3.11.a. Mass spectrum of N

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xide Impurity



the impurity, fexofenadine sample oxidation study was performed at 80 degree centigrade for

eight hours. The impurity has been found to enhance up to 65-70%. The isolation of the

impurity was performed on semi-prep chromatographic system. Combined isolated portion of

impurity was and evaporated by using rotavapour with 40 degree centigrade vacuum. The



spray ionization mass spectrometer of potential degradant found

mass to charge ratio 518 amu [M+H]+ (Fig.3.11.a). This m/z is 16 amu additional than


NO5 as elemental composition of the potential degradant.

Fig. 3.11.a. Mass spectrum of N-oxide impurity



degree centigrade for

70%. The isolation of the

prep chromatographic system. Combined isolated portion of

egree centigrade vacuum. The



tion mass spectrometer of potential degradant found

(Fig.3.11.a). This m/z is 16 amu additional than


as elemental composition of the potential degradant.

(A)

Fig. 3.11.b. Fexofenadine (B)

Tab. 3.11.a.fexofenadine and N

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(B)

Fig. 3.11.b. Fexofenadine (B)andN-Oxide imp (A) Structural formula

Tab. 3.11.a.fexofenadine and N-oxide imp.NMR .

Structural formula

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3.12 METHOD VALIDATION:

The method validation of optimized test procedure was performed as per ICH

guidelines1. The method was validated by executing the studies for the parameter are,

selectivity, linearity, recovery, precision, and robustness.

System Suitability

This parameter was verified by test procedure characteristic criteria before initiate the

analysis which determination has been done base on specification. The SST was assessed

based on retention time, the resolution between impurity-B and fexofenadine peak, ratio of

fexofenadine peak areas from two standard injections, fexofenadine peak plate count and

asymmetric factor from STD preparation. SST was checked before analysis from two

standard injections which has 2.4 ppm fexofenadine. The acceptance criteria were ratio of

fexofenadine peak aireas should be between 0.9 and 1.1, tailing factor should be LT 2.0 &

theoretical plate should be MT 5000 for fexofenadine from STD. From system suitability

solution, the resolution between impurity B and fexofenadine peak should be not less than

3.0. All parameter are found to be meeting the SST specification. (Table-3.12.a).

Tab. 3.12.a. System Suitability Test Results

Specificity:

The selectivity of the proposed test procedure was verified for its performance and

monitored the resolution of fexofenadine all impurities from each other. In this experiment

also conducted to check the interference from placebo and degradation product at retention

time of fexofenadine and fexofenadine all impurities. Drug product and placebo was stressed

as per bellow mentioned conditions.

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The finished product was stressed for base hydrolysis with 2 Normal NaOH for 24 hrs

at 60 degree centigrade, acid hydrolysis with 1N HCl for 3.4 hrs at 60°C, Hydrolytic

degradation performed at 60°C for 24 hours. Oxidation was done with 3 % H2O2 at 60 degree

centigrade for five hours. The photo stability experiment was performed as per ICH Q1B,

exposed to visible light (1.2Million Lux hours) and ultra violet light (200 watt hours per

meter2)[19]. For humidity study sample were kept at 25 degree centigrade, 90 percent relative

humidity for 10 days. Heat stressed experiment done at 105 degree centigrade for one day.

Drug product and placebo sample were treated as same for above mentioned stress condition.

The fexofenadine peak purity was tested with photo diode array detector for degraded test.

The placebo interference was verified with the placebo preparation which is equivalent

amount present in sample. The result shows that there is purity is ok for its all related

substances.

Specificity study for placebo intereference had performed by injecting placebo

preparation as per methodology. There is no intereference found at RT of FEX and its

impurities. (Fig. 3.12.a). 1.2 milligram per milliliter FEX sample was injected in the UPLC

(PDA) to confirm FEX peak purity (Fig. 3.12.b-h). More degradation was found in peroxide

degradation and minor degradation was found in acid hydrolysis, alkali hydrolysis and heat

degradation. Majorly unknown impurity was found in oxidation condition forced degradation

.Oxidative potential degradant, proton and carbon NMR. FEX was observed to be non

degrdaing in water.The optimized test procedure mass balance was calculated for to ensure its

specificity by summation of degradation sample assay and impurities and results are more

than 98.4% (Table- 3.12.b). The result shows that there is found RT of all related

substances.and nature of the optimized test procedure.

Fig.3.12.a.: Representative Chromatogram of Placebo

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Tab.3.12.b. Summary of FDstudy

Fig.3.12.a.: Representative Chromatogram of Placebo

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Fig.3.12.b.: Representative Chromatogram of Acid Stressed Sample

Fig.3.12.c.: Representative chromatogram of Alkali Stressed Sample

Fig.3.12.d.: Representative Chromatogram of Oxidation Stressed Sample

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Fig.3.12.e.: Representative chromatogram of Thermal Stressed Sample

Fig.3.12.f.: Representative chromatogram of Aqueous stressed sample

Fig.3.12.g.: Representative Chromatogram of Photolytic Stressed Sample

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Fig.3.12.h.: Representative chromatogram of Humidity stressed sample

Fig.3.12.i.: Representative chromatogram of test spiked with its impurities.

Precision of the method:

This parameter was studied by repeatability and intermediate precision. The 6 each

sample of fexofenadine ODT spiked with its 4 impurities was made and injected in

repeatability study. Sample were prepared by impurity -B and N-oxide at specification limit,

impurity A specification limit and imlimit purity C (decarboxylated impurity) specification

limit (% of impurities respecting 1.2 mg/milliliter FEX) spiking. %RSD of area for eaach

imapurity was calculeted. The test procedure intersmediate precission study were assessed by

differenet analyset, instrument and day.

Based on outcome of the study it is found that proposed test procedure is very precise.

The % RSD for the area of Impurity-A, B, C and N-oxide was maximum 3.0% and 3.6% in

repeatability and intermediate precision correspondingly. The results for each impurity are

tabulated in Table-3.12.a and 3.12.b.

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Table 3.12.a : Precision Result of Fexofenadine Impurities

Table 3.12.b: Intermediate Precision Result of Fexofenadine Impurities

Limits of Detection and Quantification of Method

The LOD and LOQ for fexofenadine HCl related substance were established by S/N

ratio which should be 3:1 and 10:1, respectively. At LOQ level six preparation fexofenadine

HCl impurities were prepared to perform the precision study. The results were interpreted in

the %RSD of the peak response. The LOD, LOQ in Table-3.12.c and Table-3.12.d.

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Table 3.12.c : LOD and LOQ Value with S/N Ratio of Fexofenadine Impurity

Table 3.12.d : Fexofenadine impurities LOQ precision

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Linearity of the Method

To perform this parameter of the proposed test procedure 6 different parts per million

solutions were prepared. The series of level covers the LOQ to 200% of specification level. A

good correlation found from responses of peak Vs standard concentrations. It was achieved in

the range of calibration, i.e. Limit of detection to 200 percent of target level. The R was more

than 0.998 and percent bias at 100 percent concentration was ± 2 percent for all four

Impurities. The results demonstrate the exceptional correlation of area of peak and concen of

Imp- A, Imp- B, Imp- C, and N- Oxide. The R2, c and y-intercept are summarized in

presented in Fig.3.12.j-m.

Fig.3.12.j.: Linearity graph for N-Oxide impurity

Fig.3.12.k.: Linearity graph for impurity-A

y = 5815x + 361.7

R² = 0.999

0

10000

20000

30000

40000

50000

0 2 4 6 8 10

Are

a

Concentration (µg/mL)

N-Oxide

y = 6080.x + 13.66

R² = 0.999

0

10000

20000

30000

40000

50000

0 2 4 6 8

Are

a


Impurity-A

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Fig.3.12.l.: Linearity graph for impurity-B

Fig.3.12.m.: Linearity graph for impurity-C

Accuracy

The recovery parameter of impurity was performed in three sample preparation at

Limit of quantification to 50 percent, 75percent, 100percent, 125percent and 150percent of

targeted concen level. All impurities % accuracy was calculated. The % accuracy of

impurities of FEX tests varies from 92.6 to 108.6% at limit of quantification, 50percent,

75percent, 100percent, 125percent and 150percent levels of test concentration. The Liquid

chromatography chromatogram is shown in figure Fig. 3.12.i. with spiked test at target

concentration of all impurities in FEX. Individual % accuracy results for impurities are

tabulated in Table-3.12.e to Table-3.12.i.

y = 5118.x + 249.3

R² = 0.998

0

2000

4000

6000

8000

10000

12000

14000

16000

0 0.5 1 1.5 2 2.5 3

Are

a


Impurity-B

y = 6830.x - 478.9

R² = 0.999

0

5000

10000

15000

20000

25000

30000

0 1 2 3 4 5

Are

a


Impurity-C

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Tab.3.12.e. Recovery Data of N-oxide Impurity

Tab.3.12.f. Recovery Data of Impurity-A Impurity

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Tab.3.12.g. Recovery Data of Impurity-B Impurity

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Tab.3.12.h. Recovery Data of Impurity-C Impurity

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Tab.3.12.i. Recovery Data of Impurity at LOQ Level

Robustness

The optimized test procedure robustness was verified by deliberate change in

predefined method parameter. In this experiment response measured as resolution of

fexofenadine and impurity-B and other SST parameter. MP buffer pH was altered to 6.8

and7.2 unit. The FR was changed to 0.36 and 0.44 milliliter per minute to check SST

performance. The column oven temp was altered to 25°C and 35°C in robustness study.ACN

proportion was changed to 90% -110% to check its effect on resolution and SST criteria.

In every the deliberate changes of chromatographic parameter, the results were met

the specification criteria for system suitability. The impurities and main peak are separated

well and all peaks are retained as per RRT. The FEX and impurity-B resolution was found to

be more than 5.4.The FEX tailing was observed 1.0 and theoretical plate count found more

than 11434 in STD injection chromatogram. The similarity factor of peak response was 1.0.

The system suitability parameters evaluated are shown in Table-3.12.j.

Tab. 3.12.j. UPLC Method

Stability in Solution and in the Mobile Phase

To study this parameter FEX sample with spiked all impurities were kept on bench

top for 2 days in closed container. Similar process was followed for STD preparation. Every

day all impurity level and STD preparation similarity factor was evaluated with new

preparation. The same process was followed to established mobile phase stability for 2 days

where every day sample spiked with all impurities were evaluated against old mobile phase.

The result shows that mobile phase is stable up to 2 days. The FEX

with impurities result is not varying more than 10%. Hence, samples are stable up to 2 days.

The similarity factor of new and old prepared standard is within the specification limit.

Therefore, STD is also stable up to 2 days on room temperat

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Tab. 3.12.j. UPLC Method - Robustness Data

Stability in Solution and in the Mobile Phase



day all impurity level and STD preparation similarity factor was evaluated with new



The result shows that mobile phase is stable up to 2 days. The FEX



Therefore, STD is also stable up to 2 days on room temperature.



day all impurity level and STD preparation similarity factor was evaluated with new STD



The result shows that mobile phase is stable up to 2 days. The FEX sample spiked



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The results (precision, specificity, linearity, accuracy, robustness and ruggedness) of

the present study indicate that the developed and validated methods are reproducible, rugged

and stability indicating.

13 chapter-iii fexofenadine odt method development and method...

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