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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
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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:
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:
Principle :
• Solute molecule having more affinity towards the stationary phase elutes later and lesser
affinity gets eluted faster.
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.
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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 Condition
Guo, et al., 2010 Simple and rapid LC-MS/MS C18 (100× 2.1 mm, 5µ)
method for quantification of methanol: ammonium acetate
FEXO in human plasma by buffer (70:30 % V/V) at flow
using Glipizied as IS rate of 1 mL/min.
Pathak, et al., 2008 RP-HPLC with fluorescence Supelco C18 column (250 ×
detector for estimation of 4.6 mm, 5µ) mobile phase
FEXO in rat plasma by ammonium acetate: ACN
application to pre-clinical (63:37 % V/V) at a flow rate
pharmacokinetics by using of 1 mL/min.
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 watery symmetry (250
al., estimation of cetirizine and mm×4.5 mm, 5 µ) 0.05M
FEXO with Montileukast in potassium dihydrogen ortho-
bulk and in pharmaceutical phosphate: ACN (35:65 %
dosage form. V/V) flow rate of 1 mL/min.10
Fexofenadine
Chemical structure:
Side effects: The most common side effects are anxiety, insomnia and miosis.
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Levocetirizine
Chemical structure:
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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.
By scanning the standard solution of the drugs over the wavelength region of 200 to
400 nm using UV-Vis Spectrophotometer.
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.
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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.
Where,
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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,
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The chromatographic conditions were optimized to develop assay method for
Fexofenadine and IS dosage forms.
Optimized method :
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Optimized chromatogram of proposed method
Assay:
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Assay results for optimized method
System suitability: System suitability test were found to be within limits for
Fexofenadine (FEXO) and IS.
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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)
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Chromatogram of FEXO (100 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
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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:
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Chromatograms of system precision (Injection-4)
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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:
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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)
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)
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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
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LOD:
Chromatogram of LOD
2 IS 17643.6 1.0
Chromatogram of LOQ
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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.
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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.
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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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