Presented by

Ms. P.Tejaswini
(Reg. No. 167N1S0408)
Under the Guidance of
Dr. M. NARENDER., M. Pharm., Ph.D.,
Associate Professor,
Department of Pharmaceutical Analysis and Quality Assurance

VIJAYA INSTITUTE OF PHARMACEUTICAL SCIENCES FOR WOMEN

Enikepadu, Vijayawada
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 Introduction.
 Aim and objective.
 Literature review.
 Materials and methods.
 Results and discussion.
 Summary and conclusion.
 References.

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 Validation of an analytical procedure is the process by which it is established, by
laboratory studies, that the performance characteristics of the procedure meet the
requirements for its intended use.

 There are many reasons for the need to validate analytical procedures. Among them
are regulatory requirements, good science, and quality control requirements.

Internal standard(IS):

 Internal standard involves the comparison of the instrument responses from the
target compounds in the sample to the responses of reference standards added to
the sample or sample extract before injection.

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 Selection of Internal standards:

 Internal standards that are similar in analytical behaviour to the compounds of

interest, and not expected to be found in the samples.

 The analyst needs to demonstrate that the measurement of the internal standard is
not affected by target analytes, surrogates, or by matrix interferences.

 This is not as useful for GC and HPLC methods with non-MS detectors, unless the
internal standards could be separated from target compounds chromatographically.

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AIM:
 In the present study, an attempt was made to provide a simple and accurate RP-HPLC
method for Fexofenadine (FEXO)in bulk and pharmaceutical formulations using an
Internal Standard(IS)method and further the method to be validated according to ICH
(Q2R1) guidelines.
Plan of work:
Step-1:
 Development of simple RP-HPLC method for quantitative estimation of Fexofenadine
by using levocetirizine as internal standard.
Step-2:

 Validation of the developed RP-HPLC method in accordance with

the analytical validation parameters such as system suitability, specificity, linearity,
accuracy, precision, robustness, limit of detection and limit of quantitation, etc,.
as per ICH (Q2R1)guidelines.
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Chromatography:

• Chromatography may be defined as method of separating a mixture of components into

individual components through equilibrium distribution between two phases.

High performance liquid chromatography(HPLC):

• HPLC is a different branch of column chromatography in which the mobile phase is

forced through the column at high speed.

Principle :

• Solute molecule having more affinity towards the stationary phase elutes later and lesser
affinity gets eluted faster.

Some of the advantages are:

 Speed analysis can be accomplished within 20 mins or less, greater sensitivity, improved
resolution of separation.
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 Reverse phase Normal phase

 Normal phase (NP-HPLC): This method is polar stationary phase and non-polar
mobile phase. Therefore the stationary phase is usually silica and typical mobile
phases are hexane, methylene chloride, chloroform, diethyl ether, and mixtures of
these solvents. Polar samples are thus retained on the polar surface of the column
material for packing longer time than less polar materials.

 Reverse phase (RP-HPLC): In this method stationary phase is non-polar

(hydrophobic) in nature, while the mobile phase is a polar, such as water and
methanol or ACN or their mixtures. It works based on the principle of hydrophobic
interactions solute molecules, hence the more non-polar the material stays longer
time in column material and elutes slowly.
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Schematic Diagram of HPLC

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 Author Year work Condition
Barabde et.al., 2015 Reported a simple and C18 column, Methanol:
accurate RP-HPLC method for water(60:40% V/V), flow rate
estimation of FEXO in tablet at 1.0 mL/min.
dosage form.
Prathyusha 2015 RP-HPLC method for Hypersil ODS C18 column,
et.al., simultaneous estimation of Buffer: acetonitrile (56:44 %
ambroxyl hydrochloride and V/V), flow rate at 1 mL/min.
fexofenadine in bulk and
dosage form.
Pallavi et.al., 2015 HPTLC method for estimation Aluminium plates 60 F 254
of FEXO as in bulk drug and used as stationary phase with
in dosage form thickness of 250 µm. mobile
phase was toluene: ethyl
acetate: ammonia (30 %)
(0.5:73:0.6%V/V/V),at220 nm.
Miura et.al., 2007 RP-HPLC method for Chiral CD-Ph column mobile
determination of FEXO phase of 0.5 % KH2PO4: ACN
enantiomers in human plasma. (65: 35 % V/V), at 220 nm.
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 Author Year work
Guo, et al., 2010 Simple and rapid LC-MS/MS
method for quantification of
FEXO in human plasma by
using Glipizied as IS
Pathak, et al., 2008 RP-HPLC with fluorescence
detector for estimation of
FEXO in rat plasma by
application to pre-clinical
pharmacokinetics by using
Diphenhydramine as IS
Nirogi, et al., 2007 RP-HPLC method with RP-column, with a flow rate of
electrospray tandem mass 0.5 mL/min.
spectroscopy for the validation
of FEXO in human plasma by
using Mosapride as IS
Ravi Shankar, et 2012 RP-HPLC for simultaneous
al., estimation of cetirizine and
FEXO with Montileukast in
bulk and in pharmaceutical
dosage form.
Condition
C18 (100× 2.1 mm, 5µ)
methanol: ammonium acetate
buffer (70:30 % V/V) at flow
rate of 1 mL/min.
Supelco C18 column (250 ×
4.6 mm, 5µ) mobile phase
ammonium acetate: ACN
(63:37 % V/V) at a flow rate
of 1 mL/min.
watery symmetry (250
mm×4.5 mm, 5 µ) 0.05M
potassium dihydrogen ortho-
phosphate: ACN (35:65 %
V/V) flow rate of 1 mL/min.10
 Fexofenadine
 Chemical structure:

 Chemical name: (±)-4-[1-Hydroxy-4-[4-(hydroxydiphenylmethyl)-1-piperidin-yl]-

butyl] -α, α-dimethyl benzene acetic acid.

 Molecular formula: C32H39NO4.

 Molecular weight: 501.68 g/mL.

 Solubility: Soluble in methanol, slightly soluble in water.

 Absorption: Oral absorption.

 Mechanism of action: Peripheral H1-blocker.

 Excretion: Excreted through feaces (80%) and urine (11-12%).

 Side effects: The most common side effects are anxiety, insomnia and miosis.
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 Levocetirizine

 Chemical structure:

 Chemical name: 2-(2-{4-[(R)-(4-Chlorophenyl)(phenyl)methyl]piperazin-1-yl}ethoxy)

acetic acid.
 Molecular formula: C21H25C1N2O3.
 Molecular weight: 388.888 g/mol.
 Solubility: Soluble in methanol and slightly soluble in water.
 Absorption: Oral absorption.
 Mechanism of action: LEVO acts as an inverse agonist that decreases activity
at histamine H1-receptors.
 Excretion: Excreted trough urine.
 Side effects: sleepiness, headache, mouth dryness, vision problems, palpitations and
fatigue.

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 Cyber lab HPLC system accomplished with UV-detector, Quantitative HPLC was
performed on an isocratic mode using Cap Cell Pack C18 column with 20 μL
injection of sample loop. The output signal was monitored and integrated using
Cyber lab LC 100 software.

 Selection of UV detection wavelength:

By scanning the standard solution of the drugs over the wavelength region of 200 to
400 nm using UV-Vis Spectrophotometer.

 Preparation of standard solution of FEXO by using IS: Accurately weigh 10 mg

of FEXO in 10 mL volumetric flasks, add 5 mL of diluent and dissolve the contents
and make up the volume to 10 mL. From this solution pipette out 1 mL of solution
into another 10 ml volumetric flask and add 5 mL of diluent. To this an equal
volume of IS (50 µg/mL) is added.
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RP-HPLC method development (Trials):
Aliquots of the mixed solutions containing FEXO and IS were prepared and a number
of trials (trails 1- 4) were performed.
Trail-1 Trail-2
Methanol: ortho-phosphate buffer (80:20 % V/V). ACN: ortho-phosphate buffer (60:40 %V/V).

Trail-3 Trail-4
ACN: ortho-phosphate (pH 4.8) (50:50 % V/V). ACN: water (70:30% V/V).

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OPTIMIZED METHOD:
• Preparation of mobile phase: A combination of acetonitrile(ACN) and water
(50:50 % V/V) was prepared, degassed with ultra sonication for about 15 min. The
resultant solution was filtered through 0.45 µ membrane filter.
• Diluent: mobile phase is used as diluent.
Experimental condition:
Parameters Chromatographic conditions
Column Cap cell pack C18 (250×4.6nm, 5µ) column
Elution mode Isocratic
Mobile phase ACN: water (50:50 % V/V)
Flow rate 1.0 mL/min
Detection wavelength 224 nm
Injection volume 20 µL
Run time 10 min
Temperature 28 oC
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Method validation:

 Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use. Results from method
validation can be used to judge the quality, reliability and consistency of analytical
results. The method was validated for different parameters like system suitability,
specificity linearity, accuracy, precision, robustness, LOD and LOQ as per ICH Q2
(R1) guidelines.

 System suitability: System suitability testing is an integral part of many analytical

procedures. The tests are based on the concept that the equipment, electronics,
analytical operations and samples to be analyzed constitute an integral system that
can be evaluated as such.

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 Linearity: The linearity of an analytical procedure is its ability (within a given
range) to obtain the test results which are directly proportional to the concentration
(amount) of analyte in the sample.
 Accuracy:The accuracy of an analytical procedure expresses the closeness of
agreement between the values which is accepted either as a conventional true value
or an accepted reference value and the value found.
 Precision: Precision of an analytical procedure expresses the closeness of
agreement (degree of scatter) between a series of measurements obtained from
multiple sampling of the same homogeneous sample under the prescribed
conditions.
 System precision: Standard solution prepared as per test method and injected six
times.
 Method precision: Prepared six sample preparations as per test method and
injected each solution.

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 Robustness: Robustness of the method was evaluated by assaying test solutions
under slight but deliberate changes in analytical conditions, such as change in flow
rate and change in wave length .

 Flow rate change: In this experiment the flow rate has changed as 0.8 ml/min, 1
ml/min and 1.2 ml/min.

 Wavelength change: In this experiment the test samples were analyzed at the
wavelength of 222 nm, 224 nm and 226 nm.

 Limit of detection (LOD): The parameter LOD was determined on the basis of
height of the signal and noise of the response.

LOD = 3.3 × D/S

Where,

D = Standard deviation of the y-intercepts of regression line and

S = The slope of the calibration curve.

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 Limit of Quantification(LOQ):

The parameter LOQ was determined on the basis of height of the signal and noise of

the response.

LOQ = 10 × D/S

Where,

D = standard deviation of the y-intercepts of regression line and

S = the slope of the calibration curve.

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 The chromatographic conditions were optimized to develop assay method for
Fexofenadine and IS dosage forms.

Optimized method :

Parameters Chromatographic conditions

Column Cap cell pack C18 (250×4.6nm, 5µ) column

Elution mode Isocratic
Mobile phase ACN: water (50:50 % V/V)
Flow rate 1.0 mL/min
Detection wavelength 224 nm
Injection volume 20 µL
Run time 10 min
Temperature 28 oC

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 Optimized chromatogram of proposed method
Assay:

HPLC chromatogram of standard mixture HPLC chromatogram of sample mixture

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 Assay results for optimized method

Formulation Lable claim %Amount found

amount (mg) (n=6)
Allerga 120 101.2%

 System suitability: System suitability test were found to be within limits for
Fexofenadine (FEXO) and IS.

System suitability parameters of optimized method

Parameters FEXO IS
Peak area 78673.2 54823.2
Retention time (min) 4.79 6.22
Tailing Factor 1.58 1.45
Theoretical plate number (N) 8413 10036
Height 75840 40521

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 Specificity

Chromatogram of standard
(Rt- 4.7, 6.2).

Chromatogram of sample
(Rt- 4.7, 6.2).

Chromatogram of blank

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Linearity:
Chromatogram of FEXO (50 µg/mL) and IS (50 µg/mL)

Chromatogram of FEXO (75 µg/mL) and IS (50 µg/mL)

Chromatogram of FEXO (100 µg/mL) and IS (50 µg/mL)

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Chromatogram of FEXO (100 µg/mL) and IS (50 µg/mL)

Chromatogram of FEXO (150 µg/mL) and IS (50 µg/mL)

Chromatogram of FEXO (175 µg/mL) and IS (50 µg/mL)

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S.No Concentration Peak area ratio Linear regression
(µg/mL) (drug/IS) Equation
FEXO IS
1 50 50 0.715
2 75 50 0.987
3 100 50 1.256 y = 0.011x+ 0.168
4 125 50 1.589 r2 = 0.997
5 150 50 1.791
6 175 50 2.081
CALIBRATION CURVE OF FEXOFENADINE
2.5
y = 0.011x + 0.168
2 r² = 0.997
Peak area ratio

1.5

0.5

0
0 50 100 150 200
Concentation (µg/mL)

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 Accuracy:
Chromatograms of 50% Standard addition sample

Chromatograms of 100% Standard addition sample

Chromatograms of 150% Standard addition sample

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S.NO Spiked Sample Sample % % mean
level area height recovery
1 50% 78697.2 39789 101.5
50% 78786.2 39651 101.5 101.3%
50% 78564.1 39845 101.1
2 100% 157394.4 79578 101.5
100% 158412.6 79268 101.6 101.5%
100% 153212.1 79155 101.5
3 150% 235091.6 119367 101.5
150% 235092.5 115427 100.7 101.3%
150% 238521.6 112789 101.7

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 Precision:
 System precision:

Chromatograms of system precision (Injection -1)

Chromatograms of system precision (Injection -2)

Chromatograms of system precision (Injection -3)

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Chromatograms of system precision (Injection-4)

Chromatograms of system precision (Injection-5)

Chromatograms of System Precision (Injection-6)

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Injection Retention time Peak area ratio Height of peak
(nm) (FEXO/IS)
1 4.7 1.752 75976
2 4.7 1.782 75642
3 4.7 1.756 75812
4 4.7 1.768 75954
5 4.7 1.781 75816
6 4.7 1.768 75819
Mean 4.7 1.761 75836

S.D - 0.004 -

%RSD - 0.227 -

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 Method precision:

Chromatograms of method Precision (Injection -1)

Chromatograms of method precision (Injection -2)

Chromatogram of method precision (Injection- 3)

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Chromatogram of Method Precision (Injection -4)

Chromatogram of method precision (Injection -5)

Chromatograms of Method Precision (Injection -6)

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Injection Retention time (min) Peak area ratio
(FEXO /IS)
1. 4.7 1.862
2. 4.7 1.873
3. 4.7 1.806
4. 4.7 1.823
5. 4.7 1.864
6. 4.7 1.887
Mean 4.7 1.852
S.D - 0.012
%RSD - 0.647

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 Robustness:
 Flow rate:

Chromatogram of robustness variation in flow rate (0.8 mL/min) low(Rt- 5.5, 7.1)

Chromatogram of robustness variation in flow rate (1 ml/min) original(Rt- 4.7, 6.2)

Chromatograms of robustness variation in flow rate (1.2 mL/min) high (Rt- 4.1, 5.4)

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 Effect of wavelength:
Chromatogram of robustness variation in wavelength (222 nm) low(Rt- 4.8, 6.3)

Chromatogram of robustness variation in wavelength (224 nm) original(Rt- 4.7, 6.2)

Chromatograms of robustness variation in wavelength (226 nm) high(Rt- 4.8, 6.2)

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Parameter Changes Peak area Peak area Mean SD %RSD
in the ratio ratio peak area
chromatog 1(Drug/IS) 2(Drug/IS) ratio
raphic
conditions

Flow rate 0.8 (low) 1.987 1.976 1..981 0.008 0.403

mL/min
1.0 1.752 1.768 1.760 0.004 0.227
(medium)

1.2 (high) 1.623 1.652 1.637 0.002 0.122

Wave 222 (low) 2.064 2.052 2.069 0.010 0.338
length
224 1.986 1.952 1.969 0.014 0.711
(nm)
(medium)

226 (high) 1.624 1.653 1.638 0.017 1.037

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 LOD:

Chromatogram of LOD

S.NO Drug Peak area LOD

(µg/mL)
1 FEXO 25643.1 1.0

2 IS 17643.6 1.0

Chromatogram of LOQ

S.NO Drug Peak LOQ(µg/mL)

area
1 FEXO 7256.2 3.0
2 IS 3781.5 1.5 38
 Results of method validation

Method validation Results Acceptance limit

parameters
Specificity - No interference
Linearity r2 = 0.997 <1
Accuracy 100.3, 100.5, 101.3 % 98-102 %
Precision 0.227 RSD < 2%
Robustness < 1% RSD < 2%
LOD 0.27 µg/mL Signal to noise ratio
should be more
than 3:1

LOQ 0.84 µg/mL Signal to noise ratio

should be more
than 10:1

Assay 101.2% 98-102%

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 In present work a new RP-HPLC method has been developed for estimation of FEXO
by using LEVO as an IS, where a well resolved peak was observed for IS from that of
analyte. A reverse phase Cap Cell Pack C18 column (250×4.6mm, 5µ) with mobile
phase consisting of ACN and water (50:50 % V/V) was used. The flow rate was 1
mL/min and the elution was monitored at 224 nm.
 The development method was found to satisfactory with good precision, linearity
and accuracy. It was validated according to ICH guidelines and all the results
present within the specified limits.
 Hence the proposed new RP-HPLC method was found to be simple, accurate,
economical, rapid and can be applied for routine analysis in quality control of
FEXO in its pharmaceutical dosage forms.

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 Barabde, G. R; Ambadekar, S. R; Anil, R. “Comparative study of estimation of
fexofenadine hydrochloride by UV-Visible spectrophotometry and HPLC method”.
Journal of Research in Pharmaceutical Science, 2016, 2 (12): 2347-2995.

 Chandrasekhar, J; Karvekar, M. D; Somashekar, P. L. “Development and validation

of HPLC method for estimation of granisetron in dosage form using ondansetron as
internal standard”. International journal of pharmaceutical research and
development, 2012, 4(03): 125-128.

 Guo, D; Zou, J; Zhu, Y. “Measurement of fexofenadine concentration in micro-

sample human plasma by a rapid and sensitive LC-MS/MS employing protein
precipitation: application to a clinical pharmacokinetic study”. Biomedical
Chromatography, 2010, 24: 335–341.

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 Kumudavalli, M.V; Jayakar, B; Kumar, M; Palanisamy, P. “RP-HPLC method
development and validation for estimation of fexofenadine in rat plasma and its
applications for pharmacokinetic studies”. International Research Journal of
Pharmacy, 2014, 5(5): 418-426.

 Moin, S; Chandrasekhar, J; Karvekar, M. D; Somashekar P. L. “Development and

validation of HPLC method for estimation of granisetron in dosage form using
Ondansetron as internal standard”. International journal of pharmaceutical
research and development, 2012, 4(3): 125 –128.

 Beckket, H; Stenlake, J. B. “Practical Pharmaceutical Chemistry”, 4th Ed., part two,

C.B.S. publications, New Delhi,1997.

 Chatwal, R. G; Anand, K. S. High performance liquid chromatography,

instrumental methods of chemical analysis, 5th ed., Himalaya publishers, Mumbai,
2010.

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 Miura, M; Uno, T; Tateishi, T; Suzuki, T. “Determination of fexofenadine
enantiomers in human plasma with high-performance liquid
chromatography”. Journal of Pharmaceutical and Biomedical Analysis,
2007, 43: 741–745.
 Nimje, H. M; Shital, T; Nimje, R. J; Oswal, S. “Stability indicating RP-HPLC
method for estimation of fexofenadine hydrochloride in pharmaceutical
formulation”. E-Journal of Chemistry 2012, 9(3): 1257-1265.

 https://en.wikipedia.org/wiki/Fexofenadine.
 https://en.wikipedia.org/wiki/Levocetirizine.

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